Coronavirus vaccine

Abstract

This disclosure relates to the field of RNA to prevent or treat coronavirus infection. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infection and inducing effective coronavirus antigen-specific immune responses such as antibody and/or T cell responses. Specifically, in one embodiment, the present disclosure relates to methods comprising administering to a subject RNA encoding a peptide or protein comprising an epitope of SARS-CoV-2 spike protein (S protein) for inducing an immune response against coronavirus S protein, in particular S protein of SARS-CoV-2, in the subject. i.e., vaccine RNA encoding vaccine antigen.

Claims

1. A pharmaceutical composition comprising an RNA wherein said RNA comprises, from a 5 to 3 direction: (i) a 5 UTR that comprises a human alpha-globin 5-UTR; (ii) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 9, wherein said nucleotide sequence: (a)includes modified uridines in place of uridines; and (b) encodes a SARS-CoV-2 Spike (S) polypeptide that is at least 95% identical to SEQ ID NO: 7 and is prefusion stabilized in that it includes at least one proline substitution at position(s) corresponding to position(s) 986 and/or 987 in SEQ ID NO: 7; and (iii) a 3-UTR that comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

2. The pharmaceutical composition of claim 1, wherein the RNA comprises a 5-cap that comprises m2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG.

3. The pharmaceutical composition of claim 1, wherein the RNA comprises a polyA sequence, wherein the polyA sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 14, which optionally comprises a modified uridine in place of one or more uridines.

4. The pharmaceutical composition of claim 3, wherein the 5-UTR comprises a sequence that is at least 90% identical to SEQ ID NO: 12, which optionally comprises a modified uridine in place of one or more uridines.

5. The pharmaceutical composition of claim 4, wherein the 3-UTR comprises a sequence that is at least 90% identical to SEQ ID NO: 13, which optionally comprises a modified uridine in place of one or more uridines.

6. The pharmaceutical composition of claim 1, wherein the RNA includes modified uridines in place of all uridines.

7. The pharmaceutical composition of claim 6, wherein the modified uridines are each N1-methyl-pseudouridine.

8. The pharmaceutical composition of claim 1, wherein the nucleotide sequence of (ii) is characterized in that its G/C content is increased by about 55% as compared to the G/C content of the nucleotide sequence of SEQ ID NO: 2.

9. The pharmaceutical composition of claim 1, wherein the RNA is formulated in lipid nanoparticles comprising: (A) a cationically ionizable lipid; (B) a sterol; (C) a neutral lipid; and (D) a polymer conjugated lipid.

10. The pharmaceutical composition of claim 9, further comprising at least one salt and/or a cryoprotectant.

11. The pharmaceutical composition of claim 10, wherein the cryoprotectant comprises sucrose.

12. The pharmaceutical composition of claim 9, wherein the composition is formulated for intramuscular administration.

13. The pharmaceutical composition of claim 9, wherein the RNA is present in the composition in an amount of about 3 ?g per dose, about 10 ?g per dose, or about 30 ?g per dose.

14. The pharmaceutical composition of claim 1, wherein the RNA comprises a polyA sequence, wherein the polyA sequence comprises at least 100 adenine nucleotides.

15. The pharmaceutical composition of claim 1, wherein the modified uridines are each N1-methyl-pseudouridine.

16. A method of vaccinating by administering a pharmaceutical composition comprising an RNA, wherein said RNA comprises, from a 5 to 3 direction: (i) a 5-UTR that comprises a human alpha-globin 5-UTR, (ii) a nucleotide sequence that is at least 90% identical to SEQ ID NO: 9, wherein said nucleotide sequence: (a) includes modified uridines in place of uridines; and (b) encodes a SARS-CoV-2 Spike (S) polypeptide that is at least 95% identical to SEQ ID NO: 7 and is prefusion stabilized in that it includes at least one proline substitution at position(s) corresponding to position(s) 986 and/or 987 in SEQ ID NO: 7; and (iii) a 3-UTR that comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.

17. The method of claim 16, wherein the step of administering comprises administering more than one dose of the pharmaceutical composition.

18. The method of claim 17, wherein the more than one dose comprises a first dose and at least a second dose, and wherein the first and second doses are spaced apart by a time period of about 21 days.

19. The method of claim 16, wherein the step of administering comprises administering at least one dose of the pharmaceutical composition, each of which independently comprises RNA in an amount within a range of about 1 ?g to about 100 ?g.

20. The method of claim 16, wherein the step of administering comprises administering at least two doses of the pharmaceutical composition, each of which comprises about 30 ?g of the RNA.

21. The method of claim 16, wherein the RNA comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 20.

22. The method of claim 21, wherein the RNA includes modified uridines in place of all uridines.

23. The method of claim 22, wherein the modified uridines are each N1-methyl-pseudouridine.

24. The method of claim 16, wherein the modified uridines are each N1-methyl-pseudouridine.

25. The method of claim 16, wherein the nucleotide sequence of (ii) is SEQ ID NO: 9.

26. The method of claim 16, wherein the step of administering comprises administering at least one dose of the pharmaceutical composition, each of which independently comprises RNA in an amount within a range of about 10 ?g to about 30 ?g.

27. The method of claim 16, wherein the administration is by intramuscular administration.

28. The method of claim 16, wherein the RNA comprises: (A) a 5-cap that comprises m.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG; or (B) a polyA sequence comprising 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence; or (C) a combination of (A) and (B).

29. The method of claim 16, wherein the RNA is formulated in lipid nanoparticles comprising: (A) a cationically ionizable lipid; (B) a sterol; (C) a neutral lipid; and (D) a polymer conjugated lipid.

30. The method of claim 28, wherein the step of administering comprises administering at least two doses of the pharmaceutical composition.

31. The method of claim 30, wherein the step of administering comprises administering a subsequent dose of the pharmaceutical composition after the second dose, wherein the subsequent dose is administered at least about 3 months after administering the second dose.

32. The method of claim 16, further comprising administering a second vaccine.

33. The method of claim 32, wherein the second vaccine is an influenza vaccine.

34. A method of manufacturing the pharmaceutical composition of claim 1, comprising combining the RNA with a cationically ionizable lipid, a sterol, a neutral lipid, and a polymer conjugated lipid to form lipid nanoparticles that encapsulate the RNA.

35. The method of claim 34, wherein the RNA comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 20.

36. The method of claim 35, wherein the nucleotide sequence includes modified uridines in place of all uridines.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Schematic overview of the S protein organization of the SARS-CoV-2 S protein.

(2) The sequence within the S1 subunit consists of the signal sequence (SS) and the receptor binding domain (RBD) which is the key subunit within the S protein which is relevant for binding to the human cellular receptor ACE2. The S2 subunit contains the S2 protease cleavage site (S2) followed by a fusion peptide (FP) for membrane fusion, heptad repeats (HR1 and HR2) with a central helix (CH) domain, the transmembrane domain (TM) and a cytoplasmic tail (CT).

(3) FIG. 2: Anticipated constructs for the development of a SARS-CoV-2 vaccine.

(4) Based on the full and wildtype S protein, we have designed different construct encoding the (1) full protein with mutations in close distance to the first heptad repeat (HRP1) that include stabilizing mutations preserving neutralisation sensitive sites, the (2) S1 domain or the (3) RB domain (RBD) only. Furthermore, to stabilize the protein fragments a fibritin domain (F) was fused to the C-terminus. All constructs start with the signal peptide (SP) to ensure Golgi transport to the cell membrane.

(5) FIG. 3: Antibody immune response against Influenza HA using the LNP-formulated modRNA.

(6) BALB/c mice were immunized twice with 1 ?g of the vaccine candidate. Total amount of viral antigen specific immunoglobulin G (IgG) was measured via ELISA. The functionality of the antibodies was assessed via VNT.

(7) FIG. 4: T cell response against Influenza HA using the LNP-formulated mod RNA platform.

(8) BALB/c mice were immunized IM with 1 ?g of the vaccine candidate, twice. The T cell response was analyzed using antigen specific peptides for T cell stimulation recovered from the spleen. IFN? release was measured after peptide stimulation using an ELISpot assay.

(9) FIG. 5: Anti-S protein IgG response 7, 14, 21 and 28 d after immunization with BNT162a1.

(10) BALB/c mice were immunized IM once with 1, 5 or 10 ?g of LNP-formulated RBL063.3. On day 7, 14, 21 and 28 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G (IgG) measured via ELISA. For day 7, day 14, day 21 and day 28, values for a serum dilution of 1:100 were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

(11) FIG. 6: Anti-S protein IgG response 7, 14, 21 and 28 d after immunization with BNT162b1.

(12) BALB/c mice were immunized IM once with 0.2, 1 or 5 ?g of LNP-formulated RBP020.3. On day 7, 14. 21 and 28 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), day 21 (1:900), and day 28 (1:2700) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

(13) FIG. 7: Neutralization of SARS-CoV-2 pseudovirus 14, 21 and 28 d after immunization with BNT162b1.

(14) BALB/c mice were immunized IM once with 0.2, 1 or 5 ?g of LNP-formulated RBP020.3. On 14, 21 and 28 d after immunization, animals were bled, and the sera were tested for SARS CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

(15) FIG. 8: Anti-S protein IgG response 7, 14 and 21 d after immunization with BNT162c1.

(16) BALB/c mice were immunized IM once with 0.2, 1 or 5 ?g of LNP-formulated RBS004.3. On day 7, 14 and 21 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), and day 21 (1:900) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

(17) FIG. 9: Neutralization of SARS-CoV-2 pseudovirus 14 and 21 d after immunization with BNT162c1.

(18) BALB/c mice were immunized IM once with 0.2, 1 or 5 ?g of LNP-formulated RBS004.3. On 14 and 21 d after immunization, animals were bled and the sera were tested for SARS CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

(19) FIG. 10: Anti-S protein IgG response 7, 14, 21 and 28 d after immunization with LNP-formulated RBL063.1.

(20) BALB/c mice were immunized IM once with 1, 5 or 10 ?g of LNP-formulated RBL063.1. On day 7, 14, 21 and 28 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:100), day 21 (1:300) and day 28 (1:900) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

(21) FIG. 11: Neutralization of SARS-CoV-2 pseudovirus 14, 21 and 28 d after immunization with LNP-formulated RBL063.1.

(22) BALB/c mice were immunized IM once with 1, 5 or 10 ?g of LNP-formulated RBL063.1. On 14, 21, and 28 d after immunization, animals were bled and the sera were tested for SARS CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

(23) FIG. 12: Anti-S protein IgG response 7, 14 and 21 d after immunization with BNT162b2 (LNP-formulated RBP020.1).

(24) BALB/c mice were immunized IM once with 0.2, 1 or 5 ?g of LNP-formulated RBP020.1. On day 7, 14, and 21 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), and day 21 (1:1100) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

(25) FIG. 13: Neutralization of SARS-CoV-2 pseudovirus 14 and 21 after immunization with BNT162b2 (LNP-formulated RBP020.1).

(26) BALB/c mice were immunized IM once with 0.2, 1 or 5 ?g of LNP-formulated RBP020.1. On day 14 and 21 after immunization, animals were bled and the sera were tested for SARS CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean +SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

(27) FIG. 14: Anti-S protein IgG response 7, 14 and 21 d after immunization with LNP-formulated RBS004.2.

(28) BALB/c mice were immunized IM once with 0.2, 1 or 5 ?g of LNP-formulated RBS004.2. On day 7, 14 and 21 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), and day 21 (1:900) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

(29) FIG. 15: Neutralization of SARS-CoV-2 pseudovirus 14 and 21 after immunization with LNP-formulated RBS004.2.

(30) BALB/c mice were immunized IM once with 0.2, 1 or 5 ?g of LNP-formulated RBS004.2. On 14, and 21 d after immunization, animals were bled, and the sera were tested for SARS CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

(31) FIG. 16: ALC-0315 activity in the screening process.

(32) FIG. 17: Luciferase expression was monitored on (A) the ventral (drainage to the liver) and (B) the right (site of injection) and dorsal (site of injection) sides of the animal after intramuscular administration in wild-type (WT) or ApoE knockout C57Bl/6 mice in the presence or absence of ApoE3. Luciferase expression was detected using Xenolight D-Luciferin Rediject at 4, 24, 72 and 96 hours post administration.

(33) FIG. 18: Luciferase activity after intravenous (IV) and intramuscular (IM) administration in wild-type (WT) or ApoE knockout C57Bl/6 mice in the presence (KO+) or absence (KO) of ApoE3. Luciferase expression was detected using Xenolight D-Luciferin Rediject at 4 hours post administration.

(34) FIG. 19: General structure of the RNA.

(35) Schematic illustration of the general structure of the RNA vaccines with 5-cap, 5- and 3-untranslated regions, coding sequences with intrinsic secretory signal peptide as well as GS-linker, and poly(A)-tail. Please note that the individual elements are not drawn exactly true to scale compared to their respective sequence lengths.

(36) UTR=Untranslated region; sec=Secretory signal peptide; RBD=Receptor Binding Domain; GS=Glycine-serine linker.

(37) FIG. 20: General structure of the RNA.

(38) Schematic illustration of the general structure of the RNA drug substances with 5-cap, 5- and 3-untranslated regions, coding sequences with intrinsic secretory signal peptide as well as GS-linker, and poly(A)-tail. Please note that the individual elements are not drawn exactly true to scale compared to their respective sequence lengths.

(39) GS=Glycine-serine linker; UTR=Untranslated region; Sec=Secretory signal peptide; RBD=Receptor Binding Domain.

(40) FIG. 21: General structure of the RNA.

(41) Schematic illustration of the general structure of the RNA vaccines with 5-cap, 5- and 3-untranslated regions, coding sequences of the Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase replicase and the SARS-CoV-2 antigen with intrinsic secretory signal peptide as well as GS-linker, and poly(A)-tail. Please note that the individual elements are not drawn exactly true to scale compared to their respective sequence lengths. UTR=Untranslated region; Sec=Secretory signal peptide; RBD=Receptor Binding Domain; GS=Glycine-serine linker.

(42) FIG. 22: ELISpot analysis 28 d after immunization with BNT162b1.

(43) BALB/c mice were immunized IM once with 1 ?g of LNP-formulated RBP020.3. On day 28 after immunization, mice were euthanized and splenocytes were prepared. ELISpot assay was performed using MACS-sorted CD4+ and CD8+ T cells. T cells were stimulated with an S protein- or RBD-specific overlapping peptide pool and IFN-? secretion was measured to assess T-cell responses. One point in the graph stands for the individual spot count of one mouse, every mouse sample was measured in duplicates (group size n=8; mean is included for the groups).

(44) FIG. 23: Cytokine concentrations in supernatants of re-stimulated splenocytes 12 d after immunization with BNT162b1.

(45) BALB/c mice were immunized IM once with 5 ?g of LNP-formulated RBP020.3. On day 12 after immunization, mice were euthanized. Splenocytes were prepared and were stimulated with an S protein-specific overlapping peptide pool. After 48 h of stimulation, supernatant was collected and cytokine concentrations were determined. One point in the graph stands for the individual cytokine concentration of one mouse, every mouse sample was measured in duplicates (group size n=8; mean is included for the groups).

(46) FIG. 24: T cell immunophenotyping in PBMCs 7 days after immunization with BNT162b1. BALB/c mice were immunized IM once with 5 ?g of LNP-formulated RBP020.3. On day 7 after immunization, mice were bled. Flow cytometry analysis of PBMCs was performed of T cells. T cells were defined as viable CD3.sup.+CD4.sup.+ and CD3.sup.+CD8.sup.+ T cells. Additional phenotyping markers are included in the figures. Tfh cells were gated from CD4.sup.+ T cells and defined as CD4.sup.+ T-bet.sup.? GATA3.sup.?CD44.sup.+CD62L.sup.?PD-1.sup.+CXCR5.sup.+ cells. One point in the graph stands for the individual cell fraction of one mouse (group size n=8; mean is included for the groups).

(47) FIG. 25: B cell immunophenotyping in draining lymph nodes 12 days after immunization with BNT162b1.

(48) BALB/c mice were immunized IM once with 5 ?g of LNP-formulated RBP020.3. On day 12 after immunization, mice were euthanized. Flow cytometry analysis of lymphocytes was performed of B cells. Activated B cells were gated within single, viable lymphocytes and defined as IgD-Dump (CD4, CD8, F4/80, GR-1).sup.? cells. Plasma cells were defined as CD138.sup.+B220.sup.low/? cells. Switched B cells were gated from non-plasma cells and defined as CD19.sup.+CD138.sup.?IgM.sup.?. Germinal center (GC) B cells were gated from switched B cells and defined as CD19.sup.+IgM.sup.?CD38.sup.?CD95.sup.+ cells and gated for IgG1 and IgG2a. One point in the graph stands for the individual cell fraction of one mouse (group size n=8; mean is included for the groups).

(49) FIG. 26: ELISpot analysis 28 d after immunization with LNP-formulated modRNA RBP020.1.

(50) BALB/c mice were immunized IM once with 5 ?g of LNP-formulated RBP020.1. On day 28 after immunization, mice were euthanized and splenocytes were prepared. ELISpot assay was performed using MACS-sorted CD4+ and CD8+ T cells. T cells were stimulated with an S protein-specific overlapping peptide pool and IFN-? secretion was measured to assess T-cell responses. One point in the graph stands for the individual spot count of one mouse, every mouse sample was measured in duplicates (group size n=8; mean is included for the groups).

(51) FIG. 27: Cytokine concentrations in supernatants of re-stimulated splenocytes 28 d after immunization with LNP-formulated modRNA RBP020.1.

(52) BALB/c mice were immunized IM once with 5 ?g of LNP-formulated RBP020.1. On day 28 after immunization, mice were euthanized. Splenocytes were prepared and were stimulated with an S protein-specific overlapping peptide pool. After 48 h of stimulation, supernatant was collected and cytokine concentrations were determined. One point in the graph stands for the individual cytokine concentration of one mouse, every mouse sample was measured in duplicates (group size n=8; mean is included for the groups).

(53) FIG. 28: ELISpot analysis 28 d after immunization with LNP-formulated saRNA RBS004.2.

(54) BALB/c mice were immunized IM once with 5 ?g of LNP-formulated RBS004.2. On day 28 after immunization, mice were euthanized and splenocytes were prepared. ELISpot assay was performed using MACS-sorted CD4+ and CD8+ T cells. T cells were stimulated with an S protein-specific overlapping peptide pool and IFN-? secretion was measured to assess T-cell responses. One point in the graph stands for the individual spot count of one mouse, every mouse sample was measured in duplicates (group size n=8; mean is included for the groups).

(55) FIG. 29: Cytokine concentrations in supernatants of re-stimulated splenocytes 28 d after immunization with LNP-formulated saRNA RBS004.2.

(56) BALB/c mice were immunized IM once with 1 ?g of LNP-formulated RBS004.2. On day 28 after immunization, mice were euthanized. Splenocytes were prepared and were stimulated with an S protein-specific overlapping peptide pool. After 48 h of stimulation, supernatant was collected and cytokine concentrations were determined. One point in the graph stands for the individual cytokine concentration of one mouse, every mouse sample was measured in duplicates (group size n=8; mean is included for the groups).

(57) FIG. 30: Schematic overview of the S protein organization of the SARS-CoV-2 S protein and novel constructs for the development of a SARS-CoV-2 vaccine.

(58) Based on the wildtype S protein, we have designed two different transmembrane-anchored RBD-based vaccine constructs encoding the RBD fragment fused to the T4 fibritin trimerization domain (F) and the autochthonus transmembrane domain (TM). Construct (1) starts with the SARS-CoV-2-S signal peptide (SP; AA 1-19 of the S protein) whereas construct (2) starts with the human Ig heavy chain signal peptide (huSec) to ensure Golgi transport to the cell membrane.

(59) FIG. 31: Anti-S protein IgG response 6, 14 and 21 d after immunization with LNP-C12 formulated modRNA coding for transmembrane-anchored RBD-based vaccine constructs.

(60) BALB/c mice were immunized IM once with 4 ?g of LNP-C12-formulated transmembrane-anchored RBD-based vaccine constructs (surrogate to BNT162b3c/BNT162b3d). On day 6, 14 and 21 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen specific immunoglobulin G (IgG) measured via ELISA. For day 6 (1:50), day 14 (1:300) and day 21 (1:900) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

(61) FIG. 32: Neutralization of SARS-CoV-2 pseudovirus 6, 14 and 21 d after immunization with LNP-C12 formulated modRNA coding for transmembrane-anchored RBD-based vaccine constructs.

(62) BALB/c mice were immunized IM once with 4 ?g of LNP-C12-formulated transmembrane-anchored RBD-based vaccine constructs (surrogate to BNT162b3c/BNT162b3d). On day 6, 14 and 21 after immunization, animals were bled and the sera were tested for SARS CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

(63) FIG. 33: Immunogenicity of BNT162b1 in rhesus macaques and comparison to human convalescent sera.

(64) Rhesus macaques were immunized IM on days 0 and 21 with 30 ?g or 100 ?g of BNT162b1 or with placebo (0.9% NaCl). Sera were obtained before immunization and 14, 21, 28, and 35 days after immunization; PBMCs were obtained before and 14 and 42 days after immunization. Sera from COVID-19 patients were obtained 20-40 days after the onset of symptoms and after at least 14 days of asymptomatic convalescence. (A) Geometric mean concentrations of IgG binding to a recombinant S1 protease fragment of SARS-CoV-2 S, in rhesus macaque sera drawn at the indicated times after immunization (n=6 per group, all measurement time points of the placebo group depicted under Control) and in human convalescent sera (n=62). (B) SARS-CoV-2 geometric mean 50% neutralization titers of the rhesus macaque sera (n=6 per group, all measurement time points of the placebo group depicted under Control) and human convalescent sera (n=38). P values were determined by a two-tailed one-way ANOVA and Dunnett's multiple comparisons test. (C) Flow cytometry analysis of CD4+ T cells producing IFN?, IL-2, TNF (T.sub.H1), IL-21 or IL-4 (T.sub.H2) cytokines in the rhesus macaque PBMCs on day 42. P values were determined by a two-tailed Kruskal-Wallis test followed by Dunn's multiple comparisons test. Each data point corresponds to an individual animal.

(65) FIG. 34: Overview of study population

(66) FIG. 35: Local Reactions Reported within 7 Days of Vaccination all Dose Levels

(67) Solicited injection-site (local) reactions were: pain at injection site (mild=does not interfere with activity; moderate=interferes with activity; severe=prevents daily activity; Grade 4=emergency room visit or hospitalization) and redness and swelling (mild=2.5 to 5.0 cm in diameter; moderate=5.5 to 10.0 cm in diameter; severe=>10.0 cm in diameter; Grade 4=necrosis or exfoliative dermatitis for redness, and necrosis for swelling). Data were collected with the use of electronic diaries for 14 days after each vaccination.

(68) FIG. 36: (A) Systemic Events Reported within 7 days after Vaccination 1: All Dose Levels; (B) Systemic Events Reported within 7 days after Vaccination 2: 10 ?g & 30 ?g Dose Levels Solicited systemic events were: nausea/vomiting (mild=no interference with activity or 1 to 2 times in 24 hours; moderate=some interference with activity or >2 times in 24 hours; severe=prevents daily activity or requires intravenous hydration; Grade 4=emergency room visit or hospitalization for hypotensive shock), diarrhea (mild, 2 to 3 loose stools in 24 hours; moderate, 4 to 5 loose stools in 24 hours; severe, >6 loose stools in 24 hours; Grade 4=emergency room visit or hospitalization), headache (mild=no interference with activity; moderate=repeated use of non-narcotic pain reliever >24 hours or some interference with activity; severe=significant, any use of narcotic pain reliever or prevents daily activity; Grade 4=emergency room visit or hospitalization), fatigue/tiredness (mild=no interference with activity; moderate=some interference with activity; severe=significant; prevents daily activity; Grade 4=emergency room visit or hospitalization), muscle pain (pain that is occurring in areas other than the injection site; mild=no interference with activity; moderate=some interference with activity; severe=significant; prevents daily activity; Grade 4=emergency room visit or hospitalization), joint pain (mild=no interference with activity; moderate=some interference with activity; severe=significant; prevents daily activity; Grade 4=emergency room visit or hospitalization), and fever (mild=100.4? F. to 101.1? F. [38.0? C. to 38.4? C.]; moderate=101.2? F. to 102.0? F. [38.5? C. to 38.9? C.]; severe=102.1? F. to 104.0? F. [39.0? C. to 40.0? C.]; Grade 4=>104.0? F. [>40.0? C.]).

(69) FIG. 37: Immunogenicity of BNT162b1RBD-Binding IgG GMCs and SARS CoV2 50% Neutralizing Titers after 1 or 2 doses

(70) Subjects in groups of 15 were immunized with the indicated dose levels of BNT162b1 (n=12) or with placebo (P, n=3) on days 1 (all dose levels and placebo) and 21 (10 ?g and 30 ?g dose levels and placebo). Sera were obtained before immunization (Day 1) and 7, 21, and 28 days after the first immunization. Human COVID-19 convalescent sera (HCS) (n=38) were obtained 20-40 days after the onset of symptoms and after at least 14 days of asymptomatic convalescence. (A) GMCs of recombinant RBD-binding IgG. Lower limit of quantitation (LLOQ) 1.15 (dotted line). (B) 50% SARS-CoV-2 neutralizing GMTs. Each data point represents a serum sample, and each vertical bar represents a geometric mean with 95% confidence interval.

(71) FIG. 38: BNT162b1 induces strong CD4 and CD8 T cell response in humans

(72) BNT162 induced T cells: INF? ELISpot ex vivo; T cell responses in 8 of 8 tested subjects. Here: subject vaccinated prime/boost with 10 ?g BNT162b1; CEF: CMV, EBV, Influenza CD8 T cell epitope mix, CEFT: CMV, EBV, Influenza, Tetanus CD4 T cell epitope mix.

(73) FIG. 39: BNT162b1-induced IgG concentrations

(74) Subjects were immunised with BNT162b1 on days 1 (all dose levels) and 22 (all dose levels except 60 ?g) (n=12 per group, from day 22 on n=11 for the 10 ?g and 50 ?g cohort). Sera were obtained on day 1 (Pre prime) and on day 8, 22 (pre boost), 29 and 43. Pre-dose responses across all dose levels were combined. Human COVID-19 convalescent sera (HCS, n=38) were obtained at least 14 days after PCR-confirmed diagnosis and at a time when the donors were no longer symptomatic. For RBD-binding IgG concentrations below the lower limit of quantification (LLOQ=1.15), LLOQ/2 values were plotted. Arrowheads indicate vaccination. Chequered bars indicate that no boost immunisation was performed. Values above bars are geometric means with 95% confidence intervals. At the time of submission, day 43 data were pending for five subjects of the 50 ag cohort and all subjects of the 60 ag cohort.

(75) FIG. 40: BNT162b1-induced virus neutralisation titers

(76) The vaccination schedule and serum sampling are the same as in FIG. 39. (A) SARS-CoV-2 50% neutralisation titers (VNT.sub.50) in immunized subjects and COVID-19 convalescent patients (HCS). For values below the lower limit of quantification (LLOQ)=20, LLOQ/2 values were plotted. Arrowheads indicate days of immunisation. Chequered bars indicate that no boost immunisation was performed. Geometric mean (values above bars) with 95% confidence interval. At the time of submission, day 43 data were not yet available for five subjects of the 50 ?g cohort and all subjects of the 60 ag cohort, (B) Correlation of RBD-binding IgG geometric mean concentrations (GMC) (as in FIG. 39) with VNT.sub.50 on day 29 (all evaluable subject sera). Nonparametric Spearman correlation. (C) Pseudovirus 50% neutralisation titers (pVNT.sub.50) across a pseudovirus panel displaying 17 SARS-CoV-2 spike protein variants including 16 RBD mutants and the dominant spike protein variant D614G (dose level 10, 30 and 50 ?g, n=1-2 each; day 29). Lower limit of quantification (LLOQ)=40. Geometric mean.

(77) FIG. 41: Frequency and magnitude of BNT162b1-induced CD4.sup.+ and CD8.sup.+ T-cell responses

(78) The vaccination schedule is as in FIG. 39. PBMCs obtained on day 1 (Pre) and on day 29 (Post, 7 days after boost) (1 and 50 ?g, n=8 each; 10 and 30 ?g, n=10 each) were enriched for CD4.sup.+ or CD8.sup.+ Tcell effectors and separately stimulated over night with an overlapping peptide pool representing the vaccine-encoded RBD for assessment in direct ex vivo IFN? ELISpot. Common pathogen T-cell epitope pools CEF (CMV, EBV, influenza virus HLA class I epitopes) and CEFT (CMV, EBV, influenza virus, tetanus toxoid HLA class II epitopes) served to assess general T-cell reactivity, medium served as negative control. Each dot represents the normalized mean spot count from duplicate wells for one study subject, after subtraction of the medium-only control. (A) Ratios above post-vaccination data points are the number of subjects with detectable CD4.sup.+ or CD8.sup.+ T cell response within the total number of tested subjects per dose cohort. (B) Exemplary CD4.sup.+ and CD8.sup.+ ELISpot of a 10-?g cohort subject. (C) RBD-specific CD4.sup.+ and CD8.sup.+ T cell responses in all prime/boost vaccinated subjects and their baseline CEFT- and CEF-specific T-cell responses. (D) Correlation of VNT.sub.50 (as in FIG. 40(A)) with CD4.sup.+ T-cell responses (as in FIG. 41) of dose cohorts 10 to 50 ?g (1 and 50 ?g, n=8 each; 10 and 30 ?g, n=10 each). Nonparametric Spearman correlation.

(79) FIG. 42: Cytokine polarisation of BNT162b1-induced T cells

(80) The vaccination schedule and PBMC sampling are as in FIG. 41. PBMCs of vaccinees and COVID-19 recovered donors (HCS n=6; in (C)) were stimulated over night with an overlapping peptide pool representing the vaccine-encoded RBD and analysed by flow cytometry ((A)-(C)) and bead-based immunoassay (D). (A) Exemplary pseudocolor flow cytometry plots of cytokine-producing CD4.sup.+ and CD8.sup.+ T cells of a 10-?g cohort subject. (B) RBD-specific CD4.sup.+ T cells producing the indicated cytokine as fraction of total cytokine-producing RBD-specific CD4.sup.+ T cells, and (C) RBD-specific CD8.sup.+ (left) or CD4.sup.+ (right) T cells producing the indicated cytokine as fraction of total circulating T cells of the same subset. One CD4 non-responder (<0.02% total cytokine producing T cells) and one CD8 non-responder (<0.01% total cytokine producing T cells) from the 30-?g cohort were excluded in (B). Values above data points are the mean fractions across all dose cohorts. (D) PBMCs from the 50-?g cohort. Each dot represents the mean from duplicate wells subtracted by the DMSO control for one study subject. Lower limits of quantification (LLOQ) were 6.3 pg/mL for TNF, 2.5 pg/mL for IL-1?, and 7.6 pg/mL for IL-12p70. Mean (B).

(81) FIG. 43: Schedule of vaccination and assessment

(82) FIG. 44: Solicited adverse events

(83) Subjects were immunized with the indicated dose levels of BNT162b1 on days 1 (all dose levels) and 22 (all dose levels except 60 ?g) (n=12 per group, n=11 for 10 ?g and 50 ?g cohort from day 22 on). (A), (B) Number of subjects with local (A) or systemic reactions (B) by day (day 1-9, 22-30) and cohort. Grading of adverse events was performed according to FDA recommendations (U.S. Department of Health and Human Services, Administration, F. and D. & Research, C. for B. E. and. Toxicity grading scale for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials. (2007). Available at: www.fda.gov/regulatory-information/search-fda-guidance-documents/toxicity-grading-scale-healthy-adult-and-adolescent-volunteers-enrolled-preventive- vaccine-clinical.).

(84) FIG. 45: Pharmacodynamic markers

(85) Subjects were immunised with the indicated dose levels of BNT162b1 on days 1 (all dose levels) and 22 (all dose levels except 60 ?g). (A) Kinetics of C-reactive protein (CRP) level and (B) Kinetics of lymphocyte counts. Dotted lines indicate upper and lower limit of reference range. For values below the lower limit of quantification (LLOQ=0.3), LLOQ/2 values were plotted (A).

(86) FIG. 46: Correlation of antibody and T-cell responses

(87) Subjects were immunised with the indicated dose levels of BNT162b1 on days 1 (all dose levels) and 22 (all dose levels except 60 ?g). (A) Correlation of RBD-specific IgG responses (from FIG. 39(A)) with CD4+ T-cell responses on day 29 (1 and 50 ?g, n=8 each; 10 and 30 ?g, n=10 each). Nonparametric Spearman correlation. (B) Correlation of CD4.sup.+ with CD8.sup.+ T-cell responses (as in FIG. 41) from day 29 of dose cohorts 10 to 50 ?g (1 and 50 ?g, n=8 each; 10 and 30 ?g, n=10 each). Parametric Pearson correlation. (C) Correlation of RBD-specific IgG responses (from FIG. 39(A)) with CD8+ T-cell responses on day 29 (1 and 50 ?g, n=8 each; 10 and 30 ?g, n=10 each). Nonparametric Spearman correlation.

(88) FIG. 47: Gating strategy for flow cytometry analysis of data shown in FIG. 42

(89) Flow cytometry gating strategy for identification of IFN?, IL-2 and IL-4 secreting T cells in study subject PBMC samples. (A) CD4+ and CD8+ T cells were gated within single, viable lymphocytes. (B), (C) Gating of IFN?, IL-2 and IL-4 in CD4+ T cells (B), and IFN? and IL-2 in CD8+ T cells (C).

(90) FIG. 48: BNT162b1 18-55 years of age: Local Reactions After Each Dose

(91) FIG. 49: BNT162b1 18-55 years of age: Systemic Events After Each Dose

(92) FIG. 50: BNT162b1 65-85 years of age: RBD-Binding IgG GMCs

(93) FIG. 51: BNT162b1 65-85 years of age: 50% SARS-CoV-2 Neutralizing GMTs

(94) FIG. 52: BNT162b2 18-55 years of age: Local Reactions After Each Dose

(95) FIG. 53: BNT162b2 18-55 years of age: Systemic Events After Each Dose

(96) FIG. 54: BNT162b2 65-85 years of age: Local Reactions After Each Dose

(97) FIG. 55: BNT162b2 65-85 years of age: Systemic Events After Each Dose

(98) FIG. 56: BNT162b2 18-55 years of age: S1-Binding IgG GMCs

(99) FIG. 57: BNT162b2 18-55 years of age: 50% SARS-CoV-2 Neutralizing GMTs

(100) FIG. 58: BNT162b2 65-85 years of age: S1-Binding IgG GMCs

(101) FIG. 59: BNT162b2 65-85 years of age: 50% SARS-CoV-2 Neutralizing GMTs

(102) FIG. 60: BNT162b2-elicited T cell responses in mice

(103) Splenocytes of BALB/c mice immunized IM with BNT162b2 or buffer were ex vivo restimulated with full-length S peptide mix or negative controls (irrelevant peptide in (A), right); no peptide in ((A), left) and in (C)). P-values were determined by a two-tailed paired t-test. (A) IFN? ELISpot of splenocytes collected 12 days after immunization of mice (n=8 per group) with 5 ?g BNT162b2 (left). IFN? ELISpot of isolated splenic CD4+ T cells or CD8+ T cells 28 days after immunization of mice (n=8 mice per group) with 1 ?g BNT162b2 (middle and right). (B) CD8+ T-cell specific cytokine release by splenocytes of mice (n=8 per group) immunized with 5 ?g BNT162b2 or buffer (control), determined by flow cytometry. S-peptide specific responses are corrected for background (no peptide). (C) Cytokine production by splenocytes obtained 28 days after immunization of mice (n=8 per group, n=7 for IL-4, IL-5, and IL-13, as one outlier was removed via routs test [Q=1%] for the S peptide stimulated samples) with 1 ?g BNT162b2, determined by bead-based multiplex analysis.

(104) FIG. 61: IFN? ELiSpot data for 5 subjects vaccinated with 10 ?g BNT162b2

(105) Background-subtracted spot counts from duplicates prior to vaccination (Pre) and on day 29 (Post7 days post boost) per 10.sup.6 cells. T cell response analysis was performed in a GCLP-compliant manner using a validated ex-vivo IFN? ELISpot assay. All tests were performed in duplicate and included negative and positive controls (medium only and anti-CD3). In addition, peptide epitopes derived from cytomegalovirus (CMV), Epstein Barr virus (EBV), and influenza virus were used as positive controls. CD4- or CD8-depleted PBMCs were stimulated for 16-20 h in pre-coated ELISpot plates (Mabtech) with overlapping peptides covering the N-terminal portion and C-terminal portion of the spike glycoprotein. For analysis of ex vivo T-cell responses, bound IFN? was visualized by an alkaline phosphatase-conjugated secondary antibody. Plates were scanned using a Robot ELISPOT Readerand analysed by ImmunoCapture V6.3 or AID ELISPOT 7.0 software. Spot counts were summarized as mean values for each duplicate. T cell counts were calculated as the sum of spot counts detected after stimulation with S pool 1 and S pool 2. T-cell responses stimulated by peptides were compared to effectors incubated with medium only as negative control using an ELISpot data analysis Tool (EDA), based on two statistical tests (distribution free resampling) according to Moodie et al. (Moodie Z. et al., J Immunol Methods 315, 2006, 121-32; Moodie Z. et al., Cancer Immunol Immunother 59, 2010, 1489-501) thus providing sensitivity while maintaining control over false positive rate. No significant changes were observed between the pre- and day 29 T cell responses against the positive control peptides from CMV, EBV, and influenza virus (not shown).

(106) FIG. 62: Example of CD4+ and CD8+ IFN? ELiSpot data

(107) IFN? ELISpot was performed as in FIG. 61 using PBMCs obtained from a subject prior to immunization and on day 29 after dose 1 of 10 ?g BNT162b2 (7 days post dose 2). HLA class I and class II peptide pools CEF (cytomegalovirus [CMV], Epstein Barr virus [EBV] (7 days post dose 2), and influenza virus, HLA class I epitope mix) and CEFT (CMV, EBV, influenza virus, and tetanus toxoid HLA class II cell epitope mix) were used as benchmarking controls to assess CD8+ and CD4+ T cell reactivity.

(108) FIG. 63: Comparison of BNT162b2-elicited and benchmark INF? ELISpot responses

(109) IFN? spot counts from day 29 (7 day post dose 2) PBMC samples obtained from 5 subjects who were immunized with 10 ?g of BNT162b2 on days 1 and 22. CEF (CMV, EBV, and influenza virus HLA class I epitope mix), and CEFT (CMV, EBV, influenza virus, and tetanus toxoid HLA class II cell epitope mix) were used as benchmarking controls to assess CD8+ and CD4+ T cell reactivity. Horizontal lines indicate median values.

(110) FIG. 64: Design and characterisation of the immunogen

(111) (A) Structure of BNT162b1. Linear diagram of RNA (left), and cartoon of LNP (right). UTR, untranslated region; SP, signal peptide. (B) Representative 2D class averages from electron microscopy of negatively stained RBD-foldon trimers. Box edge: 37 nm. (C) Density map of the ACE2/B.sup.0AT1/RBD-foldon trimer complex at 3.24 ? after focused refinement of the ACE2 extracellular domain bound to an RBD monomer. Surface color-coding by subunit. A ribbon model refined to the density shows the RBD-ACE2 binding interface, with residues potentially mediating polar interactions labeled.

(112) FIG. 65: Mouse immunogenicity

(113) (A)-(C) BALB/c mice (n=8 per group) were immunised intramuscularly (IM) with 0.2, 1 or 5 ?g of BNT162b1 or buffer. Geometric mean of each group ?95% CI, P-values compare day 28 to non-immunised (0 ?g; n=8) baseline sera (multiple comparison of mixed-effect analysis using Dunnett's multiple comparisons test) ((A), (C)). (A) RBD-binding IgG responses in sera obtained 7, 14, 21 and 28 days after immunisation, determined by ELISA. For day 0, a pre-screening of randomised animals was performed (n=4). (B) Representative surface plasmon resonance sensorgram of the binding kinetics of His-tagged RBD to immobilised mouse IgG from serum 28 days after immunisation with 5 ?g BNT162b1 (n=8). Actual binding (green) and the best fit of the data to a 1:1 binding model (black). (C) VSV-SARS-CoV-2 pseudovirus 50% serum neutralising titers (pVNT.sub.50). (D)-(F), Splenocytes of BALB/c mice immunised IM with BNT162b1 or buffer (control) were ex vivo re-stimulated with full-length S peptide mix or negative controls (no peptide in ((D), left) and in ((E), (F)); irrelevant peptide in ((D), right)). P-values were determined by a two-tailed paired t-test. (D) IFN? ELISpot of splenocytes collected 12 days after immunisation of mice (n=8 per group) with 5 ?g BNT162b1 (left). IFN? ELISpot of isolated splenic CD4.sup.+ T cells (n=7, one outlier removed by Grubbs test, ?=0.05) or CD8+ T cells (n=8) 28 days after immunisation with 1 ?g BNT162b1 (middle and right). (E) T-cell specific cytokine release by splenocytes of mice (n=8 per group) immunised with 5 ?g BNT162b1, determined by flow cytometry. S-peptide specific responses are corrected for background (no peptide). (F) Cytokine production by splenocytes obtained 28 days after immunisation of mice (n=8 per group) with 0.2 ?g BNT162b1, determined by bead-based multiplex analysis.

(114) FIG. 66: Immunogenicity of BNT162b1 in rhesus macaques and comparison to human convalescent sera

(115) (A), (B) Male rhesus macaques 2-4 years of age (n=6 per group) were immunised IM on Days 0 and 21 with 30 ?g or 100 ?g of BNT162b1 or with buffer, and serum was obtained before and 14, 21, 28, 35 and 42 days after immunisation. Human convalescent sera (HCS) were obtained from SARS-CoV-2-infected patients at least 14 days after PCR-confirmed diagnosis and at a time when acute COVID-19 symptoms had resolved (n=38). Values above bars give the geometric means. (A) Geometric mean concentrations (GMCs) of IgG binding a recombinant SARS-CoV-2 RBD. Dashed line indicates geometric mean of sera from all time points for the placebo group (1.72 U/mL). Group IgG titers for every time point were analysed for statistical significance against HCS samples using one-way ANOVA with Dunnett's multiple comparison correction, and statistical significance was confirmed in the 30 ?g dose-level group (Day 28, p<0.0001; Day 35, p=0.0016), and in the 100 ?g dose-level group (Day 28, 35 and 42, all p<0.0001). (B) SARS-CoV-2 50% neutralisation titers (VNT.sub.50). Dashed line indicates geometric mean of sera from all time points for the placebo group (10.31 U/mL). Group VNT.sub.50 for every time point were analysed for statistical significance against HCS samples using one-way ANOVA with Dunnett's multiple comparison correction, and statistical significance was confirmed in the 30 ?g dose-level group (Day 28, p<0.0001), and in the 100 ?g dose-level group (Day 28 and 35, both p<0.0001; Day 42, p=0.007).

(116) FIG. 67: Viral RNA in non-immunised and immunised rhesus macaques after SARS-CoV-2 challenge

(117) Rhesus macaques (n=6 per group) were immunised on Days 0 and 21 with 100 ?g BNT162b1 or buffer (Control) as described in FIG. 66. Forty-one to 48 days after the second immunisation, the animals were challenged with 1?10.sup.6 total pfu of SARS-CoV-2 split equally between the IN and IT routes. Three non-immunised age-matched male rhesus macaques were challenged with cell culture medium (Sentinel). Viral RNA levels were detected by RT-qPCR. Ratios above data points are the number of viral RNA positive animals within all animals per group. (A) Viral RNA in bronchoalveolar lavage (BAL) fluid obtained before, and on Days 3 and 6 after challenge. At day 6, the viral load between the control and BNT162b1-immunized animals was statistically significant (p=0.0131). (B) Viral RNA in nasal swabs obtained before challenge and on day 1, 3, and 6 after challenge. At day 3, the viral load between the control and BNT162b1-immunized animals was statistically significant (p=0.0229). Dotted lines indicate the lower limits of detection (LLOD). Negative specimens were set to 2 the LLOD. P-values were determined by categorical analysis for binomial response (undetectable viral load after challenge as success, measurable viral load after challenge as failure).

(118) FIG. 68: BNT162b1 and b2 V8 immunization reduces viral RNA in rhesus macaques after challenge with SARS-CoV-2; b2 shows earlier clearance in nose

(119) FIG. 69: Exemplary pandemic supply product packaging overview

(120) FIG. 70: Exemplary vaccine storage & handling at the point of vaccination

(121) FIG. 71: Exemplary multi-dose preparation

(122) FIG. 72. Geometric Mean Titers and 95% Cl: SARS-CoV-2 Neutralization AssayNT50Phase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b1Evaluable Immunogenicity Population

(123) FIG. 73. Geometric Mean Titers and 95% Cl: SARS-CoV-2 Neutralization AssayNT50Phase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b1Evaluable Immunogenicity Population

(124) FIG. 74. Geometric Mean Titers and 95% Cl: SARS-CoV-2 Neutralization AssayNT50Phase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b2Evaluable Immunogenicity Population

(125) FIG. 75. Geometric Mean Titers and 95% CI: SARS-CoV-2 Neutralization AssayNT50 hPhase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b2Evaluable Immunogenicity Population

(126) FIG. 76. Geometric Mean Concentrations and 95% Cl: SARS-CoV-2 RBD-binding IgG Level AssayPhase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b1Evaluable Immunogenicity Population

(127) FIG. 77. Geometric Mean Concentrations and 95% Cl: SARS-CoV-2 RBD-binding IgG Level AssayPhase 1, 2 Doses, 21 Days Apart65-85 Years of Age, BNT162b1Evaluable Immunogenicity Population

(128) FIG. 78. Geometric Mean Concentrations and 95% Cl: SARS-CoV-2 S1-binding IgG Level AssayPhase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b1Evaluable Immunogenicity Population

(129) FIG. 79. Geometric Mean Concentrations and 95% Cl: SARS-CoV-2 S1-binding IgG Level AssayPhase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b1Evaluable Immunogenicity Population

(130) FIG. 80. Geometric Mean Concentrations and 95% Cl: SARS-CoV-2 S1-binding IgG Level AssayPhase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b2Evaluable Immunogenicity Population

(131) FIG. 81. Geometric Mean Concentrations and 95% CI: SARS-CoV-2 S1-binding IgG Level AssayPhase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b2Evaluable Immunogenicity Population

(132) FIG. 82. Geometric Mean Concentrations and 95% CI: SARS-CoV-2 RBD-binding IgG Level AssayPhase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b2Evaluable Immunogenicity Population

(133) FIG. 83. Geometric Mean Concentrations and 95% CI: SARS-CoV-2 RBD-binding IgG Level AssayPhase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b2Evaluable Immunogenicity Population

(134) FIG. 84. Subjects Reporting Local Reactions, by Maximum Severity, Within 7 Days After Each DosePhase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b1Safety Population

(135) FIG. 85. Subjects Reporting Local Reactions, by Maximum Severity, Within 7 Days After Each DosePhase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b1Safety Population

(136) FIG. 86. Subjects Reporting Local Reactions, by Maximum Severity, Within 7 Days After Each DosePhase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b2Safety Population

(137) FIG. 87. Subjects Reporting Local Reactions, by Maximum Severity, Within 7 Days After Each DosePhase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b2Safety Population

(138) FIG. 88. Subjects Reporting Systemic Events, by Maximum Severity, Within 7 Days After Each DosePhase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b1Safety Population

(139) FIG. 89. Subjects Reporting Systemic Events, by Maximum Severity, Within 7 Days After Each DosePhase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b1Safety Population

(140) FIG. 90. Subjects Reporting Systemic Events, by Maximum Severity, Within 7 Days After Each DosePhase 1, 2 Doses, 21 Days Apart18-55 Years of AgeBNT162b2Safety Population

(141) FIG. 91. Subjects Reporting Systemic Events, by Maximum Severity, Within 7 Days After Each DosePhase 1, 2 Doses, 21 Days Apart65-85 Years of AgeBNT162b2Safety Population

(142) FIG. 92. Subjects Reporting Local Reactions, by Maximum Severity, Within 7 Days After Each Dose, Age Group 18 55 YearsPhase 2Safety Population

(143) FIG. 93. Subjects Reporting Local Reactions, by Maximum Severity, Within 7 Days After Each Dose, Age Group 56 85 YearsPhase 2Safety Population

(144) FIG. 94. Subjects Reporting Systemic Events, by Maximum Severity, Within 7 Days After Each Dose, Age Group 18 55 YearsPhase 2Safety Population

(145) FIG. 95. Subjects Reporting Systemic Events, by Maximum Severity, Within 7 Days After Each Dose, Age Group 56 85 YearsPhase 2Safety Population

(146) FIG. 96. Subjects Reporting Local Reactions, by Maximum Severity, Within 7 Days After Each Dose, Age Group 18 55 Years?6000 Subjects for Phase 2/3Safety Population

(147) FIG. 97. Subjects Reporting Local Reactions, by Maximum Severity, Within 7 Days After Each Dose, Age Group 56 85 Years?6000 Subjects for Phase 2/3Safety Population

(148) FIG. 98. Subjects Reporting Systemic Events, by Maximum Severity, Within 7 Days After Each Dose, Age Group 18-55 Years?6000 Subjects for Phase 2/3Safety Population

(149) FIG. 99. Subjects Reporting Systemic Events, by Maximum Severity, Within 7 Days After Each Dose, Age Group 56-85 Years?6000 Subjects for Phase 2/3Safety Population

(150) FIG. 100. Cumulative Incidence Curves for the First COVID-19 Occurrence After Dose 1Dose 1 All-Available Efficacy Population

(151) FIG. 101. BNT162b2Exemplary functional 50% SARS-CoV-2 neutralising antibody titers (VN.sub.50). Younger adults (aged 18 to 55 years) and older adults (aged 56 to 85 years) were immunized with BNT162b2 on day 1 and day 22 (n=12 per group). Sera were obtained from younger adults on day 1 (baseline) and on day 8, 22 (pre boost), 29, 43, 50 and 85. Sera were obtained from older adults on day 1 (baseline) and on day 8, 22, and 29. Human COVID-19 convalescent sera (HSC, n=38) were obtained at least 14 days after a confirmed diagnosis and at a time when the donors were no longer symptomatic. SARS-CoV-2 50% neutralization titers (VN.sub.50 titers) with 95% confidence intervals are shown for younger adults immunized with 1, 3, 10, 20, or 30 ?g BNT162b2, and older adults immunized with 20 ?g BNT162b2. Values smaller than the limit of detection (LOD) are plotted as 0.5*LOD. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). The dotted horizontal line represents the LOD. VN.sub.50=50% SARS-CoV-2 neutralizing antibody titers; HCS=human COVID-19 convalescent serum.

(152) FIG. 102. BNT162b1Exemplary fold increase from baseline in functional 50% SARS-CoV-2 neutralizing antibody titers (VN.sub.50).

(153) The vaccination schedule and serum sampling are the same as in FIG. 39 (n=12 per group). Geometric means fold increase (GMFI) from baseline in VN.sub.50 titer with 95% confidence intervals are shown for younger participants (aged 18 to 55 yrs) immunized with 1, 10, 30, 50, or 60 ?g BNT162b1. Arrowheads indicate baseline (pre-dose 1, Day 1) and dose 2 (Day 22). Dose 2 was not performed in the 60 ?g dose group. The dotted horizontal line represents the threshold for seroconversion (fold increase ?4). VN.sub.50=50% SARS-CoV-2 neutralizing antibody titers.

(154) FIG. 103. BNT162b2Exemplary fold increase from baseline in functional 50% SARS-CoV-2 neutralizing antibody titers (VN.sub.50).

(155) The vaccination schedule and serum sampling are the same as in FIG. 101. Geometric means fold increase (GMFI) from baseline in VN.sub.50 titer with 95% confidence intervals are shown for (A) younger participants (aged 18 to 55 yrs) immunized with 1, 3, 10, 20, or 30 ?g BNT162b2, and (B) older participants (aged 56 to 85 yrs) immunized with 20 ?g BNT162b2. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). The dotted horizontal line represents the threshold for seroconversion (fold increase ?4). VN.sub.50=50% SARS-CoV-2 neutralizing antibody titers.

(156) FIG. 104. Exemplary frequencies of participants with SARS-CoV-2 GMT seroconversion after immuniziation with BNT162b1.

(157) The vaccination schedule and serum sampling are the same as in FIG. 39 (n=12 per group). Seroconversion with regard to 50% SARS-CoV-2 neutralizing antibody titers (VN.sub.50) is shown for younger participants (aged 18 to 55 yrs) immunized with 1, 10, 30, 50, or 60 ?g BNT162b1. Seroconversion is defined as a minimum of a 4-fold increase of functional antibody response as compared to baseline. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). Dose 2 was not performed in the 60 ?g dose group. GMT=geometric mean titer.

(158) FIG. 105. Exemplary frequencies of participants with SARS-CoV-2 GMT seroconversion after immuniziation with BNT162b2.

(159) The vaccination schedule and serum sampling are the same as in FIG. 101. Seroconversion with regard to 50% SARS-CoV-2 neutralizing antibody titers (VN.sub.50) is shown for (A) younger participants (aged 18 to 55 yrs) dosed with 1, 3, 10, 20, or 30 ?g BNT162b2, and (B) older participants (aged 56 to 85 yrs) dosed with 20 ?g BNT162b2. Seroconversion is defined as a minimum of 4-fold increase of functional antibody response as compared to baseline. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). GMT=geometric mean titer.

(160) FIG. 106. Exemplary fold increase from baseline in S1-binding antibody concentrations after immunization with BNT162b1.

(161) The vaccination schedule and serum sampling are the same as in FIG. 39 (n=12 per group). Geometric means fold increase (GMFI) from baseline in S1-binding antibody concentrations with 95% confidence intervals are shown for younger participants (aged 18 to 55 yrs) immunized with 1, 10, 30, 50, or 60 ?g BNT162b1. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). Dose 2 was not performed in the 60 ?g dose group. The dotted horizontal line represents the threshold for seroconversion (fold increase ?4).

(162) FIG. 107. Exemplary fold increase from baseline in S1-binding antibody concentration after immunization with BNT162b2.

(163) The vaccination schedule and serum sampling are the same as in FIG. 101. Geometric means fold increase (GMFI) from baseline in S1-binding antibody concentrations with 95% confidence intervals are shown for (A) younger participants (aged 18 to 55 yrs) immunized with 1, 3, 10, 20, or 30 ?g BNT162b2, and (B) older participants (aged 56 to 85 yrs) immunized with 20 ?g BNT162b2. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). The dotted horizontal line represents the threshold for seroconversion (fold increase ?4).

(164) FIG. 108. Exemplary frequencies of participants with S1-binding IgG GMC seroconversion after immunization with BNT162b1.

(165) The vaccination schedule and serum sampling are the same as in FIG. 39 (n=12 per group). Seroconversion with regard to S1-binding antibody GMC is shown for younger participants (aged 18 to 55 yrs) immunized with 1, 10, 30, 50, or 60 ?g BNT162b1. Seroconversion is defined as at least a 4-fold increase of S1-binding IgG GMC response as compared to baseline. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). Dose 2 was not performed in the 60 ?g dose group. GMC=geometric mean concentration.

(166) FIG. 109. Exemplary frequencies of participants with S1-binding IgG GMC seroconversion after immunization with BNT162b2.

(167) The vaccination schedule and serum sampling are the same as in FIG. 101. Seroconversion with regard to S1-binding antibody GMC is shown for (A) younger participants (aged 18 to 55 yrs) immunized with 1, 3, 10, 20, or 30 ?g BNT162b2, and (B) older participants (aged 56 to 85 yrs) dosed with 20 ?g BNT162b2. Seroconversion is defined as at least a 4-fold increase of S1-binding IgG GMC response as compared to baseline. Arrowheads indicate baseline (pre-Dose 1, Day 1) and Dose 2 (Day 22). GMC=geometric mean concentration

(168) FIG. 110. Exemplary results of cytokine production produced from S-specific CD4+ T cells from younger participants immunized with BNT162b2.

(169) Peripheral blood mononuclear cell (PBMC) cell fractions isolated from blood of participants treated with varying doses of BNT162b2 were collected at baseline (pre-Dose one) and 29 days (?3 days) after Dose one and analyzed. Participants included younger participants (age 18-55 years) dosed at 1 ?g (n=8), 3 ?g (n=9), 10 ?g (n=10), 20 ?g (n=9), or 30 ?g (n=10). Bar charts show arithmetic means with 95% confidence interval. Cytokine production was calculated by summing up the fractions of all CD4.sup.+ T cells positive for either IFN?, IL-2, or IL-4, setting this sum to 100% and calculating the fraction of each specific cytokine-producing subset thereof. Two participants from the 1 ?g cohort, 1 participant from the 3 ?g cohort, and 1 participant from the 10 ?g cohort were excluded from this analysis (frequency of total cytokine-producing CD4+ T cells <0.03%). IFN=interferon; IL=interleukin; younger participants=participants aged 18 to 55 yrs; S protein=SARS-CoV-2 spike protein.

(170) FIG. 111. Exemplary results of cytokine production produced from S-specific CD4.sup.+ T cells from older participants immunized with BNT162b2.

(171) Peripheral blood mononuclear cell (PBMC) cell fractions isolated from blood of participants treated with varying doses of BNT162b2 were collected at baseline (pre-Dose one) and 29 days (?3 days) after Dose one and analyzed. Participants included older participants (age 56-85 years) dosed at 10 ?g (n=11), 20 ?g (n=8), or 30 ?g (n=9). Bar charts show arithmetic means with 95% CI. Cytokine production was calculated by summing up the fractions of all CD4.sup.+ T cells positive for either IFN?, IL-2, or IL-4, setting this sum to 100%, and calculating the fraction of each specific cytokine-producing subset thereof. Six participants from the 10 ?g cohort and 1 participant from the 20 ?g cohort were excluded from this analysis (frequency of total cytokine-producing CD4+ T cells <0.03%). IFN=interferon; IL=interleukin; older participants=participants aged 56 to 85 yrs; S protein=SARS-CoV-2 spike protein.

(172) FIG. 112. Incidence and magnitude of BNT162b2-induced T-cell responses.

(173) PBMCs obtained on day 1 (pre-prime) and day 29 (7 days post-boost) (dose cohorts 1, 10 and 20 ?g, n=9 each; 30 ?g, n=10) were enriched for CD4.sup.+ or CD8.sup.+ T cell effectors and separately stimulated over night with three overlapping peptide pools representing different portions of the wild-type sequence of SARS-CoV-2 S (N-terminal pools S pool 1 and RBD, and the C-terminal S pool 2), for assessment in direct ex vivo IFN? ELISpot. Common pathogen T-cell epitope pools CEF (immune dominant HLA class I epitopes of CMV, EBV, influenza virus) and CEFT (immune dominant HLA class II epitopes CMV, EBV, influenza virus, tetanus toxoid) were used as controls. Cell culture medium served as negative control. Each dot represents the normalised mean spot count from duplicate wells for one study participant, after subtraction of the medium-only control ((A), (C)). (A) Antigen-specific CD4+ and CD8+ T-cell responses for each dose cohort. The number of participants with a detectable T-cell response on day 29 over the total number of tested participants per dose cohort is provided. Spot count data from two participants from the 20 ?g dose cohort could not be normalised and are not plotted. (B) Example of CD4.sup.+ and CD8.sup.+ ELISpot for a 30 ?g dose cohort participant. (C) S-specific T-cell responses in all participants who recognised either S peptide pool and their baseline CEFT- and CEF-specific T-cell responses. Horizontal bars indicate median values.

(174) FIG. 113. BNT162b2-induced S-specific CD8+ and CD4+ T cells.

(175) CD4.sup.+ or CD8.sup.+ T cell effector-enriched fractions of immunised participants derived from PBMCs obtained on day 1 (pre-prime) and day 29 (7 days post-boost) (1, 10 and 20 ?g dose cohorts, n=9 each; 30 ?g dose cohort, n=10) were stimulated overnight with two overlapping peptide pools covering the wild-type SARS-CoV-2 S (S pool 1 and S pool 2) for assessment in direct ex vivo IFN? ELISpot ((A)-(C)). Each dot represents the normalised mean spot count from duplicate wells for one study participant, after subtraction of the medium-only control. T-cell responses against S pool 1 and S pool 2 per participant were combined. Spot count data from two participants from the 20 ?g dose cohort could not be normalised and are not plotted. PBMCs from vaccinated participants on day 29 (7 days post-boost) (dose cohorts 1 ?g, n=7; 10 and 30 ?g, n=10; 20 ?g, n=9) were stimulated as described above and analysed by flow cytometry ((D), (E)). (A) S-specific CD4.sup.+ and CD8.sup.+ T-cell responses for each dose cohort. Number of participants with detectable T-cell response on day 29 over the total number of tested participants per dose cohort is provided. (B) Mapping of vaccine-induced responses of participants with evaluable baseline data (n=34 for CD4.sup.+ and n=37 for CD8.sup.+ T cell responses) to different portions of S. De novo induced or amplified responses are classified as BNT162b2-induced response; no responses or pre-existing responses that were not amplified by the vaccinations are classified as no vaccine response (none). (C) Response strength to S pool 1 in individuals with or without a pre-existing response to S pool 2. Data from the 1 ?g dose cohort are excluded, as no baseline response to S pool 2 was present in this dose cohort. Horizontal bars represent median of each group. (D) Examples of pseudocolor flow cytometry plots of cytokine-producing CD4.sup.+ and CD8.sup.+ T cells from a participant prime/boost vaccinated with 30 ?g BNT162b2. (E) Frequency of vaccine-induced, S-specific IFN?.sup.+ CD4.sup.+ T cells vs. IL4.sup.+ CD4.sup.+T cells. ICS stimulation was performed using a peptide mixture of S pool 1 and S pool 2. Each data point represents one study participant (1 ?g dose cohort, n=8; 20 ?g dose cohort, n=8; 10 and 30 ?g, n=10 each). One participant from the 20 ?g dose cohort with a strong pre-existing CD4.sup.+ T cell response to S pool 2 was excluded. (F) Antigen-specific CD8.sup.+ T cell frequencies determined by pMHC class I multimer staining (% multimer.sup.+ of CD8.sup.+), ICS and ELISpot (% IFN?.sup.+ of CD8.sup.+) for the three participants analysed in FIG. 116. Signals for S pool 1 and S pool 2 were merged.

(176) FIG. 114. Correlation of antibody and T-cell responses.

(177) Data are plotted for all prime/boost vaccinated participants (dose cohorts 1, 10, 20 and 30 ?g) from day 29, with data points for participants with no detectable T cell response (open circles; (B), (C)) excluded from correlation analysis. (A) Correlation of S1-specific IgG responses with S-specific CD4.sup.+ T-cell responses. (B) Correlation of S-specific CD4.sup.+ with CD8.sup.+ T-cell responses. (C) Correlation of S1-specific IgG responses with S-specific CD8.sup.+ T-cell responses.

(178) FIG. 115. Cytokine polarisation of BNT162b2-induced T cells.

(179) PBMCs obtained on day 1 (pre-prime) and day 29 (7 days post-boost) (dose cohorts 1 ?g, n=8; 10 and 30 ?g, n=10 each; 20 ?g, n=9) and COVID-19 recovered donors (HCS, n=18; (C), (D)) were stimulated over night with three overlapping peptide pools representing different portions of the wild-type sequence of SARS-CoV-2 S (N-terminal pools S pool 1 [aa 1-643] and RBD [aa1-16 fused to aa 327-528 of S], and the C-terminal S pool 2 [aa 633-1273]), and analysed by flow cytometry. (A) Example of pseudocolor flow cytometry plots of cytokine-producing CD4.sup.+ and CD8.sup.+ T cells from a 30 ?g dose cohort participant in response to S pool 1. (B) S-specific CD4.sup.+ T cells producing the indicated cytokine as a fraction of total cytokine-producing S-specific CD4.sup.+ T cells in response to S pool 1 and S pool 2. CD4 non-responders (<0.03% total cytokine producing T cells: 1 ?g, n=2 [S pool 1] and n=1 [S pool 2]; 10 ?g, n=1) were excluded. Arithmetic mean with 95% confidence interval. (C) S-specific CD4.sup.+ (S pool 1, S pool 2 and RBD) and (D) CD8.sup.+ T cells (S pool 1, S pool 2 and RBD) producing the indicated cytokine as a fraction of total circulating T cells of the same subset. Values above data points indicate mean fractions per dose cohort. Participant PBMCs were tested as single instance ((B)-(D)).

(180) FIG. 116. Characterization of BNT162b2-induced T cells on the single epitope level.

(181) PBMCs obtained on day 1 (pre-prime) and day 29 (7 days post-boost) of three vaccinated participants (dose cohorts 10 ?g, n=1; 30 ?g, n=2) were stained with individual pMHC class I multimer cocktails and analysed for T cell epitope specificity (A) and phenotype (B; example from participant 3; YLQPRTFLL (SEQ ID NO: 40)) by flow cytometry. Peptide sequences disclosed in (A) (amino acid sequences of NYNYLYRLF (SEQ ID NO: 43), QYIKWPWYI (SEQ ID NO: 42), KWPWYIWLGF (SEQ ID NO: 44), LPFNDGVYF (SEQ ID NO: 47), QPTESIVRF (SEQ ID NO: 45), IPFAMQMAY (SEQ ID NO: 46), YLQPRTFLL (SEQ ID NO: 40), and RLQSLQTYV (SEQ ID NO: 41)) above dot plots indicate pMHC class I multimer epitope specificity, numbers above dot plots indicate the amino acids corresponding to the epitope within S. (C) Localization of identified MHC class I-restricted epitopes within S.

(182) FIG. 117. ELISA screening analysis of exemplary cohort sera to detect antibody responses directed against the recombinant SARS-CoV-2 spike protein S1 domain.

(183) ELISA was performed using serum samples collected on day 10 after two immunisations (prime/boost on days 1 and 8) with BNT162c1, or on day 17 after three administrations (prime/boost on days 1/8/15) of BNT162a1, BNT162b1, or BNT162b2 to analyse elicited antibody responses. The serum samples were tested against the S1 protein. Group mean AOD values of n=20 mice/group are shown by dots across serum dilutions ranging from 1:100 to 1:24, 300.

(184) FIG. 118. ELISA screening analysis of exemplary cohort sera to detect antibody responses directed against the recombinant SARS-CoV-2 spike protein RBD domain.

(185) ELISA was performed using serum samples collected on day 10 after two immunisations (prime/boost on days 1 and 8) with BNT162c1, or on day 17 after three administrations (prime/boost on days 1/8/15) of BNT162a1, BNT162b1, or BNT162b2 to analyse elicited antibody responses. The serum samples were tested against the RBD domain. Group mean AOD values of n=20 mice/group are shown by dots across serum dilutions ranging from 1:100 to 1:24,300.

(186) FIG. 119. Pseudovirus neturalisation activity of exemplary cohort sera plotted as pVN.sub.50 titre.

(187) Serum samples were collected on day 10 (BNT162c1, saRNA) or day 17 (all other cohorts) after first immunisation of the animals and titres of virus-neutralising antibodies were determined by pseudovirus-based neutralisation test (pVNT). Individual VNT titres resulting in 50% pseudovirus neutralisation (pVN.sub.50) are shown by dots; group mean values are indicated by horizontal bars (?SEM, standard error of the mean).

(188) FIG. 120. The virus-neutralising antibodies and specific binding antibody responses to RBD and S1 in participants.

(189) RBD=receptor binding domain. GMT=geometric mean titer. Serum samples were obtained before vaccination (day 1) and day 8, 22, 29, and 43 after the prime vaccination in younger adult group, and they were obtained before vaccination (day 1) and day 22, 29, and 43 days after the prime vaccination in older adult group. A panel of human COVID-19 convalescent serum (n=24) were obtained at least 14 days after PCR-confirmed diagnosis in COVID-19 patients. (A) GMTs of SARS-CoV-2 neutralizing antibodies. (B) GMTs of binding antibodies to RBD measured by ELISA. (C) GMTs of ELISA antibodies to S1. Each point represents a serum sample, and each vertical bar represents a geometric mean with 95% CI.

(190) FIG. 121. T-cell response in participants before and after vaccination measured by IFN-? ELISpot.

(191) IFN=interferon. PBMC=peripheral blood mononuclear cells. The S1 peptide pool covers the N-terminal half of SARS-CoV-2 spike, including RBD. S2 peptide pool covers the C-terminal of SARS-CoV-2 spike, not including RBD. CEF peptide pool consists of 32 MHC class I restricted viral peptides from human cytomegalovirus, Epstein-Barr virus and influenza virus. Panel A shows the number of specific T cell with secretion of IFN-? at day 1, 29, and 43 in the younger participants aged 18-55 years. Panel B shows the number of specific T cell with secretion of IFN-? at day 1, 29, and 43 in the older participants aged 65-85 years.

(192) FIG. 122. 50% pseudovirus neutralization titers of 16 sera from BNT162b2 vaccine recipients against VSV-SARS-CoV-2-S pseudovirus bearing the Wuhan or lineage B.1.1.7 spike protein. N=8 representative sera each from younger adults (aged 18 to 55 yrs; indicated by triangles) and older adults (aged 56 to 85 yrs; indicated by circles) drawn at day 43 (21 days after dose 2) were tested.

(193) FIG. 123. Schematic illustration of the production of VSV pseudoviruses bearing SARS-CoV-2 S protein. (1) Transfection of SARS-CoV-2-S expression plasmid into HEK293/T17 cells. (2) Infection of SARS-CoV-2 S expressing cells with VSV-G complemented input virus lacking the VSV-G in its genome (VSV?G) and encoding for reporter genes. (3) Neutralization of residual VSV-G complemented input virus by addition of anti-VSV-G antibody yields SARS-CoV-2 S pseudotyped VSV?G as a surrogate for live SARS-CoV-2.

(194) FIG. 124. Titration of SARS-CoV-2 Wuhan reference strain and lineage B.1.1.7 spike-pseudotyped VSV on Vero 76 cells using GFP-infected cells as read-out.

(195) FIG. 125. Scheme of the BNT162b2 vaccination and serum sampling.

(196) FIG. 126. Plot of the ratio of pVNT.sub.50 between SARS-CoV-2 lineage B.1.1.7 and Wuhan reference strain spike-pseudotyped VSV. Triangles represent sera from younger adults (aged 18 to 55 yrs), and circles represent sera from older adults (aged 56 to 85 yrs). The sea were drawn on day 43 (21 days after dose 2).

(197) FIG. 127. 50% pseudovirus neutralization titers (pVNT50) of 12 sera from BNT162b2 vaccine recipients against VSV-SARS-CoV-2-S pseudovirus bearing the Wuhan Hu-1 reference, lineage B.1.1.298 or lineage B.1.351 spike protein. N=12 sera from younger adults immunized with 30 ?g BNT162b2 drawn at either day 29 or day 43 (7 or 21 days after dose 2) were tested. Geometric mean titers are indicated. Statistical significance of the difference between the neutralization of the Wuhan Hu-1 reference pseudovirus and either the lineage B.1.1.298 or the lineage B.1.351 pseudovirus was calculated by a Wilcoxon matched-pairs signed rank test. Two-tailed p-values are reported. ns, not significant;***, P<0.001; LLOQ, lower limit of quantification.

(198) FIG. 128. 50% plaque reduction neutralization titers of 20 sera from BNT162b2 vaccine recipients against N501 and Y501 SARS-CoV-2. Seven sera (indicated by triangles) were drawn 2 weeks after the second dose of vaccine; 13 sera (indicated by circles) were drawn 4 weeks after the second dose.

(199) FIG. 129. Diagram of the N501Y substitution. Lleader sequence; ORFopen reading frame; RBDreceptor binding domain; Sspike glycoprotein; S1N-terminal furin cleavage fragment of S; S2C-terminal furin cleavage fragment of S; Eenvelope protein; Mmembrane protein; Nnucleoprotein; UTRuntranslated region. The nucleotide sequence of CCC ACT AAT GGT GTT (SEQ ID NO: 50) and the amino acid sequence of Pro Thr Asn Gly Val (SEQ ID NO: 49) correspond to the unmutated S protein; and the nucleotide sequence of CCC ACTTAT GGT GTT (SEQ ID NO: 50) and the amino acid sequence of Pro Thr Tyr Gly Val (SEQ ID NO: 51) correspond to an S protein comprising the N501Y substitution.

(200) FIG. 130. Plaque morphologies of N501 and Y501 SARS-CoV-2 on Vero E6 cells.

(201) FIG. 131. Scheme of the BNT162 vaccination and serum sampling.

(202) FIG. 132. Plot of the ratio of PRNT.sub.50 between Y501 and N501 viruses. Triangles represent sera drawn two weeks after the second dose; circles represent sera drawn four weeks after the second dose.

(203) FIG. 133. Engineered mutations. Nucleotide and amino acid positions are indicated. Deletions are depicted by dotted lines. Mutant nucleotides are in red. L, leader sequence; ORF, open reading frame; RBD, receptor binding domain; S, spike glycoprotein; S1, N-terminal furin cleavage fragment of S; S2, C-terminal furin cleavage fragment of S; E, envelope protein; M, membrane protein; N, nucleoprotein; UTR, untranslated region.

(204) FIG. 134. Plaque morphologies of WT (USA-WA1/2020), mutant N501Y, A69/70+N501Y+D614G, and E484K+N501Y+D614G SARS-CoV-2s on Vero E6 cells.

(205) FIG. 135. Scheme of the BNT162 vaccination and serum sampling.

(206) FIG. 136. PRNT.sub.50s of twenty BNT162b2-vaccinated human sera against wild-type (WT) and mutant SARS-CoV-2. (A) WT (USA-WA1/2020) and mutant N501Y. (B) WT and A69/70+N501Y+D614G. (C) WT and E484K+N501Y+D614G. Seven (triangles) and thirteen (circles) sera were drawn 2 and 4 weeks after the second dose of vaccination, respectively. Sera with different PRNT.sub.50s against WT and mutant viruses are connected by lines. Results in (A) were from one experiment; results in (B) and (C) were from another set of experiments. Each data point is the average of duplicate assay results.

(207) FIG. 137. Ratios of neutralization GMTs against mutant viruses to GMTs against WT virus. Triangles represent sera drawn two weeks after the second dose of vaccination; circles represent sera drawn four weeks after the second dose of vaccination.

(208) FIG. 138. Diagram of engineered spike substitutions and deletions. The genome and sequence of clinical isolate USA-WA1/2020 are used as the wild-type virus in this study. Mutations from the United Kingdom B.1.1.7, Brazilian P.1, and South African B.1.351 lineages are presented. Deletions are indicated by dotted lines. Mutated nucleotides are in red. Nucleotide and amino acid positions are indicated. Lleader sequence; ORFopen reading frame; RBDreceptor binding domain; Sspike glycoprotein; S1N-terminal furin cleavage fragment of S; S2C-terminal furin cleavage fragment of S; Eenvelope protein; Mmembrane protein; Nnucleoprotein; UTRuntranslated region.

(209) FIG. 139. Plaque morphologies of USA-WA1/2020 and mutant SARS-CoV-2's. The plaque assays were performed on Vero E6 cells in 6-well plates.

(210) FIG. 140. Scheme of BNT162 immunization and serum collection.

(211) FIG. 141. Serum Neutralization of Variant Strains of SARS-CoV-2 after the Second Dose of BNT162b2 Vaccine. Shown are the results of 50% plaque reduction neutralization testing (PRNT.sub.50) with the use of 20 samples obtained from 15 trial participants 2 weeks (circles) or 4 weeks (triangles) after the administration of the second dose of the BNT162b2 vaccine. The mutant viruses were obtained by engineering the full set of mutations in the B.1.1.7, P.1., or B.1.351 lineages or subsets of the S gene mutations in the B.1.351 lineage (B.1.351-A242-244+D614G and B.1.351-RBD-D614G) into USA-WA1/2020. Each data point represents the geometric mean PRNT.sub.50 obtained with a serum sample against the indicated virus, including data from repeat experiments, as detailed in Table 31. The data for USA-WA1/2020 are from three experiments; for B.1.1.7-spike, B.1.351-A242-244+D614G, and B.1.351-RBD-D614G viruses from one experiment each; and for P.1-spike and B.1.351-spike viruses from two experiments each. In each experiment, the neutralization titer was determined in duplicate assays, and the geometric mean was taken. LOD: limit of detection.

(212) FIG. 142. Durability of BNT162b2-induced T cell responses.

(213) PBMCs obtained on Day 1 (pre-prime), Day 29, Day 85, and Day 184 (7 days, 9 and 23 weeks post-boost, respectively), were analyzed in ex vivo IFN? ELISpot (for details see GA-RB-022-01A). Common pathogen T-cell epitope pools CEF (CMV, EBV, and influenza virus HLA class I epitopes) and CEFT (CMV, EBV, influenza virus, and tetanus toxoid HLA class II epitopes) served to assess general T-cell reactivity, cell culture medium served as negative control. Each dot represents the sum of normalized mean spot count from duplicate wells stimulated with two peptide pools corresponding to the full-length wt S protein for one study subject, after subtraction of the medium-only control. Ratios above post-vaccination data points are the number of subjects with detectable CD4+ or CD8+ T-cell responses within the total number of tested subjects per dose cohort and time-point.

(214) FIG. 143. A specific vaccine mRNA signal is detected in the LN 6h post injection using modV9 probe in dual IHC-ISH assay (darker color is associated with higher signaling). Vaccine is mostly localized to subcapsular sinus (LN in 9 and 5 positions) and B cell follicles (LN in 12 and 1 positions). Dendritic cells are visualized by CD11c staining (upper images) and only some of them uptake the vaccine. Majority of CD169+ macrophages (subcapsular sinus macrophages, middle images) are positive for the vaccine. B cells (CD19+, lower images) are the second major population showing vaccine signal.

(215) FIG. 144. A specific vaccine mRNA signal is detected in the spleen 6h post injection using modV9 probe in dual IHC-ISH assay (darker color is associated with higher signaling). Majority of the vaccine signal is detected in the white pulp. Dendritic cells are visualized by CD11c staining (upper images) and only some of them uptake the vaccine. A small portion of F4/80+ macrophages (middle images) uptake the vaccine. B cells (CD19+, lower images) are the major population showing the vaccine signal.

(216) FIG. 145. Exemplary Stability Data. Exemplary data from certain stability studies (see, for example, Example 42, are shown for a BNT162b2 LNP preparation at indicated concentrations and temperature conditions, as assessed by ELISA characterizing antibodies reactive to S1 spike protein.

DESCRIPTION OF THE SEQUENCES

(217) The following table provides a listing of certain sequences referenced herein.

(218) TABLE-US-00001 TABLE1 DESCRIPTIONOFTHESEQUENCES SEQ ID NO: Description SEQUENCE AntigenicSproteinsequences 1 Sprotein(amino MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEK acid) SNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFV FKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCAL DPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNC YFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEIL DITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP RRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGFNFSQ1LPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAG TITSGINTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQE KNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPINYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKG VKLHYT 2 Sprotein(CDS) auguuuguguuucuugugcugcugccucuugugucuucucagugugugaauuugacaacaagaacacagcugccaccagcuuauacaaauucuuuuaccagaggagug uauuauccugauaaaguguuuagaucuucugugcugcacagcacacaggaccuguuucugccauuuuuuagcaaugugacaugguuucaugcaauucaugugucugg aacaaauggaacaaaaagauuugauaauccugugcugccuuuuaaugauggaguguauuuugcuucaacagaaaagucaaauauuauuagaggauggauuuuugga acaacacuggauucuaaaacacagucucugcugauugugaauaaugcaacaaauguggugauuaaagugugugaauuucaguuuuguaaugauccuuuucugggagu guauuaucacaaaaauaauaaaucuuggauggaaucugaauuuagaguguauuccucugcaaauaauuguacauuugaauaugugucucagccuuuucugauggau cuggaaggaaaacagggcaauuuuaaaaaucugagagaauuuguguuuaaaaauauugauggauauuuuaaaauuuauucuaaacacacaccaauuaauuuaguga gagaucugccucagggauuuucugcucuggaaccucugguggaucugccaauuggcauuaauauuacaagauuucagacacugcuggcucugcacagaucuuaucugac accuggagauucuucuucuggauggacagccggagcugcagcuuauuaugugggcuaucugcagccaagaacauuucugcugaaauauaaugaaaauggaacaauuac agaugcuguggauugugcucuggauccucugucugaaacaaaauguacauuaaaaucuuuuacaguggaaaaaggcauuuaucagacaucuaauuuuagagugcagc caacagaaucuauugugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaagauuugcaucuguguaugcauggaauagaaaaaga auuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuu acaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagauugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugau uuuacaggaugugugauugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuu uugaaagagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagcca accaauggugugggauaucagccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaaaaucuacaaauuuagugaaa aauaaaugugugaauuuuaauuuuaauggauuaacaggaacaggagugcugacagaaucuaauaaaaaauuucugccuuuucagcaguuuggcagagauauugcag auaccacagaugcagugagagauccucagacauuagaaauucuggauauuacaccuuguucuuuugggggugugucugugauuacaccuggaacaaauacaucuaauc agguggcugugcuguaucaggaugugaauuguacagaagugccaguggcaauucaugcagaucagcugacaccaacauggagaguguauucuacaggaucuaaugugu uucagacaagagcaggaugucugauuggagcagaacaugugaauaauucuuaugaaugugauauuccaauuggagcaggcauuugugcaucuuaucagacacagacaa auuccccaaggagagcaagaucuguggcaucucagucuauuauugcauacaccaugucucugggagcagaaaauucuguggcauauucuaauaauucuauugcuauuc caacaaauuuuaccauuucugugacaacagaaauuuuaccugugucuaugacaaaaacaucuguggauuguaccauguacauuuguggagauucuacagaauguucu aaucugcugcugcaguauggaucuuuuuguacacagcugaauagagcuuuaacaggaauugcuguggaacaggauaaaaauacacaggaaguguuugcucaggugaaa cagauuuacaaaacaccaccaauuaaagauuuuggaggauuuaauuuuagccagauucugccugauccuucuaaaccuucuaaaagaucuuuuauugaagaucugcu guuuaauaaagugacacuggcagaugcaggauuuauuaaacaguauggagauugccugggugauauugcugcaagagaucugauuugugcucagaaauuuaauggac ugacagugcugccuccucugcugacagaugaaaugauugcucaguacacaucugcuuuacuggcuggaacaauuacaagcggauggacauuuggagcuggagcugcucu gcagauuccuuuugcaaugcagauggcuuacagauuuaauggaauuggagugacacagaauguguuauaugaaaaucagaaacugauugcaaaucaguuuaauucug caauuggcaaaauucaggauucucugucuucuacagcuucugcucugggaaaacugcaggauguggugaaucagaaugcacaggcacugaauacucuggugaaacagcu gucuagcaauuuuggggcaauuucuucugugcugaaugauauucugucuagacuggauaaaguggaagcugaagugcagauugauagacugaucacaggaagacugca gucucugcagacuuaugugacacagcagcugauuagagcugcugaaauuagagcuucugcuaaucuggcugcuacaaaaaugucugaaugugugcugggacagucaaa aagaguggauuuuuguggaaaaggauaucaucugaugucuuuuccacagucugcuccacauggagugguguuuuuacaugugacauaugugccagcacaggaaaagaa uuuuaccacagcaccagcaauuugucaugauggaaaagcacauuuuccaagagaaggaguguuugugucuaauggaacacauugguuugugacacagagaaauuuuu augaaccucagauuauuacaacagauaauacauuugugucaggaaauugugauguggugauuggaauugugaauaauacaguguaugauccacugcagccagaacug gauucuuuuaaagaagaacuggauaaauauuuuaaaaaucacacaucuccugauguggauuuaggagauauuucuggaaucaaugcaucuguggugaauauucaga aagaaauugauagacugaaugaaguggccaaaaaucugaaugaaucucugauugaucugcaggaacuuggaaaauaugaacaguacauuaaauggccuugguacauu uggcuuggauuuauugcaggauuaauugcaauugugauggugacaauuauguuauguuguaugacaucauguuguucuuguuuaaaaggauguuguucuugugga agcuguuguaaauuugaugaagaugauucugaaccuguguuaaaaggagugaaauugcauuacaca 3 SproteinRBD MFVFLVLLPLVSSQCVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR (amino QIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG acid)(V05) YQPYRVVVLSFELLHAPATVCGPK 4 SproteinRBD auguuuguguuucuugugcugcugccucuugugucuucucaguguguggugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaag (CDS)(V05) auuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuaugga gugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagauugcccccggacagacaggaaa aauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucug uacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauug uuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacagu guguggaccuaaa 5 SproteinRBD/ MFVFLVLLPLVSSQCVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVR Fibritin(amino QIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVG acid)(V05) YQPYRVVVLSFELLHAPATVCGPKGSPGSGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPG auguuuguguuucuugugcugcugccucuugugucuucucaguguguggugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaag auuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuaugga gugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagauugcccccggacagacaggaaa 6 SproteinRBD/ aauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucug Fibritin(CDS) uacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauug (V05) uuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacagu guguggaccuaaaggcucccccggcuccggcuccggaucugguuauauuccugaagcuccaagagaugggcaagcuuacguucguaaagauggcgaauggguauuacuu ucuaccuuuuuaggccggucccuggaggugcuguuccagggccccggc 7 SproteinPP MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEK (aminoacid) SNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFV (V08/V09) FKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCAL DPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNC YFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEIL DITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP RRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAG TITSGINTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISS VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQE KNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDI SGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPINYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKG VKLHYT 8 SproteinPP auguuuguguuucuugugcugcugccucuugugucuucucagugugugaauuugacaacaagaacacagcugccaccagcuuauacaaauucuuuuaccagaggagug (CDS)(V08) uauuauccugauaaaguguuuagaucuucugugcugcacagcacacaggaccuguuucugccauuuuuuagcaaugugacaugguuucaugcaauucaugugucugg aacaaauggaacaaaaagauuugauaauccugugcugccuuuuaaugauggaguguauuuugcuucaacagaaaagucaaauauuauuagaggauggauuuuugga acaacacuggauucuaaaacacagucucugcugauugugaauaaugcaacaaauguggugauuaaagugugugaauuucaguuuuguaaugauccuuuucugggagu guauuaucacaaaaauaauaaaucuuggauggaaucugaauuuagaguguauuccucugcaaauaauuguacauuugaauaugugucucagccuuuucugauggau cuggaaggaaaacagggcaauuuuaaaaaucugagagaauuuguguuuaaaaauauugauggauauuuuaaaauuuauucuaaacacacaccaauuaauuuaguga gagaucugccucagggauuuucugcucuggaaccucugguggaucugccaauuggcauuaauauuacaagauuucagacacugcuggcucugcacagaucuuaucugac accuggagauucuucuucuggauggacagccggagcugcagcuuauuaugugggcuaucugcagccaagaacauuucugcugaaauauaaugaaaauggaacaauuac agaugcuguggauugugcucuggauccucugucugaaacaaaauguacauuaaaaucuuuuacaguggaaaaaggcauuuaucagacaucuaauuuuagagugcagc caacagaaucuauugugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaaugcaacaagauuugcaucuguguaugcauggaauagaaaaaga auuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuu acaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagauugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugau uuuacaggaugugugauugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuu uugaaagagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagcca accaauggugugggauaucagccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaaaaucuacaaauuuagugaaa aauaaaugugugaauuuuaauuuuaauggauuaacaggaacaggagugcugacagaaucuaauaaaaaauuucugccuuuucagcaguuuggcagagauauugcag auaccacagaugcagugagagauccucagacauuagaaauucuggauauuacaccuuguucuuuugggggugugucugugauuacaccuggaacaaauacaucuaauc agguggcugugcuguaucaggaugugaauuguacagaagugccaguggcaauucaugcagaucagcugacaccaacauggagaguguauucuacaggaucuaaugugu uucagacaagagcaggaugucugauuggagcagaacaugugaauaauucuuaugaaugugauauuccaauuggagcaggcauuugugcaucuuaucagacacagacaa auuccccaaggagagcaagaucuguggcaucucagucuauuauugcauacaccaugucucugggagcagaaaauucuguggcauauucuaauaauucuauugcuauuc caacaaauuuuaccauuucugugacaacagaaauuuuaccugugucuaugacaaaaacaucuguggauuguaccauguacauuuguggagauucuacagaauguucu aaucugcugcugcaguauggaucuuuuuguacacagcugaauagagcuuuaacaggaauugcuguggaacaggauaaaaauacacaggaaguguuugcucaggugaaa cagauuuacaaaacaccaccaauuaaagauuuuggaggauuuaauuuuagccagauucugccugauccuucuaaaccuucuaaaagaucuuuuauugaagaucugcu guuuaauaaagugacacuggcagaugcaggauuuauuaaacaguauggagauugccugggugauauugcugcaagagaucugauuugugcucagaaauuuaauggac ugacagugcugccuccucugcugacagaugaaaugauugcucaguacacaucugcuuuacuggcuggaacaauuacaagcggauggacauuuggagcuggagcugcucu gcagauuccuuuugcaaugcagauggcuuacagauuuaauggaauuggagugacacagaauguguuauaugaaaaucagaaacugauugcaaaucaguuuaauucug caauuggcaaaauucaggauucucugucuucuacagcuucugcucugggaaaacugcaggauguggugaaucagaaugcacaggcacugaauacucuggugaaacagcu gucuagcaauuuuggggcaauuucuucugugcugaaugauauucugucuagacuggauccuccugaagcugaagugcagauugauagacugaucacaggaagacugca gucucugcagacuuaugugacacagcagcugauuagagcugcugaaauuagagcuucugcuaaucuggcugcuacaaaaaugucugaaugugugcugggacagucaaa aagaguggauuuuuguggaaaaggauaucaucugaugucuuuuccacagucugcuccacauggagugguguuuuuacaugugacauaugugccagcacaggaaaagaa uuuuaccacagcaccagcaauuugucaugauggaaaagcacauuuuccaagagaaggaguguuugugucuaauggaacacauugguuugugacacagagaaauuuuu augaaccucagauuauuacaacagauaauacauuugugucaggaaauugugauguggugauuggaauugugaauaauacaguguaugauccacugcagccagaacug gauucuuuuaaagaagaacuggauaaauauuuuaaaaaucacacaucuccugauguggauuuaggagauauuucuggaaucaaugcaucuguggugaauauucaga aagaaauugauagacugaaugaaguggccaaaaaucugaaugaaucucugauugaucugcaggaacuuggaaaauaugaacaguacauuaaauggccuugguacauu uggcuuggauuuauugcaggauuaauugcaauugugauggugacaauuauguuauguuguaugacaucauguuguucuuguuuaaaaggauguuguucuugugga agcuguuguaaauuugaugaagaugauucugaaccuguguuaaaaggagugaaauugcauuacaca 9 SproteinPP auguucguguuccuggugcugcugccucugguguccagccagugugugaaccugaccaccagaacacagcugccuccagccuacaccaacagcuuuaccagaggcguguac (CDS)(V09) uaccccgacaagguguucagauccagcgugcugcacucuacccaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccauccacguguccggcaccaaug gcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagaggcuggaucuucggcaccacacuggacag caagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugcaacgaccccuuccugggcgucuacuaccacaagaacaac aagagcuggauggaaagcgaguuccggguguacagcagcgccaacaacugcaccuucgaguacgugucccagccuuuccugauggaccuggaaggcaagcagggcaacuuc aagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuacagcaagcacaccccuaucaaccucgugcgggaucugccucagggcuucucugcucug gaaccccugguggaucugcccaucggcaucaacaucacccgguuucagacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcggauggacagcug gugccgccgcuuacuaugugggcuaccugcagccuagaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcg agacaaagugcacccugaaguccuucaccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucu gugccccuucggcgagguguucaaugccaccagauucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuccgcc agcuucagcaccuucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggagaugaagugcggcag auugccccuggacagacaggcaagaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaaccuggacuccaaagucggcg gcaacuacaauuaccuguaccggcuguuccggaaguccaaucugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcagcaccccuuguaacggcgugg aaggcuucaacugcuacuucccacugcaguccuacggcuuucagcccacaaauggcgugggcuaucagcccuacagagugguggugcugagcuucgaacugcugcaugccc cugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacggccugaccggcaccggcgugcugacagagagcaacaaga aguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagauccccagacacuggaaauccuggacaucaccccuugcagcuucggcggag ugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguaccaggacgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuac auggcggguguacuccaccggcagcaauguguuucagaccagagccggcugucugaucggagccgagcacgugaacaauagcuacgagugcgacauccccaucggcgcugg aaucugcgccagcuaccagacacagacaaacagcccucggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggcc uacuccaacaacucuaucgcuauccccaccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcg gcgauuccaccgagugcuccaaccugcugcugcaguacggcagcuucugcacccagcugaauagagcccugacagggaucgccguggaacaggacaagaacacccaagaggu guucgcccaagugaagcagaucuacaagaccccuccuaucaaggacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuucau cgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugcgcccagaaguu uaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggcuggacauuuggagcaggcgcc gcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaaccagaagcugaucgccaaccaguucaacagc gccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaaccagaaugcccaggcacugaacacccuggucaagcagcugu ccuccaacuucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccugaggccgaggugcagaucgacagacugaucacaggcagacugcagagccu ccagacauacgugacccagcagcugaucagagccgccgagauuagagccucugccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagagugga cuuuugcggcaagggcuaccaccugaugagcuucccucagucugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgc uccagccaucugccacgacggcaaagcccacuuuccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucauc accaccgacaacaccuucgugucuggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacug gacaaguacuuuaagaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgaggug gccaagaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacugauugc caucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacgaggacgauucugag cccgugcugaagggcgugaaacugcacuacaca Foldon 10 Foldon(amino GSGYIPEAPRDGQAYVRKDGEINVLLSTFLGRSLEVLFQGPG acid) 11 Foldon(CDS) ggaucugguuauauuccugaagcuccaagagaugggcaagcuuacguucguaaagauggcgaauggguauuacuuucuaccuuuuuaggccggucccuggaggugcug uuccagggccccggc 5'-UTR(hAg-Kozak) 12 5-UTR AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC 3'-UTR(FIelement) 13 3-UTR CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCC ACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACA CCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGU GCCAGCCACACC A30L70 14 A30L70 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAA

DETAILED DESCRIPTION

(219) Although the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

(220) Preferably, the terms used herein are defined as described in A multilingual glossary of biotechnological terms: (IUPAC Recommendations), H. G. W. Leuenberger, B. Nagel, and H. K?lbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

(221) The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

(222) In the following, the elements of the present disclosure will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements should be considered disclosed by this description unless the context indicates otherwise.

(223) The term about means approximately or nearly, and in the context of a numerical value or range set forth herein in one embodiment means?20%, ?10%, ?5%, or ?3% of the numerical value or range recited or claimed.

(224) The terms a and an and the and similar reference used in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., such as), provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

(225) Unless expressly specified otherwise, the term comprising is used in the context of the present document to indicate that further members may optionally be present in addition to the members of the list introduced by comprising. It is, however, contemplated as a specific embodiment of the present disclosure that the term comprising encompasses the possibility of no further members being present, i.e., for the purpose of this embodiment comprising is to be understood as having the meaning of consisting of or consisting essentially of. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present disclosure was not entitled to antedate such disclosure.

Definitions

(226) In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

(227) Terms such as reduce, decrease, inhibit or impair as used herein relate to an overall reduction or the ability to cause an overall reduction, preferably of at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or even more, in the level. These terms include a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.

(228) Terms such as increase, enhance or exceed preferably relate to an increase or enhancement by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or even more.

(229) According to the disclosure, the term peptide comprises oligo- and polypeptides and refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term protein or polypeptide refers to large peptides, in particular peptides having at least about 150 amino acids, but the terms peptide, protein and polypeptide are used herein usually as synonyms.

(230) A therapeutic protein has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In one embodiment, a therapeutic protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term therapeutic protein includes entire proteins or peptides, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Examples of therapeutically active proteins include, but are not limited to, antigens for vaccination and immunostimulants such as cytokines.

(231) Fragment, with reference to an amino acid sequence (peptide or protein), relates to a part of an amino acid sequence, i.e. a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 3-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) is obtainable e.g. by translation of a truncated open reading frame that lacks the 5-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises e.g. at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence.

(232) By variant herein is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid modification. The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. Preferably, the variant amino acid sequence has at least one amino acid modification compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid modifications, and preferably from 1 to about 10 or from 1 to about 5 amino acid modifications compared to the parent.

(233) By wild type or WT or native herein is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or protein has an amino acid sequence that has not been intentionally modified.

(234) For the purposes of the present disclosure, variants of an amino acid sequence (peptide, protein or polypeptide) comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants. The term variant includes all mutants, splice variants, posttranslationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particularthose which are naturally occurring. The term variant includes, in particular, fragments of an amino acid sequence.

(235) Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C-terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous proteins or peptides and/or to replacing amino acids with other ones having similar properties. Preferably, amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

(236) Preferably the degree of similarity, preferably identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is given preferably for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preferably for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity can be done with art known tools, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5. Sequence similarity indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. Sequence identity between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. Sequence identity between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

(237) The terms % identical, % identity or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or window of comparison, in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, ?2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.

(238) Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.

(239) In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence. Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

(240) The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins having substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

(241) In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a functional fragment or functional variant. The term functional fragment or functional variant of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to antigens or antigenic sequences, one particular function is one or more immunogenic activities displayed by the amino acid sequence from which the fragment or variant is derived. The term functional fragment or functional variant, as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., inducing an immune response. In one embodiment, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

(242) An amino acid sequence (peptide, protein or polypeptide) derived from a designated amino acid sequence (peptide, protein or polypeptide) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the antigens suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.

(243) As used herein, an instructional material or instructions includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the compositions of the invention or be shipped together with a container which contains the compositions. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.

(244) Isolated means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not isolated, but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is isolated. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

(245) The term recombinant in the context of the present invention means made through genetic engineering. Preferably, a recombinant object such as a recombinant nucleic acid in the context of the present invention is not occurring naturally.

(246) The term naturally occurring as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.

(247) Physiological pH as used herein refers to a pH of about 7.5.

(248) The term genetic modification or simply modification includes the transfection of cells with nucleic acid. The term transfection relates to the introduction of nucleic acids, in particular RNA, into a cell. For purposes of the present invention, the term transfection also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient. Thus, according to the present invention, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and/or an organism of a patient. According to the invention, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection. Generally, nucleic acid encoding antigen is transiently transfected into cells. RNA can be transfected into cells to transiently express its coded protein.

(249) The term seroconversion includes a ?4-fold rise from before vaccination to 1-month post Dose 2.

(250) Coronavirus

(251) Coronaviruses are enveloped, positive-sense, single-stranded RNA ((+) ssRNA) viruses. They have the largest genomes (26-32 kb) among known RNA viruses and are phylogenetically divided into four genera (?, ?, ?, and ?), with betacoronaviruses further subdivided into four lineages (A, B, C, and D). Coronaviruses infect a wide range of avian and mammalian species, including humans. Some human coronaviruses generally cause mild respiratory diseases, although severity can be greater in infants, the elderly, and the immunocompromised. Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV), belonging to betacoronavirus lineages C and B, respectively, are highly pathogenic. Both viruses emerged into the human population from animal reservoirs within the last 15 years and caused outbreaks with high case-fatality rates. The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that causes atypical pneumonia (coronavirus disease 2019; COVID-19) has raged in China since mid-December 2019, and has developed to be a public health emergency of international concern. SARS-CoV-2 (MN908947.3) belongs to betacoronavirus lineage B. It has at least 70% sequence similarity to SARS-CoV.

(252) In general, coronaviruses have four structural proteins, namely, envelope (E), membrane (M), nucleocapsid (N), and spike (S). The E and M proteins have important functions in the viral assembly, and the N protein is necessary for viral RNA synthesis. The critical glycoprotein S is responsible for virus binding and entry into target cells. The S protein is synthesized as a single-chain inactive precursor that is cleaved by furin-like host proteases in the producing cell into two noncovalently associated subunits, S1 and S2. The S1 subunit contains the receptor-binding domain (RBD), which recognizes the host-cell receptor. The S2 subunit contains the fusion peptide, two heptad repeats, and a transmembrane domain, all of which are required to mediate fusion of the viral and host-cell membranes by undergoing a large conformational rearrangement. The S1 and S2 subunits trimerize to form a large prefusion spike.

(253) The S precursor protein of SARS-CoV-2 can be proteolytically cleaved into S1 (685 aa) and S2 (588 aa) subunits. The S1 subunit consists of the receptor-binding domain (RBD), which mediates virus entry into sensitive cells through the host angiotensin-converting enzyme 2 (ACE2) receptor.

(254) Antigen

(255) The present invention comprises the use of RNA encoding an amino acid sequence comprising SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof. Thus, the RNA encodes a peptide or protein comprising at least an epitope SARS-CoV-2 S protein or an immunogenic variant thereof for inducing an immune response against coronavirus S protein, in particular SARS-CoV-2 S protein in a subject. The amino acid sequence comprising SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof (i.e., the antigenic peptide or protein) is also designated herein as vaccine antigen, peptide and protein antigen, antigen molecule or simply antigen. The SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof is also designated herein as antigenic peptide or protein or antigenic sequence.

(256) SARS-CoV-2 coronavirus full length spike (S) protein consist of 1273 amino acids (see SEQ ID NO: 1). In specific embodiments, full length spike (S) protein according to SEQ ID NO: 1 is modified in such a way that the prototypical prefusion conformation is stabilized. Stabilization of the prefusion conformation may be obtained by introducing two consecutive proline substitutions at AS residues 986 and 987 in the full length spike protein. Specifically, spike (S) protein stabilized protein variants are obtained in a way that the amino acid residue at position 986 is exchanged to proline and the amino acid residue at position 987 is also exchanged to proline. In one embodiment, a SARS-CoV-2 S protein variant comprises the amino acid sequence shown in SEQ ID NO: 7.

(257) In one embodiment, the vaccine antigen described herein comprises, consists essentially of or consists of a spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof.

(258) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

(259) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 3819 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 1 or 7.

(260) In one embodiment, the vaccine antigen comprises, consists essentially of or consists of SARS-CoV-2 spike S1 fragment (S1) (the S1 subunit of a spike protein (S) of SARS-CoV-2), a variant thereof, or a fragment thereof.

(261) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1.

(262) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 683 of SEQ ID NO: 1.

(263) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1.

(264) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 49 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 17 to 685 of SEQ ID NO: 1.

(265) In one embodiment, the vaccine antigen comprises, consists essentially of or consists of the receptor binding domain (RBD) of the S1 subunit of a spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof. The amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, a variant thereof, or a fragment thereof is also referred to herein as RBD or RBD domain.

(266) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1.

(267) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 979 to 1584 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1.

(268) According to certain embodiments, a signal peptide is fused, either directly or through a linker, to a SARS-CoV-2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein. Accordingly, in one embodiment, a signal peptide is fused to the above described amino acid sequences derived from SARS-CoV-2 S protein or immunogenic fragments thereof (antigenic peptides or proteins) comprised by the vaccine antigens described above.

(269) Such signal peptides are sequences, which typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N-terminus of the antigenic peptide or protein, without being limited thereto. Signal peptides as defined herein preferably allow the transport of the antigenic peptide or protein as encoded by the RNA into a defined cellular compartment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment. In one embodiment, the signal peptide sequence as defined herein includes, without being limited thereto, the signal peptide sequence of SARS-CoV-2 S protein, in particular a sequence comprising the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or a functional variant thereof.

(270) In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1. In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

(271) In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1. In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence of nucleotides 1 to 48 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 16 of SEQ ID NO: 1.

(272) In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1. In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.

(273) In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1. In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence of nucleotides 1 to 57 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 19 of SEQ ID NO: 1.

(274) The signal peptide sequence as defined herein further includes, without being limited thereto, the signal peptide sequence of an immunoglobulin, e.g., the signal peptide sequence of an immunoglobulin heavy chain variable region, wherein the immunoglobulin may be human immunoglobulin. In particular, the signal peptide sequence as defined herein includes a sequence comprising the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31 or a functional variant thereof.

(275) In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or a functional fragment of the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31. In one embodiment, a signal sequence comprises the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31.

(276) In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or a functional fragment of the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31. In one embodiment, RNA encoding a signal sequence (i) comprises the nucleotide sequence of nucleotides 54 to 119 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31.

(277) Such signal peptides are preferably used in order to promote secretion of the encoded antigenic peptide or protein. More preferably, a signal peptide as defined herein is fused to an encoded antigenic peptide or protein as defined herein.

(278) Accordingly, in particularly preferred embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a signal peptide, said signal peptide preferably being fused to the antigenic peptide or protein, more preferably to the N-terminus of the antigenic peptide or protein as described herein.

(279) In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7. In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 1 or 7.

(280) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 1 or 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 1 or 7. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or 7.

(281) In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 7.

(282) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, or a fragment of the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 15, 16, 19, 20, 24, or 25; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

(283) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.

(284) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 1 to 2049 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 683 of SEQ ID NO: 1.

(285) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.

(286) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 1 to 2055 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 685 of SEQ ID NO: 1.

(287) In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 3, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 3, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 3. In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 3.

(288) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 4, or a fragment of the nucleotide sequence of SEQ ID NO: 4, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 4; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 3, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 3. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 4; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 3.

(289) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29.

(290) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 716 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 221 of SEQ ID NO: 29.

(291) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31.

(292) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 725 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 224 of SEQ ID NO: 31.

(293) According to certain embodiments, a trimerization domain is fused, either directly or through a linker, e.g., a glycine/serine linker, to a SARS-CoV-2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein. Accordingly, in one embodiment, a trimerization domain is fused to the above described amino acid sequences derived from SARS-CoV-2 S protein or immunogenic fragments thereof (antigenic peptides or proteins) comprised by the vaccine antigens described above (which may optionally be fused to a signal peptide as described above).

(294) Such trimerization domains are preferably located at the C-terminus of the antigenic peptide or protein, without being limited thereto. Trimerization domains as defined herein preferably allow the trimerization of the antigenic peptide or protein as encoded by the RNA. Examples of trimerization domains as defined herein include, without being limited thereto, foldon, the natural trimerization domain of T4 fibritin. The C-terminal domain of T4 fibritin (foldon) is obligatory for the formation of the fibritin trimer structure and can be used as an artificial trimerization domain. In one embodiment, the trimerization domain as defined herein includes, without being limited thereto, a sequence comprising the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10 or a functional variant thereof. In one embodiment, the trimerization domain as defined herein includes, without being limited thereto, a sequence comprising the amino acid sequence of SEQ ID NO: 10 or a functional variant thereof.

(295) In one embodiment, a trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10. In one embodiment, a trimerization domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10.

(296) In one embodiment, RNA encoding a trimerization domain (i) comprises the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, or a fragment of the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10. In one embodiment, RNA encoding a trimerization domain (i) comprises the nucleotide sequence of nucleotides 7 to 87 of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10.

(297) In one embodiment, a trimerization domain comprises the amino acid sequence SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10. In one embodiment, a trimerization domain comprises the amino acid sequence of SEQ ID NO: 10. In one embodiment, RNA encoding a trimerization domain (i) comprises the nucleotide sequence of SEQ ID NO: 11, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11, or a fragment of the nucleotide sequence of SEQ ID NO: 11, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10, or a functional fragment of the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 10. In one embodiment, RNA encoding a trimerization domain (i) comprises the nucleotide sequence of SEQ ID NO: 11; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 10.

(298) Such trimerization domains are preferably used in order to promote trimerization of the encoded antigenic peptide or protein. More preferably, a trimerization domain as defined herein is fused to an antigenic peptide or protein as defined herein.

(299) Accordingly, in particularly preferred embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or protein and a trimerization domain as defined herein, said trimerization domain preferably being fused to the antigenic peptide or protein, more preferably to the C-terminus of the antigenic peptide or protein.

(300) In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5. In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 5.

(301) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 6, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6, or a fragment of the nucleotide sequence of SEQ ID NO: 6, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 6; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 6; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.

(302) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 17, 21, or 26, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26, or a fragment of the nucleotide sequence of SEQ ID NO: 17, 21, or 26, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 5, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 17, 21, or 26; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.

(303) In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 18, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 18, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 18, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 18. In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 18.

(304) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29.

(305) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 257 of SEQ ID NO: 29.

(306) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31.

(307) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 260 of SEQ ID NO: 31.

(308) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29.

(309) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 111 to 824 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20 to 257 of SEQ ID NO: 29.

(310) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.

(311) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 120 to 833 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23 to 260 of SEQ ID NO: 31.

(312) According to certain embodiments, a transmembrane domain domain is fused, either directly or through a linker, e.g., a glycine/serine linker, to a SARS-CoV-2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein. Accordingly, in one embodiment, a transmembrane domain is fused to the above described amino acid sequences derived from SARS-CoV-2 S protein or immunogenic fragments thereof (antigenic peptides or proteins) comprised by the vaccine antigens described above (which may optionally be fused to a signal peptide and/or trimerization domain as described above).

(313) Such transmembrane domains are preferably located at the C-terminus of the antigenic peptide or protein, without being limited thereto. Preferably, such transmembrane domains are located at the C-terminus of the trimerization domain, if present, without being limited thereto. In one embodiment, a trimerization domain is present between the SARS-CoV-2 S protein, a variant thereof, or a fragment thereof, i.e., the antigenic peptide or protein, and the transmembrane domain.

(314) Transmembrane domains as defined herein preferably allow the anchoring into a cellular membrane of the antigenic peptide or protein as encoded by the RNA.

(315) In one embodiment, the transmembrane domain sequence as defined herein includes, without being limited thereto, the transmembrane domain sequence of SARS-CoV-2 S protein, in particular a sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1 or a functional variant thereof.

(316) In one embodiment, a transmembrane domain sequence comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1. In one embodiment, a transmembrane domain sequence comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.

(317) In one embodiment, RNA encoding a transmembrane domain sequence (i) comprises the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, or a fragment of the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or a functional fragment of the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1. In one embodiment, RNA encoding a transmembrane domain sequence (i) comprises the nucleotide sequence of nucleotides 3619 to 3762 of SEQ ID NO: 2, 8 or 9; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.

(318) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29.

(319) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 311 of SEQ ID NO: 29.

(320) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31.

(321) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 54 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 1 to 314 of SEQ ID NO: 31.

(322) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29.

(323) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, or a fragment of the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29.

(324) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 111 to 986 of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 20 to 311 of SEQ ID NO: 29.

(325) In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31. In one embodiment, a vaccine antigen comprises the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31.

(326) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32, or a fragment of the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of nucleotides 120 to 995 of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of amino acids 23 to 314 of SEQ ID NO: 31.

(327) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 30, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30, or a fragment of the nucleotide sequence of SEQ ID NO: 30, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 29, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 29. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 30; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 29.

(328) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 32, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32, or a fragment of the nucleotide sequence of SEQ ID NO: 32, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 31, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 31, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 31, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 31. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 32; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 31.

(329) In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 28, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 28, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 28, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 28. In one embodiment, a vaccine antigen comprises the amino acid sequence of SEQ ID NO: 28.

(330) In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 27, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 27, or a fragment of the nucleotide sequence of SEQ ID NO: 27, or the nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 27; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 28, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 28, or an immunogenic fragment of the amino acid sequence of SEQ ID NO: 28, or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 28. In one embodiment, RNA encoding a vaccine antigen (i) comprises the nucleotide sequence of SEQ ID NO: 27; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 28.

(331) In one embodiment, the vaccine antigens described above comprise a contiguous sequence of SARS-CoV-2 coronavirus spike (S) protein that consists of or essentially consists of the above described amino acid sequences derived from SARS-CoV-2 S protein or immunogenic fragments thereof (antigenic peptides or proteins) comprised by the vaccine antigens described above. In one embodiment, the vaccine antigens described above comprise a contiguous sequence of SARS-CoV-2 coronavirus spike (S) protein of no more than 220 amino acids, 215 amino acids, 210 amino acids, or 205 amino acids.

(332) In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2 (RBP020.1 or RBP020.2). In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) described herein as RBP020.2.

(333) In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 21, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5. In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.

(334) In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19, or 20, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or 20; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

(335) In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 20, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, RNA encoding a vaccine antigen is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

(336) As used herein, the term vaccine refers to a composition that induces an immune response upon inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.

(337) In one embodiment, the RNA encoding the antigen molecule is expressed in cells of the subject to provide the antigen molecule. In one embodiment, expression of the antigen molecule is at the cell surface or into the extracellular space. In one embodiment, the antigen molecule is presented in the context of MHC. In one embodiment, the RNA encoding the antigen molecule is transiently expressed in cells of the subject. In one embodiment, after administration of the RNA encoding the antigen molecule, in particular after intramuscular administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in muscle occurs. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in spleen occurs. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in antigen presenting cells, preferably professional antigen presenting cells occurs. In one embodiment, the antigen presenting cells are selected from the group consisting of dendritic cells, macrophages and B cells. In one embodiment, after administration of the RNA encoding the antigen molecule, no or essentially no expression of the RNA encoding the antigen molecule in lung and/or liver occurs. In one embodiment, after administration of the RNA encoding the antigen molecule, expression of the RNA encoding the antigen molecule in spleen is at least 5-fold the amount of expression in lung.

(338) In some embodiments, the methods and agents, e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to lymph nodes and/or spleen. In some embodiments, RNA encoding a vaccine antigen is detectable in lymph nodes and/or spleen 6 hours or later following administration and preferably up to 6 days or longer. In some embodiments, the methods and agents, e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to B cell follicles, subcapsular sinus, and/or T cell zone, in particular B cell follicles and/or subcapsular sinus of lymph nodes.

(339) In some embodiments, the methods and agents, e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to B cells (CD19+), subcapsular sinus macrophages (CD169+) and/or dendritic cells (CD11c+) in the T cell zone and intermediary sinus of lymph nodes, in particular to B cells (CD19+) and/or subcapsular sinus macrophages (CD169+) of lymph nodes.

(340) In some embodiments, the methods and agents, e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to white pulp of spleen.

(341) In some embodiments, the methods and agents, e.g., mRNA compositions, described herein following administration, in particular following intramuscular administration, to a subject result in delivery of the RNA encoding a vaccine antigen to B cells, DCs (CD11c+), in particular those surrounding the B cells, and/or mcrophages of spleen, in particular to B cells and/or DCs (CD11c+).

(342) In one embodiment, the vaccine antigen is expressed in lymph node and/or spleen, in particular in the cells of lymph node and/or spleen described above.

(343) The peptide and protein antigens suitable for use according to the disclosure typically include a peptide or protein comprising an epitope of SARS-CoV-2 S protein or a functional variant thereof for inducing an immune response. The peptide or protein or epitope may be derived from a target antigen, i.e. the antigen against which an immune response is to be elicited. For example, the peptide or protein antigen or the epitope contained within the peptide or protein antigen may be a target antigen or a fragment or variant of a target antigen. The target antigen may be a coronavirus S protein, in particular SARS-CoV-2 S protein.

(344) The antigen molecule or a procession product thereof, e.g., a fragment thereof, may bind to an antigen receptor such as a BCR or TCR carried by immune effector cells, or to antibodies. A peptide and protein antigen which is provided to a subject according to the invention by administering RNA encoding the peptide and protein antigen, i.e., a vaccine antigen, preferably results in the induction of an immune response, e.g., a humoral and/or cellular immune response in the subject being provided the peptide or protein antigen. Said immune response is preferably directed against a target antigen, in particular coronavirus S protein, in particular SARS-CoV-2 S protein. Thus, a vaccine antigen may comprise the target antigen, a variant thereof, or a fragment thereof. In one embodiment, such fragment or variant is immunologically equivalent to the target antigen. In the context of the present disclosure, the term fragment of an antigen or variant of an antigen means an agent which results in the induction of an immune response which immune response targets the antigen, i.e. a target antigen. Thus, the vaccine antigen may correspond to or may comprise the target antigen, may correspond to or may comprise a fragment of the target antigen or may correspond to or may comprise an antigen which is homologous to the target antigen or a fragment thereof. Thus, according to the disclosure, a vaccine antigen may comprise an immunogenic fragment of a target antigen or an amino acid sequence being homologous to an immunogenic fragment of a target antigen. An immunogenic fragment of an antigen according to the disclosure preferably relates to a fragment of an antigen which is capable of inducing an immune response against the target antigen. The vaccine antigen may be a recombinant antigen.

(345) The term immunologically equivalent means that the immunologically equivalent molecule such as the immunologically equivalent amino acid sequence exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effects, e.g., with respect to the type of the immunological effect. In the context of the present disclosure, the term immunologically equivalent is preferably used with respect to the immunological effects or properties of antigens or antigen variants used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if said amino acid sequence when exposed to the immune system of a subject induces an immune reaction having a specificity of reacting with the reference amino acid sequence.

(346) Activation or stimulation, as used herein, refers to the state of an immune effector cell such as T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with initiation of signaling pathways, induced cytokine production, and detectable effector functions. The term activated immune effector cells refers to, among other things, immune effector cells that are undergoing cell division.

(347) The term priming refers to a process wherein an immune effector cell such as a T cell has its first contact with its specific antigen and causes differentiation into effector cells such as effector T cells.

(348) The term clonal expansion or expansion refers to a process wherein a specific entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cell recognizing said antigen is amplified. Preferably, clonal expansion leads to differentiation of the immune effector cells.

(349) The term antigen relates to an agent comprising an epitope against which an immune response can be generated. The term antigen includes, in particular, proteins and peptides. In one embodiment, an antigen is presented by cells of the immune system such as antigen presenting cells like dendritic cells or macrophages. An antigen or a procession product thereof such as a T-cell epitope is in one embodiment bound by a T- or B-cell receptor, or by an immunoglobulin molecule such as an antibody. Accordingly, an antigen or a procession product thereof may react specifically with antibodies or T lymphocytes (T cells). In one embodiment, an antigen is a viral antigen, such as a coronavirus S protein, e.g., SARS-CoV-2 S protein, and an epitope is derived from such antigen.

(350) The term viral antigen refers to any viral component having antigenic properties, i.e. being able to provoke an immune response in an individual. The viral antigen may be coronavirus S protein, e.g., SARS-CoV-2 S protein.

(351) The term expressed on the cell surface or associated with the cell surface means that a molecule such as an antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by antibodies located outside the cell. In this context, a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein.

(352) Cell surface or surface of a cell is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by e.g. antigen-specific antibodies added to the cells.

(353) The term extracellular portion or exodomain in the context of the present invention refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or a fragment thereof.

(354) The term epitope refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, the epitope may be recognized by T cells, B cells or antibodies. An epitope of an antigen may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is between about 10 and about 25 amino acids in length. The term epitope includes T cell epitopes.

(355) The term T cell epitope refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term major histocompatibility complex and the abbreviation MHC includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC are expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell. In the case of class I MHC/peptide complexes, the binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC/peptide complexes, the binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.

(356) The peptide and protein antigen can be 2-100 amino acids, including for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, a peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

(357) The peptide or protein antigen can be any peptide or protein that can induce or increase the ability of the immune system to develop antibodies and T cell responses to the peptide or protein.

(358) In one embodiment, vaccine antigen is recognized by an immune effector cell. Preferably, the vaccine antigen if recognized by an immune effector cell is able to induce in the presence of appropriate co-stimulatory signals, stimulation, priming and/or expansion of the immune effector cell carrying an antigen receptor recognizing the vaccine antigen. In the context of the embodiments of the present invention, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen presenting cell. In one embodiment, an antigen is presented by a diseased cell such as a virus-infected cell. In one embodiment, an antigen receptor is a TCR which binds to an epitope of an antigen presented in the context of MHC. In one embodiment, binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented by cells such as antigen presenting cells results in stimulation, priming and/orexpansion of said Tcells. In one embodiment, binding of a TCR when expressed by T cells and/or present on T cells to an antigen presented on diseased cells results in cytolysis and/or apoptosis of the diseased cells, wherein said T cells preferably release cytotoxic factors, e.g. perforins and granzymes.

(359) In one embodiment, an antigen receptor is an antibody or B cell receptor which binds to an epitope in an antigen. In one embodiment, an antibody or B cell receptor binds to native epitopes of an antigen.

(360) Nucleic Acids

(361) The term polynucleotide or nucleic acid, as used herein, is intended to include DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the invention, a polynucleotide is preferably isolated.

(362) Nucleic acids may be comprised in a vector. The term vector as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

(363) In one embodiment of all aspects of the invention, the RNA encoding the vaccine antigen is expressed in cells such as antigen presenting cells of the subject treated to provide the vaccine antigen.

(364) The nucleic acids described herein may be recombinant and/or isolated molecules.

(365) In the present disclosure, the term RNA relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, ribonucleotide refers to a nucleotide with a hydroxyl group at the 2-position of a ?-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered analogs of naturally-occurring RNA.

(366) In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA) that relates to a RNA transcript which encodes a peptide or protein. As established in the art, mRNA generally contains a 5 untranslated region (5-UTR), a peptide coding region and a 3 untranslated region (3-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides.

(367) In one embodiment, RNA is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA. In certain embodiments of the present disclosure, the RNA is replicon RNA or simply a replicon, in particular self-replicating RNA or self-amplifying RNA. In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or comprises elements derived from a ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jos? et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5-cap, and a 3 poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1-nsP4) are typically encoded together by a first ORF beginning near the 5 terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3 terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234). Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

(368) In one embodiment, the RNA described herein may have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.

(369) The term uracil, as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is:

(370) ##STR00001##

(371) The term uridine, as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:

(372) ##STR00002##

(373) UTP (uridine 5-triphosphate) has the following structure:

(374) ##STR00003##

(375) Pseudo-UTP (pseudouridine 5-triphosphate) has the following structure:

(376) ##STR00004##
Pseudouridine is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

(377) Another exemplary modified nucleoside is N1-methyl-pseudouridine (m1?), which has the structure:

(378) ##STR00005##

(379) N1-methyl-pseudo-UTP has the following structure:

(380) ##STR00006##

(381) Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the structure:

(382) ##STR00007##

(383) In some embodiments, one or more uridine in the RNA described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

(384) In some embodiments, RNA comprises a modified nucleoside in place of at least one uridine.

(385) In some embodiments, RNA comprises a modified nucleoside in place of each uridine.

(386) In some embodiments, the modified nucleoside is independently selected from pseudouridine (?), N1-methyl-pseudouridine (m1?), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (?). In some embodiments, the modified nucleoside comprises N1-methyl-pseudouridine (m1?). In some embodiments, the modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (?), N1-methyl-pseudouridine (m1?), and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (?) and N1-methyl-pseudouridine (m1?). In some embodiments, the modified nucleosides comprise pseudouridine (?) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise N1-methyl-pseudouridine (m1?) and 5-methyl-uridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (?), N1-methyl-pseudouridine (m1?), and 5-methyl-uridine (m5U).

(387) In some embodiments, the modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m.sup.3U), 5-methoxy-uridine (mo.sup.5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s.sup.2U), 4-thio-uridine (s.sup.4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho.sup.5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo.sup.5U), uridine 5-oxyacetic acid methyl ester (mcmo.sup.5U), 5-carboxymethyl-uridine (cm.sup.5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm.sup.5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm.sup.5U), 5-methoxycarbonylmethyl-uridine (mcm.sup.5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm.sup.5s.sup.2U), 5-aminomethyl-2-thio-uridine (nm.sup.5s.sup.2U), 5-methylaminomethyl-uridine (mnm.sup.5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm.sup.5s.sup.2U), 5-methylaminomethyl-2-seleno-uridine (mnm.sup.5se.sup.2U), 5-carbamoylmethyl-uridine (ncm.sup.5U), 5-carboxymethylaminomethyl-uridine (cmnm.sup.5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm.sup.5s.sup.2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (?m.sup.5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(?m.sup.5s.sup.2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m.sup.5s.sup.2U), 1-methyl-4-thio-pseudouridine (m.sup.1s.sup.4?), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m.sup.3?), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m.sup.5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp.sup.3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp.sup.3 ?), 5-(isopentenylaminomethyl)uridine (inm.sup.5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm.sup.5s.sup.2U), ?-thio-uridine, 2-O-methyl-uridine (Urn), 5,2-O-dimethyl-uridine (m.sup.5Um), 2-O-methyl-pseudouridine (?m), 2-thio-2-O-methyl-uridine (s.sup.2Um), 5-methoxycarbonylmethyl-2-O-methyl-uridine (mcm.sup.5Um), 5-carbamoylmethyl-2-O-methyl-uridine (ncm.sup.5Um), 5-carboxymethylaminomethyl-2-O-methyl-uridine (cmnm.sup.5Um), 3,2-O-dimethyl-uridine (m.sup.3Um), 5-(isopentenylaminomethyl)-2-O-methyl-uridine (inm.sup.5Um), 1-thio-uridine, deoxythymidine, 2-F-ara-uridine, 2-F-uridine, 2-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

(388) In one embodiment, the RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in one embodiment, in the RNA 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. In one embodiment, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (?), N1-methyl-pseudouridine (m1?), and 5-methyl-uridine (m5U). In one embodiment, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1?). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1?) in place of each uridine.

(389) In some embodiments, the RNA according to the present disclosure comprises a 5-cap. In one embodiment, the RNA of the present disclosure does not have uncapped 5-triphosphates. In one embodiment, the RNA may be modified by a 5-cap analog. The term 5-cap refers to a structure found on the 5-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via a 5- to 5-triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position. Providing an RNA with a 5-cap or 5-cap analog may be achieved by in vitro transcription, in which the 5-cap is co-transcriptionally expressed into the RNA strand, or may be attached to RNA post-transcriptionally using capping enzymes. In some embodiments, the building block cap for RNA is m.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG (also sometimes referred to as m.sub.2.sup.7,3OG(5)ppp(5)m.sup.2-OApG), which has the following structure:

(390) ##STR00008##

(391) Below is an exemplary Cap1 RNA, which comprises RNA and m.sub.2.sup.7,3PG(5)ppp(5)m.sup.2-OApG:

(392) ##STR00009##

(393) Below is another exemplary Cap1 RNA (no cap analog):

(394) ##STR00010##

(395) In some embodiments, the RNA is modified with Cap0 structures using, in one embodiment, the cap analog anti-reverse cap (ARCA Cap (m.sub.2.sup.7,3OG(5)ppp(5)G)) with the structure:

(396) ##STR00011##

(397) Below is an exemplary Cap0 RNA comprising RNA and m.sub.2.sup.7,3O(5)ppp(5)G:

(398) ##STR00012##

(399) In some embodiments, the Cap0 structures are generated using the cap analog Beta-S-ARCA (m.sub.2.sup.7,2OG(5)ppSp(5)G) with the structure:

(400) ##STR00013##

(401) Below is an exemplary Cap0 RNA comprising Beta-S-ARCA (m.sub.2.sup.7,2OG(5)ppSp(5)G) and RNA:

(402) ##STR00014##

(403) The D1 diastereomer of beta-S-ARCA or beta-S-ARCA(D1) is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time (cf., WO 2011/015347, herein incorporated by reference).

(404) A particularly preferred cap is beta-S-ARCA(D1) (m.sub.2.sup.7,2-OGppSpG) or m.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG.

(405) In some embodiments, RNA according to the present disclosure comprises a 5-UTR and/or a 3-UTR. The term untranslated region or UTR relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5 (upstream) of an open reading frame (5-UTR) and/or 3 (downstream) of an open reading frame (3-UTR). A 5-UTR, if present, is located at the 5 end, upstream of the start codon of a protein-encoding region. A 5-UTR is downstream of the 5-cap (if present), e.g. directly adjacent to the 5-cap. A 3-UTR, if present, is located at the 3 end, downstream of the termination codon of a protein-encoding region, but the term 3-UTR does preferably not include the poly(A) sequence. Thus, the 3-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.

(406) In some embodiments, RNA comprises a 5-UTR comprising the nucleotide sequence of SEQ ID NO: 12, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 12.

(407) In some embodiments, RNA comprises a 3-UTR comprising the nucleotide sequence of SEQ ID NO: 13, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 13.

(408) A particularly preferred 5-UTR comprises the nucleotide sequence of SEQ ID NO: 12. A particularly preferred 3-UTR comprises the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the RNA according to the present disclosure comprises a 3-poly(A) sequence.

(409) As used herein, the term poly(A) sequence or poly-A tail refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3-end of an RNA molecule. Poly(A) sequences are known to those of skill in the art and may follow the 3-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. RNAs disclosed herein can have a poly(A) sequence attached to the free 3-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

(410) It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

(411) The poly(A) sequence may be of any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, essentially consists of means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, consists of means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term A nucleotide or A refers to adenylate. In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.

(412) In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1 may be used in the present invention. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly(A) sequence contained in an RNA molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

(413) In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3-end, i.e., the poly(A) sequence is not masked or followed at its 3-end by a nucleotide other than A.

(414) In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

(415) In some embodiments, RNA comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 14. A particularly preferred poly(A) sequence comprises comprises the nucleotide sequence of SEQ ID NO: 14.

(416) According to the disclosure, vaccine antigen is preferably administered as single-stranded, 5-capped mRNA that is translated into the respective protein upon entering cells of a subject being administered the RNA. Preferably, the RNA contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5-cap, 5-UTR, 3-UTR, poly(A) sequence).

(417) In one embodiment, beta-S-ARCA(D1) is utilized as specific capping structure at the 5-end of the RNA. In one embodiment, m.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG is utilized as specific capping structure at the 5-end of the RNA. In one embodiment, the 5-UTR sequence is derived from the human alpha-globin mRNA and optionally has an optimized Kozak sequence to increase translational efficiency. In one embodiment, a combination of two sequence elements (FI element) derived from the amino terminal enhancer of split (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I) are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA. In one embodiment, two re-iterated 3-UTRs derived from the human beta-globin mRNA are placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA. In one embodiment, a poly(A) sequence measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues is used. This poly(A) sequence was designed to enhance RNA stability and translational efficiency.

(418) In one embodiment of all aspects of the invention, RNA encoding a vaccine antigen is expressed in cells of the subject treated to provide the vaccine antigen. In one embodiment of all aspects of the invention, the RNA is transiently expressed in cells of the subject. In one embodiment of all aspects of the invention, the RNA is in vitro transcribed RNA. In one embodiment of all aspects of the invention, expression of the vaccine antigen is at the cell surface. In one embodiment of all aspects of the invention, the vaccine antigen is expressed and presented in the context of MHC. In one embodiment of all aspects of the invention, expression of the vaccine antigen is into the extracellular space, i.e., the vaccine antigen is secreted.

(419) In the context of the present disclosure, the term transcription relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein.

(420) According to the present invention, the term transcription comprises in vitro transcription, wherein the term in vitro transcription relates to a process wherein RNA, in particular mRNA, is in vitro synthesized in a cell-free system, preferably using appropriate cell extracts. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term vector. According to the present invention, the RNA used in the present invention preferably is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

(421) With respect to RNA, the term expression or translation relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

(422) In one embodiment, after administration of the RNA described herein, e.g., formulated as RNA lipid particles, at least a portion of the RNA is delivered to a target cell. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce the peptide or protein it enodes. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell or macrophage. RNA particles such as RNA lipid particles described herein may be used for delivering RNA to such target cell. Accordingly, the present disclosure also relates to a method for delivering RNA to a target cell in a subject comprising the administration of the RNA particles described herein to the subject. In one embodiment, the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA. Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

(423) In one embodiment, the RNA encoding vaccine antigen to be administered according to the invention is non-immunogenic. RNA encoding immunostimulant may be administered according to the invention to provide an adjuvant effect. The RNA encoding immunostimulant may be standard RNA or non-immunogenic RNA.

(424) The term non-immunogenic RNA as used herein refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., than would have been induced by standard RNA (stdRNA). In one preferred embodiment, non-immunogenic RNA, which is also termed modified RNA (modRNA) herein, is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and removing double-stranded RNA (dsRNA).

(425) For rendering the non-immunogenic RNA non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In one embodiment, the modified nucleosides comprises a replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In one embodiment, the modified nucleobase is a modified uracil. In one embodiment, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m.sup.3U), 5-methoxy-uridine (mo.sup.5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s.sup.2U), 4-thio-uridine (s.sup.4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho.sup.5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo.sup.5U), uridine 5-oxyacetic acid methyl ester (mcmo.sup.5U), 5-carboxymethyl-uridine (cm.sup.5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm.sup.5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm.sup.5U), 5-methoxycarbonylmethyl-uridine (mcm.sup.5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm.sup.5s.sup.2U), 5-aminomethyl-2-thio-uridine (nm.sup.5s.sup.2U), 5-methylaminomethyl-uridine (mnm.sup.5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm.sup.5s.sup.2U), 5-methylaminomethyl-2-seleno-uridine (mnm.sup.5se.sup.2U), 5-carbamoylmethyl-uridine (ncm.sup.5U), 5-carboxymethylaminomethyl-uridine (cmnm.sup.5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm.sup.5s.sup.2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (?m.sup.5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(?m.sup.5s.sup.2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m.sup.5s.sup.2U), 1-methyl-4-thio-pseudouridine (m.sup.1s.sup.4?), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m.sup.3), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m.sup.5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp.sup.3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp.sup.3 ?), 5-(isopentenylaminomethyl)uridine (inm.sup.5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm.sup.5s.sup.2U), ?-thio-uridine, 2-O-methyl-uridine (Um), 5,2-O-dimethyl-uridine (m.sup.5Um), 2-O-methyl-pseudouridine (?m), 2-thio-2-O-methyl-uridine (s.sup.2Um), 5-methoxycarbonylmethyl-2-O-methyl-uridine (mcm.sup.5Um), 5-carbamoylmethyl-2-O-methyl-uridine (ncm.sup.5Um), 5-carboxymethylaminomethyl-2-O-methyl-uridine (cmnm.sup.5Um), 3,2-O-dimethyl-uridine (m.sup.3Um), 5-(isopentenylaminomethyl)-2-O-methyl-uridine (inm.sup.5Um), 1-thio-uridine, deoxythymidine, 2-F-ara-uridine, 2-F-uridine, 2-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine. In one particularly preferred embodiment, the nucleoside comprising a modified nucleobase is pseudouridine (?), N1-methyl-pseudouridine (m1?) or 5-methyl-uridine (m5U), in particular N1-methyl-pseudouridine.

(426) In one embodiment, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines. During synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase significant amounts of aberrant products, including double-stranded RNA (dsRNA) are produced due to unconventional activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using E. coli RNaseIII that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In one embodiment, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material.

(427) As the term is used herein, remove or removal refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the non-separated mixture of first and second substances.

(428) In one embodiment, the removal of dsRNA from non-immunogenic RNA comprises a removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNA composition is dsRNA. In one embodiment, the non-immunogenic RNA is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA composition comprises a purified preparation of single-stranded nucleoside modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside modified RNA is substantially free of double stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

(429) In one embodiment, the non-immunogenic RNA is translated in a cell more efficiently than standard RNA with the same sequence. In one embodiment, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In one embodiment, translation is enhanced by a 3-fold factor. In one embodiment, translation is enhanced by a 4-fold factor. In one embodiment, translation is enhanced by a 5-fold factor. In one embodiment, translation is enhanced by a 6-fold factor. In one embodiment, translation is enhanced by a 7-fold factor. In one embodiment, translation is enhanced by an 8-fold factor. In one embodiment, translation is enhanced by a 9-fold factor. In one embodiment, translation is enhanced by a 10-fold factor. In one embodiment, translation is enhanced by a 15-fold factor. In one embodiment, translation is enhanced by a 20-fold factor. In one embodiment, translation is enhanced by a 50-fold factor. In one embodiment, translation is enhanced by a 100-fold factor. In one embodiment, translation is enhanced by a 200-fold factor. In one embodiment, translation is enhanced by a 500-fold factor. In one embodiment, translation is enhanced by a 1000-fold factor. In one embodiment, translation is enhanced by a 2000-fold factor. In one embodiment, the factor is 10-1000-fold. In one embodiment, the factor is 10-100-fold. In one embodiment, the factor is 10-200-fold. In one embodiment, the factor is 10-300-fold. In one embodiment, the factor is 10-500-fold. In one embodiment, the factor is 20-1000-fold. In one embodiment, the factor is 30-1000-fold. In one embodiment, the factor is 50-1000-fold. In one embodiment, the factor is 100-1000-fold. In one embodiment, the factor is 200-1000-fold. In one embodiment, translation is enhanced by any other significant amount or range of amounts.

(430) In one embodiment, the non-immunogenic RNA exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In one embodiment, the non-immunogenic RNA exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In one embodiment, innate immunogenicity is reduced by a 3-fold factor. In one embodiment, innate immunogenicity is reduced by a 4-fold factor. In one embodiment, innate immunogenicity is reduced by a 5-fold factor. In one embodiment, innate immunogenicity is reduced by a 6-fold factor. In one embodiment, innate immunogenicity is reduced by a 7-fold factor. In one embodiment, innate immunogenicity is reduced by a 8-fold factor. In one embodiment, innate immunogenicity is reduced by a 9-fold factor. In one embodiment, innate immunogenicity is reduced by a 10-fold factor. In one embodiment, innate immunogenicity is reduced by a 15-fold factor. In one embodiment, innate immunogenicity is reduced by a 20-fold factor. In one embodiment, innate immunogenicity is reduced by a 50-fold factor. In one embodiment, innate immunogenicity is reduced by a 100-fold factor. In one embodiment, innate immunogenicity is reduced by a 200-fold factor. In one embodiment, innate immunogenicity is reduced by a 500-fold factor. In one embodiment, innate immunogenicity is reduced by a 1000-fold factor. In one embodiment, innate immunogenicity is reduced by a 2000-fold factor.

(431) The term exhibits significantly less innate immunogenicity refers to a detectable decrease in innate immunogenicity. In one embodiment, the term refers to a decrease such that an effective amount of the non-immunogenic RNA can be administered without triggering a detectable innate immune response. In one embodiment, the term refers to a decrease such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In one embodiment, the decrease is such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA. Immunogenicity is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system. As used herein endogenous refers to any material from or produced inside an organism, cell, tissue or system.

(432) As used herein, the term exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.

(433) The term expression as used herein is defined as the transcription and/or translation of a particular nucleotide sequence.

(434) As used herein, the terms linked, fused, or fusion are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

(435) Codon-Optimization/Increase in G/C Content

(436) In some embodiment, the amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof described herein is encoded by a coding sequence which is codon-optimized and/or the G/C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence. In one embodiment, the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.

(437) The term codon-optimized refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present invention, coding regions are preferably codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of rare codons.

(438) In some embodiments of the invention, the guanosine/cytosine (G/C) content of the coding region of the RNA described herein is increased compared to the G/C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favourable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and/or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleotides.

(439) In various embodiments, the G/C content of the coding region of the RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of the wild type RNA.

(440) Embodiments of Administered RNAs

(441) In some embodiments, compositions or medical preparations described herein comprise RNA encoding an amino acid sequence comprising SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof. Likewise, methods described herein comprise administration of such RNA.

(442) The active platform for use herein is based on an antigen-coding RNA vaccine to induce robust neutralising antibodies and accompanying/concomitant T cell response to achieve protective immunization with preferably minimal vaccine doses. The RNA administered is preferably in-vitro transcribed RNA.

(443) Three different RNA platforms are particularly preferred, namely non-modified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA) and self-amplifying RNA (saRNA). In one particularly preferred embodiment, the RNA is in vitro transcribed RNA.

(444) As described herein, embodiments of each of these platforms are assessed herein (see, for example Example 2), representing a novel and powerful approach to and system for rapid vaccine development. This described approach and system achieved remarkable and efficient success, enabling development of an effective clinical candidate within several months of provision of antigen (e.g., SARS-CoV-2 S1 protein and/or RBD thereof) sequence (as described herein, relevant sequence information (e.g., GenBank: MN908947.3) became available in January 2020). Insights and advantages embodied in this described approach and system include, for example, ability to directly compare one or more features of different strategies to achieve rapid, efficient, and effective development. Among other things, the present disclosure encompasses insights that identify the source of a problem with more typical strategies for vaccine development. Moreover, findings included herein establish a variety of advantages and benefits, particularly in rapid vaccine development and notably of special benefit in a pandemic.

(445) As described herein, in some embodiments, vaccine candidates are assessed for titer of antibodies induced in a model organism (e.g., mouse; see e.g., Example 2) directed to an encoded antigen (e.g., S1 protein) or portion thereof (e.g., RBD). In some embodiments, vaccine candidates are assessed for pseudoviral neutralization (see e.g., Example 2) activity of induced antibodies. In some embodiments, vaccine candidates are characterized for nature of T cell response induced (e.g., T.sub.H1 vs T.sub.H2 character; see, e.g., Example 4). In some embodiments, vaccine candidates are assessed in more than one model organism (see. E.g., Examples 2, Example 4, etc)

(446) In the following, embodiments of these three different RNA platforms are described, wherein certain terms used when describing elements thereof have the following meanings:

(447) S1S2 protein/S1S2 RBD: Sequences encoding the respective antigen of SARS-CoV-2.

(448) nsP1, nsP2, nsP3, and nsP4: Wildtype sequences encoding the Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase replicase and a subgenomic promotor plus conserved sequence elements supporting replication and translation.

(449) virUTR: Viral untranslated region encoding parts of the subgenomic promotor as well as replication and translation supporting sequence elements.

(450) hAg-Kozak: 5-UTR sequence of the human alpha-globin mRNA with an optimized Kozak sequence to increase translational efficiency.

(451) Sec: Sec corresponds to the intrinsic S1S2 protein secretory signal peptide (sec), which guides translocation of the nascent polypeptide chain into the endoplasmatic reticulum.

(452) Glycine-serine linker (GS): Sequences coding for short linker peptides predominantly consisting of the amino acids glycine (G) and serine (S), as commonly used for fusion proteins.

(453) Fibritin: Partial sequence of T4 fibritin (foldon), used as artificial trimerization domain.

(454) TM: TM sequence corresponds to the transmembrane part of the S1S2 protein.

(455) FI element: The 3-UTR is a combination of two sequence elements derived from the amino terminal enhancer of split (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression.

(456) A30L70: A poly(A)-tail measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another 70 adenosine residues designed to enhance RNA stability and translational efficiency in dendritic cells.

(457) In general, vaccine RNA described herein may comprise, from 5 to 3, one of the following structures: Cap-5-UTR-Vaccine Antigen-Encoding Sequence-3-UTR-Poly(A)

(458) or beta-S-ARCA(D1)-hAg-Kozak-Vaccine Antigen-Encoding Sequence-FI-A30L70.

(459) In general, a vaccine antigen described herein may comprise, from N-terminus to C-terminus, one of the following structures: Signal Sequence-RBD-Trimerization Domain

(460) or Signal Sequence-RBD-Trimerization Domain-Transmembrane Domain.

(461) RBD and Trimerization Domain may be separated by a linker, in particular a GS linker such as a linker having the amino acid sequence GSPGSGSGS (SEQ ID NO: 33). Trimerization Domain and Transmembrane Domain may be separated by a linker, in particular a GS linker such as a linker having the amino acid sequence GSGSGS (SEQ ID NO: 34).

(462) Signal Sequence may be a signal sequence as described herein. RBD may be a RBD domain as described herein. Trimerization Domain may be a trimerization domain as described herein. Transmembrane Domain may be a transmembrane domain as described herein.

(463) In one embodiment,

(464) Signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence,

(465) RBD comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence,

(466) Trimerization Domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence; and

(467) Transmembrane Domain comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to this amino acid sequence.

(468) In one embodiment, Signal sequence comprises the amino acid sequence of amino acids 1 to 16 or 1 to 19 of SEQ ID NO: 1 or the amino acid sequence of amino acids 1 to 22 of SEQ ID NO: 31, RBD comprises the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 1, Trimerization Domain comprises the amino acid sequence of amino acids 3 to 29 of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10; and Transmembrane Domain comprises the amino acid sequence of amino acids 1207 to 1254 of SEQ ID NO: 1.

(469) The above described RNA or RNA encoding the above described vaccine antigen may be non-modified uridine containing mRNA (uRNA), nucleoside modified mRNA (modRNA) or self-amplifying RNA (saRNA). In one embodiment, the above described RNA or RNA encoding the above described vaccine antigen is nucleoside modified mRNA (modRNA).

(470) Non-Modified Uridine Messenger RNA (uRNA)

(471) The active principle of the non-modified messenger RNA (uRNA) drug substance is a single-stranded mRNA that is translated upon entering a cell. In addition to the sequence encoding the coronavirus vaccine antigen (i.e. open reading frame), each uRNA preferably contains common structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5-cap, 5-UTR, 3-UTR, poly(A)-tail). The preferred 5 cap structure is beta-S-ARCA(D1) (m.sub.2.sup.7,2-OGppSpG). The preferred 5-UTR and 3-UTR comprise the nucleotide sequence of SEQ ID NO: 12 and the nucleotide sequence of SEQ ID NO: 13, respectively. The preferred poly(A)-tail comprises the sequence of SEQ ID NO: 14.

(472) Different embodiment of this platform are as follows:

(473) TABLE-US-00002 RBL063.1 (SEQ ID NO: 15; SEQ ID NO: 7) Structure beta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded Viral spike protein (S1S2 protein) of the SARS-CoV-2 antigen (S1S2 full-length protein, sequence variant) RBL063.2 (SEQ ID NO: 16; SEQ ID NO: 7) Structure beta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded Viral spike protein (S1S2 protein) of the SARS-CoV-2 antigen (S1S2 full-length protein, sequence variant) BNT162a1; RBL063.3 (SEQ ID NO: 17; SEQ ID NO: 5) Structure beta-S-ARCA(D1)-hAg-Kozak-RBD-GS-Fibritin-FI-A30L70 Encoded Viral spike protein (S protein) of the SARS-CoV-2 (partial antigen sequence, Receptor Binding Domain (RBD) of S1S2 protein)

(474) FIG. 19 schematizes the general structure of the antigen-encoding RNAs.

(475) TABLE-US-00003 NucleotideSequenceofRBL063.1(SEQIDNO:15;SEQIDNO:7) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinbold letters.Inaddition,thesequenceofthetranslatedproteinisshowninitalicletters belowthecodingnucleotidesequence(*=stopcodon). 102030405052 GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC hAg-Kozak 62728292102112 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGAAUUUGACAACA MFVFLVLLPLVSSQCVNLTT Sprotein 122132142152162172 AGAACACAGCUGCCACCAGCUUAUACAAAUUCUUUUACCAGAGGAGUGUAUUAUCCUGAU RTQLPPAYTNSFTRGVYYPD Sprotein 182192202212222232 AAAGUGUUUAGAUCUUCUGUGCUGCACAGCACACAGGACCUGUUUCUGCCAUUUUUUAGC KVFRSSVLHSTQDLFLPFFS Sprotein 242252262272282292 AAUGUGACAUGGUUUCAUGCAAUUCAUGUGUCUGGAACAAAUGGAACAAAAAGAUUUGAU NVTWFHAIHVSGTNGTKRFD Sprotein 302312322332342352 AAUCCUGUGCUGCCUUUUAAUGAUGGAGUGUAUUUUGCUUCAACAGAAAAGUCAAAUAUU NPVLPFNDGVYEASTEKSNI Sprotein 362372382392402412 AUUAGAGGAUGGAUUUUUGGAACAACACUGGAUUCUAAAACACAGUCUCUGCUGAUUGUG IRGWIFGTTLDSKTQSLLIV Sprotein 422432442452462472 AAUAAUGCAACAAAUGUGGUGAUUAAAGUGUGUGAAUUUCAGUUUUGUAAUGAUCCUUUU NNATNVVIKVCEFQFCNDPF Sprotein 482492502512522532 CUGGGAGUGUAUUAUCACAAAAAUAAUAAAUCUUGGAUGGAAUCUGAAUUUAGAGUGUAU LGVYYHKNNKSWMESEFRVY Sprotein 542552562572582592 UCCUCUGCAAAUAAUUGUACAUUUGAAUAUGUGUCUCAGCCUUUUCUGAUGGAUCUGGAA SSANNCTFEYVSQPFLMDLE Sprotein 602612622632642652 GGAAAACAGGGCAAUUUUAAAAAUCUGAGAGAAUUUGUGUUUAAAAAUAUUGAUGGAUAU GKQGNFKNLREFVFKNIDGY Sprotein 662672682692702712 UUUAAAAUUUAUUCUAAACACACACCAAUUAAUUUAGUGAGAGAUCUGCCUCAGGGAUUU FKIYSKHTPINLVRDLPQGF Sprotein 722732742752762772 UCUGCUCUGGAACCUCUGGUGGAUCUGCCAAUUGGCAUUAAUAUUACAAGAUUUCAGACA SALEPLVDLPIGINITRFQT Sprotein 782792802812822832 CUGCUGGCUCUGCACAGAUCUUAUCUGACACCUGGAGAUUCUUCUUCUGGAUGGACAGCC LLALHRSYLTPGDSSSGWTA Sprotein 842852862872882892 GGAGCUGCAGCUUAUUAUGUGGGCUAUCUGCAGCCAAGAACAUUUCUGCUGAAAUAUAAU GAAAYYVGYLQPRTFLLKYN Sprotein 902912922932942952 GAAAAUGGAACAAUUACAGAUGCUGUGGAUUGUGCUCUGGAUCCUCUGUCUGAAACAAAA ENGTITDAVDCALDPLSETK Sprotein 96297298299210021012 UGUACAUUAAAAUCUUUUACAGUGGAAAAAGGCAUUUAUCAGACAUCUAAUUUUAGAGUG CTLKSFTVEKGIYQTSNFRV Sprotein 102210321042105210621072 CAGCCAACAGAAUCUAUUGUGAGAUUUCCAAAUAUUACAAAUCUGUGUCCAUUUGGAGAA QPTESIVRFPNITNLCPFGE Sprotein 108210921102111211221132 GUGUUUAAUGCAACAAGAUUUGCAUCUGUGUAUGCAUGGAAUAGAAAAAGAAUUUCUAAU VFNATRFASVYAWNRKRISN Sprotein 114211521162117211821192 UGUGUGGCUGAUUAUUCUGUGCUGUAUAAUAGUGCUUCUUUUUCCACAUUUAAAUGUUAU CVADYSVLYNSASFSTFKCY Sprotein 120212121222123212421252 GGAGUGUCUCCAACAAAAUUAAAUGAUUUAUGUUUUACAAAUGUGUAUGCUGAUUCUUUU GVSPTKLNDLCFTNVYADSF Sprotein 126212721282129213021312 GUGAUCAGAGGUGAUGAAGUGAGACAGAUUGCCCCCGGACAGACAGGAAAAAUUGCUGAU VIRGDEVRQIAPGQTGKIAD Sprotein 132213321342135213621372 UACAAUUACAAACUGCCUGAUGAUUUUACAGGAUGUGUGAUUGCUUGGAAUUCUAAUAAU YNYKLPDDFTGCVIAWNSNN Sprotein 138213921402141214221432 UUAGAUUCUAAAGUGGGAGGAAAUUACAAUUAUCUGUACAGACUGUUUAGAAAAUCAAAU LDSKVGGNYNYLYRLFRKSN Sprotein 144214521462147214821492 CUGAAACCUUUUGAAAGAGAUAUUUCAACAGAAAUUUAUCAGGCUGGAUCAACACCUUGU LKPPERDISTEIYQAGSTPC Sprotein 150215121522153215421552 AAUGGAGUGGAAGGAUUUAAUUGUUAUUUUCCAUUACAGAGCUAUGGAUUUCAGCCAACC NGVEGFNCYFPLQSYGFQPT Sprotein 156215721582159216021612 AAUGGUGUGGGAUAUCAGCCAUAUAGAGUGGUGGUGCUGUCUUUUGAACUGCUGCAUGCA NGVGYQPYRVVVLSFELLHA Sprotein 162216321642165216621672 CCUGCAACAGUGUGUGGACCUAAAAAAUCUACAAAUUUAGUGAAAAAUAAAUGUGUGAAU PATVCGPKKSTNLVKNKCVN Sprotein 168216921702171217221732 UUUAAUUUUAAUGGAUUAACAGGAACAGGAGUGCUGACAGAAUCUAAUAAAAAAUUUCUG FNFNGLTGTGVLTESNKKFL Sprotein 174217521762177217821792 CCUUUUCAGCAGUUUGGCAGAGAUAUUGCAGAUACCACAGAUGCAGUGAGAGAUCCUCAG PFQQFGRDIADTTDAVRDPQ Sprotein 180218121822183218421852 ACAUUAGAAAUUCUGGAUAUUACACCUUGUUCUUUUGGGGGUGUGUCUGUGAUUACACCU TLEILDITPCSFGGVSVITP Sprotein 186218721882189219021912 GGAACAAAUACAUCUAAUCAGGUGGCUGUGCUGUAUCAGGAUGUGAAUUGUACAGAAGUG GTNTSNQVAVLYQDVNCTEV Sprotein 192219321942195219621972 CCAGUGGCAAUUCAUGCAGAUCAGCUGACACCAACAUGGAGAGUGUAUUCUACAGGAUCU PVAIHADQLTPTWRVYSTGS Sprotein 198219922002201220222032 AAUGUGUUUCAGACAAGAGCAGGAUGUCUGAUUGGAGCAGAACAUGUGAAUAAUUCUUAU NVFQTRAGCLIGAEHVNNSY Sprotein 204220522062207220822092 GAAUGUGAUAUUCCAAUUGGAGCAGGCAUUUGUGCAUCUUAUCAGACACAGACAAAUUCC ECDIPIGAGICASYQTQTNS Sprotein 210221122122213221422152 CCAAGGAGAGCAAGAUCUGUGGCAUCUCAGUCUAUUAUUGCAUACACCAUGUCUCUGGGA PRRARSVASQSIIAYTMSLG Sprotein 216221722182219222022212 GCAGAAAAUUCUGUGGCAUAUUCUAAUAAUUCUAUUGCUAUUCCAACAAAUUUUACCAUU AENSVAYSNNSIAIPTNFTI Sprotein 222222322242225222622272 UCUGUGACAACAGAAAUUUUACCUGUGUCUAUGACAAAAACAUCUGUGGAUUGUACCAUG SVTTEILPVSMTKTSVDCTM Sprotein 228222922302231223222332 UACAUUUGUGGAGAUUCUACAGAAUGUUCUAAUCUGCUGCUGCAGUAUGGAUCUUUUUGU YICGDSTECSNLLLQYGSFC Sprotein 234223522362237223822392 ACACAGCUGAAUAGAGCUUUAACAGGAAUUGCUGUGGAACAGGAUAAAAAUACACAGGAA TQLNRALTGIAVEQDKNTQE Sprotein 240224122422243224422452 GUGUUUGCUCAGGUGAAACAGAUUUACAAAACACCACCAAUUAAAGAUUUUGGAGGAUUU VFAQVKQIYKTPPIKDFGGF Sprotein 246224722482249225022512 AAUUUUAGCCAGAUUCUGCCUGAUCCUUCUAAACCUUCUAAAAGAUCUUUUAUUGAAGAU NFSQILPDPSKPSKRSFIED Sprotein 252225322542255225622572 CUGCUGUUUAAUAAAGUGACACUGGCAGAUGCAGGAUUUAUUAAACAGUAUGGAGAUUGC LLFNKVTLADAGFIKQYGDC Sprotein 258225922602261226222632 CUGGGUGAUAUUGCUGCAAGAGAUCUGAUUUGUGCUCAGAAAUUUAAUGGACUGACAGUG LGDIAARDLICAQKFNGLTV Sprotein 264226522662267226822692 CUGCCUCCUCUGCUGACAGAUGAAAUGAUUGCUCAGUACACAUCUGCUUUACUGGCUGGA LPPLLTDEMIAQYTSALLAG Sprotein 270227122722273227422752 ACAAUUACAAGCGGAUGGACAUUUGGAGCUGGAGCUGCUCUGCAGAUUCCUUUUGCAAUG TITSGWTFGAGAALQIPFAM Sprotein 276227722782279228022812 CAGAUGGCUUACAGAUUUAAUGGAAUUGGAGUGACACAGAAUGUGUUAUAUGAAAAUCAG QMAYRFNGIGVTQNVLYENQ Sprotein 282228322842285228622872 AAACUGAUUGCAAAUCAGUUUAAUUCUGCAAUUGGCAAAAUUCAGGAUUCUCUGUCUUCU KLIANQFNSAIGKIQDSLSS Sprotein 288228922902291229222932 ACAGCUUCUGCUCUGGGAAAACUGCAGGAUGUGGUGAAUCAGAAUGCACAGGCACUGAAU TASALGKLQDVVNQNAQALN Sprotein 294229522962297229822992 ACUCUGGUGAAACAGCUGUCUAGCAAUUUUGGGGCAAUUUCUUCUGUGCUGAAUGAUAUU TLVKQLSSNFGAISSVLNDI Sprotein 300230123022303230423052 CUGUCUAGACUGGAUCCTCCTGAAGCUGAAGUGCAGAUUGAUAGACUGAUCACAGGAAGA LSRLDPPEAEVQIDRLITGR Sprotein 306230723082309231023112 CUGCAGUCUCUGCAGACUUAUGUGACACAGCAGCUGAUUAGAGCUGCUGAAAUUAGAGCU LQSLQTYVTQQLIRAAEIRA Sprotein 312231323142315231623172 UCUGCUAAUCUGGCUGCUACAAAAAUGUCUGAAUGUGUGCUGGGACAGUCAAAAAGAGUG SANLAATKMSECVLGQSKRV Sprotein 318231923202321232223232 GAUUUUUGUGGAAAAGGAUAUCAUCUGAUGUCUUUUCCACAGUCUGCUCCACAUGGAGUG DFCGKGYHLMSEPQSAPHGV Sprotein 324232523262327232823292 GUGUUUUUACAUGUGACAUAUGUGCCAGCACAGGAAAAGAAUUUUACCACAGCACCAGCA VFLHVTYVPAQEKNFTTAPA Sprotein 330233123322333233423352 AUUUGUCAUGAUGGAAAAGCACAUUUUCCAAGAGAAGGAGUGUUUGUGUCUAAUGGAACA ICHDGKAHFPREGVFVSNGT Sprotein 336233723382339234023412 CAUUGGUUUGUGACACAGAGAAAUUUUUAUGAACCUCAGAUUAUUACAACAGAUAAUACA HWFVTQRNFYEPQIITTDNT Sprotein 342234323442345234623472 UUUGUGUCAGGAAAUUGUGAUGUGGUGAUUGGAAUUGUGAAUAAUACAGUGUAUGAUCCA FVSGNCDVVIGIVNNTVYDP Sprotein 348234923502351235223532 CUGCAGCCAGAACUGGAUUCUUUUAAAGAAGAACUGGAUAAAUAUUUUAAAAAUCACACA LQPELDSFKEELDKYFKNHT Sprotein 354235523562357235823592 UCUCCUGAUGUGGAUUUAGGAGAUAUUUCUGGAAUCAAUGCAUCUGUGGUGAAUAUUCAG SPDVDLGDISGINASVVNIQ Sprotein 360236123622363236423652 AAAGAAAUUGAUAGACUGAAUGAAGUGGCCAAAAAUCUGAAUGAAUCUCUGAUUGAUCUG KEIDRLNEVAKNLNESLIDL Sprotein 366236723682369237023712 CAGGAACUUGGAAAAUAUGAACAGUACAUUAAAUGGCCUUGGUACAUUUGGCUUGGAUUU QELGKYEQYIKWPWYIWLGF Sprotein 372237323742375237623772 AUUGCAGGAUUAAUUGCAAUUGUGAUGGUGACAAUUAUGUUAUGUUGUAUGACAUCAUGU IAGLIAIVMVTIMLCCMTSC Sprotein 378237923802381238223832 UGUUCUUGUUUAAAAGGAUGUUGUUCUUGUGGAAGCUGUUGUAAAUUUGAUGAAGAUGAU CSCLKGCCSCGSCCKFDEDD Sprotein 38423852386238723877 UCUGAACCUGUGUUAAAAGGAGUGAAAUUGCAUUACACAUGAUGA SEPVLKGVKLHYT** Sprotein 388738973907391739273937 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 394739573967397739873997 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 400740174027403740474057 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 406740774087409741074117 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 412741374147415741674172 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC FIelement 418241924202421242224232 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 42424252426242724282 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(476) TABLE-US-00004 NucleotideSequenceofRBL063.2(SEQIDNO:16;SEQIDNO:7) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinbold letters.Inaddition,thesequenceofthetranslatedproteinisshowninitalicletters belowthecodingnuleotidesequence(*=stopcodon). 102030405052 GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC hAg-Kozak 62728292102112 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGAAUUUGACAACA MFVFLVLLPLVSSQCVNLTT Sprotein 122132142152162172 AGAACACAGCUGCCACCAGCUUAUACAAAUUCUUUUACCAGAGGAGUGUAUUAUCCUGAU RTQLPPAYTNSFTRGVYYPD Sprotein 182192202212222232 AAAGUGUUUAGAUCUUCUGUGCUGCACAGCACACAGGACCUGUUUCUGCCAUUUUUUAGC KVFRSSVLHSTQDLFLPFFS Sprotein 242252262272282292 AAUGUGACAUGGUUUCAUGCAAUUCAUGUGUCUGGAACAAAUGGAACAAAAAGAUUUGAU NVTWFHAIHVSGTNGTKRFD Sprotein 302312322332342352 AAUCCUGUGCUGCCUUUUAAUGAUGGAGUGUAUUUUGCUUCAACAGAAAAGUCAAAUAUU NPVLPFNDGVYEASTEKSNI Sprotein 362372382392402412 AUUAGAGGAUGGAUUUUUGGAACAACACUGGAUUCUAAAACACAGUCUCUGCUGAUUGUG IRGWIFGTTLDSKTQSLLIV Sprotein 422432442452462472 AAUAAUGCAACAAAUGUGGUGAUUAAAGUGUGUGAAUUUCAGUUUUGUAAUGAUCCUUUU NNATNVVIKVCEFQFCNDPF Sprotein 482492502512522532 CUGGGAGUGUAUUAUCACAAAAAUAAUAAAUCUUGGAUGGAAUCUGAAUUUAGAGUGUAU LGVYYHKNNKSWMESEFRVY Sprotein 542552562572582592 UCCUCUGCAAAUAAUUGUACAUUUGAAUAUGUGUCUCAGCCUUUUCUGAUGGAUCUGGAA SSANNCTFEYVSQPFLMDLE Sprotein 602612622632642652 GGAAAACAGGGCAAUUUUAAAAAUCUGAGAGAAUUUGUGUUUAAAAAUAUUGAUGGAUAU GKQGNFKNLREFVFKNIDGY Sprotein 662672682692702712 UUUAAAAUUUAUUCUAAACACACACCAAUUAAUUUAGUGAGAGAUCUGCCUCAGGGAUUU FKIYSKHTPINLVRDLPQGF Sprotein 722732742752762772 UCUGCUCUGGAACCUCUGGUGGAUCUGCCAAUUGGCAUUAAUAUUACAAGAUUUCAGACA SALEPLVDLPIGINITRFQT Sprotein 782792802812822832 CUGCUGGCUCUGCACAGAUCUUAUCUGACACCUGGAGAUUCUUCUUCUGGAUGGACAGCC LLALHRSYLTPGDSSSGWTA Sprotein 842852862872882892 GGAGCUGCAGCUUAUUAUGUGGGCUAUCUGCAGCCAAGAACAUUUCUGCUGAAAUAUAAU GAAAYYVGYLQPRTFLLKYN Sprotein 902912922932942952 GAAAAUGGAACAAUUACAGAUGCUGUGGAUUGUGCUCUGGAUCCUCUGUCUGAAACAAAA ENGTITDAVDCALDPLSETK Sprotein 96297298299210021012 UGUACAUUAAAAUCUUUUACAGUGGAAAAAGGCAUUUAUCAGACAUCUAAUUUUAGAGUG CTLKSFTVEKGIYQTSNFRV Sprotein 102210321042105210621072 CAGCCAACAGAAUCUAUUGUGAGAUUUCCAAAUAUUACAAAUCUGUGUCCAUUUGGAGAA QPTESIVRFPNITNLCPFGE Sprotein 108210921102111211221132 GUGUUUAAUGCAACAAGAUUUGCAUCUGUGUAUGCAUGGAAUAGAAAAAGAAUUUCUAAU VFNATRFASVYAWNRKRISN Sprotein 114211521162117211821192 UGUGUGGCUGAUUAUUCUGUGCUGUAUAAUAGUGCUUCUUUUUCCACAUUUAAAUGUUAU CVADYSVLYNSASFSTFKCY Sprotein 120212121222123212421252 GGAGUGUCUCCAACAAAAUUAAAUGAUUUAUGUUUUACAAAUGUGUAUGCUGAUUCUUUU GVSPTKLNDLCFTNVYADSF Sprotein 126212721282129213021312 GUGAUCAGAGGUGAUGAAGUGAGACAGAUUGCCCCCGGACAGACAGGAAAAAUUGCUGAU VIRGDEVRQIAPGQTGKIAD Sprotein 132213321342135213621372 UACAAUUACAAACUGCCUGAUGAUUUUACAGGAUGUGUGAUUGCUUGGAAUUCUAAUAAU YNYKLPDDFTGCVIAWNSNN Sprotein 138213921402141214221432 UUAGAUUCUAAAGUGGGAGGAAAUUACAAUUAUCUGUACAGACUGUUUAGAAAAUCAAAU LDSKVGGNYNYLYRLFRKSN Sprotein 144214521462147214821492 CUGAAACCUUUUGAAAGAGAUAUUUCAACAGAAAUUUAUCAGGCUGGAUCAACACCUUGU LKPPERDISTEIYQAGSTPC Sprotein 150215121522153215421552 AAUGGAGUGGAAGGAUUUAAUUGUUAUUUUCCAUUACAGAGCUAUGGAUUUCAGCCAACC NGVEGFNCYFPLQSYGFQPT Sprotein 156215721582159216021612 AAUGGUGUGGGAUAUCAGCCAUAUAGAGUGGUGGUGCUGUCUUUUGAACUGCUGCAUGCA NGVGYQPYRVVVLSFELLHA Sprotein 162216321642165216621672 CCUGCAACAGUGUGUGGACCUAAAAAAUCUACAAAUUUAGUGAAAAAUAAAUGUGUGAAU PATVCGPKKSTNLVKNKCVN Sprotein 168216921702171217221732 UUUAAUUUUAAUGGAUUAACAGGAACAGGAGUGCUGACAGAAUCUAAUAAAAAAUUUCUG FNFNGLTGTGVLTESNKKFL Sprotein 174217521762177217821792 CCUUUUCAGCAGUUUGGCAGAGAUAUUGCAGAUACCACAGAUGCAGUGAGAGAUCCUCAG PFQQFGRDIADTTDAVRDPQ Sprotein 180218121822183218421852 ACAUUAGAAAUUCUGGAUAUUACACCUUGUUCUUUUGGGGGUGUGUCUGUGAUUACACCU TLEILDITPCSFGGVSVITP Sprotein 186218721882189219021912 GGAACAAAUACAUCUAAUCAGGUGGCUGUGCUGUAUCAGGAUGUGAAUUGUACAGAAGUG GTNTSNQVAVLYQDVNCTEV Sprotein 192219321942195219621972 CCAGUGGCAAUUCAUGCAGAUCAGCUGACACCAACAUGGAGAGUGUAUUCUACAGGAUCU PVAIHADQLTPTWRVYSTGS Sprotein 198219922002201220222032 AAUGUGUUUCAGACAAGAGCAGGAUGUCUGAUUGGAGCAGAACAUGUGAAUAAUUCUUAU NVFQTRAGCLIGAEHVNNSY Sprotein 204220522062207220822092 GAAUGUGAUAUUCCAAUUGGAGCAGGCAUUUGUGCAUCUUAUCAGACACAGACAAAUUCC ECDIPIGAGICASYQTQTNS Sprotein 210221122122213221422152 CCAAGGAGAGCAAGAUCUGUGGCAUCUCAGUCUAUUAUUGCAUACACCAUGUCUCUGGGA PRRARSVASQSIIAYTMSLG Sprotein 216221722182219222022212 GCAGAAAAUUCUGUGGCAUAUUCUAAUAAUUCUAUUGCUAUUCCAACAAAUUUUACCAUU AENSVAYSNNSIAIPTNFTI Sprotein 222222322242225222622272 UCUGUGACAACAGAAAUUUUACCUGUGUCUAUGACAAAAACAUCUGUGGAUUGUACCAUG SVTTEILPVSMTKTSVDCTM Sprotein 228222922302231223222332 UACAUUUGUGGAGAUUCUACAGAAUGUUCUAAUCUGCUGCUGCAGUAUGGAUCUUUUUGU YICGDSTECSNLLLQYGSFC Sprotein 234223522362237223822392 ACACAGCUGAAUAGAGCUUUAACAGGAAUUGCUGUGGAACAGGAUAAAAAUACACAGGAA TQLNRALTGIAVEQDKNTQE Sprotein 240224122422243224422452 GUGUUUGCUCAGGUGAAACAGAUUUACAAAACACCACCAAUUAAAGAUUUUGGAGGAUUU VFAQVKQIYKTPPIKDFGGF Sprotein 246224722482249225022512 AAUUUUAGCCAGAUUCUGCCUGAUCCUUCUAAACCUUCUAAAAGAUCUUUUAUUGAAGAU NFSQILPDPSKPSKRSFIED Sprotein 252225322542255225622572 CUGCUGUUUAAUAAAGUGACACUGGCAGAUGCAGGAUUUAUUAAACAGUAUGGAGAUUGC LLFNKVTLADAGFIKQYGDC Sprotein 258225922602261226222632 CUGGGUGAUAUUGCUGCAAGAGAUCUGAUUUGUGCUCAGAAAUUUAAUGGACUGACAGUG LGDIAARDLICAQKFNGLTV Sprotein 264226522662267226822692 CUGCCUCCUCUGCUGACAGAUGAAAUGAUUGCUCAGUACACAUCUGCUUUACUGGCUGGA LPPLLTDEMIAQYTSALLAG Sprotein 270227122722273227422752 ACAAUUACAAGCGGAUGGACAUUUGGAGCUGGAGCUGCUCUGCAGAUUCCUUUUGCAAUG TITSGWTFGAGAALQIPFAM Sprotein 276227722782279228022812 CAGAUGGCUUACAGAUUUAAUGGAAUUGGAGUGACACAGAAUGUGUUAUAUGAAAAUCAG QMAYRFNGIGVTQNVLYENQ Sprotein 282228322842285228622872 AAACUGAUUGCAAAUCAGUUUAAUUCUGCAAUUGGCAAAAUUCAGGAUUCUCUGUCUUCU KLIANQFNSAIGKIQDSLSS Sprotein 288228922902291229222932 ACAGCUUCUGCUCUGGGAAAACUGCAGGAUGUGGUGAAUCAGAAUGCACAGGCACUGAAU TASALGKLQDVVNQNAQALN Sprotein 294229522962297229822992 ACUCUGGUGAAACAGCUGUCUAGCAAUUUUGGGGCAAUUUCUUCUGUGCUGAAUGAUAUU TLVKQLSSNFGAISSVLNDI Sprotein 300230123022303230423052 CUGUCUAGACUGGAUCCTCCTGAAGCUGAAGUGCAGAUUGAUAGACUGAUCACAGGAAGA LSRLDPPEAEVQIDRLITGR Sprotein 306230723082309231023112 CUGCAGUCUCUGCAGACUUAUGUGACACAGCAGCUGAUUAGAGCUGCUGAAAUUAGAGCU LQSLQTYVTQQLIRAAEIRA Sprotein 312231323142315231623172 UCUGCUAAUCUGGCUGCUACAAAAAUGUCUGAAUGUGUGCUGGGACAGUCAAAAAGAGUG SANLAATKMSECVLGQSKRV Sprotein 318231923202321232223232 GAUUUUUGUGGAAAAGGAUAUCAUCUGAUGUCUUUUCCACAGUCUGCUCCACAUGGAGUG DFCGKGYHLMSEPQSAPHGV Sprotein 324232523262327232823292 GUGUUUUUACAUGUGACAUAUGUGCCAGCACAGGAAAAGAAUUUUACCACAGCACCAGCA VFLHVTYVPAQEKNFTTAPA Sprotein 330233123322333233423352 AUUUGUCAUGAUGGAAAAGCACAUUUUCCAAGAGAAGGAGUGUUUGUGUCUAAUGGAACA ICHDGKAHFPREGVFVSNGT Sprotein 336233723382339234023412 CAUUGGUUUGUGACACAGAGAAAUUUUUAUGAACCUCAGAUUAUUACAACAGAUAAUACA HWFVTQRNFYEPQIITTDNT Sprotein 342234323442345234623472 UUUGUGUCAGGAAAUUGUGAUGUGGUGAUUGGAAUUGUGAAUAAUACAGUGUAUGAUCCA FVSGNCDVVIGIVNNTVYDP Sprotein 348234923502351235223532 CUGCAGCCAGAACUGGAUUCUUUUAAAGAAGAACUGGAUAAAUAUUUUAAAAAUCACACA LQPELDSFKEELDKYFKNHT Sprotein 354235523562357235823592 UCUCCUGAUGUGGAUUUAGGAGAUAUUUCUGGAAUCAAUGCAUCUGUGGUGAAUAUUCAG SPDVDLGDISGINASVVNIQ Sprotein 360236123622363236423652 AAAGAAAUUGAUAGACUGAAUGAAGUGGCCAAAAAUCUGAAUGAAUCUCUGAUUGAUCUG KEIDRLNEVAKNLNESLIDL Sprotein 366236723682369237023712 CAGGAACUUGGAAAAUAUGAACAGUACAUUAAAUGGCCUUGGUACAUUUGGCUUGGAUUU QELGKYEQYIKWPWYIWLGF Sprotein 372237323742375237623772 AUUGCAGGAUUAAUUGCAAUUGUGAUGGUGACAAUUAUGUUAUGUUGUAUGACAUCAUGU IAGLIAIVMVTIMLCCMTSC Sprotein 378237923802381238223832 UGUUCUUGUUUAAAAGGAUGUUGUUCUUGUGGAAGCUGUUGUAAAUUUGAUGAAGAUGAU CSCLKGCCSCGSCCKFDEDD Sprotein 38423852386238723877 UCUGAACCUGUGUUAAAAGGAGUGAAAUUGCAUUACACAUGAUGA SEPVLKGVKLHYT** Sprotein 388738973907391739273937 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 394739573967397739873997 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 400740174027403740474057 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 406740774087409741074117 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 412741374147415741674172 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC FIelement 418241924202421242224232 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 42424252426242724282 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(477) TABLE-US-00005 NucleotideSequenceofRBL063.3(SEQIDNO:17;SEQIDNO:5) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinbold letters.Inaddition,thesequenceofthetranslatedproteinisshowninitalicletters belowthecodingnucleotidesequence(*=stopcodon). 102030405052 GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC hAg-Kozak 62728292102112 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGGUGAGAUUUCCA MFVFLVLLPLVSSQCVVRFP RBD(Sprotein) 122132142152162172 AAUAUUACAAAUCUGUGUCCAUUUGGAGAAGUGUUUAAUGCAACAAGAUUUGCAUCUGUG NITNLCPFGEVFNATRFASV RBD(Sprotein) 182192202212222232 UAUGCAUGGAAUAGAAAAAGAAUUUCUAAUUGUGUGGCUGAUUAUUCUGUGCUGUAUAAU YAWNRKRISNCVADYSVLYN RBD(Sprotein) 242252262272282292 AGUGCUUCUUUUUCCACAUUUAAAUGUUAUGGAGUGUCUCCAACAAAAUUAAAUGAUUUA SASFSTFKCYGVSPTKLNDL RBD(Sprotein) 302312322332342352 UGUUUUACAAAUGUGUAUGCUGAUUCUUUUGUGAUCAGAGGUGAUGAAGUGAGACAGAUU CFTNVYADSFVIRGDEVRQI RBD(Sprotein) 362372382392402412 GCCCCCGGACAGACAGGAAAAAUUGCUGAUUACAAUUACAAACUGCCUGAUGAUUUUACA APGQTGKIADYNYKLPDDFT RBD(Sprotein) 422432442452462472 GGAUGUGUGAUUGCUUGGAAUUCUAAUAAUUUAGAUUCUAAAGUGGGAGGAAAUUACAAU GCVIAWNSNNLDSKVGGNYN RBD(Sprotein) 482492502512522532 UAUCUGUACAGACUGUUUAGAAAAUCAAAUCUGAAACCUUUUGAAAGAGAUAUUUCAACA YLYRLFRKSNLKPFERDIST RBD(Sprotein) 542552562572582592 GAAAUUUAUCAGGCUGGAUCAACACCUUGUAAUGGAGUGGAAGGAUUUAAUUGUUAUUUU EIYQAGSTPCNGVEGFNCYF RBD(Sprotein) 602612622632642652 CCAUUACAGAGCUAUGGAUUUCAGCCAACCAAUGGUGUGGGAUAUCAGCCAUAUAGAGUG PLQSYGEQPTNGVGYQPYRV RBD(Sprotein) 662672682692702706 GUGGUGCUGUCUUUUGAACUGCUGCAUGCACCUGCAACAGUGUGUGGACCUAAA VVLSFELLHAPATVCGPK RBD(Sprotein) 716726733 GGCUCCCCCGGCUCCGGCUCCGGAUCU GSPGSGSGS GSlinker 743753763773783793 GGUUAUAUUCCUGAAGCUCCAAGAGAUGGGCAAGCUUACGUUCGUAAAGAUGGCGAAUGG GYIPEAPRDGQAYVRKDGEW fibritin 803813823833843853 GUAUUACUUUCUACCUUUUUAGGCCGGUCCCUGGAGGUGCUGUUCCAGGGCCCCGGCUGA VLLSTFLGRSLEVLFQGPG* fibritin 856 UGA * fibritin 866876886896906916 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 926936946956966976 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 9869961006101610261036 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 104610561066107610861096 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 110611161126113611461151 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC FIelement 116111711181119112011211 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 12211231124112511261 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(478) Nucleoside Modified Messenger RNA (modRNA)

(479) The active principle of the nucleoside modified messenger RNA (modRNA) drug substance is as well a single-stranded mRNA that is translated upon entering a cell. In addition to the sequence encoding the coronavirus vaccine antigen (i.e. open reading frame), each modRNA contains common structural elements optimized for maximal efficacy of the RNA as the uRNA (5-cap, 5-UTR, 3-UTR, poly(A)-tail). Compared to the uRNA, modRNA contains 1-methyl-pseudouridine instead of uridine. The preferred 5 cap structure is m.sub.2.sup.7,3-OGppp(m.sub.12-O)ApG. The preferred 5-UTR and 3-UTR comprise the nucleotide sequence of SEQ ID NO: 12 and the nucleotide sequence of SEQ ID NO: 13, respectively. The preferred poly(A)-tail comprises the sequence of SEQ ID NO: 14. An additional purification step is applied for modRNA to reduce dsRNA contaminants generated during the in vitro transcription reaction.

(480) Different embodiment of this platform are as follows:

(481) TABLE-US-00006 BNT162b2; RBP020.1 (SEQ ID NO: 19; SEQ ID NO: 7) Structure m.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG)- hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) BNT162b2; RBP020.2 (SEQ ID NO: 20; SEQ ID NO: 7) Structure m.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG)- hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) BNT162b1; RBP020.3 (SEQ ID NO: 21; SEQ ID NO: 5) Structure m.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG)- hAg-Kozak-RBD-GS-Fibritin-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (partial sequence, Receptor Binding Domain (RBD) of S1S2 protein fused to fibritin)

(482) FIG. 20 schematizes the general structure of the antigen-encoding RNAs.

(483) TABLE-US-00007 NucleotideSequenceofRBP020.1(SEQIDNO:19;SEQIDNO:7) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinboldletters. Inaddition,thesequenceofthetranslatedproteinisshowninitaliclettersbelowthe codingnucleotidesequence(*=stopcodon). 102030405053 AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC hAg-Kozak 63738393103113 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGAAUUUGACAACA MFVFLVLLPLVSSQCVNLTT Sprotein 123133143153163173 AGAACACAGCUGCCACCAGCUUAUACAAAUUCUUUUACCAGAGGAGUGUAUUAUCCUGAU RTQLPPAYTNSFTRGVYYPD Sprotein 183193203213223233 AAAGUGUUUAGAUCUUCUGUGCUGCACAGCACACAGGACCUGUUUCUGCCAUUUUUUAGC KVFRSSVLHSTQDLFLPFFS Sprotein 243253263273283293 AAUGUGACAUGGUUUCAUGCAAUUCAUGUGUCUGGAACAAAUGGAACAAAAAGAUUUGAU NVTWFHAIHVSGTNGTKRFD Sprotein 303313323333343353 AAUCCUGUGCUGCCUUUUAAUGAUGGAGUGUAUUUUGCUUCAACAGAAAAGUCAAAUAUU NPVLPFNDGVYEASTEKSNI Sprotein 363373383393403413 AUUAGAGGAUGGAUUUUUGGAACAACACUGGAUUCUAAAACACAGUCUCUGCUGAUUGUG IRGWIFGTTLDSKTQSLLIV Sprotein 423433443453463473 AAUAAUGCAACAAAUGUGGUGAUUAAAGUGUGUGAAUUUCAGUUUUGUAAUGAUCCUUUU NNATNVVIKVCEFQFCNDPF Sprotein 483493503513523533 CUGGGAGUGUAUUAUCACAAAAAUAAUAAAUCUUGGAUGGAAUCUGAAUUUAGAGUGUAU LGVYYHKNNKSWMESEFRVY Sprotein 543553563573583593 UCCUCUGCAAAUAAUUGUACAUUUGAAUAUGUGUCUCAGCCUUUUCUGAUGGAUCUGGAA SSANNCTFEYVSQPFLMDLE Sprotein 603613623633643653 GGAAAACAGGGCAAUUUUAAAAAUCUGAGAGAAUUUGUGUUUAAAAAUAUUGAUGGAUAU GKQGNFKNLREFVFKNIDGY Sprotein 663673683693703713 UUUAAAAUUUAUUCUAAACACACACCAAUUAAUUUAGUGAGAGAUCUGCCUCAGGGAUUU FKIYSKHTPINLVRDLPQGF Sprotein 723733743753763773 UCUGCUCUGGAACCUCUGGUGGAUCUGCCAAUUGGCAUUAAUAUUACAAGAUUUCAGACA SALEPLVDLPIGINITRFQT Sprotein 783793803813823833 CUGCUGGCUCUGCACAGAUCUUAUCUGACACCUGGAGAUUCUUCUUCUGGAUGGACAGCC LLALHRSYLTPGDSSSGWTA Sprotein 843853863873883893 GGAGCUGCAGCUUAUUAUGUGGGCUAUCUGCAGCCAAGAACAUUUCUGCUGAAAUAUAAU GAAAYYVGYLQPRTFLLKYN Sprotein 93913923933943953 GAAAAUGGAACAAUUACAGAUGCUGUGGAUUGUGCUCUGGAUCCUCUGUCUGAAACAAAA ENGTITDAVDCALDPLSETK Sprotein 96397398399310031013 UGUACAUUAAAAUCUUUUACAGUGGAAAAAGGCAUUUAUCAGACAUCUAAUUUUAGAGUG CTLKSFTVEKGIYQTSNFRV Sprotein 102310331043105310631073 CAGCCAACAGAAUCUAUUGUGAGAUUUCCAAAUAUUACAAAUCUGUGUCCAUUUGGAGAA QPTESIVRFPNITNLCPFGE Sprotein 108310931103111311231133 GUGUUUAAUGCAACAAGAUUUGCAUCUGUGUAUGCAUGGAAUAGAAAAAGAAUUUCUAAU VFNATRFASVYAWNRKRISN Sprotein 114311531163117311831193 UGUGUGGCUGAUUAUUCUGUGCUGUAUAAUAGUGCUUCUUUUUCCACAUUUAAAUGUUAU CVADYSVLYNSASFSTFKCY Sprotein 120312131223123312431253 GGAGUGUCUCCAACAAAAUUAAAUGAUUUAUGUUUUACAAAUGUGUAUGCUGAUUCUUUU GVSPTKLNDLCFTNVYADSF Sprotein 126212721282129213021312 GUGAUCAGAGGUGAUGAAGUGAGACAGAUUGCCCCCGGACAGACAGGAAAAAUUGCUGAU VIRGDEVRQIAPGQTGKIAD Sprotein 132313331343135313631373 UACAAUUACAAACUGCCUGAUGAUUUUACAGGAUGUGUGAUUGCUUGGAAUUCUAAUAAU YNYKLPDDFTGCVIAWNSNN Sprotein 138313931403141314231433 UUAGAUUCUAAAGUGGGAGGAAAUUACAAUUAUCUGUACAGACUGUUUAGAAAAUCAAAU LDSKVGGNYNYLYRLFRKSN Sprotein 144314531463147314831493 CUGAAACCUUUUGAAAGAGAUAUUUCAACAGAAAUUUAUCAGGCUGGAUCAACACCUUGU LKPPERDISTEIYQAGSTPC Sprotein 150315131523153315431553 AAUGGAGUGGAAGGAUUUAAUUGUUAUUUUCCAUUACAGAGCUAUGGAUUUCAGCCAACC NGVEGFNCYFPLQSYGFQPT Sprotein 156315731583159316031613 AAUGGUGUGGGAUAUCAGCCAUAUAGAGUGGUGGUGCUGUCUUUUGAACUGCUGCAUGCA NGVGYQPYRVVVLSFELLHA Sprotein 162316331643165316631673 CCUGCAACAGUGUGUGGACCUAAAAAAUCUACAAAUUUAGUGAAAAAUAAAUGUGUGAAU PATVCGPKKSTNLVKNKCVN Sprotein 168316931703171317231733 UUUAAUUUUAAUGGAUUAACAGGAACAGGAGUGCUGACAGAAUCUAAUAAAAAAUUUCUG FNFNGLTGTGVLTESNKKFL Sprotein 174317531763177317831793 CCUUUUCAGCAGUUUGGCAGAGAUAUUGCAGAUACCACAGAUGCAGUGAGAGAUCCUCAG PFQQFGRDIADTTDAVRDPQ Sprotein 180318131823183318431853 ACAUUAGAAAUUCUGGAUAUUACACCUUGUUCUUUUGGGGGUGUGUCUGUGAUUACACCU TLEILDITPCSFGGVSVITP Sprotein 186318731883189319031913 GGAACAAAUACAUCUAAUCAGGUGGCUGUGCUGUAUCAGGAUGUGAAUUGUACAGAAGUG GTNTSNQVAVLYQDVNCTEV Sprotein 192319331943195319631973 CCAGUGGCAAUUCAUGCAGAUCAGCUGACACCAACAUGGAGAGUGUAUUCUACAGGAUCU PVAIHADQLTPTWRVYSTGS Sprotein 198319932003201320232033 AAUGUGUUUCAGACAAGAGCAGGAUGUCUGAUUGGAGCAGAACAUGUGAAUAAUUCUUAU NVFQTRAGCLIGAEHVNNSY Sprotein 204320532063207320832093 GAAUGUGAUAUUCCAAUUGGAGCAGGCAUUUGUGCAUCUUAUCAGACACAGACAAAUUCC ECDIPIGAGICASYQTQTNS Sprotein 210321132123213321432153 CCAAGGAGAGCAAGAUCUGUGGCAUCUCAGUCUAUUAUUGCAUACACCAUGUCUCUGGGA PRRARSVASQSIIAYTMSLG Sprotein 216321732183219322032213 GCAGAAAAUUCUGUGGCAUAUUCUAAUAAUUCUAUUGCUAUUCCAACAAAUUUUACCAUU AENSVAYSNNSIAIPTNFTI Sprotein 222322332243225322632273 UCUGUGACAACAGAAAUUUUACCUGUGUCUAUGACAAAAACAUCUGUGGAUUGUACCAUG SVTTEILPVSMTKTSVDCTM Sprotein 228322932303231323232333 UACAUUUGUGGAGAUUCUACAGAAUGUUCUAAUCUGCUGCUGCAGUAUGGAUCUUUUUGU YICGDSTECSNLLLQYGSFC Sprotein 234323532363237323832393 ACACAGCUGAAUAGAGCUUUAACAGGAAUUGCUGUGGAACAGGAUAAAAAUACACAGGAA TQLNRALTGIAVEQDKNTQE Sprotein 240324132423243324432453 GUGUUUGCUCAGGUGAAACAGAUUUACAAAACACCACCAAUUAAAGAUUUUGGAGGAUUU VFAQVKQIYKTPPIKDFGGF Sprotein 246324732483249325032513 AAUUUUAGCCAGAUUCUGCCUGAUCCUUCUAAACCUUCUAAAAGAUCUUUUAUUGAAGAU NFSQILPDPSKPSKRSFIED Sprotein 252325332543255325632573 CUGCUGUUUAAUAAAGUGACACUGGCAGAUGCAGGAUUUAUUAAACAGUAUGGAGAUUGC LLFNKVTLADAGFIKQYGDC Sprotein 258325932603261326232633 CUGGGUGAUAUUGCUGCAAGAGAUCUGAUUUGUGCUCAGAAAUUUAAUGGACUGACAGUG LGDIAARDLICAQKFNGLTV Sprotein 264326532663267326832693 CUGCCUCCUCUGCUGACAGAUGAAAUGAUUGCUCAGUACACAUCUGCUUUACUGGCUGGA LPPLLTDEMIAQYTSALLAG Sprotein 270327132723273327432753 ACAAUUACAAGCGGAUGGACAUUUGGAGCUGGAGCUGCUCUGCAGAUUCCUUUUGCAAUG TITSGWTFGAGAALQIPFAM Sprotein 276327732783279328032813 CAGAUGGCUUACAGAUUUAAUGGAAUUGGAGUGACACAGAAUGUGUUAUAUGAAAAUCAG QMAYRFNGIGVTQNVLYENQ Sprotein 282328332843285328632873 AAACUGAUUGCAAAUCAGUUUAAUUCUGCAAUUGGCAAAAUUCAGGAUUCUCUGUCUUCU KLIANQFNSAIGKIQDSLSS Sprotein 288328932903291329232933 ACAGCUUCUGCUCUGGGAAAACUGCAGGAUGUGGUGAAUCAGAAUGCACAGGCACUGAAU TASALGKLQDVVNQNAQALN Sprotein 294329532963297329832993 ACUCUGGUGAAACAGCUGUCUAGCAAUUUUGGGGCAAUUUCUUCUGUGCUGAAUGAUAUU TLVKQLSSNFGAISSVLNDI Sprotein 300330133023303330433053 CUGUCUAGACUGGAUCCTCCTGAAGCUGAAGUGCAGAUUGAUAGACUGAUCACAGGAAGA LSRLDPPEAEVQIDRLITGR Sprotein 306330733083309331033113 CUGCAGUCUCUGCAGACUUAUGUGACACAGCAGCUGAUUAGAGCUGCUGAAAUUAGAGCU LQSLQTYVTQQLIRAAEIRA Sprotein 312331333143315331633173 UCUGCUAAUCUGGCUGCUACAAAAAUGUCUGAAUGUGUGCUGGGACAGUCAAAAAGAGUG SANLAATKMSECVLGQSKRV Sprotein 318331933203321332233233 GAUUUUUGUGGAAAAGGAUAUCAUCUGAUGUCUUUUCCACAGUCUGCUCCACAUGGAGUG DFCGKGYHLMSEPQSAPHGV Sprotein 324332533263327332833293 GUGUUUUUACAUGUGACAUAUGUGCCAGCACAGGAAAAGAAUUUUACCACAGCACCAGCA VFLHVTYVPAQEKNFTTAPA Sprotein 330333133323333333433353 AUUUGUCAUGAUGGAAAAGCACAUUUUCCAAGAGAAGGAGUGUUUGUGUCUAAUGGAACA ICHDGKAHFPREGVFVSNGT Sprotein 336333733383339334033413 CAUUGGUUUGUGACACAGAGAAAUUUUUAUGAACCUCAGAUUAUUACAACAGAUAAUACA HWFVTQRNFYEPQIITTDNT Sprotein 342334333443345334633473 UUUGUGUCAGGAAAUUGUGAUGUGGUGAUUGGAAUUGUGAAUAAUACAGUGUAUGAUCCA FVSGNCDVVIGIVNNTVYDP Sprotein 348334933503351335233533 CUGCAGCCAGAACUGGAUUCUUUUAAAGAAGAACUGGAUAAAUAUUUUAAAAAUCACACA LQPELDSFKEELDKYFKNHT Sprotein 354335533563357335833593 UCUCCUGAUGUGGAUUUAGGAGAUAUUUCUGGAAUCAAUGCAUCUGUGGUGAAUAUUCAG SPDVDLGDISGINASVVNIQ Sprotein 360336133623363336433653 AAAGAAAUUGAUAGACUGAAUGAAGUGGCCAAAAAUCUGAAUGAAUCUCUGAUUGAUCUG KEIDRLNEVAKNLNESLIDL Sprotein 366336733683369337033713 CAGGAACUUGGAAAAUAUGAACAGUACAUUAAAUGGCCUUGGUACAUUUGGCUUGGAUUU QELGKYEQYIKWPWYIWLGF Sprotein 372337333743375337633773 AUUGCAGGAUUAAUUGCAAUUGUGAUGGUGACAAUUAUGUUAUGUUGUAUGACAUCAUGU IAGLIAIVMVTIMLCCMTSC Sprotein 378337933803381338233833 UGUUCUUGUUUAAAAGGAUGUUGUUCUUGUGGAAGCUGUUGUAAAUUUGAUGAAGAUGAU CSCLKGCCSCGSCCKFDEDD Sprotein 38433853386338733878 UCUGAACCUGUGUUAAAAGGAGUGAAAUUGCAUUACACAUGAUGA SEPVLKGVKLHYT** Sprotein 388838983908391839283938 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 394839583968397839883998 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 408840184028403840484058 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 406840784088409841084118 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 412841384148415841684173 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC FIelement 418341934203421342234233 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 42434253426342734283 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(484) TABLE-US-00008 NucleotideSequenceofRBP020.2(SEQIDNO:20;SEQIDNO:7) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinboldletters. Inaddition,thesequenceofthetranslatedproteinisshowninitaliclettersbelowthe codingnucleotidesequence(*=stopcodon). 102030405053 AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC hAg-Kozak 63738393103113 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGAAUUUGACAACA MFVFLVLLPLVSSQCVNLTT Sprotein 123133143153163173 AGAACACAGCUGCCACCAGCUUAUACAAAUUCUUUUACCAGAGGAGUGUAUUAUCCUGAU RTQLPPAYTNSFTRGVYYPD Sprotein 183193203213223233 AAAGUGUUUAGAUCUUCUGUGCUGCACAGCACACAGGACCUGUUUCUGCCAUUUUUUAGC KVFRSSVLHSTQDLFLPFFS Sprotein 243253263273283293 AAUGUGACAUGGUUUCAUGCAAUUCAUGUGUCUGGAACAAAUGGAACAAAAAGAUUUGAU NVTWFHAIHVSGTNGTKRFD Sprotein 303313323333343353 AAUCCUGUGCUGCCUUUUAAUGAUGGAGUGUAUUUUGCUUCAACAGAAAAGUCAAAUAUU NPVLPFNDGVYEASTEKSNI Sprotein 363373383393403413 AUUAGAGGAUGGAUUUUUGGAACAACACUGGAUUCUAAAACACAGUCUCUGCUGAUUGUG IRGWIFGTTLDSKTQSLLIV Sprotein 423433443453463473 AAUAAUGCAACAAAUGUGGUGAUUAAAGUGUGUGAAUUUCAGUUUUGUAAUGAUCCUUUU NNATNVVIKVCEFQFCNDPF Sprotein 483493503513523533 CUGGGAGUGUAUUAUCACAAAAAUAAUAAAUCUUGGAUGGAAUCUGAAUUUAGAGUGUAU LGVYYHKNNKSWMESEFRVY Sprotein 543553563573583593 UCCUCUGCAAAUAAUUGUACAUUUGAAUAUGUGUCUCAGCCUUUUCUGAUGGAUCUGGAA SSANNCTFEYVSQPFLMDLE Sprotein 603613623633643653 GGAAAACAGGGCAAUUUUAAAAAUCUGAGAGAAUUUGUGUUUAAAAAUAUUGAUGGAUAU GKQGNFKNLREFVFKNIDGY Sprotein 663673683693703713 UUUAAAAUUUAUUCUAAACACACACCAAUUAAUUUAGUGAGAGAUCUGCCUCAGGGAUUU FKIYSKHTPINLVRDLPQGF Sprotein 723733743753763773 UCUGCUCUGGAACCUCUGGUGGAUCUGCCAAUUGGCAUUAAUAUUACAAGAUUUCAGACA SALEPLVDLPIGINITRFQT Sprotein 783793803813823833 CUGCUGGCUCUGCACAGAUCUUAUCUGACACCUGGAGAUUCUUCUUCUGGAUGGACAGCC LLALHRSYLTPGDSSSGWTA Sprotein 843853863873883893 GGAGCUGCAGCUUAUUAUGUGGGCUAUCUGCAGCCAAGAACAUUUCUGCUGAAAUAUAAU GAAAYYVGYLQPRTFLLKYN Sprotein 93913923933943953 GAAAAUGGAACAAUUACAGAUGCUGUGGAUUGUGCUCUGGAUCCUCUGUCUGAAACAAAA ENGTITDAVDCALDPLSETK Sprotein 96397398399310031013 UGUACAUUAAAAUCUUUUACAGUGGAAAAAGGCAUUUAUCAGACAUCUAAUUUUAGAGUG CTLKSFTVEKGIYQTSNFRV Sprotein 102310331043105310631073 CAGCCAACAGAAUCUAUUGUGAGAUUUCCAAAUAUUACAAAUCUGUGUCCAUUUGGAGAA QPTESIVRFPNITNLCPFGE Sprotein 108310931103111311231133 GUGUUUAAUGCAACAAGAUUUGCAUCUGUGUAUGCAUGGAAUAGAAAAAGAAUUUCUAAU VFNATRFASVYAWNRKRISN Sprotein 114311531163117311831193 UGUGUGGCUGAUUAUUCUGUGCUGUAUAAUAGUGCUUCUUUUUCCACAUUUAAAUGUUAU CVADYSVLYNSASFSTFKCY Sprotein 120312131223123312431253 GGAGUGUCUCCAACAAAAUUAAAUGAUUUAUGUUUUACAAAUGUGUAUGCUGAUUCUUUU GVSPTKLNDLCFTNVYADSF Sprotein 126212721282129213021312 GUGAUCAGAGGUGAUGAAGUGAGACAGAUUGCCCCCGGACAGACAGGAAAAAUUGCUGAU VIRGDEVRQIAPGQTGKIAD Sprotein 132313331343135313631373 UACAAUUACAAACUGCCUGAUGAUUUUACAGGAUGUGUGAUUGCUUGGAAUUCUAAUAAU YNYKLPDDFTGCVIAWNSNN Sprotein 138313931403141314231433 UUAGAUUCUAAAGUGGGAGGAAAUUACAAUUAUCUGUACAGACUGUUUAGAAAAUCAAAU LDSKVGGNYNYLYRLFRKSN Sprotein 144314531463147314831493 CUGAAACCUUUUGAAAGAGAUAUUUCAACAGAAAUUUAUCAGGCUGGAUCAACACCUUGU LKPPERDISTEIYQAGSTPC Sprotein 150315131523153315431553 AAUGGAGUGGAAGGAUUUAAUUGUUAUUUUCCAUUACAGAGCUAUGGAUUUCAGCCAACC NGVEGFNCYFPLQSYGFQPT Sprotein 156315731583159316031613 AAUGGUGUGGGAUAUCAGCCAUAUAGAGUGGUGGUGCUGUCUUUUGAACUGCUGCAUGCA NGVGYQPYRVVVLSFELLHA Sprotein 162316331643165316631673 CCUGCAACAGUGUGUGGACCUAAAAAAUCUACAAAUUUAGUGAAAAAUAAAUGUGUGAAU PATVCGPKKSTNLVKNKCVN Sprotein 168316931703171317231733 UUUAAUUUUAAUGGAUUAACAGGAACAGGAGUGCUGACAGAAUCUAAUAAAAAAUUUCUG FNFNGLTGTGVLTESNKKFL Sprotein 174317531763177317831793 CCUUUUCAGCAGUUUGGCAGAGAUAUUGCAGAUACCACAGAUGCAGUGAGAGAUCCUCAG PFQQFGRDIADTTDAVRDPQ Sprotein 180318131823183318431853 ACAUUAGAAAUUCUGGAUAUUACACCUUGUUCUUUUGGGGGUGUGUCUGUGAUUACACCU TLEILDITPCSFGGVSVITP Sprotein 186318731883189319031913 GGAACAAAUACAUCUAAUCAGGUGGCUGUGCUGUAUCAGGAUGUGAAUUGUACAGAAGUG GTNTSNQVAVLYQDVNCTEV Sprotein 192319331943195319631973 CCAGUGGCAAUUCAUGCAGAUCAGCUGACACCAACAUGGAGAGUGUAUUCUACAGGAUCU PVAIHADQLTPTWRVYSTGS Sprotein 198319932003201320232033 AAUGUGUUUCAGACAAGAGCAGGAUGUCUGAUUGGAGCAGAACAUGUGAAUAAUUCUUAU NVFQTRAGCLIGAEHVNNSY Sprotein 204320532063207320832093 GAAUGUGAUAUUCCAAUUGGAGCAGGCAUUUGUGCAUCUUAUCAGACACAGACAAAUUCC ECDIPIGAGICASYQTQTNS Sprotein 210321132123213321432153 CCAAGGAGAGCAAGAUCUGUGGCAUCUCAGUCUAUUAUUGCAUACACCAUGUCUCUGGGA PRRARSVASQSIIAYTMSLG Sprotein 216321732183219322032213 GCAGAAAAUUCUGUGGCAUAUUCUAAUAAUUCUAUUGCUAUUCCAACAAAUUUUACCAUU AENSVAYSNNSIAIPTNFTI Sprotein 222322332243225322632273 UCUGUGACAACAGAAAUUUUACCUGUGUCUAUGACAAAAACAUCUGUGGAUUGUACCAUG SVTTEILPVSMTKTSVDCTM Sprotein 228322932303231323232333 UACAUUUGUGGAGAUUCUACAGAAUGUUCUAAUCUGCUGCUGCAGUAUGGAUCUUUUUGU YICGDSTECSNLLLQYGSFC Sprotein 234323532363237323832393 ACACAGCUGAAUAGAGCUUUAACAGGAAUUGCUGUGGAACAGGAUAAAAAUACACAGGAA TQLNRALTGIAVEQDKNTQE Sprotein 240324132423243324432453 GUGUUUGCUCAGGUGAAACAGAUUUACAAAACACCACCAAUUAAAGAUUUUGGAGGAUUU VFAQVKQIYKTPPIKDFGGF Sprotein 246324732483249325032513 AAUUUUAGCCAGAUUCUGCCUGAUCCUUCUAAACCUUCUAAAAGAUCUUUUAUUGAAGAU NFSQILPDPSKPSKRSFIED Sprotein 252325332543255325632573 CUGCUGUUUAAUAAAGUGACACUGGCAGAUGCAGGAUUUAUUAAACAGUAUGGAGAUUGC LLFNKVTLADAGFIKQYGDC Sprotein 258325932603261326232633 CUGGGUGAUAUUGCUGCAAGAGAUCUGAUUUGUGCUCAGAAAUUUAAUGGACUGACAGUG LGDIAARDLICAQKFNGLTV Sprotein 264326532663267326832693 CUGCCUCCUCUGCUGACAGAUGAAAUGAUUGCUCAGUACACAUCUGCUUUACUGGCUGGA LPPLLTDEMIAQYTSALLAG Sprotein 270327132723273327432753 ACAAUUACAAGCGGAUGGACAUUUGGAGCUGGAGCUGCUCUGCAGAUUCCUUUUGCAAUG TITSGWTFGAGAALQIPFAM Sprotein 276327732783279328032813 CAGAUGGCUUACAGAUUUAAUGGAAUUGGAGUGACACAGAAUGUGUUAUAUGAAAAUCAG QMAYRFNGIGVTQNVLYENQ Sprotein 282328332843285328632873 AAACUGAUUGCAAAUCAGUUUAAUUCUGCAAUUGGCAAAAUUCAGGAUUCUCUGUCUUCU KLIANQFNSAIGKIQDSLSS Sprotein 288328932903291329232933 ACAGCUUCUGCUCUGGGAAAACUGCAGGAUGUGGUGAAUCAGAAUGCACAGGCACUGAAU TASALGKLQDVVNQNAQALN Sprotein 294329532963297329832993 ACUCUGGUGAAACAGCUGUCUAGCAAUUUUGGGGCAAUUUCUUCUGUGCUGAAUGAUAUU TLVKQLSSNFGAISSVLNDI Sprotein 300330133023303330433053 CUGUCUAGACUGGAUCCTCCTGAAGCUGAAGUGCAGAUUGAUAGACUGAUCACAGGAAGA LSRLDPPEAEVQIDRLITGR Sprotein 306330733083309331033113 CUGCAGUCUCUGCAGACUUAUGUGACACAGCAGCUGAUUAGAGCUGCUGAAAUUAGAGCU LQSLQTYVTQQLIRAAEIRA Sprotein 312331333143315331633173 UCUGCUAAUCUGGCUGCUACAAAAAUGUCUGAAUGUGUGCUGGGACAGUCAAAAAGAGUG SANLAATKMSECVLGQSKRV Sprotein 318331933203321332233233 GAUUUUUGUGGAAAAGGAUAUCAUCUGAUGUCUUUUCCACAGUCUGCUCCACAUGGAGUG DFCGKGYHLMSEPQSAPHGV Sprotein 324332533263327332833293 GUGUUUUUACAUGUGACAUAUGUGCCAGCACAGGAAAAGAAUUUUACCACAGCACCAGCA VFLHVTYVPAQEKNFTTAPA Sprotein 330333133323333333433353 AUUUGUCAUGAUGGAAAAGCACAUUUUCCAAGAGAAGGAGUGUUUGUGUCUAAUGGAACA ICHDGKAHFPREGVFVSNGT Sprotein 336333733383339334033413 CAUUGGUUUGUGACACAGAGAAAUUUUUAUGAACCUCAGAUUAUUACAACAGAUAAUACA HWFVTQRNFYEPQIITTDNT Sprotein 342334333443345334633473 UUUGUGUCAGGAAAUUGUGAUGUGGUGAUUGGAAUUGUGAAUAAUACAGUGUAUGAUCCA FVSGNCDVVIGIVNNTVYDP Sprotein 348334933503351335233533 CUGCAGCCAGAACUGGAUUCUUUUAAAGAAGAACUGGAUAAAUAUUUUAAAAAUCACACA LQPELDSFKEELDKYFKNHT Sprotein 354335533563357335833593 UCUCCUGAUGUGGAUUUAGGAGAUAUUUCUGGAAUCAAUGCAUCUGUGGUGAAUAUUCAG SPDVDLGDISGINASVVNIQ Sprotein 360336133623363336433653 AAAGAAAUUGAUAGACUGAAUGAAGUGGCCAAAAAUCUGAAUGAAUCUCUGAUUGAUCUG KEIDRLNEVAKNLNESLIDL Sprotein 366336733683369337033713 CAGGAACUUGGAAAAUAUGAACAGUACAUUAAAUGGCCUUGGUACAUUUGGCUUGGAUUU QELGKYEQYIKWPWYIWLGF Sprotein 372337333743375337633773 AUUGCAGGAUUAAUUGCAAUUGUGAUGGUGACAAUUAUGUUAUGUUGUAUGACAUCAUGU IAGLIAIVMVTIMLCCMTSC Sprotein 378337933803381338233833 UGUUCUUGUUUAAAAGGAUGUUGUUCUUGUGGAAGCUGUUGUAAAUUUGAUGAAGAUGAU CSCLKGCCSCGSCCKFDEDD Sprotein 38433853386338733878 UCUGAACCUGUGUUAAAAGGAGUGAAAUUGCAUUACACAUGAUGA SEPVLKGVKLHYT** Sprotein 388838983908391839283938 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 394839583968397839883998 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 408840184028403840484058 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 406840784088409841084118 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 412841384148415841684173 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC FIelement 418341934203421342234233 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 42434253426342734283 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(485) TABLE-US-00009 NucleotideSequenceofRBP020.3(SEQIDNO:21;SEQIDNO:5) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinboldletters. Inaddition,thesequenceofthetranslatedproteinisshowninitaliclettersbelowthe codingnucleotidesequence(*=stopcodon). 102030405053 GGGCGAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC hAg-Kozak 637383931031123 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGGUGAGAUUUCCA MFVFLVLLPLVSSQCVVRFP RBD(Sprotein) 123133143153163173 AAUAUUACAAAUCUGUGUCCAUUUGGAGAAGUGUUUAAUGCAACAAGAUUUGCAUCUGUG NITNLCPFGEVFNATRFASV RBD(Sprotein) 183193203213223233 UAUGCAUGGAAUAGAAAAAGAAUUUCUAAUUGUGUGGCUGAUUAUUCUGUGCUGUAUAAU YAWNRKRISNCVADYSVLYN RBD(Sprotein) 243253263273283293 AGUGCUUCUUUUUCCACAUUUAAAUGUUAUGGAGUGUCUCCAACAAAAUUAAAUGAUUUA SASFSTFKCYGVSPTKLNDL RBD(Sprotein) 303313323333343353 UGUUUUACAAAUGUGUAUGCUGAUUCUUUUGUGAUCAGAGGUGAUGAAGUGAGACAGAUU CFTNVYADSFVIRGDEVRQI RBD(Sprotein) 363373383393403413 GCCCCCGGACAGACAGGAAAAAUUGCUGAUUACAAUUACAAACUGCCUGAUGAUUUUACA APGQTGKIADYNYKLPDDFT RBD(Sprotein) 423433443453463473 GGAUGUGUGAUUGCUUGGAAUUCUAAUAAUUUAGAUUCUAAAGUGGGAGGAAAUUACAAU GCVIAWNSNNLDSKVGGNYN RBD(Sprotein) 48343503513523533 UAUCUGUACAGACUGUUUAGAAAAUCAAAUCUGAAACCUUUUGAAAGAGAUAUUUCAACA YLYRLFRKSNLKPFERDIST RBD(Sprotein) 543553563573583593 GAAAUUUAUCAGGCUGGAUCAACACCUUGUAAUGGAGUGGAAGGAUUUAAUUGUUAUUUU EIYQAGSTPCNGVEGFNCYF RBD(Sprotein) 603613623633643653 CCAUUACAGAGCUAUGGAUUUCAGCCAACCAAUGGUGUGGGAUAUCAGCCAUAUAGAGUG PLQSYGEQPTNGVGYQPYRV RBD(Sprotein) 663673683693703707 GUGGUGCUGUCUUUUGAACUGCUGCAUGCACCUGCAACAGUGUGUGGACCUAAA VVLSFELLHAPATVCGPK RBD(Sprotein) 717727734 GGCUCCCCCGGCUCCGGCUCCGGAUCU GSPGSGSGS GSlinker 744754764774784794 GGUUAUAUUCCUGAAGCUCCAAGAGAUGGGCAAGCUUACGUUCGUAAAGAUGGCGAAUGG GYIPEAPRDGQAYVRKDGEW fibritin 804814824834844854 GUAUUACUUUCUACCUUUUUAGGCCGGUCCCUGGAGGUGCUGUUCCAGGGCCCCGGCUGA VLLSTFLGRSLEVLFQGPG* fibritin 857 UGA * fibritin 867877887897907917 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 927937947957967977 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 9879971007101710271037 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 104710571067107710871097 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 110711171127113711471152 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC FIelement 116211721182119212021212 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 12221232124212521262 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(486) Further embodiments of the nucleoside modified messenger RNA (modRNA) platform are as follows:

(487) TABLE-US-00010 BNT162b3c(SEQIDNO:29;SEQIDNO:30) Structurem.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A3OL70 EncodedantigenViralspikeprotein(S1S2protein)oftheSARS-CoV-2(partialsequence, ReceptorBindingDomain(RBD)ofS1S2proteinfusedtoFibritinfusedtoTransmembrane Domain(TM)ofS1S2protein);intrinsicS1S2proteinsecretorysignalpeptide(aa1-19)at theN-terminusoftheantigensequence agaauaaacuaguauucuucugguccccacagacucagagagaacccgccaccaug 56 Met 1 uuuguguuucuugugcugcugccucuugugucuucucagugugugaau 104 PheValPheLeuValLeuLeuProLeuValSerSerGlnCysValAsn 51015 uugacagugagauuuccaaauauuacaaaucuguguccauuuggagaa 152 LeuThrValArgPheProAsnIleThrAsnLeuCysProPheGlyGlu 202530 guguuuaaugcaacaagauuugcaucuguguaugcauggaauagaaaa 200 ValPheAsnAlaThrArgPheAlaSerValTyrAlaTrpAsnArgLys 354045 agaauuucuaauuguguggcugauuauucugugcuguauaauagugcu 248 ArgIleSerAsnCysValAlaAspTyrSerValLeuTyrAsnSerAla 50556065 ucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaau 296 SerPheSerThrPheLysCysTyrGlyValSerProThrLysLeuAsn 707580 gauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggu 344 AspLeuCysPheThrAsnValTyrAlaAspSerPheValIleArgGly 859095 gaugaagugagacagauugcccccggacagacaggaaaaauugcugau 392 AspGluValArgGlnIleAlaProGlyGlnThrGlyLysIleAlaAsp 100105110 uacaauuacaaacugccugaugauuuuacaggaugugugauugcuugg 440 TyrAsnTyrLysLeuProAspAspPheThrGlyCysValIleAlaTrp 115120125 aauucuaauaauuuagauucuaaagugggaggaaauuacaauuaucug 488 AsnSerAsnAsnLeuAspSerLysValGlyGlyAsnTyrAsnTyrLeu 130135140145 uacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauu 536 TyrArgLeuPheArgLysSerAsnLeuLysProPheGluArgAspIle 150155160 ucaacagaaauuuaucaggcuggaucaacaccuuguaauggaguggaa 584 SerThrGluIleTyrGlnAlaGlySerThrProCysAsnGlyValGlu 165170175 ggauuuaauuguuauuuuccauuacagagcuauggauuucagccaacc 632 GlyPheAsnCysTyrPheProLeuGlnSerTyrGlyPheGlnProThr 180185190 aauggugugggauaucagccauauagagugguggugcugucuuuugaa 680 AsnGlyValGlyTyrGlnProTyrArgValValValLeuSerPheGlu 195200205 cugcugcaugcaccugcaacaguguguggaccuaaaggcucccccggc 728 LeuLeuHisAlaProAlaThrValCysGlyProLysGlySerProGly 210215220225 uccggcuccggaucugguuauauuccugaagcuccaagagaugggcaa 776 SerGlySerGlySerGlyTyrIleProGluAlaProArgAspGlyGln 230235240 gcuuacguucguaaagauggcgaauggguauuacuuucuaccuuuuua 824 AlaTyrValArgLysAspGlyGluTrpValLeuLeuSerThrPheLeu 245250255 ggaagcggcagcggaucugaacaguacauuaaauggccuugguacauu 872 GlySerGlySerGlySerGluGlnTyrIleLysTrpProTrpTyrIle 260265270 uggcuuggauuuauugcaggauuaauugcaauugugauggugacaauu 920 TrpLeuGlyPheIleAlaGlyLeuIleAlaIleValMetValThrIle 275280285 auguuauguuguaugacaucauguuguucuuguuuaaaaggauguugu 968 MetLeuCysCysMetThrSerCysCysSerCysLeuLysGlyCysCys 290295300305 ucuuguggaagcuguuguugaugacucgagcugguacugcaugcacgcaau 1019 SerCysGlySerCysCys 310 gcuagcugccccuuucccguccuggguaccccgagucucccccgaccucgggucccaggu 1079 augcucccaccuccaccugccccacucaccaccucugcuaguuccagacaccucccaagc 1139 acgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaaacagcaguga 1199 uuaaccuuuagcaauaaacgaaaguuuaacuaagcuauacuaaccccaggguuggucaau 1259 uucgugccagccacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagc 1319 auaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1379 aaaaaaaaaaaaaaaaaa 1397

(488) TABLE-US-00011 BNT162b3d(SEQIDNO:31;SEQIDNO:32) Structure m.sub.2.sup.7,3-OGppp(m.sub.1.sup.2-O)ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A30L70 Encodedantigen Viralspikeprotein(S1S2protein)oftheSARS-CoV-2(partialsequence, ReceptorBindingDomain(RBD)ofS1S2proteinfusedtoFibritinfused toTransmembraneDomain(TM)ofS1S2protein);immunoglobulinsecretory signalpeptide(aa1-22)attheN-terminusoftheantigensequence agaauaaacuaguauucuucugguccccacagacucagagagaacccgccaccaug 56 Met 1 gauuggauuuggagaauccuguuccucgugggagccgcuacaggagcc 104 AspTrpIleTrpArgIleLeuPheLeuValGlyAlaAlaThrGlyAla 51015 cacucccagaugcaggugagauuuccaaauauuacaaaucugugucca 152 HisSerGlnMetGlnValArgPheProAsnIleThrAsnLeuCysPro 202530 uuuggagaaguguuuaaugcaacaagauuugcaucuguguaugcaugg 200 PheGlyGluValPheAsnAlaThrArgPheAlaSerValTyrAlaTrp 354045 aauagaaaaagaauuucuaauuguguggcugauuauucugugcuguau 248 AsnArgLysArgIleSerAsnCysValAlaAspTyrSerValLeuTyr 50556065 aauagugcuucuuuuuccacauuuaaauguuauggagugucuccaaca 296 AsnSerAlaSerPheSerThrPheLysCysTyrGlyValSerProThr 707580 aaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugug 344 LysLeuAsnAspLeuCysPheThrAsnValTyrAlaAspSerPheVal 859095 aucagaggugaugaagugagacagauugcccccggacagacaggaaaa 392 IleArgGlyAspGluValArgGlnIleAlaProGlyGlnThrGlyLys 100105110 auugcugauuacaauuacaaacugccugaugauuuuacaggaugugug 440 IleAlaAspTyrAsnTyrLysLeuProAspAspPheThrGlyCysVal 115120125 auugcuuggaauucuaauaauuuagauucuaaagugggaggaaauuac 488 IleAlaTrpAsnSerAsnAsnLeuAspSerLysValGlyGlyAsnTyr 130135140145 aauuaucuguacagacuguuuagaaaaucaaaucugaaaccuuuugaa 536 AsnTyrLeuTyrArgLeuPheArgLysSerAsnLeuLysProPheGlu 150155160 agagauauuucaacagaaauuuaucaggcuggaucaacaccuuguaau 584 ArgAspIleSerThrGluIleTyrGlnAlaGlySerThrProCysAsn 165170175 ggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuu 632 GlyValGluGlyPheAsnCysTyrPheProLeuGlnSerTyrGlyPhe 180185190 cagccaaccaauggugugggauaucagccauauagagugguggugcug 680 GlnProThrAsnGlyValGlyTyrGlnProTyrArgValValValLeu 195200205 ucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaggc 728 SerPheGluLeuLeuHisAlaProAlaThrValCysGlyProLysGly 210215220225 ucccccggcuccggcuccggaucugguuauauuccugaagcuccaaga 776 SerProGlySerGlySerGlySerGlyTyrIleProGluAlaProArg 230235240 gaugggcaagcuuacguucguaaagauggcgaauggguauuacuuucu 824 AspGlyGlnAlaTyrValArgLysAspGlyGluTrpValLeuLeuSer 245250255 accuuuuuaggaagcggcagcggaucugaacaguacauuaaauggccu 872 ThrPheLeuGlySerGlySerGlySerGluGlnTyrIleLysTrpPro 260265270 ugguacauuuggcuuggauuuauugcaggauuaauugcaauugugaug 920 TrpTyrIleTrpLeuGlyPheIleAlaGlyLeuIleAlaIleValMet 275280285 gugacaauuauguuauguuguaugacaucauguuguucuuguuuaaaa 968 ValThrIleMetLeuCysCysMetThrSerCysCysSerCysLeuLys 290295300305 ggauguuguucuuguggaagcuguuguugaugacucgagcugguacugca 1018 GlyCysCysSerCysGlySerCysCys 310 ugcacgcaaugcuagcugccccuuucccguccuggguaccccgagucucccccgaccucg 1078 ggucccagguaugcucccaccuccaccugccccacucaccaccucugcuaguuccagaca 1138 ccucccaagcacgcagcaaugcagcucaaaacgcuuagccuagccacacccccacgggaa 1198 acagcagugauuaaccuuuagcaauaaacgaaaguuuaacuaagcuauacuaaccccagg 1258 guuggucaauuucgugccagccacacccuggagcuagcaaaaaaaaaaaaaaaaaaaaaa 1318 aaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa 1378 aaaaaaaaaaaaaaaaaaaaaaaaaaaa 1406

(489) Self-amplifying RNA (saRNA)

(490) The active principle of the self-amplifying mRNA (saRNA) drug substance is a single-stranded RNA, which self-amplifies upon entering a cell, and the coronavirus vaccine antigen is translated thereafter. In contrast to uRNA and modRNA that preferably code for a single protein, the coding region of saRNA contains two open reading frames (ORFs). The 5-ORF encodes the RNA-dependent RNA polymerase such as Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase (replicase). The replicase ORF is followed 3 by a subgenomic promoter and a second ORF encoding the antigen. Furthermore, saRNA UTRs contain 5 and 3 conserved sequence elements (CSEs) required for self-amplification. The saRNA contains common structural elements optimized for maximal efficacy of the RNA as the uRNA (5-cap, 5-UTR, 3-UTR, poly(A)-tail). The saRNA preferably contains uridine. The preferred 5 cap structure is beta-S-ARCA(D1) (m.sub.2.sup.7,2-OGppSpG).

(491) Cytoplasmic delivery of saRNA initiates an alphavirus-like life cycle. However, the saRNA does not encode for alphaviral structural proteins that are required for genome packaging or cell entry, therefore generation of replication competent viral particles is very unlikely to not possible. Replication does not involve any intermediate steps that generate DNA. The use/uptake of saRNA therefore poses no risk of genomic integration or other permanent genetic modification within the target cell. Furthermore, the saRNA itself prevents its persistent replication by effectively activating innate immune response via recognition of dsRNA intermediates.

(492) Different embodiment of this platform are as follows:

(493) TABLE-US-00012 RBS004.1 (SEQ ID NO: 24; SEQ ID NO: 7) Structure beta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) RBS004.2 (SEQ ID NO: 25; SEQ ID NO: 7) Structure beta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) BNT162C1; RBS004.3 (SEQ ID NO: 26; SEQ ID NO: 5) Structure beta-S-ARCA(D1)-replicase-RBD-GS-Fibritin-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 (partial sequence, Receptor Binding Domain (RBD) of S1S2 protein) RBS004.4 (SEQ ID NO: 27; SEQ ID NO: 28) Structure beta-S-ARCA(D1)-replicase-RBD-GS-Fibritin-TM-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 (partial sequence, Receptor Binding Domain (RBD) of S1S2 protein)

(494) FIG. 21 schematizes the general structure of the antigen-encoding RNAs.

(495) TABLE-US-00013 NucleotideSequenceofRBS004.1(SEQIDNO:24;SEQIDNO:7) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinbold letters.Inaddition,thesequenceofthetranslatedproteinisshowninitalicletters belowthecodingnucleotidesequence(*=stopcodon). 1020304045 GAUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA 5UTR 5565758595105 AUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGG MEKVHVDIEEDSPFLRALQR nsp1 115125135145155165 AGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUGCUAAUGCC SFPQFEVEAKQVTDNDHANA nsp1 175185195205215225 AGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACG RAFSHLASKLIETEVDPSDT nsp1 235245255265275285 AUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGU ILDIGSAPARRMYSKHKYHC nsp1 295305315325335345 AUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUG ICPMRCAEDPDRLYKYATKL nsp1 355365375385395405 AAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUCGCC KKNCKEITDKELDKKMKELA nsp1 415425435445455465 GCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCG AVMSDPDLETETMCLHDDES nsp1 475485495505515525 UGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACA CRYEGQVAVYQDVYAVDGPT nsp1 535545555565575585 AGUCUCUAUCACCAAGCCAAUAAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACC SLYHQANKGVRVAYWIGFDT nsp1 595605615625635645 ACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCAUCAUACUCUACCAACUGGGCC TPFMFKNLAGAYPSYSTNWA nsp1 655665675685695705 GACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAGCGG DETVLTARNIGLCSSDVMER nsp1 715725735745755765 UCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCAACAAUGUUCUA SRRGMSILRKKYLKPSNNVL nsp1 775785795805815825 UUCUCUGUUGGCUCGACCAUCUACCACGAAAAGAGGGACUUACUGAGGAGCUGGCACCUG FSVGSTIYHEKRDLLRSWHL nsp1 835845855865875885 CCGUCUGUAUUUCACUUACGUGGCAAGCAAAAUUACACAUGUCGGUGUGAGACUAUAGUU PSVFHLRGKQNYTCRCETIV nsp1 895905915925935945 AGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCU SCDGYVVKRIAISPGLYGKP nsp1 9559659759859951005 UCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACA SGYAATMHREGELCCKVTDT nsp1 101510251035104510551065 UUGAACGGGGAGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGAC LNGERVSFPVCTYVPATLCD nsp1 107510851095110511151125 CAAAUGACUGGCAUACUGGCAACAGAUGUCAGUGCGGACGACGCGCAAAAACUGCUGGUU QMTGILATDVSADDAQKLLV nsp1 113511451155116511751185 GGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAACACCAAUACCAUGAAA GLNQRIVVNGRTQRNTNTMK nsp1 119812051215122512351245 AAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAA NYLLPVVAQAFARWAKEYKE nsp1 125512651275128512951305 GAUCAAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGUUGU DQEDERPLGLRDRQLVMGCC nsp1 131513251335134513551365 UGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUC WAFRRHKITSIYKRPDTQTI nsp1 137513851395140514151425 AUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCAGGAUAGGCAGUAACACAUUG IKVNSDFHSFVLPRIGSNTL nsp1 143514451455146514751485 GAGAUCGGGCUGAGAACAAGAAUCAGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCACCU EIGLRTRIRKMLEEHKEPSP nsp1 149515051515152515351545 CUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUG LITAEDVQEAKCAADEAKEV nsp1 155515651575158515951605 CGUGAAGCCGAGGAGUUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCC REAEELRAALPPLAADVEEP nsp1 16151625163516451650 ACUCUGGAAGCCGAUGUCGACUUGAUGUUACAAGAGGCUGGGGCC TLEADVDLMLQEAGA nsp1 166016701680169017001710 GGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGCUGGCGAGGACAAG GSVETPRGLIKVTSYAGEDK nsp2 172017301740175017601770 AUCGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGC IGSYAVLSPQAVLKSEKLSC nsp2 178017901800181018201830 AUCCACCCUCUCGCUGAACAAGUCAUAGUGAUAACACACUCUGGCCGAAAAGGGCGUUAU IHPLAEQVIVITHSGRKGRY nsp2 184018501860187018801890 GCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGACAUGCAAUACCCGUCCAG AVEPYHGKVVVPEGHAIPVQ nsp2 190019101920193019401950 GACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUAAAC DFQALSESATIVYNEREFVN nsp2 196019701980199020002010 AGGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUAC RYLHHIATHGGALNTDEEYY nsp2 202020302040205020602070 AAAACUGUCAAGCCCAGCGAGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAG KTVKPSEHDGEYLYDIDRKQ nsp2 208020902100211021202130 UGCGUCAAGAAAGAGCUAGUCACUGGGCUAGGGCUCACAGGCGAGCUGGUCGAUCCUCCC CVKKELVTGLGLTGELVDPP nsp2 214021502160217021802190 UUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCA FHEFAYESLRTRPAAPYQVP nsp2 220022102220223022402250 ACCAUAGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUC TIGVYGVPGSGKSGIIKSAV nsp2 226022702280229023002310 ACCAAAAAAGAUCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGAC TKKDLVVSAKKENCAEIIRD nsp2 232023302340235023602370 GUCAAGAAAAUGAAAGGGCUGGACGUCAAUGCCAGAACUGUGGACUCAGUGCUCUUGAAU VKKMKGLDVNARTVDSVLLN nsp2 238023902400241024202430 GGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACGAGGCUUUUGCUUGUCAUGCAGGU GCKHPVETLYIDEAFACHAG nsp2 244024502460247024802490 ACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGAGAUCCC TLRALIAIIRPKKAVLCGDP nsp2 250025102520253025402550 AAACAGUGCGGUUUUUUUAACAUGAUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGC KQCGEENMMCLKVHFNHEIC nsp2 256025702580259026002610 ACACAAGUCUUCCACAAAAGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCGGUCGUC TQVFHKSISRRCTKSVTSVV nsp2 262026302640265026602670 UCAACCUUGUUUUACGACAAAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUG STLFYDKKMRTTNPKETKIV nsp2 268026902700271027202730 AUUGACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGA IDTTGSTKPKQDDLILTCFR nsp2 274027502760277027802790 GGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCC GWVKQLQIDYKGNEIMTAAA nsp2 280028102820283028402850 UCUCAAGGGCUGACCCGUAAAGGUGUGUAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCU SQGLTRKGVYAVRYKVNENP nsp2 286028702880289029002910 CUGUACGCACCCACCUCAGAACAUGUGAACGUCCUACUGACCCGCACGGAGGACCGCAUC LYAPTSEHVNVLLTRTEDRI nsp2 292029302940295029602970 GUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUACCCUGGG VWKTLAGDPWIKTLTAKYPG nsp2 298029903000301030203030 AAUUUCACUGCCACGAUAGAGGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUC NFTATIEEWQAEHDAIMRHI nsp2 304030503060307030803090 UUGGAGAGACCGGACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAG LERPDPTDVFQNKANVCWAK nsp2 310031103120313031403150 GCUUUAGUGCCGGUGCUGAAGACCGCUGGCAUAGACAUGACCACUGAACAAUGGAACACU ALVPVLKTAGIDMTTEQWNT nsp2 316031703180319032003210 GUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGAACCAACUAUGC VDYFETDKAHSAEIVLNQLC nsp2 322032303240325032603270 GUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUA VRFFGLDLDSGLFSAPTVPL nsp2 328032903300331033203330 UCCAUUAGGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAA SIRNNHWDNSPSPNMYGLNK nsp2 334033503360337033803390 GAAGUGGUCCGUCAGCUCUCUCGCAGGUACCCACAACUGCCUCGGGCAGUUGCCACUGGU EVVRQLSRRYPQLPRAVATG nsp2 340034103420343034403450 AGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAUCCGCGCAUAAACCUAGUA RVYDMNTGTLRNYDPRINLV nsp2 346034703480349035003510 CCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGU PVNRRLPHALVLHHNEHPQS nsp2 352035303540355035603570 GACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUCGGGGAAAAG DFSSFVSKLKGRTVLVVGEK nsp2 358035903600361036203630 UUGUCCGUCCCAGGCAAAAUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGA LSVPGKMVDWLSDRPEATER nsp2 364036503660367036803690 GCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAU ARLDLGIPGDVPKYDIIFVN nsp2 370037103720373037403750 GUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUUAAGCUA VRTPYKYHHYQQCEDHAIKL nsp2 376037703780379038003810 AGCAUGUUGACCAAGAAAGCAUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUA SMLTKKACLHLNPGGTCVSI nsp2 382038303840385038603870 GGUUAUGGUUACGCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUC GYGYADRASESIIGAIARQF nsp2 388038903900391039203930 AAGUUUUCCCGAGUAUGCAAACCGAAAUCCUCACUUGAGGAGACGGAAGUUCUGUUUGUA KFSRVCKPKSSLEETEVLFV nsp2 394039503960397039803990 UUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUAUCAUCAACCUUG FIGYDRKARTHNPYKLSSTL nsp2 40004010402040304032 ACCAACAUUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGU TNIYTGSRLHEAGC nsp2 404240524062407240824092 GCACCCUCAUAUCAUGUGGUGCGAGGGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUA APSYHVVRGDIATATEGVII nsp3 410241124122413241424152 AAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGCGGAGCGCUGUAUAAGAAA NAANSKGQPGGGVCGALYKK nsp3 416241724182419242024212 UUCCCGGAAAGUUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGU FPESFDLQPIEVGKARLVKG nsp3 422242324242425242624272 GCAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUUCAACAAAGUUUCGGAGGUUGAA AAKHIIHAVGPNFNKVSEVE nsp3 428242924302431243224332 GGUGACAAACAGUUGGCAGAGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAU GDKQLAEAYESIAKIVNDNN nsp3 434243524362437243824392 UACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCAUCUUUUCCGGGAACAAAGAUCGA YKSVAIPLLSTGIFSGNKDR nsp3 440244124422443244424452 CUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAUGUAGCC LTQSLNHLLTALDTTDADVA nsp3 446244724482449245024512 AUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAA IYCRDKKWEMTLKEAVARRE nsp3 452245324542455245624572 GCAGUGGAGGAGAUAUGCAUAUCCGACGAUUCUUCAGUGACAGAACCUGAUGCAGAGCUG AVEEICISDDSSVTEPDAEL nsp3 458245924602461246224632 GUGAGGGUGCAUCCCAAGAGUUCUUUGGCUGGAAGGAAGGGCUACAGCACAAGCGAUGGC VRVHPKSSLAGRKGYSTSDG nsp3 464246524662467246824692 AAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAGGAUAUAGCAGAA KTFSYLEGTKFHQAAKDIAE nsp3 470247124722473247424752 AUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUC INAMWPVATEANEQVCMYIL nsp3 476247724782479248024812 GGAGAAAGCAUGAGCAGUAUUAGGUCGAAAUGCCCCGUCGAGGAGUCGGAAGCCUCCACA GESMSSIRSKCPVEESEAST nsp3 482248324842485248624872 CCACCUAGCACGCUGCCUUGCUUGUGCAUCCAUGCCAUGACUCCAGAAAGAGUACAGCGC PPSTLPCLCIHAMTPERVQR nsp3 48824892491249224932 CUAAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCCUUUCCAUUGCCGAAGUAU LKASRPEQITVCSSFPLPKY nsp3 494249524962497249824992 AGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUG RITGVQKIQCSQPILFSPKV nsp3 500250125022503250425052 CCUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCG PAYIHPRKYLVETPPVDETP nsp3 506250725082509251025112 GAGCCAUCGGCAGAGAACCAAUCCACAGAGGGGACACCUGAACAACCACCACUUAUAACC EPSAENQSTEGTPEQPPLIT nsp3 512251325142515251625172 GAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUCAUCAUCGAAGAAGAAGAAGAAGAU EDETRTRTPEPIIIEEEEED nsp3 518251925202521252225232 AGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCAGACAUU SISLLSDGPTHQVLQVEADI nsp3 524252525262527252825292 CACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGACUUUGAU HGPPSVSSSSWSIPHASDED nsp3 530253125322533253425352 GUGGACAGUUUAUCCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCAACG VDSLSILDTLEGASVTSGAT nsp3 536253725382539254025412 UCAGCCGAGACUAACUCUUACUUCGCAAAGAGUAUGGAGUUUCUGGCGCGACCGGUGCCU SAETNSYFAKSMEFLARPVP nsp3 542254325442545254625472 GCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACAAGAACACCGUCA APRTVFRNPPHPAPRTRTPS nsp3 548254925502551255225532 CUUGCACCCAGCAGGGCCUGCUCCAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAU LAPSRACSRTSLVSTPPGVN nsp3 554255525562557255825592 AGGGUGAUCACUAGAGAGGAGCUCGAAGCGCUUACCCCGUCACGCACUCCUAGCAGGUCG RVITREELEALTPSRTPSRS nsp3 560256125622563256425652 GUCUCCAGAACCAGCCUGGUCUCCAACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAG VSRTSLVSNPPGVNRVITRE nsp3 566256725682569257025703 GAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAUGCGGGUGCA EFEAFVAQQQ*RFDAGA nsp3 571357235733574357535763 UACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACG YIFSSDTGQGHLQQKSVRQT nsp4 577357835793580358135823 GUGCUAUCCGAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUC VLSEVVLERTELEISYAPRL nsp4 583358435853586358735883 GACCAAGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAAUCCCACACCUGCUAAC DQEKEELLRKKLQLNPTPAN nsp4 589359035913592359335943 AGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUAACAGCUAGACGUAUU RSRYQSRKVENMKAITARRI nsp4 595359635973598359936003 CUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUG LQGLGHYLKAEGKVECYRTL nsp4 601360236033604360536063 CAUCCUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCA HPVPLYSSSVNRAFSSPKVA nsp4 607360836093610361136123 GUGGAAGCCUGUAACGCCAUGUUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUU VEACNAMLKENFPTVASYCI nsp4 613361436153616361736183 AUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGAGCUUCAUGCUGCUUAGACACU IPEYDAYLDMVDGASCCLDT nsp4 619362036213622362336243 GCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAACCC ASFCPAKLRSFPKKHSYLEP nsp4 625362636273628362936303 ACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCU TIRSAVPSAIQNTLQNVLAA nsp4 631363236333634363536363 GCCACAAAAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCG ATKRNCNVTQMRELPVLDSA nsp4 637363836393640364136423 GCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCGUGUAAUAAUGAAUAUUGGGAAACGUUU AFNVECFKKYACNNEYWETF nsp4 643364436453646364736483 AAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUACCAAAUUAAAA KENPIRLTEENVVNYITKLK nsp4 649365036513652365336543 GGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUA GPKAAALFAKTHNLNMLQDI nsp4 655365636573658365936603 CCAAUGGACAGGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAA PMDRFVMDLKRDVKVTPGTK nsp4 661366236633664366536663 CAUACUGAAGAACGGCCCAAGGUACAGGUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCG HTEERPKVQVIQAADPLATA nsp4 667366836693670367136723 UAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCGGUCCUGCUUCCGAAC YLCGIHRELVRRLNAVLLPN nsp4 673367436753676367736783 AUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUC IHTLEDMSAEDFDAIIAEHF nsp4 679368036813682368336843 CAGCCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGAC QPGDCVLETDIASFDKSEDD nsp4 685368636873688368936903 GCCAUGGCUCUGACCGCGUUAAUGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUG AMALTALMILEDLGVDAELL nsp4 691369236933694369536963 ACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUACAUUUGCCCACUAAAACUAAA TLIEAAFGEISSIHLPTKTK nsp4 697369836993700370137023 UUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACAGUC FKFGAMMKSGMFLTLFVNTV nsp4 703370437053706370737083 AUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGAUCACCAUGUGCA INIVIASRVLRERLTGSPCA nsp4 709371037113712371337143 GCAUUCAUUGGAGAUGACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGAC AFIGDDNIVKGVKSDKLMAD nsp4 715371637173718371937203 AGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAGAUUAUAGAUGCUGUGGUGGGCGAGAAA RCATWLNMEVKIIDAVVGEK nsp4 721372237233724372537263 GCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGU APYFCGGEILCDSVTGTACR nsp4 727372837293730373137323 GUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUAGGCAAACCUCUGGCAGCAGACGAUGAA VADPLKRLFKLGKPLAADDE nsp4 733373437353736373737383 CAUGAUGAUGACAGGAGAAGGGCAUUGCAUGAGGAGUCAACACGCUGGAACCGAGUGGGU HDDDRRRALHEESTRWNRVG nsp4 739374037413742374337443 AUUCUUUCAGAGCUGUGCAAGGCAGUAGAAUCAAGGUAUGAAACCGUAGGAACUUCCAUC ILSELCKAVESRYETVGTSI nsp4 745374637473748374937503 AUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUCAGCUACCUGAGAGGG IVMAMTTLASSVKSFSYLRG nsp4 751375237527 GCCCCUAUAACUCUCUACGGCUAA APITLYG* nsp4 75377547755775677568 CCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGACUAGU virUTR 757875887598760876187628 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGAAUUUGACAACA MFVFLVLLPLVSSQCVNLTT Sprotein 763876487658766876787688 AGAACACAGCUGCCACCAGCUUAUACAAAUUCUUUUACCAGAGGAGUGUAUUAUCCUGAU RTQLPPAYTNSFTRGVYYPD Sprotein 769877087718772877387748 AAAGUGUUUAGAUCUUCUGUGCUGCACAGCACACAGGACCUGUUUCUGCCAUUUUUUAGC KVFRSSVLHSTQDLFLPFFS Sprotein 775877687778778877987808 AAUGUGACAUGGUUUCAUGCAAUUCAUGUGUCUGGAACAAAUGGAACAAAAAGAUUUGAU NVTWFHAIHVSGTNGTKRFD Sprotein 781878287838784878587868 AAUCCUGUGCUGCCUUUUAAUGAUGGAGUGUAUUUUGCUUCAACAGAAAAGUCAAAUAUU NPVLPFNDGVYEASTEKSNI Sprotein 787878887898790879187928 AUUAGAGGAUGGAUUUUUGGAACAACACUGGAUUCUAAAACACAGUCUCUGCUGAUUGUG IRGWIFGTTLDSKTQSLLIV Sprotein 793879487958796879787988 AAUAAUGCAACAAAUGUGGUGAUUAAAGUGUGUGAAUUUCAGUUUUGUAAUGAUCCUUUU NNATNVVIKVCEFQFCNDPF Sprotein 799880088018802880388048 CUGGGAGUGUAUUAUCACAAAAAUAAUAAAUCUUGGAUGGAAUCUGAAUUUAGAGUGUAU LGVYYHKNNKSWMESEFRVY Sprotein 805880688078808880988108 UCCUCUGCAAAUAAUUGUACAUUUGAAUAUGUGUCUCAGCCUUUUCUGAUGGAUCUGGAA SSANNCTFEYVSQPFLMDLE Sprotein 811881288138814881588168 GGAAAACAGGGCAAUUUUAAAAAUCUGAGAGAAUUUGUGUUUAAAAAUAUUGAUGGAUAU GKQGNFKNLREFVFKNIDGY Sprotein 817881888198820882188228 UUUAAAAUUUAUUCUAAACACACACCAAUUAAUUUAGUGAGAGAUCUGCCUCAGGGAUUU FKIYSKHTPINLVRDLPQGF Sprotein 823882488258826882788288 UCUGCUCUGGAACCUCUGGUGGAUCUGCCAAUUGGCAUUAAUAUUACAAGAUUUCAGACA SALEPLVDLPIGINITRFQT Sprotein 829883088318832883388348 CUGCUGGCUCUGCACAGAUCUUAUCUGACACCUGGAGAUUCUUCUUCUGGAUGGACAGCC LLALHRSYLTPGDSSSGWTA Sprotein 835883688378838883988408 GGAGCUGCAGCUUAUUAUGUGGGCUAUCUGCAGCCAAGAACAUUUCUGCUGAAAUAUAAU GAAAYYVGYLQPRTFLLKYN Sprotein 841884288438844884588468 GAAAAUGGAACAAUUACAGAUGCUGUGGAUUGUGCUCUGGAUCCUCUGUCUGAAACAAAA ENGTITDAVDCALDPLSETK Sprotein 847884888498850885188528 UGUACAUUAAAAUCUUUUACAGUGGAAAAAGGCAUUUAUCAGACAUCUAAUUUUAGAGUG CTLKSFTVEKGIYQTSNFRV Sprotein 853885488558856885788588 CAGCCAACAGAAUCUAUUGUGAGAUUUCCAAAUAUUACAAAUCUGUGUCCAUUUGGAGAA QPTESIVRFPNITNLCPFGE Sprotein 859886088618862886388648 GUGUUUAAUGCAACAAGAUUUGCAUCUGUGUAUGCAUGGAAUAGAAAAAGAAUUUCUAAU VFNATRFASVYAWNRKRISN Sprotein 865886688678868886988708 UGUGUGGCUGAUUAUUCUGUGCUGUAUAAUAGUGCUUCUUUUUCCACAUUUAAAUGUUAU CVADYSVLYNSASFSTFKCY Sprotein 871887288738874887588768 GGAGUGUCUCCAACAAAAUUAAAUGAUUUAUGUUUUACAAAUGUGUAUGCUGAUUCUUUU GVSPTKLNDLCFTNVYADSF Sprotein 877887888798880888188828 GUGAUCAGAGGUGAUGAAGUGAGACAGAUUGCCCCCGGACAGACAGGAAAAAUUGCUGAU VIRGDEVRQIAPGQTGKIAD Sprotein 883888488858886888788888 UACAAUUACAAACUGCCUGAUGAUUUUACAGGAUGUGUGAUUGCUUGGAAUUCUAAUAAU YNYKLPDDFTGCVIAWNSNN Sprotein 889889088918892889388948 UUAGAUUCUAAAGUGGGAGGAAAUUACAAUUAUCUGUACAGACUGUUUAGAAAAUCAAAU LDSKVGGNYNYLYRLFRKSN Sprotein 895889688978898889989008 CUGAAACCUUUUGAAAGAGAUAUUUCAACAGAAAUUUAUCAGGCUGGAUCAACACCUUGU LKPPERDISTEIYQAGSTPC Sprotein 901890289038904890589068 AAUGGAGUGGAAGGAUUUAAUUGUUAUUUUCCAUUACAGAGCUAUGGAUUUCAGCCAACC NGVEGFNCYFPLQSYGFQPT Sprotein 907890889098910891189128 AAUGGUGUGGGAUAUCAGCCAUAUAGAGUGGUGGUGCUGUCUUUUGAACUGCUGCAUGCA NGVGYQPYRVVVLSFELLHA Sprotein 913891489158916891789188 CCUGCAACAGUGUGUGGACCUAAAAAAUCUACAAAUUUAGUGAAAAAUAAAUGUGUGAAU PATVCGPKKSTNLVKNKCVN Sprotein 919892089218922892389248 UUUAAUUUUAAUGGAUUAACAGGAACAGGAGUGCUGACAGAAUCUAAUAAAAAAUUUCUG FNFNGLTGTGVLTESNKKFL Sprotein 925892689278928892989308 CCUUUUCAGCAGUUUGGCAGAGAUAUUGCAGAUACCACAGAUGCAGUGAGAGAUCCUCAG PFQQFGRDIADTTDAVRDPQ Sprotein 931893289338934893589368 ACAUUAGAAAUUCUGGAUAUUACACCUUGUUCUUUUGGGGGUGUGUCUGUGAUUACACCU TLEILDITPCSFGGVSVITP Sprotein 937893889398940894189428 GGAACAAAUACAUCUAAUCAGGUGGCUGUGCUGUAUCAGGAUGUGAAUUGUACAGAAGUG GTNTSNQVAVLYQDVNCTEV Sprotein 943894489458946894789488 CCAGUGGCAAUUCAUGCAGAUCAGCUGACACCAACAUGGAGAGUGUAUUCUACAGGAUCU PVAIHADQLTPTWRVYSTGS Sprotein 949895089518952895389548 AAUGUGUUUCAGACAAGAGCAGGAUGUCUGAUUGGAGCAGAACAUGUGAAUAAUUCUUAU NVFQTRAGCLIGAEHVNNSY Sprotein 955895689578958895989608 GAAUGUGAUAUUCCAAUUGGAGCAGGCAUUUGUGCAUCUUAUCAGACACAGACAAAUUCC ECDIPIGAGICASYQTQTNS Sprotein 961896289638964896589668 CCAAGGAGAGCAAGAUCUGUGGCAUCUCAGUCUAUUAUUGCAUACACCAUGUCUCUGGGA PRRARSVASQSIIAYTMSLG Sprotein 967896889698970897189728 GCAGAAAAUUCUGUGGCAUAUUCUAAUAAUUCUAUUGCUAUUCCAACAAAUUUUACCAUU AENSVAYSNNSIAIPTNFTI Sprotein 973897489758976897789788 UCUGUGACAACAGAAAUUUUACCUGUGUCUAUGACAAAAACAUCUGUGGAUUGUACCAUG SVTTEILPVSMTKTSVDCTM Sprotein 979898089818982898389848 UACAUUUGUGGAGAUUCUACAGAAUGUUCUAAUCUGCUGCUGCAGUAUGGAUCUUUUUGU YICGDSTECSNLLLQYGSFC Sprotein 985898689878988898989908 ACACAGCUGAAUAGAGCUUUAACAGGAAUUGCUGUGGAACAGGAUAAAAAUACACAGGAA TQLNRALTGIAVEQDKNTQE Sprotein 991899289938994899589968 GUGUUUGCUCAGGUGAAACAGAUUUACAAAACACCACCAAUUAAAGAUUUUGGAGGAUUU VFAQVKQIYKTPPIKDFGGF Sprotein 997899889998100081001810028 AAUUUUAGCCAGAUUCUGCCUGAUCCUUCUAAACCUUCUAAAAGAUCUUUUAUUGAAGAU NFSQILPDPSKPSKRSFIED Sprotein 100381004810058100681007810088 CUGCUGUUUAAUAAAGUGACACUGGCAGAUGCAGGAUUUAUUAAACAGUAUGGAGAUUGC LLFNKVTLADAGFIKQYGDC Sprotein 100981010810118101281013810148 CUGGGUGAUAUUGCUGCAAGAGAUCUGAUUUGUGCUCAGAAAUUUAAUGGACUGACAGUG LGDIAARDLICAQKFNGLTV Sprotein 101581016810178101881019810208 CUGCCUCCUCUGCUGACAGAUGAAAUGAUUGCUCAGUACACAUCUGCUUUACUGGCUGGA LPPLLTDEMIAQYTSALLAG Sprotein 102181022810238102481025810268 ACAAUUACAAGCGGAUGGACAUUUGGAGCUGGAGCUGCUCUGCAGAUUCCUUUUGCAAUG TITSGWTFGAGAALQIPFAM Sprotein 102781028810298103081031810328 CAGAUGGCUUACAGAUUUAAUGGAAUUGGAGUGACACAGAAUGUGUUAUAUGAAAAUCAG QMAYRFNGIGVTQNVLYENQ Sprotein 103381034810358103681037810388 AAACUGAUUGCAAAUCAGUUUAAUUCUGCAAUUGGCAAAAUUCAGGAUUCUCUGUCUUCU KLIANQFNSAIGKIQDSLSS Sprotein 103981040810418104281043810448 ACAGCUUCUGCUCUGGGAAAACUGCAGGAUGUGGUGAAUCAGAAUGCACAGGCACUGAAU TASALGKLQDVVNQNAQALN Sprotein 104581046810478104881049810508 ACUCUGGUGAAACAGCUGUCUAGCAAUUUUGGGGCAAUUUCUUCUGUGCUGAAUGAUAUU TLVKQLSSNFGAISSVLNDI Sprotein 105181052810538105481055810568 CUGUCUAGACUGGAUCCTCCTGAAGCUGAAGUGCAGAUUGAUAGACUGAUCACAGGAAGA LSRLDPPEAEVQIDRLITGR Sprotein 105781058810598106081061810628 CUGCAGUCUCUGCAGACUUAUGUGACACAGCAGCUGAUUAGAGCUGCUGAAAUUAGAGCU LQSLQTYVTQQLIRAAEIRA Sprotein 106381064810658106681067810688 UCUGCUAAUCUGGCUGCUACAAAAAUGUCUGAAUGUGUGCUGGGACAGUCAAAAAGAGUG SANLAATKMSECVLGQSKRV Sprotein 106981070810718107281073810748 GAUUUUUGUGGAAAAGGAUAUCAUCUGAUGUCUUUUCCACAGUCUGCUCCACAUGGAGUG DFCGKGYHLMSEPQSAPHGV Sprotein 107581076810778107881079810808 GUGUUUUUACAUGUGACAUAUGUGCCAGCACAGGAAAAGAAUUUUACCACAGCACCAGCA VFLHVTYVPAQEKNFTTAPA Sprotein 108181082810838108481085810868 AUUUGUCAUGAUGGAAAAGCACAUUUUCCAAGAGAAGGAGUGUUUGUGUCUAAUGGAACA ICHDGKAHFPREGVFVSNGT Sprotein 108781088810898109081091810928 CAUUGGUUUGUGACACAGAGAAAUUUUUAUGAACCUCAGAUUAUUACAACAGAUAAUACA HWFVTQRNFYEPQIITTDNT Sprotein 109381094810958109681097810988 UUUGUGUCAGGAAAUUGUGAUGUGGUGAUUGGAAUUGUGAAUAAUACAGUGUAUGAUCCA FVSGNCDVVIGIVNNTVYDP Sprotein 109981100811018110281103811048 CUGCAGCCAGAACUGGAUUCUUUUAAAGAAGAACUGGAUAAAUAUUUUAAAAAUCACACA LQPELDSFKEELDKYFKNHT Sprotein 110581106811078110881109811108 UCUCCUGAUGUGGAUUUAGGAGAUAUUUCUGGAAUCAAUGCAUCUGUGGUGAAUAUUCAG SPDVDLGDISGINASVVNIQ Sprotein 111181112811138111481115811168 AAAGAAAUUGAUAGACUGAAUGAAGUGGCCAAAAAUCUGAAUGAAUCUCUGAUUGAUCUG KEIDRLNEVAKNLNESLIDL Sprotein 111781118811198112081121811228 CAGGAACUUGGAAAAUAUGAACAGUACAUUAAAUGGCCUUGGUACAUUUGGCUUGGAUUU QELGKYEQYIKWPWYIWLGF Sprotein 112381124811258112681127811288 AUUGCAGGAUUAAUUGCAAUUGUGAUGGUGACAAUUAUGUUAUGUUGUAUGACAUCAUGU IAGLIAIVMVTIMLCCMTSC Sprotein 112981130811318113281133811348 UGUUCUUGUUUAAAAGGAUGUUGUUCUUGUGGAAGCUGUUGUAAAUUUGAUGAAGAUGAU CSCLKGCCSCGSCCKFDEDD Sprotein 1135811368113781138811393 UCUGAACCUGUGUUAAAAGGAGUGAAAUUGCAUUACACAUGAUGA SEPVLKGVKLHYT** Sprotein 114031141311423114331144311453 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 114631147311483114931150311513 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 115231153311543115531156311573 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 115831159311603116131162311633 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 116431165311663116731168311693 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGCGGCCGCAUGAAUAC FIelement 117031171311723117331174311753 AGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAU FIelement 117631177311783117931180311807 UUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUC FIelement 118171182711837118471185711867 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 1187711887118971190711917 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(496) TABLE-US-00014 NucleotideSequenceofRBS004.2(SEQIDNO:25;SEQIDNO:7) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinbold letters.Inaddition,thesequenceofthetranslatedproteinisshowninitalicletters belowthecodingnucleotidesequence(*=stopcodon). 1020304045 GAUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA 5UTR 5565758595105 AUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGG MEKVHVDIEEDSPFLRALQR nsp1 115125135145155165 AGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUGCUAAUGCC SFPQFEVEAKQVTDNDHANA nsp1 175185195205215225 AGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACG RAFSHLASKLIETEVDPSDT nsp1 235245255265275285 AUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGU ILDIGSAPARRMYSKHKYHC nsp1 295305315325335345 AUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUG ICPMRCAEDPDRLYKYATKL nsp1 355365375385395405 AAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUCGCC KKNCKEITDKELDKKMKELA nsp1 415425435445455465 GCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCG AVMSDPDLETETMCLHDDES nsp1 475485495505515525 UGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACA CRYEGQVAVYQDVYAVDGPT nsp1 535545555565575585 AGUCUCUAUCACCAAGCCAAUAAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACC SLYHQANKGVRVAYWIGFDT nsp1 595605615625635645 ACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCAUCAUACUCUACCAACUGGGCC TPFMFKNLAGAYPSYSTNWA nsp1 655665675685695705 GACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAGCGG DETVLTARNIGLCSSDVMER nsp1 715725735745755765 UCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCAACAAUGUUCUA SRRGMSILRKKYLKPSNNVL nsp1 775785795805815825 UUCUCUGUUGGCUCGACCAUCUACCACGAAAAGAGGGACUUACUGAGGAGCUGGCACCUG FSVGSTIYHEKRDLLRSWHL nsp1 835845855865875885 CCGUCUGUAUUUCACUUACGUGGCAAGCAAAAUUACACAUGUCGGUGUGAGACUAUAGUU PSVFHLRGKQNYTCRCETIV nsp1 895905915925935945 AGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCU SCDGYVVKRIAISPGLYGKP nsp1 9559659759859951005 UCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACA SGYAATMHREGELCCKVTDT nsp1 101510251035104510551065 UUGAACGGGGAGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGAC LNGERVSFPVCTYVPATLCD nsp1 107510851095110511151125 CAAAUGACUGGCAUACUGGCAACAGAUGUCAGUGCGGACGACGCGCAAAAACUGCUGGUU QMTGILATDVSADDAQKLLV nsp1 113511451155116511751185 GGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAACACCAAUACCAUGAAA GLNQRIVVNGRTQRNTNTMK nsp1 119812051215122512351245 AAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAA NYLLPVVAQAFARWAKEYKE nsp1 125512651275128512951305 GAUCAAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGUUGU DQEDERPLGLRDRQLVMGCC nsp1 131513251335134513551365 UGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUC WAFRRHKITSIYKRPDTQTI nsp1 137513851395140514151425 AUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCAGGAUAGGCAGUAACACAUUG IKVNSDFHSFVLPRIGSNTL nsp1 143514451455146514751485 GAGAUCGGGCUGAGAACAAGAAUCAGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCACCU EIGLRTRIRKMLEEHKEPSP nsp1 149515051515152515351545 CUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUG LITAEDVQEAKCAADEAKEV nsp1 155515651575158515951605 CGUGAAGCCGAGGAGUUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCC REAEELRAALPPLAADVEEP nsp1 16151625163516451650 ACUCUGGAAGCCGAUGUCGACUUGAUGUUACAAGAGGCUGGGGCC TLEADVDLMLQEAGA nsp1 166016701680169017001710 GGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGCUGGCGAGGACAAG GSVETPRGLIKVTSYAGEDK nsp2 172017301740175017601770 AUCGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGC IGSYAVLSPQAVLKSEKLSC nsp2 178017901800181018201830 AUCCACCCUCUCGCUGAACAAGUCAUAGUGAUAACACACUCUGGCCGAAAAGGGCGUUAU IHPLAEQVIVITHSGRKGRY nsp2 184018501860187018801890 GCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGACAUGCAAUACCCGUCCAG AVEPYHGKVVVPEGHAIPVQ nsp2 190019101920193019401950 GACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUAAAC DFQALSESATIVYNEREFVN nsp2 196019701980199020002010 AGGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUAC RYLHHIATHGGALNTDEEYY nsp2 202020302040205020602070 AAAACUGUCAAGCCCAGCGAGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAG KTVKPSEHDGEYLYDIDRKQ nsp2 208020902100211021202130 UGCGUCAAGAAAGAGCUAGUCACUGGGCUAGGGCUCACAGGCGAGCUGGUCGAUCCUCCC CVKKELVTGLGLTGELVDPP nsp2 214021502160217021802190 UUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCA FHEFAYESLRTRPAAPYQVP nsp2 220022102220223022402250 ACCAUAGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUC TIGVYGVPGSGKSGIIKSAV nsp2 226022702280229023002310 ACCAAAAAAGAUCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGAC TKKDLVVSAKKENCAEIIRD nsp2 232023302340235023602370 GUCAAGAAAAUGAAAGGGCUGGACGUCAAUGCCAGAACUGUGGACUCAGUGCUCUUGAAU VKKMKGLDVNARTVDSVLLN nsp2 238023902400241024202430 GGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACGAGGCUUUUGCUUGUCAUGCAGGU GCKHPVETLYIDEAFACHAG nsp2 244024502460247024802490 ACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGAGAUCCC TLRALIAIIRPKKAVLCGDP nsp2 250025102520253025402550 AAACAGUGCGGUUUUUUUAACAUGAUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGC KQCGEENMMCLKVHFNHEIC nsp2 256025702580259026002610 ACACAAGUCUUCCACAAAAGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCGGUCGUC TQVFHKSISRRCTKSVTSVV nsp2 262026302640265026602670 UCAACCUUGUUUUACGACAAAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUG STLFYDKKMRTTNPKETKIV nsp2 268026902700271027202730 AUUGACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGA IDTTGSTKPKQDDLILTCFR nsp2 274027502760277027802790 GGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCC GWVKQLQIDYKGNEIMTAAA nsp2 280028102820283028402850 UCUCAAGGGCUGACCCGUAAAGGUGUGUAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCU SQGLTRKGVYAVRYKVNENP nsp2 286028702880289029002910 CUGUACGCACCCACCUCAGAACAUGUGAACGUCCUACUGACCCGCACGGAGGACCGCAUC LYAPTSEHVNVLLTRTEDRI nsp2 292029302940295029602970 GUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUACCCUGGG VWKTLAGDPWIKTLTAKYPG nsp2 298029903000301030203030 AAUUUCACUGCCACGAUAGAGGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUC NFTATIEEWQAEHDAIMRHI nsp2 304030503060307030803090 UUGGAGAGACCGGACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAG LERPDPTDVFQNKANVCWAK nsp2 310031103120313031403150 GCUUUAGUGCCGGUGCUGAAGACCGCUGGCAUAGACAUGACCACUGAACAAUGGAACACU ALVPVLKTAGIDMTTEQWNT nsp2 316031703180319032003210 GUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGAACCAACUAUGC VDYFETDKAHSAEIVLNQLC nsp2 322032303240325032603270 GUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUA VRFFGLDLDSGLFSAPTVPL nsp2 328032903300331033203330 UCCAUUAGGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAA SIRNNHWDNSPSPNMYGLNK nsp2 334033503360337033803390 GAAGUGGUCCGUCAGCUCUCUCGCAGGUACCCACAACUGCCUCGGGCAGUUGCCACUGGU EVVRQLSRRYPQLPRAVATG nsp2 340034103420343034403450 AGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAUCCGCGCAUAAACCUAGUA RVYDMNTGTLRNYDPRINLV nsp2 346034703480349035003510 CCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGU PVNRRLPHALVLHHNEHPQS nsp2 352035303540355035603570 GACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUCGGGGAAAAG DFSSFVSKLKGRTVLVVGEK nsp2 358035903600361036203630 UUGUCCGUCCCAGGCAAAAUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGA LSVPGKMVDWLSDRPEATER nsp2 364036503660367036803690 GCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAU ARLDLGIPGDVPKYDIIFVN nsp2 370037103720373037403750 GUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUUAAGCUA VRTPYKYHHYQQCEDHAIKL nsp2 376037703780379038003810 AGCAUGUUGACCAAGAAAGCAUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUA SMLTKKACLHLNPGGTCVSI nsp2 382038303840385038603870 GGUUAUGGUUACGCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUC GYGYADRASESIIGAIARQF nsp2 388038903900391039203930 AAGUUUUCCCGAGUAUGCAAACCGAAAUCCUCACUUGAGGAGACGGAAGUUCUGUUUGUA KFSRVCKPKSSLEETEVLFV nsp2 394039503960397039803990 UUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUAUCAUCAACCUUG FIGYDRKARTHNPYKLSSTL nsp2 40004010402040304032 ACCAACAUUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGU TNIYTGSRLHEAGC nsp2 404240524062407240824092 GCACCCUCAUAUCAUGUGGUGCGAGGGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUA APSYHVVRGDIATATEGVII nsp3 410241124122413241424152 AAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGCGGAGCGCUGUAUAAGAAA NAANSKGQPGGGVCGALYKK nsp3 416241724182419242024212 UUCCCGGAAAGUUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGU FPESFDLQPIEVGKARLVKG nsp3 422242324242425242624272 GCAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUUCAACAAAGUUUCGGAGGUUGAA AAKHIIHAVGPNFNKVSEVE nsp3 428242924302431243224332 GGUGACAAACAGUUGGCAGAGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAU GDKQLAEAYESIAKIVNDNN nsp3 434243524362437243824392 UACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCAUCUUUUCCGGGAACAAAGAUCGA YKSVAIPLLSTGIFSGNKDR nsp3 440244124422443244424452 CUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAUGUAGCC LTQSLNHLLTALDTTDADVA nsp3 446244724482449245024512 AUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAA IYCRDKKWEMTLKEAVARRE nsp3 452245324542455245624572 GCAGUGGAGGAGAUAUGCAUAUCCGACGAUUCUUCAGUGACAGAACCUGAUGCAGAGCUG AVEEICISDDSSVTEPDAEL nsp3 458245924602461246224632 GUGAGGGUGCAUCCCAAGAGUUCUUUGGCUGGAAGGAAGGGCUACAGCACAAGCGAUGGC VRVHPKSSLAGRKGYSTSDG nsp3 464246524662467246824692 AAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAGGAUAUAGCAGAA KTFSYLEGTKFHQAAKDIAE nsp3 470247124722473247424752 AUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUC INAMWPVATEANEQVCMYIL nsp3 476247724782479248024812 GGAGAAAGCAUGAGCAGUAUUAGGUCGAAAUGCCCCGUCGAGGAGUCGGAAGCCUCCACA GESMSSIRSKCPVEESEAST nsp3 482248324842485248624872 CCACCUAGCACGCUGCCUUGCUUGUGCAUCCAUGCCAUGACUCCAGAAAGAGUACAGCGC PPSTLPCLCIHAMTPERVQR nsp3 488248924902491249224932 CUAAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCCUUUCCAUUGCCGAAGUAU LKASRPEQITVCSSFPLPKY nsp3 494249524962497249824992 AGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUG RITGVQKIQCSQPILFSPKV nsp3 500250125022503250425052 CCUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCG PAYIHPRKYLVETPPVDETP nsp3 506250725082509251025112 GAGCCAUCGGCAGAGAACCAAUCCACAGAGGGGACACCUGAACAACCACCACUUAUAACC EPSAENQSTEGTPEQPPLIT nsp3 512251325142515251625172 GAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUCAUCAUCGAAGAAGAAGAAGAAGAU EDETRTRTPEPIIIEEEEED nsp3 518251925202521252225232 AGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCAGACAUU SISLLSDGPTHQVLQVEADI nsp3 524252525262527252825292 CACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGACUUUGAU HGPPSVSSSSWSIPHASDED nsp3 530253125322533253425352 GUGGACAGUUUAUCCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCAACG VDSLSILDTLEGASVTSGAT nsp3 536253725382539254025412 UCAGCCGAGACUAACUCUUACUUCGCAAAGAGUAUGGAGUUUCUGGCGCGACCGGUGCCU SAETNSYFAKSMEFLARPVP nsp3 542254325442545254625472 GCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACAAGAACACCGUCA APRTVFRNPPHPAPRTRTPS nsp3 548254925502551255225532 CUUGCACCCAGCAGGGCCUGCUCCAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAU LAPSRACSRTSLVSTPPGVN nsp3 554255525562557255825592 AGGGUGAUCACUAGAGAGGAGCUCGAAGCGCUUACCCCGUCACGCACUCCUAGCAGGUCG RVITREELEALTPSRTPSRS nsp3 560256125622563256425652 GUCUCCAGAACCAGCCUGGUCUCCAACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAG VSRTSLVSNPPGVNRVITRE nsp3 566256725682569257025703 GAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAUGCGGGUGCA EFEAFVAQQQ*RFDAGA nsp3 571357235733574357535763 UACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACG YIFSSDTGQGHLQQKSVRQT nsp4 577357835793580358135823 GUGCUAUCCGAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUC VLSEVVLERTELEISYAPRL nsp4 583358435853586358735883 GACCAAGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAAUCCCACACCUGCUAAC DQEKEELLRKKLQLNPTPAN nsp4 589359035913592359335943 AGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUAACAGCUAGACGUAUU RSRYQSRKVENMKAITARRI nsp4 595359635973598359936003 CUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUG LQGLGHYLKAEGKVECYRTL nsp4 601360236033604360536063 CAUCCUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCA HPVPLYSSSVNRAFSSPKVA nsp4 607360836093610361136123 GUGGAAGCCUGUAACGCCAUGUUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUU VEACNAMLKENFPTVASYCI nsp4 613361436153616361736183 AUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGAGCUUCAUGCUGCUUAGACACU IPEYDAYLDMVDGASCCLDT nsp4 619362036213622362336243 GCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAACCC ASFCPAKLRSFPKKHSYLEP nsp4 625362636273628362936303 ACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCU TIRSAVPSAIQNTLQNVLAA nsp4 631363236333634363536363 GCCACAAAAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCG ATKRNCNVTQMRELPVLDSA nsp4 637363836393640364136423 GCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCGUGUAAUAAUGAAUAUUGGGAAACGUUU AFNVECFKKYACNNEYWETF nsp4 643364436453646364736483 AAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUACCAAAUUAAAA KENPIRLTEENVVNYITKLK nsp4 649365036513652365336543 GGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUA GPKAAALFAKTHNLNMLQDI nsp4 655365636573658365936603 CCAAUGGACAGGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAA PMDRFVMDLKRDVKVTPGTK nsp4 661366236633664366536663 CAUACUGAAGAACGGCCCAAGGUACAGGUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCG HTEERPKVQVIQAADPLATA nsp4 667366836693670367136723 UAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCGGUCCUGCUUCCGAAC YLCGIHRELVRRLNAVLLPN nsp4 673367436753676367736783 AUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUC IHTLEDMSAEDFDAIIAEHF nsp4 679368036813682368336843 CAGCCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGAC QPGDCVLETDIASFDKSEDD nsp4 685368636873688368936903 GCCAUGGCUCUGACCGCGUUAAUGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUG AMALTALMILEDLGVDAELL nsp4 691369236933694369536963 ACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUACAUUUGCCCACUAAAACUAAA TLIEAAFGEISSIHLPTKTK nsp4 697369836993700370137023 UUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACAGUC FKFGAMMKSGMFLTLFVNTV nsp4 703370437053706370737083 AUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGAUCACCAUGUGCA INIVIASRVLRERLTGSPCA nsp4 709371037113712371337143 GCAUUCAUUGGAGAUGACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGAC AFIGDDNIVKGVKSDKLMAD nsp4 715371637173718371937203 AGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAGAUUAUAGAUGCUGUGGUGGGCGAGAAA RCATWLNMEVKIIDAVVGEK nsp4 721372237233724372537263 GCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGU APYFCGGEILCDSVTGTACR nsp4 727372837293730373137323 GUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUAGGCAAACCUCUGGCAGCAGACGAUGAA VADPLKRLFKLGKPLAADDE nsp4 733373437353736373737383 CAUGAUGAUGACAGGAGAAGGGCAUUGCAUGAGGAGUCAACACGCUGGAACCGAGUGGGU HDDDRRRALHEESTRWNRVG nsp4 739374037413742374337443 AUUCUUUCAGAGCUGUGCAAGGCAGUAGAAUCAAGGUAUGAAACCGUAGGAACUUCCAUC ILSELCKAVESRYETVGTSI nsp4 745374637473748374937503 AUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUCAGCUACCUGAGAGGG IVMAMTTLASSVKSFSYLRG nsp4 751375237527 GCCCCUAUAACUCUCUACGGCUAA APITLYG* nsp4 75377547755775677568 CCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGACUAGU virUTR 757875887598760876187628 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGAAUUUGACAACA MFVFLVLLPLVSSQCVNLTT Sprotein 763876487658766876787688 AGAACACAGCUGCCACCAGCUUAUACAAAUUCUUUUACCAGAGGAGUGUAUUAUCCUGAU RTQLPPAYTNSFTRGVYYPD Sprotein 769877087718772877387748 AAAGUGUUUAGAUCUUCUGUGCUGCACAGCACACAGGACCUGUUUCUGCCAUUUUUUAGC KVFRSSVLHSTQDLFLPFFS Sprotein 775877687778778877987808 AAUGUGACAUGGUUUCAUGCAAUUCAUGUGUCUGGAACAAAUGGAACAAAAAGAUUUGAU NVTWFHAIHVSGTNGTKRFD Sprotein 781878287838784878587868 AAUCCUGUGCUGCCUUUUAAUGAUGGAGUGUAUUUUGCUUCAACAGAAAAGUCAAAUAUU NPVLPFNDGVYEASTEKSNI Sprotein 787878887898790879187928 AUUAGAGGAUGGAUUUUUGGAACAACACUGGAUUCUAAAACACAGUCUCUGCUGAUUGUG IRGWIFGTTLDSKTQSLLIV Sprotein 793879487958796879787988 AAUAAUGCAACAAAUGUGGUGAUUAAAGUGUGUGAAUUUCAGUUUUGUAAUGAUCCUUUU NNATNVVIKVCEFQFCNDPF Sprotein 799880088018802880388048 CUGGGAGUGUAUUAUCACAAAAAUAAUAAAUCUUGGAUGGAAUCUGAAUUUAGAGUGUAU LGVYYHKNNKSWMESEFRVY Sprotein 805880688078808880988108 UCCUCUGCAAAUAAUUGUACAUUUGAAUAUGUGUCUCAGCCUUUUCUGAUGGAUCUGGAA SSANNCTFEYVSQPFLMDLE Sprotein 811881288138814881588168 GGAAAACAGGGCAAUUUUAAAAAUCUGAGAGAAUUUGUGUUUAAAAAUAUUGAUGGAUAU GKQGNFKNLREFVFKNIDGY Sprotein 817881888198820882188228 UUUAAAAUUUAUUCUAAACACACACCAAUUAAUUUAGUGAGAGAUCUGCCUCAGGGAUUU FKIYSKHTPINLVRDLPQGF Sprotein 823882488258826882788288 UCUGCUCUGGAACCUCUGGUGGAUCUGCCAAUUGGCAUUAAUAUUACAAGAUUUCAGACA SALEPLVDLPIGINITRFQT Sprotein 829883088318832883388348 CUGCUGGCUCUGCACAGAUCUUAUCUGACACCUGGAGAUUCUUCUUCUGGAUGGACAGCC LLALHRSYLTPGDSSSGWTA Sprotein 835883688378838883988408 GGAGCUGCAGCUUAUUAUGUGGGCUAUCUGCAGCCAAGAACAUUUCUGCUGAAAUAUAAU GAAAYYVGYLQPRTFLLKYN Sprotein 841884288438844884588468 GAAAAUGGAACAAUUACAGAUGCUGUGGAUUGUGCUCUGGAUCCUCUGUCUGAAACAAAA ENGTITDAVDCALDPLSETK Sprotein 847884888498850885188528 UGUACAUUAAAAUCUUUUACAGUGGAAAAAGGCAUUUAUCAGACAUCUAAUUUUAGAGUG CTLKSFTVEKGIYQTSNFRV Sprotein 853885488558856885788588 CAGCCAACAGAAUCUAUUGUGAGAUUUCCAAAUAUUACAAAUCUGUGUCCAUUUGGAGAA QPTESIVRFPNITNLCPFGE Sprotein 859886088618862886388648 GUGUUUAAUGCAACAAGAUUUGCAUCUGUGUAUGCAUGGAAUAGAAAAAGAAUUUCUAAU VFNATRFASVYAWNRKRISN Sprotein 865886688678868886988708 UGUGUGGCUGAUUAUUCUGUGCUGUAUAAUAGUGCUUCUUUUUCCACAUUUAAAUGUUAU CVADYSVLYNSASFSTFKCY Sprotein 871887288738874887588768 GGAGUGUCUCCAACAAAAUUAAAUGAUUUAUGUUUUACAAAUGUGUAUGCUGAUUCUUUU GVSPTKLNDLCFTNVYADSF Sprotein 877887888798880888188828 GUGAUCAGAGGUGAUGAAGUGAGACAGAUUGCCCCCGGACAGACAGGAAAAAUUGCUGAU VIRGDEVRQIAPGQTGKIAD Sprotein 883888488858886888788888 UACAAUUACAAACUGCCUGAUGAUUUUACAGGAUGUGUGAUUGCUUGGAAUUCUAAUAAU YNYKLPDDFTGCVIAWNSNN Sprotein 889889088918892889388948 UUAGAUUCUAAAGUGGGAGGAAAUUACAAUUAUCUGUACAGACUGUUUAGAAAAUCAAAU LDSKVGGNYNYLYRLFRKSN Sprotein 895889688978898889989008 CUGAAACCUUUUGAAAGAGAUAUUUCAACAGAAAUUUAUCAGGCUGGAUCAACACCUUGU LKPPERDISTEIYQAGSTPC Sprotein 901890289038904890589068 AAUGGAGUGGAAGGAUUUAAUUGUUAUUUUCCAUUACAGAGCUAUGGAUUUCAGCCAACC NGVEGFNCYFPLQSYGFQPT Sprotein 907890889098910891189128 AAUGGUGUGGGAUAUCAGCCAUAUAGAGUGGUGGUGCUGUCUUUUGAACUGCUGCAUGCA NGVGYQPYRVVVLSFELLHA Sprotein 913891489158916891789188 CCUGCAACAGUGUGUGGACCUAAAAAAUCUACAAAUUUAGUGAAAAAUAAAUGUGUGAAU PATVCGPKKSTNLVKNKCVN Sprotein 919892089218922892389248 UUUAAUUUUAAUGGAUUAACAGGAACAGGAGUGCUGACAGAAUCUAAUAAAAAAUUUCUG FNFNGLTGTGVLTESNKKFL Sprotein 925892689278928892989308 CCUUUUCAGCAGUUUGGCAGAGAUAUUGCAGAUACCACAGAUGCAGUGAGAGAUCCUCAG PFQQFGRDIADTTDAVRDPQ Sprotein 931893289338934893589368 ACAUUAGAAAUUCUGGAUAUUACACCUUGUUCUUUUGGGGGUGUGUCUGUGAUUACACCU TLEILDITPCSFGGVSVITP Sprotein 937893889398940894189428 GGAACAAAUACAUCUAAUCAGGUGGCUGUGCUGUAUCAGGAUGUGAAUUGUACAGAAGUG GTNTSNQVAVLYQDVNCTEV Sprotein 943894489458946894789488 CCAGUGGCAAUUCAUGCAGAUCAGCUGACACCAACAUGGAGAGUGUAUUCUACAGGAUCU PVAIHADQLTPTWRVYSTGS Sprotein 949895089518952895389548 AAUGUGUUUCAGACAAGAGCAGGAUGUCUGAUUGGAGCAGAACAUGUGAAUAAUUCUUAU NVFQTRAGCLIGAEHVNNSY Sprotein 955895689578958895989608 GAAUGUGAUAUUCCAAUUGGAGCAGGCAUUUGUGCAUCUUAUCAGACACAGACAAAUUCC ECDIPIGAGICASYQTQTNS Sprotein 961896289638964896589668 CCAAGGAGAGCAAGAUCUGUGGCAUCUCAGUCUAUUAUUGCAUACACCAUGUCUCUGGGA PRRARSVASQSIIAYTMSLG Sprotein 967896889698970897189728 GCAGAAAAUUCUGUGGCAUAUUCUAAUAAUUCUAUUGCUAUUCCAACAAAUUUUACCAUU AENSVAYSNNSIAIPTNFTI Sprotein 973897489758976897789788 UCUGUGACAACAGAAAUUUUACCUGUGUCUAUGACAAAAACAUCUGUGGAUUGUACCAUG SVTTEILPVSMTKTSVDCTM Sprotein 979898089818982898389848 UACAUUUGUGGAGAUUCUACAGAAUGUUCUAAUCUGCUGCUGCAGUAUGGAUCUUUUUGU YICGDSTECSNLLLQYGSFC Sprotein 985898689878988898989908 ACACAGCUGAAUAGAGCUUUAACAGGAAUUGCUGUGGAACAGGAUAAAAAUACACAGGAA TQLNRALTGIAVEQDKNTQE Sprotein 991899289938994899589968 GUGUUUGCUCAGGUGAAACAGAUUUACAAAACACCACCAAUUAAAGAUUUUGGAGGAUUU VFAQVKQIYKTPPIKDFGGF Sprotein 997899889998100081001810028 AAUUUUAGCCAGAUUCUGCCUGAUCCUUCUAAACCUUCUAAAAGAUCUUUUAUUGAAGAU NFSQILPDPSKPSKRSFIED Sprotein 100381004810058100681007810088 CUGCUGUUUAAUAAAGUGACACUGGCAGAUGCAGGAUUUAUUAAACAGUAUGGAGAUUGC LLFNKVTLADAGFIKQYGDC Sprotein 100981010810118101281013810148 CUGGGUGAUAUUGCUGCAAGAGAUCUGAUUUGUGCUCAGAAAUUUAAUGGACUGACAGUG LGDIAARDLICAQKFNGLTV Sprotein 101581016810178101881019810208 CUGCCUCCUCUGCUGACAGAUGAAAUGAUUGCUCAGUACACAUCUGCUUUACUGGCUGGA LPPLLTDEMIAQYTSALLAG Sprotein 102181022810238102481025810268 ACAAUUACAAGCGGAUGGACAUUUGGAGCUGGAGCUGCUCUGCAGAUUCCUUUUGCAAUG TITSGWTFGAGAALQIPFAM Sprotein 102781028810298103081031810328 CAGAUGGCUUACAGAUUUAAUGGAAUUGGAGUGACACAGAAUGUGUUAUAUGAAAAUCAG QMAYRFNGIGVTQNVLYENQ Sprotein 103381034810358103681037810388 AAACUGAUUGCAAAUCAGUUUAAUUCUGCAAUUGGCAAAAUUCAGGAUUCUCUGUCUUCU KLIANQFNSAIGKIQDSLSS Sprotein 103981040810418104281043810448 ACAGCUUCUGCUCUGGGAAAACUGCAGGAUGUGGUGAAUCAGAAUGCACAGGCACUGAAU TASALGKLQDVVNQNAQALN Sprotein 104581046810478104881049810508 ACUCUGGUGAAACAGCUGUCUAGCAAUUUUGGGGCAAUUUCUUCUGUGCUGAAUGAUAUU TLVKQLSSNFGAISSVLNDI Sprotein 105181052810538105481055810568 CUGUCUAGACUGGAUCCTCCTGAAGCUGAAGUGCAGAUUGAUAGACUGAUCACAGGAAGA LSRLDPPEAEVQIDRLITGR Sprotein 105781058810598106081061810628 CUGCAGUCUCUGCAGACUUAUGUGACACAGCAGCUGAUUAGAGCUGCUGAAAUUAGAGCU LQSLQTYVTQQLIRAAEIRA Sprotein 106381064810658106681067810688 UCUGCUAAUCUGGCUGCUACAAAAAUGUCUGAAUGUGUGCUGGGACAGUCAAAAAGAGUG SANLAATKMSECVLGQSKRV Sprotein 106981070810718107281073810748 GAUUUUUGUGGAAAAGGAUAUCAUCUGAUGUCUUUUCCACAGUCUGCUCCACAUGGAGUG DFCGKGYHLMSEPQSAPHGV Sprotein 107581076810778107881079810808 GUGUUUUUACAUGUGACAUAUGUGCCAGCACAGGAAAAGAAUUUUACCACAGCACCAGCA VFLHVTYVPAQEKNFTTAPA Sprotein 108181082810838108481085810868 AUUUGUCAUGAUGGAAAAGCACAUUUUCCAAGAGAAGGAGUGUUUGUGUCUAAUGGAACA ICHDGKAHFPREGVFVSNGT Sprotein 108781088810898109081091810928 CAUUGGUUUGUGACACAGAGAAAUUUUUAUGAACCUCAGAUUAUUACAACAGAUAAUACA HWFVTQRNFYEPQIITTDNT Sprotein 109381094810958109681097810988 UUUGUGUCAGGAAAUUGUGAUGUGGUGAUUGGAAUUGUGAAUAAUACAGUGUAUGAUCCA FVSGNCDVVIGIVNNTVYDP Sprotein 109981100811018110281103811048 CUGCAGCCAGAACUGGAUUCUUUUAAAGAAGAACUGGAUAAAUAUUUUAAAAAUCACACA LQPELDSFKEELDKYFKNHT Sprotein 110581106811078110881109811108 UCUCCUGAUGUGGAUUUAGGAGAUAUUUCUGGAAUCAAUGCAUCUGUGGUGAAUAUUCAG SPDVDLGDISGINASVVNIQ Sprotein 111181112811138111481115811168 AAAGAAAUUGAUAGACUGAAUGAAGUGGCCAAAAAUCUGAAUGAAUCUCUGAUUGAUCUG KEIDRLNEVAKNLNESLIDL Sprotein 111781118811198112081121811228 CAGGAACUUGGAAAAUAUGAACAGUACAUUAAAUGGCCUUGGUACAUUUGGCUUGGAUUU QELGKYEQYIKWPWYIWLGF Sprotein 112381124811258112681127811288 AUUGCAGGAUUAAUUGCAAUUGUGAUGGUGACAAUUAUGUUAUGUUGUAUGACAUCAUGU IAGLIAIVMVTIMLCCMTSC Sprotein 112981130811318113281133811348 UGUUCUUGUUUAAAAGGAUGUUGUUCUUGUGGAAGCUGUUGUAAAUUUGAUGAAGAUGAU CSCLKGCCSCGSCCKFDEDD Sprotein 1135811368113781138811393 UCUGAACCUGUGUUAAAAGGAGUGAAAUUGCAUUACACAUGAUGA SEPVLKGVKLHYT** Sprotein 114031141311423114331144311453 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 114631147311483114931150311513 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 115231153311543115531156311573 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 115831159311603116131162311633 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 116431165311663116731168311693 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGCGGCCGCAUGAAUAC FIelement 117031171311723117331174311753 AGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAU FIelement 117631177311783117931180311807 UUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUC FIelement 118171182711837118471185711867 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 1187711887118971190711917 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(497) TABLE-US-00015 NucleotideSequenceofRBS004.3(SEQIDNO:26;SEQIDNO:5) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinbold letters.Inaddition,thesequenceofthetranslatedproteinisshowninitalicletters belowthecodingnucleotidesequence(*=stopcodon). 1020304045 GAUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA 5UTR 5565758595105 AUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGG MEKVHVDIEEDSPFLRALQR nsp1 115125135145155165 AGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUGCUAAUGCC SFPQFEVEAKQVTDNDHANA nsp1 175185195205215225 AGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACG RAFSHLASKLIETEVDPSDT nsp1 235245255265275285 AUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGU ILDIGSAPARRMYSKHKYHC nsp1 295305315325335345 AUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUG ICPMRCAEDPDRLYKYATKL nsp1 355365375385395405 AAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUCGCC KKNCKEITDKELDKKMKELA nsp1 415425435445455465 GCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCG AVMSDPDLETETMCLHDDES nsp1 475485495505515525 UGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACA CRYEGQVAVYQDVYAVDGPT nsp1 535545555565575585 AGUCUCUAUCACCAAGCCAAUAAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACC SLYHQANKGVRVAYWIGFDT nsp1 595605615625635645 ACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCAUCAUACUCUACCAACUGGGCC TPFMFKNLAGAYPSYSTNWA nsp1 655665675685695705 GACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAGCGG DETVLTARNIGLCSSDVMER nsp1 715725735745755765 UCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCAACAAUGUUCUA SRRGMSILRKKYLKPSNNVL nsp1 775785795805815825 UUCUCUGUUGGCUCGACCAUCUACCACGAAAAGAGGGACUUACUGAGGAGCUGGCACCUG FSVGSTIYHEKRDLLRSWHL nsp1 835845855865875885 CCGUCUGUAUUUCACUUACGUGGCAAGCAAAAUUACACAUGUCGGUGUGAGACUAUAGUU PSVFHLRGKQNYTCRCETIV nsp1 895905915925935945 AGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCU SCDGYVVKRIAISPGLYGKP nsp1 9559659759859951005 UCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACA SGYAATMHREGELCCKVTDT nsp1 101510251035104510551065 UUGAACGGGGAGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGAC LNGERVSFPVCTYVPATLCD nsp1 107510851095110511151125 CAAAUGACUGGCAUACUGGCAACAGAUGUCAGUGCGGACGACGCGCAAAAACUGCUGGUU QMTGILATDVSADDAQKLLV nsp1 113511451155116511751185 GGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAACACCAAUACCAUGAAA GLNQRIVVNGRTQRNTNTMK nsp1 119812051215122512351245 AAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAA NYLLPVVAQAFARWAKEYKE nsp1 125512651275128512951305 GAUCAAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGUUGU DQEDERPLGLRDRQLVMGCC nsp1 131513251335134513551365 UGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUC WAFRRHKITSIYKRPDTQTI nsp1 137513851395140514151425 AUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCAGGAUAGGCAGUAACACAUUG IKVNSDFHSFVLPRIGSNTL nsp1 143514451455146514751485 GAGAUCGGGCUGAGAACAAGAAUCAGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCACCU EIGLRTRIRKMLEEHKEPSP nsp1 149515051515152515351545 CUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUG LITAEDVQEAKCAADEAKEV nsp1 155515651575158515951605 CGUGAAGCCGAGGAGUUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCC REAEELRAALPPLAADVEEP nsp1 16151625163516451650 ACUCUGGAAGCCGAUGUCGACUUGAUGUUACAAGAGGCUGGGGCC TLEADVDLMLQEAGA nsp1 166016701680169017001710 GGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGCUGGCGAGGACAAG GSVETPRGLIKVTSYAGEDK nsp2 172017301740175017601770 AUCGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGC IGSYAVLSPQAVLKSEKLSC nsp2 178017901800181018201830 AUCCACCCUCUCGCUGAACAAGUCAUAGUGAUAACACACUCUGGCCGAAAAGGGCGUUAU IHPLAEQVIVITHSGRKGRY nsp2 184018501860187018801890 GCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGACAUGCAAUACCCGUCCAG AVEPYHGKVVVPEGHAIPVQ nsp2 190019101920193019401950 GACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUAAAC DFQALSESATIVYNEREFVN nsp2 196019701980199020002010 AGGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUAC RYLHHIATHGGALNTDEEYY nsp2 202020302040205020602070 AAAACUGUCAAGCCCAGCGAGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAG KTVKPSEHDGEYLYDIDRKQ nsp2 208020902100211021202130 UGCGUCAAGAAAGAGCUAGUCACUGGGCUAGGGCUCACAGGCGAGCUGGUCGAUCCUCCC CVKKELVTGLGLTGELVDPP nsp2 214021502160217021802190 UUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCA FHEFAYESLRTRPAAPYQVP nsp2 220022102220223022402250 ACCAUAGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUC TIGVYGVPGSGKSGIIKSAV nsp2 226022702280229023002310 ACCAAAAAAGAUCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGAC TKKDLVVSAKKENCAEIIRD nsp2 232023302340235023602370 GUCAAGAAAAUGAAAGGGCUGGACGUCAAUGCCAGAACUGUGGACUCAGUGCUCUUGAAU VKKMKGLDVNARTVDSVLLN nsp2 238023902400241024202430 GGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACGAGGCUUUUGCUUGUCAUGCAGGU GCKHPVETLYIDEAFACHAG nsp2 244024502460247024802490 ACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGAGAUCCC TLRALIAIIRPKKAVLCGDP nsp2 250025102520253025402550 AAACAGUGCGGUUUUUUUAACAUGAUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGC KQCGEENMMCLKVHFNHEIC nsp2 256025702580259026002610 ACACAAGUCUUCCACAAAAGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCGGUCGUC TQVFHKSISRRCTKSVTSVV nsp2 262026302640265026602670 UCAACCUUGUUUUACGACAAAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUG STLFYDKKMRTTNPKETKIV nsp2 268026902700271027202730 AUUGACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGA IDTTGSTKPKQDDLILTCFR nsp2 274027502760277027802790 GGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCC GWVKQLQIDYKGNEIMTAAA nsp2 280028102820283028402850 UCUCAAGGGCUGACCCGUAAAGGUGUGUAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCU SQGLTRKGVYAVRYKVNENP nsp2 286028702880289029002910 CUGUACGCACCCACCUCAGAACAUGUGAACGUCCUACUGACCCGCACGGAGGACCGCAUC LYAPTSEHVNVLLTRTEDRI nsp2 292029302940295029602970 GUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUACCCUGGG VWKTLAGDPWIKTLTAKYPG nsp2 298029903000301030203030 AAUUUCACUGCCACGAUAGAGGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUC NFTATIEEWQAEHDAIMRHI nsp2 304030503060307030803090 UUGGAGAGACCGGACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAG LERPDPTDVFQNKANVCWAK nsp2 310031103120313031403150 GCUUUAGUGCCGGUGCUGAAGACCGCUGGCAUAGACAUGACCACUGAACAAUGGAACACU ALVPVLKTAGIDMTTEQWNT nsp2 316031703180319032003210 GUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGAACCAACUAUGC VDYFETDKAHSAEIVLNQLC nsp2 322032303240325032603270 GUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUA VRFFGLDLDSGLFSAPTVPL nsp2 328032903300331033203330 UCCAUUAGGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAA SIRNNHWDNSPSPNMYGLNK nsp2 334033503360337033803390 GAAGUGGUCCGUCAGCUCUCUCGCAGGUACCCACAACUGCCUCGGGCAGUUGCCACUGGU EVVRQLSRRYPQLPRAVATG nsp2 340034103420343034403450 AGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAUCCGCGCAUAAACCUAGUA RVYDMNTGTLRNYDPRINLV nsp2 346034703480349035003510 CCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGU PVNRRLPHALVLHHNEHPQS nsp2 352035303540355035603570 GACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUCGGGGAAAAG DFSSFVSKLKGRTVLVVGEK nsp2 358035903600361036203630 UUGUCCGUCCCAGGCAAAAUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGA LSVPGKMVDWLSDRPEATER nsp2 364036503660367036803690 GCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAU ARLDLGIPGDVPKYDIIFVN nsp2 370037103720373037403750 GUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUUAAGCUA VRTPYKYHHYQQCEDHAIKL nsp2 376037703780379038003810 AGCAUGUUGACCAAGAAAGCAUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUA SMLTKKACLHLNPGGTCVSI nsp2 382038303840385038603870 GGUUAUGGUUACGCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUC GYGYADRASESIIGAIARQF nsp2 388038903900391039203930 AAGUUUUCCCGAGUAUGCAAACCGAAAUCCUCACUUGAGGAGACGGAAGUUCUGUUUGUA KFSRVCKPKSSLEETEVLFV nsp2 394039503960397039803990 UUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUAUCAUCAACCUUG FIGYDRKARTHNPYKLSSTL nsp2 40004010402040304032 ACCAACAUUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGU TNIYTGSRLHEAGC nsp2 404240524062407240824092 GCACCCUCAUAUCAUGUGGUGCGAGGGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUA APSYHVVRGDIATATEGVII nsp3 410241124122413241424152 AAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGCGGAGCGCUGUAUAAGAAA NAANSKGQPGGGVCGALYKK nsp3 416241724182419242024212 UUCCCGGAAAGUUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGU FPESFDLQPIEVGKARLVKG nsp3 422242324242425242624272 GCAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUUCAACAAAGUUUCGGAGGUUGAA AAKHIIHAVGPNFNKVSEVE nsp3 428242924302431243224332 GGUGACAAACAGUUGGCAGAGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAU GDKQLAEAYESIAKIVNDNN nsp3 434243524362437243824392 UACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCAUCUUUUCCGGGAACAAAGAUCGA YKSVAIPLLSTGIFSGNKDR nsp3 440244124422443244424452 CUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAUGUAGCC LTQSLNHLLTALDTTDADVA nsp3 446244724482449245024512 AUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAA IYCRDKKWEMTLKEAVARRE nsp3 452245324542455245624572 GCAGUGGAGGAGAUAUGCAUAUCCGACGAUUCUUCAGUGACAGAACCUGAUGCAGAGCUG AVEEICISDDSSVTEPDAEL nsp3 458245924602461246224632 GUGAGGGUGCAUCCCAAGAGUUCUUUGGCUGGAAGGAAGGGCUACAGCACAAGCGAUGGC VRVHPKSSLAGRKGYSTSDG nsp3 464246524662467246824692 AAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAGGAUAUAGCAGAA KTFSYLEGTKFHQAAKDIAE nsp3 470247124722473247424752 AUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUC INAMWPVATEANEQVCMYIL nsp3 476247724782479248024812 GGAGAAAGCAUGAGCAGUAUUAGGUCGAAAUGCCCCGUCGAGGAGUCGGAAGCCUCCACA GESMSSIRSKCPVEESEAST nsp3 482248324842485248624872 CCACCUAGCACGCUGCCUUGCUUGUGCAUCCAUGCCAUGACUCCAGAAAGAGUACAGCGC PPSTLPCLCIHAMTPERVQR nsp3 488248924902491249224932 CUAAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCCUUUCCAUUGCCGAAGUAU LKASRPEQITVCSSFPLPKY nsp3 494249524962497249824992 AGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUG RITGVQKIQCSQPILFSPKV nsp3 500250125022503250425052 CCUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCG PAYIHPRKYLVETPPVDETP nsp3 506250725082509251025112 GAGCCAUCGGCAGAGAACCAAUCCACAGAGGGGACACCUGAACAACCACCACUUAUAACC EPSAENQSTEGTPEQPPLIT nsp3 512251325142515251625172 GAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUCAUCAUCGAAGAAGAAGAAGAAGAU EDETRTRTPEPIIIEEEEED nsp3 518251925202521252225232 AGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCAGACAUU SISLLSDGPTHQVLQVEADI nsp3 524252525262527252825292 CACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGACUUUGAU HGPPSVSSSSWSIPHASDED nsp3 530253125322533253425352 GUGGACAGUUUAUCCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCAACG VDSLSILDTLEGASVTSGAT nsp3 536253725382539254025412 UCAGCCGAGACUAACUCUUACUUCGCAAAGAGUAUGGAGUUUCUGGCGCGACCGGUGCCU SAETNSYFAKSMEFLARPVP nsp3 542254325442545254625472 GCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACAAGAACACCGUCA APRTVFRNPPHPAPRTRTPS nsp3 548254925502551255225532 CUUGCACCCAGCAGGGCCUGCUCCAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAU LAPSRACSRTSLVSTPPGVN nsp3 554255525562557255825592 AGGGUGAUCACUAGAGAGGAGCUCGAAGCGCUUACCCCGUCACGCACUCCUAGCAGGUCG RVITREELEALTPSRTPSRS nsp3 560256125622563256425652 GUCUCCAGAACCAGCCUGGUCUCCAACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAG VSRTSLVSNPPGVNRVITRE nsp3 566256725682569257025703 GAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAUGCGGGUGCA EFEAFVAQQQ*RFDAGA nsp3 571357235733574357535763 UACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACG YIFSSDTGQGHLQQKSVRQT nsp4 577357835793580358135823 GUGCUAUCCGAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUC VLSEVVLERTELEISYAPRL nsp4 583358435853586358735883 GACCAAGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAAUCCCACACCUGCUAAC DQEKEELLRKKLQLNPTPAN nsp4 589359035913592359335943 AGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUAACAGCUAGACGUAUU RSRYQSRKVENMKAITARRI nsp4 595359635973598359936003 CUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUG LQGLGHYLKAEGKVECYRTL nsp4 601360236033604360536063 CAUCCUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCA HPVPLYSSSVNRAFSSPKVA nsp4 607360836093610361136123 GUGGAAGCCUGUAACGCCAUGUUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUU VEACNAMLKENFPTVASYCI nsp4 613361436153616361736183 AUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGAGCUUCAUGCUGCUUAGACACU IPEYDAYLDMVDGASCCLDT nsp4 619362036213622362336243 GCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAACCC ASFCPAKLRSFPKKHSYLEP nsp4 625362636273628362936303 ACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCU TIRSAVPSAIQNTLQNVLAA nsp4 631363236333634363536363 GCCACAAAAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCG ATKRNCNVTQMRELPVLDSA nsp4 637363836393640364136423 GCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCGUGUAAUAAUGAAUAUUGGGAAACGUUU AFNVECFKKYACNNEYWETF nsp4 643364436453646364736483 AAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUACCAAAUUAAAA KENPIRLTEENVVNYITKLK nsp4 649365036513652365336543 GGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUA GPKAAALFAKTHNLNMLQDI nsp4 655365636573658365936603 CCAAUGGACAGGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAA PMDRFVMDLKRDVKVTPGTK nsp4 661366236633664366536663 CAUACUGAAGAACGGCCCAAGGUACAGGUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCG HTEERPKVQVIQAADPLATA nsp4 667366836693670367136723 UAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCGGUCCUGCUUCCGAAC YLCGIHRELVRRLNAVLLPN nsp4 673367436753676367736783 AUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUC IHTLEDMSAEDFDAIIAEHF nsp4 679368036813682368336843 CAGCCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGAC QPGDCVLETDIASFDKSEDD nsp4 685368636873688368936903 GCCAUGGCUCUGACCGCGUUAAUGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUG AMALTALMILEDLGVDAELL nsp4 691369236933694369536963 ACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUACAUUUGCCCACUAAAACUAAA TLIEAAFGEISSIHLPTKTK nsp4 697369836993700370137023 UUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACAGUC FKFGAMMKSGMFLTLFVNTV nsp4 703370437053706370737083 AUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGAUCACCAUGUGCA INIVIASRVLRERLTGSPCA nsp4 709371037113712371337143 GCAUUCAUUGGAGAUGACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGAC AFIGDDNIVKGVKSDKLMAD nsp4 715371637173718371937203 AGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAGAUUAUAGAUGCUGUGGUGGGCGAGAAA RCATWLNMEVKIIDAVVGEK nsp4 721372237233724372537263 GCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGU APYFCGGEILCDSVTGTACR nsp4 727372837293730373137323 GUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUAGGCAAACCUCUGGCAGCAGACGAUGAA VADPLKRLFKLGKPLAADDE nsp4 733373437353736373737383 CAUGAUGAUGACAGGAGAAGGGCAUUGCAUGAGGAGUCAACACGCUGGAACCGAGUGGGU HDDDRRRALHEESTRWNRVG nsp4 739374037413742374337443 AUUCUUUCAGAGCUGUGCAAGGCAGUAGAAUCAAGGUAUGAAACCGUAGGAACUUCCAUC ILSELCKAVESRYETVGTSI nsp4 745374637473748374937503 AUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUCAGCUACCUGAGAGGG IVMAMTTLASSVKSFSYLRG nsp4 751375237527 GCCCCUAUAACUCUCUACGGCUAA APITLYG* nsp4 75377547755775677568 CCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGACUAGU virUTR 757875887598760876187628 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGGUGAGAUUUCCA MFVFLVLLPLVSSQCVVRFP RBD(Sprotein) 763876487658766876787688 AAUAUUACAAAUCUGUGUCCAUUUGGAGAAGUGUUUAAUGCAACAAGAUUUGCAUCUGUG NITNLCPFGEVFNATRFASV RBD(Sprotein) 769877087718772877387748 UAUGCAUGGAAUAGAAAAAGAAUUUCUAAUUGUGUGGCUGAUUAUUCUGUGCUGUAUAAU YAWNRKRISNCVADYSVLYN RBD(Sprotein) 775877687778778877987808 AGUGCUUCUUUUUCCACAUUUAAAUGUUAUGGAGUGUCUCCAACAAAAUUAAAUGAUUUA SASFSTFKCYGVSPTKLNDL RBD(Sprotein) 781878287838784878587868 UGUUUUACAAAUGUGUAUGCUGAUUCUUUUGUGAUCAGAGGUGAUGAAGUGAGACAGAUU CFTNVYADSFVIRGDEVRQI RBD(Sprotein) 787878887898790879187928 GCCCCCGGACAGACAGGAAAAAUUGCUGAUUACAAUUACAAACUGCCUGAUGAUUUUACA APGQTGKIADYNYKLPDDFT RBD(Sprotein) 793879487958796879787988 GGAUGUGUGAUUGCUUGGAAUUCUAAUAAUUUAGAUUCUAAAGUGGGAGGAAAUUACAAU GCVIAWNSNNLDSKVGGNYN RBD(Sprotein) 799880088018802880388048 UAUCUGUACAGACUGUUUAGAAAAUCAAAUCUGAAACCUUUUGAAAGAGAUAUUUCAACA YLYRLFRKSNLKPFERDIST RBD(Sprotein) 805880688078808880988108 GAAAUUUAUCAGGCUGGAUCAACACCUUGUAAUGGAGUGGAAGGAUUUAAUUGUUAUUUU EIYQAGSTPCNGVEGFNCYF RBD(Sprotein) 811881288138814881588168 CCAUUACAGAGCUAUGGAUUUCAGCCAACCAAUGGUGUGGGAUAUCAGCCAUAUAGAGUG PLQSYGEQPTNGVGYQPYRV RBD(Sprotein) 817881888198820882188222 GUGGUGCUGUCUUUUGAACUGCUGCAUGCACCUGCAACAGUGUGUGGACCUAAA VVLSFELLHAPATVCGPK RBD(Sprotein) 823282428249 GGCUCCCCCGGCUCCGGCUCCGGAUCU GSPGSGSGS GSlinker 825982698279828982998309 GGUUAUAUUCCUGAAGCUCCAAGAGAUGGGCAAGCUUACGUUCGUAAAGAUGGCGAAUGG GYIPEAPRDGQAYVRKDGEW fibritin 831983298339834983598369 GUAUUACUUUCUACCUUUUUAGGCCGGUCCCUGGAGGUGCUGUUCCAGGGCCCCGGCUGA VLLSTFLGRSLEVLFQGPG* fibritin 8372 UGA * fibritin 838283928402841284228432 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 844284528462847284828492 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 850285128522853285428552 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 856285728582859286028612 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 862286328642865286628672 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGCGGCCGCAUGAAUAC FIelement 868286928702871287228732 AGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAU FIelement 874287528762877287828786 UUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUC FIelement 879688068816882688368846 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 88568866887688868896 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(498) TABLE-US-00016 NucleotideSequenceofRBS004.4(SEQIDNO:27;SEQIDNO:28) Nucleotidesequenceisshownwithindividualsequenceelementsasindicatedinbold letters.Inaddition,thesequenceofthetranslatedproteinisshowninitalicletters belowthecodingnucleotidesequence(*=stopcodon). 1020304045 GAUGGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA 5UTR 5565758595105 AUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGG MEKVHVDIEEDSPFLRALQR nsp1 115125135145155165 AGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUGCUAAUGCC SFPQFEVEAKQVTDNDHANA nsp1 175185195205215225 AGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACG RAFSHLASKLIETEVDPSDT nsp1 235245255265275285 AUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGU ILDIGSAPARRMYSKHKYHC nsp1 295305315325335345 AUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUG ICPMRCAEDPDRLYKYATKL nsp1 355365375385395405 AAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUCGCC KKNCKEITDKELDKKMKELA nsp1 415425435445455465 GCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCG AVMSDPDLETETMCLHDDES nsp1 475485495505515525 UGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACA CRYEGQVAVYQDVYAVDGPT nsp1 535545555565575585 AGUCUCUAUCACCAAGCCAAUAAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACC SLYHQANKGVRVAYWIGFDT nsp1 595605615625635645 ACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCAUCAUACUCUACCAACUGGGCC TPFMFKNLAGAYPSYSTNWA nsp1 655665675685695705 GACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAGCGG DETVLTARNIGLCSSDVMER nsp1 715725735745755765 UCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCAACAAUGUUCUA SRRGMSILRKKYLKPSNNVL nsp1 775785795805815825 UUCUCUGUUGGCUCGACCAUCUACCACGAAAAGAGGGACUUACUGAGGAGCUGGCACCUG FSVGSTIYHEKRDLLRSWHL nsp1 835845855865875885 CCGUCUGUAUUUCACUUACGUGGCAAGCAAAAUUACACAUGUCGGUGUGAGACUAUAGUU PSVFHLRGKQNYTCRCETIV nsp1 895905915925935945 AGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCU SCDGYVVKRIAISPGLYGKP nsp1 9559659759859951005 UCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACA SGYAATMHREGELCCKVTDT nsp1 101510251035104510551065 UUGAACGGGGAGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGAC LNGERVSFPVCTYVPATLCD nsp1 107510851095110511151125 CAAAUGACUGGCAUACUGGCAACAGAUGUCAGUGCGGACGACGCGCAAAAACUGCUGGUU QMTGILATDVSADDAQKLLV nsp1 113511451155116511751185 GGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAACACCAAUACCAUGAAA GLNQRIVVNGRTQRNTNTMK nsp1 119812051215122512351245 AAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAA NYLLPVVAQAFARWAKEYKE nsp1 125512651275128512951305 GAUCAAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGUUGU DQEDERPLGLRDRQLVMGCC nsp1 131513251335134513551365 UGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUC WAFRRHKITSIYKRPDTQTI nsp1 137513851395140514151425 AUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCAGGAUAGGCAGUAACACAUUG IKVNSDFHSFVLPRIGSNTL nsp1 143514451455146514751485 GAGAUCGGGCUGAGAACAAGAAUCAGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCACCU EIGLRTRIRKMLEEHKEPSP nsp1 149515051515152515351545 CUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUG LITAEDVQEAKCAADEAKEV nsp1 155515651575158515951605 CGUGAAGCCGAGGAGUUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCC REAEELRAALPPLAADVEEP nsp1 16151625163516451650 ACUCUGGAAGCCGAUGUCGACUUGAUGUUACAAGAGGCUGGGGCC TLEADVDLMLQEAGA nsp1 166016701680169017001710 GGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGCUGGCGAGGACAAG GSVETPRGLIKVTSYAGEDK nsp2 172017301740175017601770 AUCGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGC IGSYAVLSPQAVLKSEKLSC nsp2 178017901800181018201830 AUCCACCCUCUCGCUGAACAAGUCAUAGUGAUAACACACUCUGGCCGAAAAGGGCGUUAU IHPLAEQVIVITHSGRKGRY nsp2 184018501860187018801890 GCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGACAUGCAAUACCCGUCCAG AVEPYHGKVVVPEGHAIPVQ nsp2 190019101920193019401950 GACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUAAAC DFQALSESATIVYNEREFVN nsp2 196019701980199020002010 AGGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUAC RYLHHIATHGGALNTDEEYY nsp2 202020302040205020602070 AAAACUGUCAAGCCCAGCGAGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAG KTVKPSEHDGEYLYDIDRKQ nsp2 208020902100211021202130 UGCGUCAAGAAAGAGCUAGUCACUGGGCUAGGGCUCACAGGCGAGCUGGUCGAUCCUCCC CVKKELVTGLGLTGELVDPP nsp2 214021502160217021802190 UUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCA FHEFAYESLRTRPAAPYQVP nsp2 220022102220223022402250 ACCAUAGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUC TIGVYGVPGSGKSGIIKSAV nsp2 226022702280229023002310 ACCAAAAAAGAUCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGAC TKKDLVVSAKKENCAEIIRD nsp2 232023302340235023602370 GUCAAGAAAAUGAAAGGGCUGGACGUCAAUGCCAGAACUGUGGACUCAGUGCUCUUGAAU VKKMKGLDVNARTVDSVLLN nsp2 238023902400241024202430 GGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACGAGGCUUUUGCUUGUCAUGCAGGU GCKHPVETLYIDEAFACHAG nsp2 244024502460247024802490 ACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGAGAUCCC TLRALIAIIRPKKAVLCGDP nsp2 250025102520253025402550 AAACAGUGCGGUUUUUUUAACAUGAUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGC KQCGEENMMCLKVHFNHEIC nsp2 256025702580259026002610 ACACAAGUCUUCCACAAAAGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCGGUCGUC TQVFHKSISRRCTKSVTSVV nsp2 262026302640265026602670 UCAACCUUGUUUUACGACAAAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUG STLFYDKKMRTTNPKETKIV nsp2 268026902700271027202730 AUUGACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGA IDTTGSTKPKQDDLILTCFR nsp2 274027502760277027802790 GGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCC GWVKQLQIDYKGNEIMTAAA nsp2 280028102820283028402850 UCUCAAGGGCUGACCCGUAAAGGUGUGUAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCU SQGLTRKGVYAVRYKVNENP nsp2 286028702880289029002910 CUGUACGCACCCACCUCAGAACAUGUGAACGUCCUACUGACCCGCACGGAGGACCGCAUC LYAPTSEHVNVLLTRTEDRI nsp2 292029302940295029602970 GUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUACCCUGGG VWKTLAGDPWIKTLTAKYPG nsp2 298029903000301030203030 AAUUUCACUGCCACGAUAGAGGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUC NFTATIEEWQAEHDAIMRHI nsp2 304030503060307030803090 UUGGAGAGACCGGACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAG LERPDPTDVFQNKANVCWAK nsp2 310031103120313031403150 GCUUUAGUGCCGGUGCUGAAGACCGCUGGCAUAGACAUGACCACUGAACAAUGGAACACU ALVPVLKTAGIDMTTEQWNT nsp2 316031703180319032003210 GUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGAACCAACUAUGC VDYFETDKAHSAEIVLNQLC nsp2 322032303240325032603270 GUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUA VRFFGLDLDSGLFSAPTVPL nsp2 328032903300331033203330 UCCAUUAGGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAA SIRNNHWDNSPSPNMYGLNK nsp2 334033503360337033803390 GAAGUGGUCCGUCAGCUCUCUCGCAGGUACCCACAACUGCCUCGGGCAGUUGCCACUGGU EVVRQLSRRYPQLPRAVATG nsp2 340034103420343034403450 AGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAUCCGCGCAUAAACCUAGUA RVYDMNTGTLRNYDPRINLV nsp2 346034703480349035003510 CCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGU PVNRRLPHALVLHHNEHPQS nsp2 352035303540355035603570 GACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUCGGGGAAAAG DFSSFVSKLKGRTVLVVGEK nsp2 358035903600361036203630 UUGUCCGUCCCAGGCAAAAUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGA LSVPGKMVDWLSDRPEATER nsp2 364036503660367036803690 GCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAU ARLDLGIPGDVPKYDIIFVN nsp2 370037103720373037403750 GUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUUAAGCUA VRTPYKYHHYQQCEDHAIKL nsp2 376037703780379038003810 AGCAUGUUGACCAAGAAAGCAUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUA SMLTKKACLHLNPGGTCVSI nsp2 382038303840385038603870 GGUUAUGGUUACGCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUC GYGYADRASESIIGAIARQF nsp2 388038903900391039203930 AAGUUUUCCCGAGUAUGCAAACCGAAAUCCUCACUUGAGGAGACGGAAGUUCUGUUUGUA KFSRVCKPKSSLEETEVLFV nsp2 394039503960397039803990 UUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUAUCAUCAACCUUG FIGYDRKARTHNPYKLSSTL nsp2 40004010402040304032 ACCAACAUUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGU TNIYTGSRLHEAGC nsp2 404240524062407240824092 GCACCCUCAUAUCAUGUGGUGCGAGGGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUA APSYHVVRGDIATATEGVII nsp3 410241124122413241424152 AAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGCGGAGCGCUGUAUAAGAAA NAANSKGQPGGGVCGALYKK nsp3 416241724182419242024212 UUCCCGGAAAGUUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGU FPESFDLQPIEVGKARLVKG nsp3 422242324242425242624272 GCAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUUCAACAAAGUUUCGGAGGUUGAA AAKHIIHAVGPNFNKVSEVE nsp3 428242924302431243224332 GGUGACAAACAGUUGGCAGAGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAU GDKQLAEAYESIAKIVNDNN nsp3 434243524362437243824392 UACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCAUCUUUUCCGGGAACAAAGAUCGA YKSVAIPLLSTGIFSGNKDR nsp3 440244124422443244424452 CUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAUGUAGCC LTQSLNHLLTALDTTDADVA nsp3 446244724482449245024512 AUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAA IYCRDKKWEMTLKEAVARRE nsp3 452245324542455245624572 GCAGUGGAGGAGAUAUGCAUAUCCGACGAUUCUUCAGUGACAGAACCUGAUGCAGAGCUG AVEEICISDDSSVTEPDAEL nsp3 458245924602461246224632 GUGAGGGUGCAUCCCAAGAGUUCUUUGGCUGGAAGGAAGGGCUACAGCACAAGCGAUGGC VRVHPKSSLAGRKGYSTSDG nsp3 464246524662467246824692 AAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAGGAUAUAGCAGAA KTFSYLEGTKFHQAAKDIAE nsp3 470247124722473247424752 AUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUC INAMWPVATEANEQVCMYIL nsp3 476247724782479248024812 GGAGAAAGCAUGAGCAGUAUUAGGUCGAAAUGCCCCGUCGAGGAGUCGGAAGCCUCCACA GESMSSIRSKCPVEESEAST nsp3 482248324842485248624872 CCACCUAGCACGCUGCCUUGCUUGUGCAUCCAUGCCAUGACUCCAGAAAGAGUACAGCGC PPSTLPCLCIHAMTPERVQR nsp3 488248924902491249224932 CUAAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCCUUUCCAUUGCCGAAGUAU LKASRPEQITVCSSFPLPKY nsp3 494249524962497249824992 AGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUG RITGVQKIQCSQPILFSPKV nsp3 500250125022503250425052 CCUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCG PAYIHPRKYLVETPPVDETP nsp3 506250725082509251025112 GAGCCAUCGGCAGAGAACCAAUCCACAGAGGGGACACCUGAACAACCACCACUUAUAACC EPSAENQSTEGTPEQPPLIT nsp3 512251325142515251625172 GAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUCAUCAUCGAAGAAGAAGAAGAAGAU EDETRTRTPEPIIIEEEEED nsp3 518251925202521252225232 AGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCAGACAUU SISLLSDGPTHQVLQVEADI nsp3 524252525262527252825292 CACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGACUUUGAU HGPPSVSSSSWSIPHASDED nsp3 530253125322533253425352 GUGGACAGUUUAUCCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCAACG VDSLSILDTLEGASVTSGAT nsp3 536253725382539254025412 UCAGCCGAGACUAACUCUUACUUCGCAAAGAGUAUGGAGUUUCUGGCGCGACCGGUGCCU SAETNSYFAKSMEFLARPVP nsp3 542254325442545254625472 GCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACAAGAACACCGUCA APRTVFRNPPHPAPRTRTPS nsp3 548254925502551255225532 CUUGCACCCAGCAGGGCCUGCUCCAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAU LAPSRACSRTSLVSTPPGVN nsp3 554255525562557255825592 AGGGUGAUCACUAGAGAGGAGCUCGAAGCGCUUACCCCGUCACGCACUCCUAGCAGGUCG RVITREELEALTPSRTPSRS nsp3 560256125622563256425652 GUCUCCAGAACCAGCCUGGUCUCCAACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAG VSRTSLVSNPPGVNRVITRE nsp3 566256725682569257025703 GAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAUGCGGGUGCA EFEAFVAQQQ*RFDAGA nsp3 571357235733574357535763 UACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACG YIFSSDTGQGHLQQKSVRQT nsp4 577357835793580358135823 GUGCUAUCCGAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUC VLSEVVLERTELEISYAPRL nsp4 583358435853586358735883 GACCAAGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAAUCCCACACCUGCUAAC DQEKEELLRKKLQLNPTPAN nsp4 589359035913592359335943 AGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUAACAGCUAGACGUAUU RSRYQSRKVENMKAITARRI nsp4 595359635973598359936003 CUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUG LQGLGHYLKAEGKVECYRTL nsp4 601360236033604360536063 CAUCCUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCA HPVPLYSSSVNRAFSSPKVA nsp4 607360836093610361136123 GUGGAAGCCUGUAACGCCAUGUUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUU VEACNAMLKENFPTVASYCI nsp4 613361436153616361736183 AUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGAGCUUCAUGCUGCUUAGACACU IPEYDAYLDMVDGASCCLDT nsp4 619362036213622362336243 GCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAACCC ASFCPAKLRSFPKKHSYLEP nsp4 625362636273628362936303 ACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCU TIRSAVPSAIQNTLQNVLAA nsp4 631363236333634363536363 GCCACAAAAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCG ATKRNCNVTQMRELPVLDSA nsp4 637363836393640364136423 GCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCGUGUAAUAAUGAAUAUUGGGAAACGUUU AFNVECFKKYACNNEYWETF nsp4 643364436453646364736483 AAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUACCAAAUUAAAA KENPIRLTEENVVNYITKLK nsp4 649365036513652365336543 GGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUA GPKAAALFAKTHNLNMLQDI nsp4 655365636573658365936603 CCAAUGGACAGGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAA PMDRFVMDLKRDVKVTPGTK nsp4 661366236633664366536663 CAUACUGAAGAACGGCCCAAGGUACAGGUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCG HTEERPKVQVIQAADPLATA nsp4 667366836693670367136723 UAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCGGUCCUGCUUCCGAAC YLCGIHRELVRRLNAVLLPN nsp4 673367436753676367736783 AUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUC IHTLEDMSAEDFDAIIAEHF nsp4 679368036813682368336843 CAGCCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGAC QPGDCVLETDIASFDKSEDD nsp4 685368636873688368936903 GCCAUGGCUCUGACCGCGUUAAUGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUG AMALTALMILEDLGVDAELL nsp4 691369236933694369536963 ACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUACAUUUGCCCACUAAAACUAAA TLIEAAFGEISSIHLPTKTK nsp4 697369836993700370137023 UUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACAGUC FKFGAMMKSGMFLTLFVNTV nsp4 703370437053706370737083 AUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGAUCACCAUGUGCA INIVIASRVLRERLTGSPCA nsp4 709371037113712371337143 GCAUUCAUUGGAGAUGACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGAC AFIGDDNIVKGVKSDKLMAD nsp4 715371637173718371937203 AGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAGAUUAUAGAUGCUGUGGUGGGCGAGAAA RCATWLNMEVKIIDAVVGEK nsp4 721372237233724372537263 GCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGU APYFCGGEILCDSVTGTACR nsp4 727372837293730373137323 GUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUAGGCAAACCUCUGGCAGCAGACGAUGAA VADPLKRLFKLGKPLAADDE nsp4 733373437353736373737383 CAUGAUGAUGACAGGAGAAGGGCAUUGCAUGAGGAGUCAACACGCUGGAACCGAGUGGGU HDDDRRRALHEESTRWNRVG nsp4 739374037413742374337443 AUUCUUUCAGAGCUGUGCAAGGCAGUAGAAUCAAGGUAUGAAACCGUAGGAACUUCCAUC ILSELCKAVESRYETVGTSI nsp4 745374637473748374937503 AUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUCAGCUACCUGAGAGGG IVMAMTTLASSVKSFSYLRG nsp4 751375237527 GCCCCUAUAACUCUCUACGGCUAA APITLYG* nsp4 75377547755775677568 CCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGACUAGU virUTR 757875887598760876187628 AUGUUUGUGUUUCUUGUGCUGCUGCCUCUUGUGUCUUCUCAGUGUGUGGUGAGAUUUCCA MFVFLVLLPLVSSQCVVRFP RBD(Sprotein) 763876487658766876787688 AAUAUUACAAAUCUGUGUCCAUUUGGAGAAGUGUUUAAUGCAACAAGAUUUGCAUCUGUG NITNLCPFGEVFNATRFASV RBD(Sprotein) 769877087718772877387748 UAUGCAUGGAAUAGAAAAAGAAUUUCUAAUUGUGUGGCUGAUUAUUCUGUGCUGUAUAAU YAWNRKRISNCVADYSVLYN RBD(Sprotein) 775877687778778877987808 AGUGCUUCUUUUUCCACAUUUAAAUGUUAUGGAGUGUCUCCAACAAAAUUAAAUGAUUUA SASFSTFKCYGVSPTKLNDL RBD(Sprotein) 781878287838784878587868 UGUUUUACAAAUGUGUAUGCUGAUUCUUUUGUGAUCAGAGGUGAUGAAGUGAGACAGAUU CFTNVYADSFVIRGDEVRQI RBD(Sprotein) 787878887898790879187928 GCCCCCGGACAGACAGGAAAAAUUGCUGAUUACAAUUACAAACUGCCUGAUGAUUUUACA APGQTGKIADYNYKLPDDFT RBD(Sprotein) 793879487958796879787988 GGAUGUGUGAUUGCUUGGAAUUCUAAUAAUUUAGAUUCUAAAGUGGGAGGAAAUUACAAU GCVIAWNSNNLDSKVGGNYN RBD(Sprotein) 799880088018802880388048 UAUCUGUACAGACUGUUUAGAAAAUCAAAUCUGAAACCUUUUGAAAGAGAUAUUUCAACA YLYRLFRKSNLKPFERDIST RBD(Sprotein) 805880688078808880988108 GAAAUUUAUCAGGCUGGAUCAACACCUUGUAAUGGAGUGGAAGGAUUUAAUUGUUAUUUU EIYQAGSTPCNGVEGFNCYF RBD(Sprotein) 811881288138814881588168 CCAUUACAGAGCUAUGGAUUUCAGCCAACCAAUGGUGUGGGAUAUCAGCCAUAUAGAGUG PLQSYGEQPTNGVGYQPYRV RBD(Sprotein) 817881888198820882188222 GUGGUGCUGUCUUUUGAACUGCUGCAUGCACCUGCAACAGUGUGUGGACCUAAA VVLSFELLHAPATVCGPK RBD(Sprotein) 823282428249 GGCUCCCCCGGCUCCGGCUCCGGAUCU GSPGSGSGS GSlinker 825982698279828982998309 GGUUAUAUUCCUGAAGCUCCAAGAGAUGGGCAAGCUUACGUUCGUAAAGAUGGCGAAUGG GYIPEAPRDGQAYVRKDGEW fibritin 831983298330 GUAUUACUUUCUACCUUUUUA VLLSTEL fibritin 83408345 GGAAGCGGCAGCGGA GSGSG GSlinker 835583658375838583958405 UCUGAACAGUACAUUAAAUGGCCUUGGUACAUUUGGCUUGGAUUUAUUGCAGGAUUAAUU SEQYIKWPWYIWLGFIAGLI TM 841584258435844584558465 GCAAUUGUGAUGGUGACAAUUAUGUUAUGUUGUAUGACAUCAUGUUGUUCUUGUUUAAAA AIVMVTIMLCCMTSCCSCLK TM 847584858495850585158525 GGAUGUUGUUCUUGUGGAAGCUGUUGUAAAUUUGAUGAAGAUGAUUCUGAACCUGUGUUA GCCSCGSCCKFDEDDSEPVL TM 853585458555 AAAGGAGUGAAAUUGCAUUACACAUGAUGA KGVKLHYT** TM 856585758585859586058615 CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG FIelement 862586358645865586658675 AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC FIelement 868586958705871587258735 UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAG FIelement 874587558765877587858795 CCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAA FIelement 880588158825883588458855 GCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCGCGGCCGCAUGAAUAC FIelement 886588758885889589058915 AGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAU FIelement 892589358945895589658969 UUUUAUUUUAUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUC FIelement 897989898999900990199029 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAA Poly(A) 90399049905990699079 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Poly(A)

(499) In some embodiments, vaccine RNA described herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 16, 17, 19, 20, 21, 24, 25, 26, 27, 30, and 32. A particularly preferred vaccine RNA described herein comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 15, 17, 19, 21, 25, 26, 30, and 32 such as selected from the group consisting of SEQ ID NO: 17, 19, 21, 26, 30, and 32.

(500) RNA described herein is preferably formulated in lipid nanoparticles (LNP). In one embodiment, the LNP comprise a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA. In one embodiment, the cationic lipid is ALC-0315, the neutral lipid is DSPC, the steroid is cholesterol, and the polymer conjugated lipid is ALC-0159. The preferred mode of administration is intramuscular administration, more preferably in aqueous cryoprotectant buffer for intramuscular administration. The drug product is a preferably a preservative-free, sterile dispersion of RNA formulated in lipid nanoparticles (LNP) in aqueous cryoprotectant buffer for intramuscular administration.

(501) In different embodiments, the drug product comprises the components shown below, preferably at the proportions or concentrations shown below:

(502) TABLE-US-00017 Component Function Proportion (mol %) ALC-0315 .sub.[1] Functional lipid 47.5 ALC-O159 .sub.[2] Functional lipid 1.8 DSPC .sub.[3] Structural lipid 10.0 Cholesterol, synthetic Structural lipid 40.7 Component Function Concentration (mg/mL) Drug Substance Active 0.5 ALC-0315 .sub.[1] Functional lipid 7.17 ALC-0159 .sub.[2] Functional lipid 0.89 DSPC .sub.[3] Structural lipid 1.56 Cholesterol, synthetic Structural lipid 3.1 Sucrose Cryoprotectant 102.69 NaCl Buffer 6.0 KCl Buffer 0.15 Na.sub.2HPO.sub.4 Buffer 1.08 KH.sub.2PO.sub.4 Buffer 0.18 Water for injection Solvent/Vehicle q.s. Drug Substance Active 1.0 ALC-0315 .sub.[1] Functional lipid 13.56 ALC-0159 .sub.[2] Functional lipid 1.77 DSPC .sub.[3] Structural lipid 3.11 Cholesterol, synthetic Structural lipid 6.20 Sucrose Cryoprotectant 102.69 NaCl Buffer 6.0 KCl Buffer 0.15 Na.sub.2HPO.sub.4 Buffer 1.08 KH.sub.2PO.sub.4 Buffer 0.15 Water for injection Solvent/Vehicle q.s. .sub.[1] ALC-0315 = ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)/6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate .sub.[2] ALC-0159 = 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide/2-[2-(?-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide .sub.[3] DSPC = 1,2-Distearoyl-sn-glycero-3-phosphocholine q.s. = quantum satis (as much as may suffice)

(503) ##STR00015##

(504) In one embodiment, the ratio of mRNA to total lipid (N/P) is between 6.0 and 6.5 such as about 6.0 or about 6.3.

(505) Nucleic Acid Containing Particles

(506) Nucleic acids described herein such as RNA encoding a vaccine antigen may be administered formulated as particles.

(507) In the context of the present disclosure, the term particle relates to a structured entity formed by molecules or molecule complexes. In one embodiment, the term particle relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure dispersed in a medium. In one embodiment, a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof.

(508) Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. In one embodiment, a nucleic acid particle is a nanoparticle.

(509) As used in the present disclosure, nanoparticle refers to a particle having an average diameter suitable for parenteral administration.

(510) A nucleic acid particle can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

(511) Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and/or the cationic polymer combine together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.

(512) In one embodiment, particles described herein further comprise at least one lipid or lipid-like material other than a cationic or cationically ionizable lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof

(513) In some embodiments, nucleic acid particles comprise more than one type of nucleic acid molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features,

(514) Nucleic acid particles described herein may have an average diameter that in one embodiment ranges from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about 300 nm.

(515) Nucleic acid particles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the nucleic acid particles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

(516) With respect to RNA lipid particles, the N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, RNA is considered to be completely bound to nanoparticles.

(517) Nucleic acid particles described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles.

(518) The term colloid as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term colloid only refers to the particles in the mixture and not the entire suspension.

(519) For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted. The most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media).

(520) In the film hydration method, lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included.

(521) Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.

(522) The term ethanol injection technique refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example lipid vesicle formation such as liposome formation. Generally, the RNA lipoplex particles described herein are obtainable by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in one embodiment, formed as follows: an ethanol solution comprising lipids, such as cationic lipids and additional lipids, is injected into an aqueous solution under stirring. In one embodiment, the RNA lipoplex particles described herein are obtainable without a step of extrusion.

(523) The term extruding or extrusion refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores.

(524) Other methods having organic solvent free characteristics may also be used according to the present disclosure for preparing a colloid.

(525) LNPs typically comprise four components: ionizable cationic lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer conjugated lipid such as polyethylene glycol (PEG)-lipids. Each component is responsible for payload protection, and enables effective intracellular delivery. LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with nucleic acid in an aqueous buffer.

(526) The term average diameter refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z.sub.average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here average diameter, diameter or size for particles is used synonymously with this value of the Z.sub.average.

(527) The polydispersity index is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the average diameter. Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.

(528) Different types of nucleic acid containing particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

(529) The present disclosure describes particles comprising nucleic acid, at least one cationic or cationically ionizable lipid or lipid-like material, and/or at least one cationic polymer which associate with nucleic acid to form nucleic acid particles and compositions comprising such particles. The nucleic acid particles may comprise nucleic acid which is complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, in particular infectious viral particles, i.e., they are not able to virally infect cells. Suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers are those that form nucleic acid particles and are included by the term particle forming components or particle forming agents. The term particle forming components or particle forming agents relates to any components which associate with nucleic acid to form nucleic acid particles. Such components include any component which can be part of nucleic acid particles.

(530) Cationic Polymer

(531) Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically condense the negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. In addition, some investigators have synthesized polymers specifically for nucleic acid delivery. Poly(S3-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

(532) A polymer, as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties such as those described herein.

(533) If more than one type of repeat unit is present within the polymer, then the polymer is said to be a copolymer. It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a block copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

(534) In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.

(535) In certain embodiments, polymer may be protamine or polyalkyleneimine, in particular protamine.

(536) The term protamine refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term protamine refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

(537) According to the disclosure, the term protamine as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

(538) In one embodiment, the polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75.10.sup.2 to 10.sup.7 Da, preferably 1000 to 10.sup.5 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

(539) Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).

(540) Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In one embodiment, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

(541) Particles described herein may also comprise polymers other than cationic polymers, i.e., non-cationic polymers and/or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

(542) Lipid and Lipid-Like Material

(543) The terms lipid and lipid-like material are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

(544) As used herein, the term amphiphilic refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

(545) The term lipid-like material, lipid-like compound or lipid-like molecule relates to substances that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. As used herein, the term lipid is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.

(546) Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.

(547) In certain embodiments, the amphiphilic compound is a lipid. The term lipid refers to a group of organic compounds that are characterized by being insoluble in water, but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

(548) Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides. Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word triacylglycerol is sometimes used synonymously with triglyceride. In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.

(549) The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived tails by ester linkages and to one head group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

(550) Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.

(551) Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.

(552) Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

(553) Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

(554) According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

(555) Cationic or Cationically Ionizable Lipids or Lipid-Like Materials

(556) The nucleic acid particles described herein may comprise at least one cationic or cationically ionizable lipid or lipid-like material as particle forming agent. Cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In one embodiment, cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

(557) As used herein, a cationic lipid or cationic lipid-like material refers to a lipid or lipid-like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

(558) In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

(559) For purposes of the present disclosure, such cationically ionizable lipids or lipid-like materials are comprised by the term cationic lipid or lipid-like material unless contradicted by the circumstances.

(560) In one embodiment, the cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.

(561) Examples of cationic lipids include, but are not limited to 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N(N,N-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), I,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5-(cholest-5-en-3-beta-oxy)-3-oxapentoxy)-3-dimethyl-1-(cis,cis-9,12-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (?)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (?)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (?)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (?AE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3?)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl) 8,8-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N.sub.12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200).

(562) In some embodiments, the cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle.

(563) Additional Lipids or Lipid-Like Materials

(564) Particles described herein may also comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. Optimizing the formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may enhance particle stability and efficacy of nucleic acid delivery.

(565) An additional lipid or lipid-like material may be incorporated which may or may not affect the overall charge of the nucleic acid particles. In certain embodiments, the additional lipid or lipid-like material is a non-cationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. As used herein, an anionic lipid refers to any lipid that is negatively charged at a selected pH. As used herein, a neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2-hydroxyethyl ether, cholesteryl-4-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

(566) Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains. In certain preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol.

(567) In certain embodiments, the nucleic acid particles include both a cationic lipid and an additional lipid.

(568) In one embodiment, particles described herein include a polymer conjugated lipid such as a pegylated lipid. The term pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.

(569) Without wishing to be bound by theory, the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

(570) In some embodiments, the non-cationic lipid, in particular neutral lipid, (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in the particle.

(571) Lipoplex Particles

(572) In certain embodiments of the present disclosure, the RNA described herein may be present in RNA lipoplex particles.

(573) In the context of the present disclosure, the term RNA lipoplex particle relates to a particle that contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, a RNA lipoplex particle is a nanoparticle.

(574) In certain embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

(575) In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

(576) RNA lipoplex particles described herein have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

(577) The RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration. The RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM.

(578) Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.

(579) Lipid Nanoparticles (LNPs)

(580) In one embodiment, nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs). The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

(581) In one embodiment, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

(582) In one embodiment, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle. In one embodiment, the LNP comprises from 40 to 55 mol percent, from 40 to 50 mol percent, from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol percent, from 47 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid. In one embodiment, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the cationic lipid.

(583) In one embodiment, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent. In one embodiment, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent.

(584) In one embodiment, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 35 to 45 mol percent or from 38 to 43 mol percent. In one embodiment, the steroid is present in a concentration of about 40, 41, 42, 43, 44, 45 or 46 mol percent.

(585) In one embodiment, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer conjugated lipid.

(586) In one embodiment, the LNP comprises from 40 to 50 mol percent a cationic lipid; from 5 to 15 mol percent of a neutral lipid; from 35 to 45 mol percent of a steroid; from 1 to 10 mol percent of a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

(587) In one embodiment, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle.

(588) In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In one embodiment, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In one embodiment, the neutral lipid is DSPC.

(589) In one embodiment, the steroid is cholesterol.

(590) In one embodiment, the polymer conjugated lipid is a pegylated lipid. In one embodiment, the pegylated lipid has the following structure:

(591) ##STR00016##

(592) or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

(593) R.sup.12 and R.sup.13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In one embodiment, R.sup.12 and R.sup.13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In one embodiment, w has a mean value ranging from 40 to 55. In one embodiment, the average w is about 45. In one embodiment, R.sup.12 and R.sup.13 are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.

(594) In one embodiment, the pegylated lipid is DMG-PEG 2000, e.g., having the following structure:

(595) ##STR00017##

(596) In some embodiments, the cationic lipid component of the LNPs has the structure of Formula (III):

(597) ##STR00018## or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O, and the other of L.sup.1 or L.sup.2 is O(C?O), (C?O)O, C(?O), O, S(O).sub.x, SS, C(?O)S, SC(?O), NR.sup.aC(?O), C(?O)NR.sup.a, NR.sup.aC(?O)NR.sup.a, OC(?O)NR.sup.a or NR.sup.aC(?O)O or a direct bond; G.sup.1 and G.sup.2 are each independently unsubstituted C.sub.1-C.sub.12 alkylene or C.sub.1-C.sub.12 alkenylene; G.sup.3 is C.sub.1-C.sub.24 alkylene, C.sub.1-C.sub.24 alkenylene, C.sub.3-C.sub.5 cycloalkylene, C.sub.3-C.sub.5 cycloalkenylene; R.sup.a is H or C.sub.1-C.sub.12 alkyl; R.sup.1 and R.sup.2 are each independently C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24 alkenyl; R.sup.3 is H, OR.sup.5, CN, C(?O)OR.sup.4, OC(?O)R.sup.4 or NR.sup.5C(?O)R.sup.4; R.sup.4 is C.sub.1-C.sub.12 alkyl; R.sup.5 is H or C.sub.1-C.sub.6 alkyl; and x is 0, 1 or 2.

(598) In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

(599) ##STR00019## wherein: A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R.sup.6 is, at each occurrence, independently H, OH or C.sub.1-C.sub.24 alkyl; n is an integer ranging from 1 to 15.

(600) In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

(601) In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID):

(602) ##STR00020##

(603) wherein y and z are each independently integers ranging from 1 to 12.

(604) In any of the foregoing embodiments of Formula (III), one of L.sup.1 or L.sup.2 is O(C?O). For example, in some embodiments each of L.sup.1 and L.sup.2 are O(C?O). In some different embodiments of any of the foregoing, L and L.sup.2 are each independently (C?O)O or O(C?O). For example, in some embodiments each of L.sup.1 and L.sup.2 is (C?O)O.

(605) In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

(606) ##STR00021##

(607) In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

(608) ##STR00022##

(609) In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

(610) In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

(611) In some of the foregoing embodiments of Formula (III), R.sup.6 is H. In other of the foregoing embodiments, R.sup.6 is C.sub.1-C.sub.24 alkyl. In other embodiments, R.sup.6 is OH.

(612) In some embodiments of Formula (III), G.sup.3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G.sup.3 is linear C.sub.1-C.sub.24 alkylene or linear C.sub.1-C.sub.24 alkenylene.

(613) In some other foregoing embodiments of Formula (III), R.sup.1 or R.sup.2, or both, is C.sub.6-C.sub.24 alkenyl. For example, in some embodiments, R.sup.1 and R.sup.2 each, independently have the following structure:

(614) ##STR00023##

(615) wherein: R.sup.7a and R.sup.7b are, at each occurrence, independently H or C.sub.1-C.sub.12 alkyl; and a is an integer from 2 to 12, wherein R.sup.7a, R.sup.7b and a are each selected such that R.sup.1 and R.sup.2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

(616) In some of the foregoing embodiments of Formula (III), at least one occurrence of R.sup.7a is H. For example, in some embodiments, R.sup.7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R.sup.7b is C.sub.1-C.sub.8 alkyl. For example, in some embodiments, C.sub.1-C.sub.8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

(617) In different embodiments of Formula (III), R.sup.1 or R.sup.2, or both, has one of the following structures:

(618) ##STR00024##

(619) In some of the foregoing embodiments of Formula (III), R.sup.3 is OH, CN, C(?O)OR.sup.4, OC(?O)R.sup.4 or NHC(?O)R.sup.4. In some embodiments, R.sup.4 is methyl or ethyl.

(620) In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in the table below.

(621) Representative Compounds of Formula (III).

(622) TABLE-US-00018 No. Structure III-1 III-2 III-3 III-4 III-5 III-6 0 III-7 III-8 III-9 III-10 III-11 III-12 III-13 III-14 III-15 III-16 0 III-17 III-18 III-19 III-20 III-21 III-22 III-23 III-24 III-25 III-26 0 III-27 III-28 III-29 III-30 III-31 III-32 III-33 III-34 III-35 III-36 0

(623) In some embodiments, the LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is ALC-0159.

(624) In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to about 50 mole percent. In one embodiment, the neutral lipid is present in the LNP in an amount from about 5 to about 15 mole percent. In one embodiment, the steroid is present in the LNP in an amount from about 35 to about 45 mole percent. In one embodiment, the pegylated lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In some embodiments, the LNP comprises compound III-3 in an amount from about 40 to about 50 mole percent, DSPC in an amount from about 5 to about 15 mole percent, cholesterol in an amount from about 35 to about 45 mole percent, and ALC-0159 in an amount from about 1 to about 10 mole percent.

(625) In some embodiments, the LNP comprises compound III-3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent.

(626) In various different embodiments, the cationic lipid has one of the structures set forth in the table below.

(627) TABLE-US-00019 No. Structure A B C D E F

(628) In some embodiments, the LNP comprises a cationic lipid shown in the above table, e.g., a cationic lipid of Formula (B) or Formula (D), in particular a cationic lipid of Formula (D), RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000.

(629) In one embodiment, the LNP comprises a cationic lipid that is an ionizable lipid-like material (lipidoid). In one embodiment, the cationic lipid has the following structure:

(630) ##STR00067##

(631) The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In one embodiment, the N/P value is about 6.

(632) LNP described herein may have an average diameter that in one embodiment ranges from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm.

(633) RNA Targeting

(634) Some aspects of the disclosure involve the targeted delivery of the RNA disclosed herein (e.g., RNA encoding vaccine antigens and/or immunostimulants).

(635) In one embodiment, the disclosure involves targeting lung. Targeting lung is in particular preferred if the RNA administered is RNA encoding vaccine antigen. RNA may be delivered to lung, for example, by administering the RNA which may be formulated as particles as described herein, e.g., lipid particles, by inhalation.

(636) In one embodiment, the disclosure involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen is in particular preferred if the RNA administered is RNA encoding vaccine antigen.

(637) In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen.

(638) The lymphatic system is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating lymph. The primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and the bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, which include lymph nodes and the spleen, maintain mature na?ve lymphocytes and initiate an adaptive immune response.

(639) RNA may be delivered to spleen by so-called lipoplex formulations, in which the RNA is bound to liposomes comprising a cationic lipid and optionally an additional or helper lipid to form injectable nanoparticle formulations. The liposomes may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. RNA lipoplex particles may be prepared by mixing the liposomes with RNA. Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.

(640) The electric charge of the RNA lipoplex particles of the present disclosure is the sum of the electric charges present in the at least one cationic lipid and the electric charges present in the RNA. The charge ratio is the ratio of the positive charges present in the at least one cationic lipid to the negative charges present in the RNA. The charge ratio of the positive charges present in the at least one cationic lipid to the negative charges present in the RNA is calculated by the following equation: charge ratio=[(cationic lipid concentration (mol))*(the total number of positive charges in the cationic lipid)]/[(RNA concentration (mol))*(the total number of negative charges in RNA)].

(641) The spleen targeting RNA lipoplex particles described herein at physiological pH preferably have a net negative charge such as a charge ratio of positive charges to negative charges from about 1.9:2 to about 1:2, or about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In specific embodiments, the charge ratio of positive charges to negative charges in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

(642) Immunostimulants may be provided to a subject by administering to the subject RNA encoding an immunostimulant in a formulation for preferential delivery of RNA to liver or liver tissue. The delivery of RNA to such target organ or tissue is preferred, in particular, if it is desired to express large amounts of the immunostimulant and/or if systemic presence of the immunostimulant, in particular in significant amounts, is desired or required.

(643) RNA delivery systems have an inherent preference to the liver. This pertains to lipid-based particles, cationic and neutral nanoparticles, in particular lipid nanoparticles such as liposomes, nanomicelles and lipophilic ligands in bioconjugates. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or the lipid metabolism (liposomes and lipid or cholesterol conjugates).

(644) For in vivo delivery of RNA to the liver, a drug delivery system may be used to transport the RNA into the liver by preventing its degradation. For example, polyplex nanomicelles consisting of a poly(ethylene glycol) (PEG)-coated surface and an mRNA-containing core is a useful system because the nanomicelles provide excellent in vivo stability of the RNA, under physiological conditions. Furthermore, the stealth property provided by the polyplex nanomicelle surface, composed of dense PEG palisades, effectively evades host immune defenses.

(645) Examples of suitable immunostimulants for targeting liver are cytokines involved in T cell proliferation and/or maintenance. Examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.

(646) In another embodiment, RNA encoding an immunostimulant may be administered in a formulation for preferential delivery of RNA to the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. The delivery of an immunostimulant to such target tissue is preferred, in particular, if presence of the immunostimulant in this organ or tissue is desired (e.g., for inducing an immune response, in particular in case immunostimulants such as cytokines are required during T-cell priming or for activation of resident immune cells), while it is not desired that the immunostimulant is present systemically, in particular in significant amounts (e.g., because the immunostimulant has systemic toxicity).

(647) Examples of suitable immunostimulants are cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-?, or IFN-3, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended-PK cytokines.

(648) Immunostimulants

(649) In one embodiment, the RNA encoding vaccine antigen may be non-immunogenic. In this and other embodiments, the RNA encoding vaccine antigen may be co-administered with an immunostimulant or RNA encoding an immunostimulant. The methods and agents described herein are particularly effective if the immunostimulant is attached to a pharmacokinetic modifying group (hereafter referred to as extended-pharmacokinetic (PK) immunostimulant). The methods and agents described herein are particularly effective if the immunostimulant is administered in the form of RNA encoding an immunostimulant. In one embodiment, said RNA is targeted to the liver for systemic availability. Liver cells can be efficiently transfected and are able to produce large amounts of protein.

(650) An immunostimulant is any substance that stimulates the immune system by inducing activation or increasing activity of any of the immune system's components, in particular immune effector cells. The immunostimulant may be pro-inflammatory.

(651) According to one aspect, the immunostimulant is a cytokine or a variant thereof. Examples of cytokines include interferons, such as interferon-alpha (IFN-?) or interferon-gamma (IFN-?), interleukins, such as IL2, IL7, IL12, IL15 and IL23, colony stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor. According to another aspect, the immunostimulant includes an adjuvant-type immunostimulatory agent such as APC Toll-like Receptor agonists or costimulatory/cell adhesion membrane proteins. Examples of Toll-like Receptor agonists include costimulatory/adhesion proteins such as CD80, CD86, and ICAM-1.

(652) Cytokines are a category of small proteins (?5-20 kDa) that are important in cell signaling. Their release has an effect on the behavior of cells around them. Cytokines are involved in autocrine signaling, paracrine signaling and endocrine signaling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine may be produced by more than one type of cell. Cytokines act through receptors, and are especially important in the immune system; cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways.

(653) According to the disclosure, a cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof. A cytokine may be human cytokine and may be derived from any vertebrate, especially any mammal. One particularly preferred cytokine is interferon-?.

(654) Interferons

(655) Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and also tumor cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defenses.

(656) Based on the type of receptor through which they signal, interferons are typically divided among three classes: type I interferon, type II interferon, and type III interferon.

(657) All type I interferons bind to a specific cell surface receptor complex known as the IFN-?/? receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains.

(658) The type I interferons present in humans are IFN?, IFN?, IFN?, IFN? and IFN?. In general, type I interferons are produced when the body recognizes a virus that has invaded it. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, which leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA.

(659) The IFN? proteins are produced mainly by plasmacytoid dendritic cells (pDCs). They are mainly involved in innate immunity against viral infection. The genes responsible for their synthesis come in 13 subtypes that are called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21. These genes are found together in a cluster on chromosome 9.

(660) The IFN? proteins are produced in large quantities by fibroblasts. They have antiviral activity that is involved mainly in innate immune response. Two types of IFN? have been described, IFN31 and IFN33. The natural and recombinant forms of IFN31 have antiviral, antibacterial, and anticancer properties.

(661) Type II interferon (IFN? in humans) is also known as immune interferon and is activated by IL12. Furthermore, type II interferons are released by cytotoxic T cells and T helper cells. Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Although discovered more recently than type I and type II IFNs, recent information demonstrates the importance of type III IFNs in some types of virus or fungal infections.

(662) In general, type I and II interferons are responsible for regulating and activating the immune response.

(663) According to the disclosure, a type I interferon is preferably IFN? or IFN?, more preferably IFN?.

(664) According to the disclosure, an interferon may be a naturally occurring interferon or a functional fragment or variant thereof. An interferon may be human interferon and may be derived from any vertebrate, especially any mammal.

(665) Interleukins

(666) Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that can be divided into four major groups based on distinguishing structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes more than 50 interleukins and related proteins.

(667) According to the disclosure, an interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. An interleukin may be human interleukin and may be derived from any vertebrate, especially any mammal.

(668) Extended-PK Group

(669) Immunostimulant polypeptides described herein can be prepared as fusion or chimeric polypeptides that include an immunostimulant portion and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulant). The immunostimulant may be fused to an extended-PK group, which increases circulation half-life. Non-limiting examples of extended-PK groups are described infra. It should be understood that other PK groups that increase the circulation half-life of immunostimulants such as cytokines, or variants thereof, are also applicable to the present disclosure. In certain embodiments, the extended-PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

(670) As used herein, the term PK is an acronym for pharmacokinetic and encompasses properties of a compound including, by way of example, absorption, distribution, metabolism, and elimination by a subject. As used herein, an extended-PK group refers to a protein, peptide, or moiety that increases the circulation half-life of a biologically active molecule when fused to or administered together with the biologically active molecule. Examples of an extended-PK group include serum albumin (e.g., HSA), Immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005/0287153 and 2007/0003549). Other exemplary extended-PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 July; 16(7):903-15 which is herein incorporated by reference in its entirety. As used herein, an extended-PK immunostimulant refers to an immunostimulant moiety in combination with an extended-PK group. In one embodiment, the extended-PK immunostimulant is a fusion protein in which an immunostimulant moiety is linked or fused to an extended-PK group.

(671) In certain embodiments, the serum half-life of an extended-PK immunostimulant is increased relative to the immunostimulant alone (i.e., the immunostimulant not fused to an extended-PK group). In certain embodiments, the serum half-life of the extended-PK immunostimulant is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer relative to the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended-PK immunostimulant is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5 fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended-PK immunostimulant is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

(672) As used herein, half-life refers to the time taken for the serum or plasma concentration of a compound such as a peptide or protein to reduce by 50%, in vivo, for example due to degradation and/or clearance or sequestration by natural mechanisms. An extended-PK immunostimulant suitable for use herein is stabilized in vivo and its half-life increased by, e.g., fusion to serum albumin (e.g., HSA or MSA), which resist degradation and/or clearance or sequestration. The half-life can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art, and may for example generally involve the steps of suitably administering a suitable dose of the amino acid sequence or compound to a subject; collecting blood samples or other samples from said subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in said blood sample; and calculating, from (a plot of) the data thus obtained, the time until the level or concentration of the amino acid sequence or compound has been reduced by 50% compared to the initial level upon dosing. Further details are provided in, e.g., standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

(673) In certain embodiments, the extended-PK group includes serum albumin, or fragments thereof or variants of the serum albumin or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term albumin). Polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form albumin fusion proteins. Such albumin fusion proteins are described in U.S. Publication No. 20070048282.

(674) As used herein, albumin fusion protein refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a protein such as a therapeutic protein, in particular an immunostimulant. The albumin fusion protein may be generated by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding an albumin. The therapeutic protein and albumin, once part of the albumin fusion protein, may each be referred to as a portion, region or moiety of the albumin fusion protein (e.g., a therapeutic protein portion or an albumin protein portion). In a highly preferred embodiment, an albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to a mature form of the therapeutic protein) and at least one molecule of albumin (including but not limited to a mature form of albumin). In one embodiment, an albumin fusion protein is processed by a host cell such as a cell of the target organ for administered RNA, e.g. a liver cell, and secreted into the circulation. Processing of the nascent albumin fusion protein that occurs in the secretory pathways of the host cell used for expression of the RNA may include, but is not limited to signal peptide cleavage; formation of disulfide bonds; proper folding; addition and processing of carbohydrates (such as for example, N- and O-linked glycosylation); specific proteolytic cleavages; and/or assembly into multimeric proteins. An albumin fusion protein is preferably encoded by RNA in a non-processed form which in particular has a signal peptide at its N-terminus and following secretion by a cell is preferably present in the processed form wherein in particular the signal peptide has been cleaved off. In a most preferred embodiment, the processed form of an albumin fusion protein refers to an albumin fusion protein product which has undergone N-terminal signal peptide cleavage, herein also referred to as a mature albumin fusion protein. In preferred embodiments, albumin fusion proteins comprising a therapeutic protein have a higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the time period between when the therapeutic protein is administered in vivo and carried into the bloodstream and when the therapeutic protein is degraded and cleared from the bloodstream, into an organ, such as the kidney or liver, that ultimately clears the therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of the therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

(675) As used herein, albumin refers collectively to albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, albumin refers to human albumin or fragments or variants thereof especially the mature form of human albumin, or albumin from other vertebrates or fragments thereof, or variants of these molecules. The albumin may be derived from any vertebrate, especially any mammal, for example human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be from a different animal than the therapeutic protein portion.

(676) In certain embodiments, the albumin is human serum albumin (HSA), or fragments or variants thereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.

(677) The terms, human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms, albumin and serum albumin are broader, and encompass human serum albumin (and fragments and variants thereof) as well as albumin from other species (and fragments and variants thereof).

(678) As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of the therapeutic protein refers to a fragment of albumin sufficient in length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein so that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability in the non-fusion state.

(679) The albumin portion of the albumin fusion proteins may comprise the full length of the albumin sequence, or may include one or more fragments thereof that are capable of stabilizing or prolonging the therapeutic activity or plasma stability. Such fragments may be of 10 or more amino acids in length or may include about 15, 20, 25, 30, 50, or more contiguous amino acids from the albumin sequence or may include part or all of specific domains of albumin. For instance, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

(680) Generally speaking, an albumin fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.

(681) According to the disclosure, albumin may be naturally occurring albumin or a fragment or variant thereof. Albumin may be human albumin and may be derived from any vertebrate, especially any mammal.

(682) Preferably, the albumin fusion protein comprises albumin as the N-terminal portion, and a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising albumin as the C-terminal portion, and a therapeutic protein as the N-terminal portion may also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused at the N- and C-termini are the same therapeutic proteins. In another preferred embodiment, the therapeutic proteins fused at the N- and C-termini are different therapeutic proteins. In one embodiment, the different therapeutic proteins are both cytokines.

(683) In one embodiment, the therapeutic protein(s) is (are) joined to the albumin through (a) peptide linker(s). A linker peptide between the fused portions may provide greater physical separation between the moieties and thus maximize the accessibility of the therapeutic protein portion, for instance, for binding to its cognate receptor. The linker peptide may consist of amino acids such that it is flexible or more rigid. The linker sequence may be cleavable by a protease or chemically.

(684) As used herein, the term Fc region refers to the portion of a native immunoglobulin formed by the respective Fc domains (or Fc moieties) of its two heavy chains. As used herein, the term Fc domain refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain wherein the Fc domain does not comprise an Fv domain. In certain embodiments, an Fc domain begins in the hinge region just upstream of the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of: a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). An Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy-chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and/or CH3 domains as well as fragments of such peptides comprising only, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derived from an immunoglobulin of any species and/or any subtype, including, but not limited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody. The Fc domain encompasses native Fc and Fc variant molecules. As set forth herein, it will be understood by one of ordinary skill in the art that any Fc domain may be modified such that it varies in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., Fc?R binding).

(685) The Fc domains of a polypeptide described herein may be derived from different immunoglobulin molecules. For example, an Fc domain of a polypeptide may comprise a CH2 and/or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, an Fc domain can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

(686) In certain embodiments, an extended-PK group includes an Fc domain or fragments thereof or variants of the Fc domain or fragments thereof (all of which for the purpose of the present disclosure are comprised by the term Fc domain). The Fc domain does not contain a variable region that binds to antigen. Fc domains suitable for use in the present disclosure may be obtained from a number of different sources. In certain embodiments, an Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is from a human IgG1 constant region. It is understood, however, that the Fc domain may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species. Moreover, the Fc domain (or a fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

(687) A variety of Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains comprising an Fc domain sequence can be selected lacking a particular effector function and/or with a particular modification to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published and suitable Fc domain sequences (e.g. hinge, CH2, and/or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art recognized techniques.

(688) In certain embodiments, the extended-PK group is a serum albumin binding protein such as those described in US2005/0287153, US2007/0003549, US2007/0178082, US2007/0269422, US2010/0113339, WO2009/083804, and WO2009/133208, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is transferrin, as disclosed in U.S. Pat. Nos. 7,176,278 and 8,158,579, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is a serum immunoglobulin binding protein such as those disclosed in US2007/0178082, US2014/0220017, and US2017/0145062, which are herein incorporated by reference in their entirety. In certain embodiments, the extended-PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012/0094909, which is herein incorporated by reference in its entirety. Methods of making fibronectin-based scaffold domain proteins are also disclosed in US2012/0094909. A non-limiting example of a Fn3-based extended-PK group is Fn3(HSA), i.e., a Fn3 protein that binds to human serum albumin.

(689) In certain aspects, the extended-PK immunostimulant, suitable for use according to the disclosure, can employ one or more peptide linkers. As used herein, the term peptide linker refers to a peptide or polypeptide sequence which connects two or more domains (e.g., the extended-PK moiety and an immunostimulant moiety) in a linear amino acid sequence of a polypeptide chain. For example, peptide linkers may be used to connect an immunostimulant moiety to a HSA domain.

(690) Linkers suitable for fusing the extended-PK group to e.g. an immunostimulant are well known in the art. Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine-polypeptide linker, i.e., a peptide that consists of glycine and serine residues.

(691) In addition to, or in place of, the heterologous polypeptides described above, an immunostimulant polypeptide described herein can contain sequences encoding a marker or reporter. Examples of marker or reporter genes include ?-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin-B-hosphotransferase (HPH), thymidine kinase (TK), ?-galactosidase, and xanthine guanine phosphoribosyltransferase (XGPRT).

(692) Pharmaceutical Compositions

(693) The agents described herein may be administered in pharmaceutical compositions or medicaments and may be administered in the form of any suitable pharmaceutical composition.

(694) In one embodiment, the pharmaceutical composition described herein is an immunogenic composition for inducing an immune response against coronavirus in a subject. For example, in one embodiment, the immunogenic composition is a vaccine.

(695) In one embodiment of all aspects of the invention, the components described herein such as RNA encoding a vaccine antigen may be administered in a pharmaceutical composition which may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers etc. In one embodiment, the pharmaceutical composition is for therapeutic or prophylactic treatments, e.g., for use in treating or preventing a coronavirus infection.

(696) The term pharmaceutical composition relates to a formulation comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation.

(697) The pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants. The term adjuvant relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The cytokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFN?, IFN?, GM-CSF, LT-a. Further known adjuvants are aluminium hydroxide, Freund's adjuvant or oil such as Montanide? ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.

(698) The pharmaceutical compositions according to the present disclosure are generally applied in a pharmaceutically effective amount and in a pharmaceutically acceptable preparation.

(699) The term pharmaceutically acceptable refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

(700) The term pharmaceutically effective amount or therapeutically effective amount refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (oreffectively higher doses achieved by a different, more localized route of administration) may be used.

(701) The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.

(702) Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal. The term excipient as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

(703) The term diluent relates a diluting and/or thinning agent. Moreover, the term diluent includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water.

(704) The term carrier refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carrier include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure includes isotonic saline.

(705) Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

(706) Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

(707) In one embodiment, pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, parenteral administration refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration.

(708) The term co-administering as used herein means a process whereby different compounds or compositions (e.g., RNA encoding an antigen and RNA encoding an immunostimulant) are administered to the same patient. The different compounds or compositions may be administered simultaneously, at essentially the same time, or sequentially.

(709) The pharmaceutical compositions and products described herein may be provided as a frozen concentrate for solution for injection, e.g., at a concentration of 0.50 mg/mL. In one embodiment, for preparation of solution for injection, a drug product is thawed and diluted with isotonic sodium chloride solution (e.g., 0.9% NaCl, saline), e.g., by a one-step dilution process. In some embodiments, bacteriostatic sodium chloride solution (e.g., 0.9% NaCl, saline) cannot be used as a diluent. In some embodiments, a diluted drug product is an off-white suspension. The concentration of the final solution for injection varies depending on the respective dose level to be administered.

(710) In one embodiment, administration is performed within 6 h after begin of preparation due to the risk of microbial contamination and considering the multiple-dose approach of the preparation process. In one embodiment, in this period of 6 h, two conditions are allowed: room temperature for preparation, handling and transfer as well as 2 to 8? C. for storage. Compositions described herein may be shipped and/or stored under temperature-controlled conditions, e.g., temperature conditions of about 4-5? C. or below, about ?20? C. or below, ?70? C.?10? C. (e.g., ?80? C. to ?60? C.), e.g., utilizing a cooling system (e.g., that may be or include dry ice) to maintain the desired temperature. In one embodiment, compositions described herein are shipped in temperature-controlled thermal shippers. Such shippers may contain a GPS-enabled thermal sensor to track the location and temperature of each shipment. The compositions can be stored by refilling with, e.g., dry ice.

(711) Treatments

(712) The present invention provides methods and agents for inducing an adaptive immune response against coronavirus in a subject comprising administering an effective amount of a composition comprising RNA encoding a coronavirus vaccine antigen described herein. In one embodiment, the methods and agents described herein provide immunity in a subject to coronavirus, coronavirus infection, or to a disease or disorder associated with coronavirus. The present invention thus provides methods and agents for treating or preventing the infection, disease, or disorder associated with coronavirus.

(713) In one embodiment, the methods and agents described herein are administered to a subject having an infection, disease, or disorder associated with coronavirus. In one embodiment, the methods and agents described herein are administered to a subject at risk for developing the infection, disease, or disorder associated with coronavirus. For example, the methods and agents described herein may be administered to a subject who is at risk for being in contact with coronavirus. In one embodiment, the methods and agents described herein are administered to a subject who lives in, traveled to, or is expected to travel to a geographic region in which coronavirus is prevalent. In one embodiment, the methods and agents described herein are administered to a subject who is in contact with or expected to be in contact with another person who lives in, traveled to, or is expected to travel to a geographic region in which coronavirus is prevalent. In one embodiment, the methods and agents described herein are administered to a subject who has knowingly been exposed to coronavirus through their occupation, or other contact. In one embodiment, a coronavirus is SARS-CoV-2. In some embodiments, methods and agents described herein are administered to a subject with evidence of prior exposure to and/or infection with SARS-CoV-2 and/or an antigen or epitope thereof or cross-reactive therewith. For example, in some embodiments, methods and agents described herein are administered to a subject in whom antibodies, B cells, and/or T cells reactive with one or more epitopes of a SARS-CoV-2 spike protein are detectable and/or have been detected.

(714) For a composition to be useful as a vaccine, the composition must induce an immune response against the coronavirus antigen in a cell, tissue or subject (e.g., a human). In some embodiments, the composition induces an immune response against the coronavirus antigen in a cell, tissue or subject (e.g., a human). In some instances, the vaccine induces a protective immune response in a mammal. The therapeutic compounds or compositions of the invention may be administered prophylactically (i.e., to prevent a disease or disorder) or therapeutically (i.e., to treat a disease or disorder) to subjects suffering from, or at risk of (or susceptible to) developing a disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term prevent encompasses any activity, which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

(715) The present disclosure reports various characterization of provided compositions (see, e.g., Example 2; see also Examples thereafter) and furthermore establishes parameters for vaccines effective in humans.

(716) In some embodiments, administration of an immunogenic composition or vaccine of the present invention may be performed by single administration or boosted by multiple administrations.

(717) In some embodiments, an amount the RNA described herein from 0.1 ?g to 300 ?g, 0.5 ?g to 200 ?g, or 1 ?g to 100 ?g, such as about 1 ?g, about 3 ?g, about 10 ag, about 30 ag, about 50 ?g, or about 100 ?g may be administered per dose. In one embodiment, the invention envisions administration of a single dose. In one embodiment, the invention envisions administration of a priming dose followed by one or more booster doses. The booster dose or the first booster dose may be administered 7 to 28 days or 14 to 24 days following administration of the priming dose.

(718) In some embodiments, an amount of the RNA described herein of 60 ?g or lower, 50 ?g or lower, 40 ?g or lower, 30 ?g or lower, 20 ?g or lower, 10 ?g or lower, 5 ?g or lower, 2.5 ?g or lower, or 1 ?g or lower may be administered per dose.

(719) In some embodiments, an amount of the RNA described herein of at least 0.25 ?g, at least 0.5 ?g, at least 1 ?g, at least 2 ?g, at least 3 ?g, at least 4 ?g, at least 5 ?g, at least 10 ?g, at least 20 ?g, at least 30 ?g, or at least 40 ?g may be administered per dose.

(720) In some embodiments, an amount of the RNA described herein of 0.25 ?g to 60 ?g, 0.5 ?g to 55 ?g, 1 ?g to 50 ?g, 5 ?g to 40 ?g, or 10 ?g to 30 ?g may be administered per dose.

(721) In one embodiment, an amount of the RNA described herein of about 30 ?g is administered per dose. In one embodiment, at least two of such doses are administered. For example, a second dose may be administered about 21 days following administration of the first dose.

(722) In some embodiments, the efficacy of the RNA vaccine described herein (e.g., administered in two doses, wherein a second dose may be administered about 21 days following administration of the first dose, and administered, for example, in an amount of about 30 ag per dose) is at least 70%, at least 80%, at least 90, or at least 95% beginning 7 days after administration of the second dose (e.g., beginning 28 days after administration of the first dose if a second dose is administered 21 days following administration of the first dose). In some embodiments, such efficacy is observed in populations of age of at least 50, at least 55, at least 60, at least 65, at least 70, or older. In some embodiments, the efficacy of the RNA vaccine described herein (e.g., administered in two doses, wherein a second dose may be administered about 21 days following administration of the first dose, and administered, for example, in an amount of about 30 ?g per dose) beginning 7 days after administration of the second dose (e.g., beginning 28 days after administration of the first dose if a second dose is administered 21 days following administration of the first dose) in populations of age of at least 65, such as 65 to 80, 65 to 75, or 65 to 70, is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%. Such efficacy may be observed over time periods of up to 1 month, 2 months, 3 months, 6 months or even longer.

(723) In one embodiment, vaccine efficacy is defined as the percent reduction in the number of subjects with evidence of infection (vaccinated subjects vs. non-vaccinated subjects).

(724) In one embodiment, efficacy is assessed through surveillance for potential cases of COVID-19. If, at any time, a patient develops acute respiratory illness, for the purposes herein, the patient can be considered to potentially have COVID-19 illness. The assessments can include a nasal (midturbinate) swab, which may be tested using a reverse transcription-polymerase chain reaction (RT-PCR) test to detect SARS-CoV-2. In addition, clinical information and results from local standard-of-care tests can be assessed.

(725) In some embodiments, efficacy assessments may utilize a definition of SARS-CoV-2-related cases wherein: Confirmed COVID-19: presence of at least 1 of the following symptoms and SARS-CoV-2 NAAT (nucleic acid amplification-based test) positive during, or within 4 days before or after, the symptomatic period: fever; new or increased cough; new or increased shortness of breath; chills; new or increased muscle pain; new loss of taste or smell; sore throat; diarrhea; vomiting.

(726) Alternatively or additionally, in some embodiments, efficacy assessments may utilize a definition of SARS-CoV-2-related cases wherein one or more of the following additional symptoms defined by the CDC can be considered: fatigue; headache; nasal congestion or runny nose; nausea.

(727) In some embodiments, efficacy assessments may utilize a definition of SARS-CoV-2-related severe cases Confirmed severe COVID-19: confirmed COVID-19 and presence of at least 1 of the following: clinical signs at rest indicative of severe systemic illness (e.g., RR ?30 breaths per minute, HR ?125 beats per minute, SpO.sub.2593% on room air at sea level, or PaO.sub.2/FiO.sub.2<300 mm Hg); respiratory failure (which can be defined as needing high-flow oxygen, noninvasive ventilation, mechanical ventilation, or ECMO); evidence of shock (e.g., SBP <90 mm Hg, DBP <60 mm Hg, or requiring vasopressors); significant acute renal, hepatic, or neurologic dysfunction; admission to an ICU; death.

(728) Alternatively or additionally, in some embodiments a serological definition can be used for patients without clinical presentation of COVID-19: e.g., confirmed seroconversion to SARS-CoV-2 without confirmed COVID-19: e.g., positive N-binding antibody result in a patient with a prior negative N-binding antibody result.

(729) In some embodiments, any or all of the following assays can be performed on serum samples: SARS-CoV-2 neutralization assay; S1-binding IgG level assay; RBD-binding IgG level assay; N-binding antibody assay.

(730) In one embodiment, methods and agents described herein are administered to a paediatric population. In various embodiments, the paediatric population comprises or consists of subjects under 18 years, e.g., 5 to less than 18 years of age, 12 to less than 18 years of age, 16 to less than 18 years of age, 12 to less than 16 years of age, or 5 to less than 12 years of age. In various embodiments, the paediatric population comprises or consists of subjects under 5 years, e.g., 2 to less than 5 years of age, 12 to less than 24 months of age, 7 to less than 12 months of age, or less than 6 months of age.

(731) In one embodiment, the paediatric population comprises or consists of subjects 12 to less than 18 years of age including subjects 16 to less than 18 years of age and/or subjects 12 to less than 16 years of age. In this embodiment, treatments may comprise 2 vaccinations 21 days apart, wherein, in one embodiment, the vaccine is administered in an amount of 30 ?g RNA per dose, e.g., by intramuscular administration.

(732) In one embodiment, the paediatric population comprises or consists of subjects 5 to less than 18 years of age including subjects 12 to less than 18 years of age and/or subjects 5 to less than 12 years of age. In this embodiment, treatments may comprise 2 vaccinations 21 days apart, wherein, in various embodiments, the vaccine is administered in an amount of 10 ?g, 20 g, or 30 ?g RNA per dose, e.g., by intramuscular administration.

(733) In one embodiment, the paediatric population comprises or consists of subjects less than 5 years of age including subjects 2 to less than 5 years of age, subjects 12 to less than 24 months of age, subjects 7 to less than 12 months of age, subjects 6 to less than 12 months of age and/or subjects less than 6 months of age. In this embodiment, treatments may comprise 2 vaccinations, e.g., 21 to 42 days apart, e.g., 21 days apart, wherein, in various embodiments, the vaccine is administered in an amount of 10 ?g, 20 g, or 30 ?g RNA per dose, e.g., by intramuscular administration.

(734) In some embodiments, efficacy for mRNA compositions described in pediatric populations (e.g., described herein) may be assessed by various metrics described herein (including, e.g., but not limited to COVID-19 incidence per 1000 person-years in subjects with no serological or virological evidence of past SARS-CoV-2 infection; geometric mean ratio (GMR) of SARS CoV-2 neutrlizing titers measured, e.g., 7 days after a second dose; etc.)

(735) In some embodiments, pediatric populations described herein (e.g., from 12 to less than 16 years of age) may be monitored for occurrence of multisystem inflammatory syndrome (MIS) (e.g., inflammation in different body parts such as, e.g., heart, lung, kidneys, brain, skin, eyes, and/or gastrointestinal organs), after administration of an RNA composition (e.g., mRNA) described herein. Exemplary symptoms of MIS in children may include, but are not limited to fever, abdominal pain, vomiting, diarrhea, neck pain, rash, bloodshot eyes, feeling extra tried, and combinations thereof.

(736) In one embodiment, RNA administered as described above is nucleoside modified messenger RNA (modRNA) described herein as BNT162b1 (RBP020.3), BNT162b2 (RBP020.1 or RBP020.2). In one embodiment, RNA administered as described above is nucleoside modified messenger RNA (modRNA) described herein as RBP020.2.

(737) In one embodiment, RNA administered as described above is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 21, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 5. In one embodiment, RNA administered as described above is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 21; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 5.

(738) In one embodiment, RNA administered as described above is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 19, or 20, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, RNA administered as described above is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 19, or 20; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

(739) In one embodiment, RNA administered as described above is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20, a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 20, and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO: 7. In one embodiment, RNA administered as described above is nucleoside modified messenger RNA (modRNA) and (i) comprises the nucleotide sequence of SEQ ID NO: 20; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7.

(740) In one embodiment, RNA administered is nucleoside modified messenger RNA (modRNA), (i) comprises the nucleotide sequence of SEQ ID NO: 20; and/or (ii) encodes an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 7, and is administered in an amount of about 30 ?g per dose. In one embodiment, at least two of such doses are administered. For example, a second dose may be administered about 21 days following administration of the first dose.

(741) In some embodiments, populations to be treated with RNA described herein comprise, essentially consist of, or consist of subjects of age of at least 50, at least 55, at least 60, or at least 65. In some embodiments, populations to be treated with RNA described herein comprise, essentially consist of, or consist of subjects of age of between 55 to 90, 60 to 85, or 65 to 85.

(742) In some embodiments, the period of time between the doses administered is at least 7 days, at least 14 days, or at least 21 days. In some embodiments, the period of time between the doses administered is between 7 days and 28 days such as between 14 days and 23 days. In some embodiments, no more than 5 doses, no more than 4 doses, or no more than 3 doses of the RNA described herein may be administered to a subject.

(743) In some embodiments, the methods and agents described herein are administered (in a regimen, e.g., at a dose, frequency of doses and/or number of doses) such that adverse events (AE), i.e., any unwanted medical occurrence in a patient, e.g., any unfavourable and unintended sign, symptom, or disease associated with the use of a medicinal product, whether or not related to the medicinal product, are mild or moderate in intensity. In some embodiments, the methods and agents described herein are administered such that adverse events (AE) can be managed with interventions such as treatment with, e.g., paracetamol or other drugs that provide analgesic, antipyretic (fever-reducing) and/or anti-inflammatory effects, e.g., nonsteroidal anti-inflammatory drugs (NSAIDs), e.g., aspirin, ibuprofen, and naproxen. Paracetamol or acetaminophen which is not classified as a NSAID exerts weak anti-inflammatory effects and can be administered as analgesic according to the invention. In some embodiments, the methods and agents described herein provide a neutralizing effect in a subject to coronavirus, coronavirus infection, or to a disease or disorder associated with coronavirus.

(744) In some embodiments, the methods and agents described herein following administration to a subject induce an immune response that blocks or neutralizes coronavirus in the subject. In some embodiments, the methods and agents described herein following administration to a subject induce the generation of antibodies such as IgG antibodies that block or neutralize coronavirus in the subject. In some embodiments, the methods and agents described herein following administration to a subject induce an immune response that blocks or neutralizes coronavirus S protein binding to ACE2 in the subject. In some embodiments, the methods and agents described herein following administration to a subject induce the generation of antibodies that block or neutralize coronavirus S protein binding to ACE2 in the subject.

(745) In some embodiments, the methods and agents described herein following administration to a subject induce geometric mean concentrations (GMCs) of RBD domain-binding antibodies such as IgG antibodies of at least 500 U/ml, 1000 U/ml, 2000 U/ml, 3000 U/ml, 4000 U/ml, 5000 U/ml, 10000 U/ml, 15000 U/ml, 20000 U/ml, 25000 U/ml, 30000 U/ml or even higher. In some embodiments, the elevated GMCs of RBD domain-binding antibodies persist for at least 14 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12 months or even longer. In some embodiments, the methods and agents described herein following administration to a subject induce geometric mean titers (GMTs) of neutralizing antibodies such as IgG antibodies of at least 100 U/ml, 200 U/ml, 300 U/ml, 400 U/ml, 500 U/ml, 1000 U/ml, 1500 U/ml, or even higher. In some embodiments, the elevated GMTs of neutralizing antibodies persist for at least 14 days, 21 days, 28 days, 1 month, 3 months, 6 months, 12 months or even longer.

(746) As used herein, the term neutralization refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the viral infection of cells. As used herein, the term neutralization with respect to coronavirus, in particular coronavirus S protein, refers to an event in which binding agents such as antibodies bind to the RBD domain of the S protein, thereby inhibiting the viral infection of cells. In particular, the term neutralization refers to an event in which binding agents eliminate or significantly reduce virulence (e.g. ability of infecting cells) of viruses of interest.

(747) The type of immune response generated in response to an antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. Immune responses can be broadly divided into two types: Th1 and Th2. Th1 immune activation is optimized for intracellular infections such as viruses, whereas Th2 immune responses are optimized for humoral (antibody) responses. Th1 cells produce interleukin 2 (IL-2), tumor necrosis factor (TNF?) and interferon gamma (IFN?). Th2 cells produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. Th1 immune activation is the most highly desired in many clinical situations. Vaccine compositions specialized in eliciting Th2 or humoral immune responses are generally not effective against most viral diseases.

(748) In some embodiments, the methods and agents described herein following administration to a subject induce or promote a Th1-mediated immune response in the subject. In some embodiments, the methods and agents described herein following administration to a subject induce or promote a cytokine profile that is typical for a Th1-mediated immune response in the subject. In some embodiments, the methods and agents described herein following administration to a subject induce or promote the production of interleukin 2 (IL-2), tumor necrosis factor (TNF?) and/or interferon gamma (IFN?) in the subject. In some embodiments, the methods and agents described herein following administration to a subject induce or promote the production of interleukin 2 (IL-2) and interferon gamma (IFN?) in the subject. In some embodiments, the methods and agents described herein following administration to a subject do not induce or promote a Th2-mediated immune response in the subject, or induce or promote a Th2-mediated immune response in the subject to a significant lower extent compared to the induction or promotion of a Th1-mediated immune response. In some embodiments, the methods and agents described herein following administration to a subject do not induce or promote a cytokine profile that is typical for a Th2-mediated immune response in the subject, or induce or promote a cytokine profile that is typical for a Th2-mediated immune response in the subject to a significant lower extent compared to the induction or promotion of a cytokine profile that is typical for a Th1-mediated immune response. In some embodiments, the methods and agents described herein following administration to a subject do not induce or promote the production of IL-4, IL-5, IL-6, IL-9, IL-10 and/or IL-13, or induce or promote the production of IL-4, IL-5, IL-6, IL-9, IL-10 and/or IL-13 in the subject to a significant lower extent compared to the induction or promotion of interleukin 2 (IL-2), tumor necrosis factor (TNF?) and/or interferon gamma (IFN?) in the subject. In some embodiments, the methods and agents described herein following administration to a subject do not induce or promote the production of IL-4, or induce or promote the production of IL-4 in the subject to a significant lower extent compared to the induction or promotion of interleukin 2 (IL-2) and interferon gamma (IFN?) in the subject.

(749) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a panel of different S protein variants such as SARS-CoV-2 S protein variants, in particular naturally occurring S protein variants. In some embodiments, the panel of different S protein variants comprises at least 5, at least 10, at least 15, or even more S protein variants. In some embodiments, such S protein variants comprise variants having amino acid modifications in the RBD domain and/or variants having amino acid modifications outside the RBD domain. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 321 (Q) in SEQ ID NO: 1 is S. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 321 (Q) in SEQ ID NO: 1 is L. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 341 (V) in SEQ ID NO: 1 is I. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 348 (A) in SEQ ID NO: 1 is T. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 354 (N) in SEQ ID NO: 1 is D. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 359 (S) in SEQ ID NO: 1 is N. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 367 (V) in SEQ ID NO: 1 is F. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 378 (K) in SEQ ID NO: 1 is S. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 378 (K) in SEQ ID NO: 1 is R. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 408 (R) in SEQ ID NO: 1 is I. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 409 (Q) in SEQ ID NO: 1 is E. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 435 (A) in SEQ ID NO: 1 is S. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 439 (N) in SEQ ID NO: 1 is K. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 458 (K) in SEQ ID NO: 1 is R. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 472 (I) in SEQ ID NO: 1 is V. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 476 (G) in SEQ ID NO: 1 is S. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 477 (S) in SEQ ID NO: 1 is N. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 483 (V) in SEQ ID NO: 1 is A. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 508 (Y) in SEQ ID NO: 1 is H. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 519 (H) in SEQ ID NO: 1 is P. In one embodiment, such S protein variant comprises SARS-CoV-2 S protein or a naturally occurring variant thereof wherein the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G.

(750) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a mutation at a position corresponding to position 501(N) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.

(751) Said S protein variant comprising a mutation at a position corresponding to position 501 (N) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), and 244 (L). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted.

(752) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets VOC-202012/01.

(753) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T7161, S982A, and D1118H.

(754) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets 501.V2.

(755) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y and A701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said S protein variant may also comprise a D.fwdarw.G mutation at a position corresponding to position 614 in SEQ ID NO: 1.

(756) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a deletion at a position corresponding to positions 69 (H) and 70 (V) in SEQ ID NO: 1.

(757) In some embodiments, a S protein variant comprising a deletion at a position corresponding to positions 69 (H) and 70 (V) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

(758) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets VOC-202012/01.

(759) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T7161, S982A, and D1118H.

(760) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets Cluster 5.

(761) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: deletion 69-70, Y453F, I692V, M1229I, and optionally S1147L.

(762) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a mutation at a position corresponding to position 614 (D) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G.

(763) In some embodiments, a S protein variant comprising a mutation at a position corresponding to position 614 (D) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

(764) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets VOC-202012/01.

(765) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T7161, S982A, and D1118H.

(766) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, D614G and A701V, and optionally: L18F, R246I, K417N, and deletion 242-244.

(767) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a mutation at positions corresponding to positions 501 (N) and 614 (D) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y and the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G.

(768) In some embodiments, a S protein variant comprising a mutation at positions corresponding to positions 501 (N) and 614 (D) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), and 1229 (M). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I.

(769) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets VOC-202012/01.

(770) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T7161, S982A, and D1118H.

(771) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, D614G and A701V, and optionally: L18F, R246I, K417N, and deletion 242-244.

(772) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a mutation at a position corresponding to position 484 (E) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K.

(773) In some embodiments, a S protein variant comprising a mutation at a position corresponding to position 484 (E) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 20 (T) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 138 (D) in SEQ ID NO: 1 is Y.

(774) In one embodiment, the amino acid corresponding to position 190 (R) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is T. In one embodiment, the amino acid corresponding to position 655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) in SEQ ID NO: 1 is F.

(775) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets 501.V2.

(776) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y and A701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said S protein variant may also comprise a D.fwdarw.G mutation at a position corresponding to position 614 in SEQ ID NO: 1.

(777) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets B.1.1.28.

(778) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets B.1.1.248.

(779) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T10271, and V1176F.

(780) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a mutation at positions corresponding to positions 501 (N) and 484 (E) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y and the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K.

(781) In some embodiments, a S protein variant comprising a mutation at positions corresponding to positions 501 (N) and 484 (E) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 20 (T) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is T. In one embodiment, the amino acid corresponding to position 655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) in SEQ ID NO: 1 is F.

(782) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets 501.V2.

(783) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y and A701V, and optionally: L18F, R246I, K417N, and deletion 242-244. Said S protein variant may also comprise a D.fwdarw.G mutation at a position corresponding to position 614 in SEQ ID NO: 1. In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets B.1.1.248.

(784) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T10271, and V1176F.

(785) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a mutation at positions corresponding to positions 501 (N), 484 (E) and 614 (D) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y, the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K and the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G.

(786) In some embodiments, a S protein variant comprising a mutation at positions corresponding to positions 501 (N), 484 (E) and 614 (D) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 417 (K), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 20 (T) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is T. In one embodiment, the amino acid corresponding to position 655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) in SEQ ID NO: 1 is F.

(787) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V, and D614G, and optionally: L18F, R246I, K417N, and deletion 242-244.

(788) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a deletion at a position corresponding to positions 242 (L), 243 (A) and 244 (L) in SEQ ID NO: 1.

(789) In some embodiments, a S protein variant comprising a deletion at a position corresponding to positions 242 (L), 243 (A) and 244 (L) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 417 (K), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 417 (K), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 20 (T) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is T. In one embodiment, the amino acid corresponding to position 655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) in SEQ ID NO: 1 is F.

(790) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets 501.V2.

(791) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V and deletion 242-244, and optionally: L18F, R246I, and K417N. Said S protein variant may also comprise a D.fwdarw.G mutation at a position corresponding to position 614 in SEQ ID NO: 1.

(792) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a mutation at a position corresponding to position 417 (K) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is T.

(793) In some embodiments, a S protein variant comprising a mutation at a position corresponding to position 417 (K) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 501 (N), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 484 (E), 701 (A), 18 (L), 246 (R), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 20 (T) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) in SEQ ID NO: 1 is F.

(794) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets 501.V2.

(795) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V, and K417N, and optionally: L18F, R246I, and deletion 242-244. Said S protein variant may also comprise a D.fwdarw.G mutation at a position corresponding to position 614 in SEQ ID NO: 1.

(796) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets B.1.1.248.

(797) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T10271, and V1176F.

(798) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant such as SARS-CoV-2 S protein variant, in particular naturally occurring S protein variant comprising a mutation at positions corresponding to positions 417 (K) and 484 (E) and/or 501 (N) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is N, and the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K and/or the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 417 (K) in SEQ ID NO: 1 is T, and the amino acid corresponding to position 484 (E) in SEQ ID NO: 1 is K and/or the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y.

(799) In some embodiments, a S protein variant comprising a mutation at positions corresponding to positions 417 (K) and 484 (E) and/or 501 (N) in SEQ ID NO: 1 may comprise one or more further mutations. Such one or more further mutations may be selected from mutations at positions corresponding to the following positions in SEQ ID NO: 1: 69 (H), 70 (V), 144 (Y), 570 (A), 614 (D), 681 (P), 716 (T), 982 (S), 1118 (D), 80 (D), 215 (D), 701 (A), 18 (L), 246 (R), 242 (L), 243 (A), 244 (L), 453 (Y), 692 (I), 1147 (S), 1229 (M), 20 (T), 26 (P), 138 (D), 190 (R), 655 (H), 1027 (T), and 1176 (V). In one embodiment, the amino acid corresponding to position 69 (H) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 70 (V) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 144 (Y) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 570 (A) in SEQ ID NO: 1 is D. In one embodiment, the amino acid corresponding to position 614 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 681 (P) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 716 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 982 (S) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 1118 (D) in SEQ ID NO: 1 is H. In one embodiment, the amino acid corresponding to position 80 (D) in SEQ ID NO: 1 is A. In one embodiment, the amino acid corresponding to position 215 (D) in SEQ ID NO: 1 is G. In one embodiment, the amino acid corresponding to position 701 (A) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 18 (L) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 246 (R) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 242 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 243 (A) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 244 (L) in SEQ ID NO: 1 is deleted. In one embodiment, the amino acid corresponding to position 453 (Y) in SEQ ID NO: 1 is F. In one embodiment, the amino acid corresponding to position 692 (I) in SEQ ID NO: 1 is V. In one embodiment, the amino acid corresponding to position 1147 (S) in SEQ ID NO: 1 is L. In one embodiment, the amino acid corresponding to position 1229 (M) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 20 (T) in SEQ ID NO: 1 is N. In one embodiment, the amino acid corresponding to position 26 (P) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 138 (D) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 190 (R) in SEQ ID NO: 1 is S. In one embodiment, the amino acid corresponding to position 655 (H) in SEQ ID NO: 1 is Y. In one embodiment, the amino acid corresponding to position 1027 (T) in SEQ ID NO: 1 is I. In one embodiment, the amino acid corresponding to position 1176 (V) in SEQ ID NO: 1 is F.

(800) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets 501.V2.

(801) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: D80A, D215G, E484K, N501Y, A701V, and K417N and optionally: L18F, R246I, and deletion 242-244. Said S protein variant may also comprise a D.fwdarw.G mutation at a position corresponding to position 614 in SEQ ID NO: 1.

(802) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets B.1.1.248.

(803) In some embodiments, the methods and agents described herein following administration to a subject induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a S protein variant comprising the following mutations at positions corresponding to the following positions in SEQ ID NO: 1: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, H655Y, T10271, and V1176F.

(804) The term amino acid corresponding to position . . . as used herein refers to an amino acid position number corresponding to an amino acid position number in SARS-CoV-2 S protein, in particular the amino acid sequence shown in SEQ ID NO: 1. Corresponding amino acid positions in other coronavirus S protein variants such as SARS-CoV-2 S protein variants may be found by alignment with SARS-CoV-2 S protein, in particular the amino acid sequence shown in SEQ ID NO: 1. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present invention. Standard sequence alignment programs such as ALIGN, ClustalW or similar, typically at default settings may be used.

(805) In some embodiments, the panel of different S protein variants to which an antibody response is targeted comprises at least 5, at least 10, at least 15, or even more S protein variants selected from the group consisting of the Q321S, V3411, A348T, N354D, S359N, V367F, K378S, R408I, Q409E, A435S, K458R, 1472V, G476S, V483A, Y508H, H519P and D614G variants described above. In some embodiments, the panel of different S protein variants to which an antibody response is targeted comprises all S protein variants from the group consisting of the Q321S, V3411, A348T, N354D, S359N, V367F, K378S, R408I, Q409E, A435S, K458R, 1472V, G476S, V483A, Y508H, H519P and D614G variants described above.

(806) In some embodiments, the panel of different S protein variants to which an antibody response is targeted comprises at least 5, at least 10, at least 15, or even more S protein variants selected from the group consisting of the Q321L, V3411, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, 1472V, G476S, S477N, V483A, Y508H, H519P and D614G variants described above. In some embodiments, the panel of different S protein variants to which an antibody response is targeted comprises all S protein variants from the group consisting of the Q321L, V3411, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, 1472V, G476S, S477N, V483A, Y508H, H519P and D614G variants described above.

(807) In some embodiments, a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof, e.g., as encoded by the RNA described herein, comprises one or more of the mutations described herein for S protein variants such as SARS-CoV-2 S protein variants, in particular naturally occurring S protein variants. In one embodiment, a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof, e.g., as encoded by the RNA described herein, comprises a mutation at a position corresponding to position 501 (N) in SEQ ID NO: 1. In one embodiment, the amino acid corresponding to position 501 (N) in SEQ ID NO: 1 is Y. In some embodiments, a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof, e.g., as encoded by the RNA described herein, comprises one or more mutations, such as all mutations, of a SARS-CoV-2 S protein of a SARS-CoV-2 variant selected from the group consisting of VOC-202012/01, 501.V2, Cluster 5 and B.1.1.248. In some embodiments, a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof, e.g., as encoded by the RNA described herein, comprises an amino acid sequence with alanine substitution at position 80, glycine substitution at position 215, lysine substitution at position 484, tyrosine substitution at position 501, valine substitution at position 701, phenylalanine substitution at position 18, isoleucine substitution at position 246, asparagine substitution at position 417, glycine substitution at position 614, deletions at positions 242 to 244, and proline substitutions at positions 986 and 987 of SEQ ID NO:1.

(808) In some embodiments, the methods and agents, e.g., mRNA compositions, described herein following administration to a subject induce a cell-mediated immune response (e.g., CD4+ and/or CD8+ T cell response). In some embodiments, T cells are induced that recognize one or more eptiopes (e.g., MHC class I-restricted epitopes) selected from the group consisting of LPFNDGVYF (SEQ ID NO: 47), GVYFASTEK (SEQ ID NO: 52), YLQPRTFLL (SEQ ID NO: 40), QPTESIVRF (SEQ ID NO: 45), CVADYSVLY (SEQ ID NO: 53), KCYGVSPTK (SEQ ID NO: 54), NYNYLYRLF (SEQ ID NO: 43), FQPTNGVGY (SEQ ID NO: 55), IPFAMQMAY (SEQ ID NO: 46), RLQSLQTYV (SEQ ID NO: 41), GTHWFVTQR (SEQ ID NO: 56), VYDPLQPEL (SEQ ID NO: 57), QYIKWPWYI (SEQ ID NO: 42), and KWPWYIWLGF (SEQ ID NO: 44). In one embodiment, T cells are induced that recognize the eptiope YLQPRTFLL (SEQ ID NO: 40). In one embodiment, T cells are induced that recognize the eptiope NYNYLYRLF (SEQ ID NO: 43). In one embodiment, T cells are induced that recognize the eptiope QYIKWPWYI (SEQ ID NO: 42). In one embodiment, T cells are induced that recognize the eptiope KCYGVSPTK (SEQ ID NO: 54). In one embodiment, T cells are induced that recognize the eptiope RLQSLQTYV (SEQ ID NO: 41). In some embodiments, the methods and agents, e.g., mRNA compositions, described herein are administered according to a regimen which achieves such induction of T cells.

(809) In some embodiments, the methods and agents, e.g., mRNA compositions, described herein following administration to a subject induce a cell-mediated immune response (e.g., CD4+ and/or CD8+ T cell response) that is detectable 15 weeks or later, 16 weeks or later, 17 weeks or later, 18 weeks or later, 19 weeks or later, 20 weeks or later, 21 weeks or later, 22 weeks or later, 23 weeks or later, 24 weeks or later or 25 weeks or later after administration, e.g., using two doses of the RNA described herein (wherein the second dose may be administered about 21 days following administration of the first dose). In some embodiments, the methods and agents, e.g., mRNA compositions, described herein are administered according to a regimen which achieves such induction of a cell-mediated immune response.

(810) In one embodiment, vaccination against Coronavirus described herein, e.g., using RNA described herein which may be administered in the amounts and regimens described herein, e.g., at two doses of 30 ?g per dose e.g. administered 21 days apart, may be repated after a certain period of time, e.g., once it is observed that protection against Coronavirus infection diminishes, using the same or a different vaccine as used forthe first vaccination. Such certain period of time may be at least 6 months, 1 year, two years etc. In one embodiment, the same RNA as used for the first vaccination is used for the second or further vaccination, however, at a lower dose or a lower frequency of administration. For example, the first vaccination may comprise vaccination using a dose of about 30 ?g per dose, wherein in one embodiment, at least two of such doses are administered, (for example, a second dose may be administered about 21 days following administration of the first dose) and the second or further vaccination may comprise vaccination using a dose of less than about 30 ?g per dose, wherein in one embodiment, only one of such doses is administered. In one embodiment, a different RNA as used for the first vaccination is used for the second or further vaccination, e.g., BNT162b2 is used for the first vaccination and BNT162B1 or BNT162b3 is used for the second or further vaccination.

(811) In one embodiment, the vaccination regimen comprises a first vaccination using at least two doses of the RNA described herein, e.g., two doses of the RNA described herein (wherein the second dose may be administered about 21 days following administration of the first dose), and a second vaccination using a single dose or multiple doses, e.g., two doses, of the RNA described herein. In various embodiments, the second vaccination is administered 3 to 24 months, 6 to 18 months, 6 to 12 months, or 5 to 7 months after administration of the first vaccination, e.g., after the initial two-dose regimen. The amount of RNA used in each dose of the second vaccination may be equal or different to the amount of RNA used in each dose of the first vaccination. In one embodiment, the amount of RNA used in each dose of the second vaccination is equal to the amount of RNA used in each dose of the first vaccination. In one embodiment, the amount of RNA used in each dose of the second vaccination and the amount of RNA used in each dose of the first vaccination is about 30 ?g per dose. In one embodiment, the same RNA as used for the first vaccination is used for the second vaccination. In one embodiment, the RNA used for the first vaccination and for the second vaccination is BNT162b2. In one embodiment, a different RNA as used for the first vaccination is used for the second vaccination. In one embodiment, the RNA used for the first vaccination is BNT162b2 and the RNA used for the second vaccination is RNA encoding a SARS-CoV-2 S protein of a SARS-CoV-2 variant strain, e.g., a strain discussed herein. In one embodiment, the RNA used for the first vaccination is BNT162b2 and the RNA used for the second vaccination is RNA encoding a SARS-CoV-2 S protein of a SARS-CoV-2 variant strain that is prevalent or rapidly spreading at the time of the second vaccination. In one embodiment, the RNA used for the first vaccination is BNT162b2 and the RNA used for the second vaccination is RNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprising one or more of the mutations described herein for S protein variants such as SARS-CoV-2 S protein variants, in particular naturally occurring S protein variants. In one embodiment, the RNA used for the first vaccination is BNT162b2 and the RNA used for the second vaccination is RNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof comprising one or more mutations, such as all mutations, of a SARS-CoV-2 S protein of a SARS-CoV-2 variant selected from the group consisting of VOC-202012/01, 501.V2, Cluster 5 and B.1.1.248. In one embodiment, the RNA used for the first vaccination encodes a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1 and the RNA used for the second vaccination is RNA encoding a polypeptide comprising an amino acid sequence with alanine substitution at position 80, glycine substitution at position 215, lysine substitution at position 484, tyrosine substitution at position 501, valine substitution at position 701, phenylalanine substitution at position 18, isoleucine substitution at position 246, asparagine substitution at position 417, glycine substitution at position 614, deletions at positions 242 to 244, and proline substitutions at positions 986 and 987 of SEQ ID NO:1.

(812) In one embodiment, the vaccination regimen comprises a first vaccination using two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 21 days apart and a second vaccination using a single dose or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 6 to 12 months after administration of the first vaccination, i.e., after the initial two-dose regimen. In one embodiment, each RNA dose comprises 30 ?g RNA.

(813) In one embodiment, the vaccination regimen comprises a first vaccination using two doses of RNA encoding a polypeptide comprising an amino acid sequence with proline residue substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 21 days apart and a second vaccination using a single dose or multiple doses of RNA encoding a polypeptide comprising an amino acid sequence with alanine substitution at position 80, glycine substitution at position 215, lysine substitution at position 484, tyrosine substitution at position 501, valine substitution at position 701, phenylalanine substitution at position 18, isoleucine substitution at position 246, asparagine substitution at position 417, glycine substitution at position 614, deletions at positions 242 to 244, and proline substitutions at positions 986 and 987 of SEQ ID NO:1 administered about 6 to 12 months after administration of the first vaccination, i.e., after the initial two-dose regimen. In one embodiment, each RNA dose comprises 30 ?g RNA.

(814) In one embodiment, the second vaccination results in a boosting of the immune response.

(815) In one embodiment, the RNA described herein is co-administered with other vaccines. In some embodiments, an RNA composition described herein is co-administered with an influenza vaccine. In some embodiments, an RNA composition provided herein and other injectable vaccine(s) are administered at different times. In some embodiments, an RNA composition provided herein is administered at the same time as other injectable vaccine(s). In some such embodiments, an RNA composition provided herein and at least one another injectable vaccine(s) are administered at different injection sites. In some embodiments, an RNA composition provided herein is not mixed with any other vaccine in the same syringe. In some embodiments, an RNA composition provided herein is not combined with other coronavirus vaccines as part of vaccination against coronavirus, e.g., SARS-CoV-2.

(816) The term disease refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, disease is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.

(817) In the present context, the term treatment, treating or therapeutic intervention relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

(818) The term therapeutic treatment relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease.

(819) The terms prophylactic treatment or preventive treatment relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms prophylactic treatment or preventive treatment are used herein interchangeably.

(820) The terms individual and subject are used herein interchangeably. They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that can be afflicted with or is susceptible to a disease or disorder but may or may not have the disease or disorder. In many embodiments, the individual is a human being. Unless otherwise stated, the terms individual and subject do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In some embodiments, the term subject includes humans of age of at least 50, at least 55, at least 60, at least 65, at least 70, or older. In some embodiments, the term subject includes humans of age of at least 65, such as 65 to 80, 65 to 75, or 65 to 70. In embodiments of the present disclosure, the individual or subject is a patient.

(821) The term patient means an individual or subject for treatment, in particular a diseased individual or subject.

(822) In one embodiment of the disclosure, the aim is to provide an immune response against coronavirus, and to prevent or treat coronavirus infection.

(823) A pharmaceutical composition comprising RNA encoding a peptide or protein comprising an epitope may be administered to a subject to elicit an immune response against an antigen comprising said epitope in the subject which may be therapeutic or partially orfully protective. A person skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective reaction to a disease is produced by immunizing a subject with an antigen or an epitope, which is immunologically relevant with respect to the disease to be treated. Accordingly, pharmaceutical compositions described herein are applicable for inducing or enhancing an immune response. Pharmaceutical compositions described herein are thus useful in a prophylactic and/or therapeutic treatment of a disease involving an antigen or epitope.

(824) As used herein, immune response refers to an integrated bodily response to an antigen or a cell expressing an antigen and refers to a cellular immune response and/or a humoral immune response. The immune system is divided into a more primitive innate immune system, and acquired or adaptive immune system of vertebrates, each of which contains humoral and cellular components.

(825) Cell-mediated immunity, cellular immunity, cellular immune response, or similar terms are meant to include a cellular response directed to cells characterized by expression of an antigen, in particular characterized by presentation of an antigen with class I or class II MHC. The cellular response relates to immune effector cells, in particular to cells called T cells or T lymphocytes which act as either helpers or killers. The helper T cells (also termed CD4+ T cells) play a central role by regulating the immune response and the killer cells (also termed cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTLs) kill diseased cells such as virus-infected cells, preventing the production of more diseased cells.

(826) An immune effector cell includes any cell which is responsive to vaccine antigen. Such responsiveness includes activation, differentiation, proliferation, survival and/or indication of one or more immune effector functions. The cells include, in particular, cells with lytic potential, in particular lymphoid cells, and are preferably T cells, in particular cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes triggers the destruction of target cells. For example, cytotoxic T cells trigger the destruction of target cells by either or both of the following means. First, upon activation T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and trigger a caspase cascade in the cytoplasm that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between the T cells and target cells.

(827) The term effector functions in the context of the present invention includes any functions mediated by components of the immune system that result, for example, in the neutralization of a pathogenic agent such as a virus and/or in the killing of diseased cells such as virus-infected cells. In one embodiment, the effector functions in the context of the present invention are T cell mediated effector functions. Such functions comprise in the case of a helper T cell (CD4+ T cell) the release of cytokines and/or the activation of CD8+ lymphocytes (CTLs) and/or B cells, and in the case of CTL the elimination of cells, i.e., cells characterized by expression of an antigen, for example, via apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN-? and TNF-?, and specific cytolytic killing of antigen expressing target cells.

(828) The term immune effector cell or immunoreactive cell in the context of the present invention relates to a cell which exerts effector functions during an immune reaction. An immune effector cell in one embodiment is capable of binding an antigen such as an antigen presented in the context of MHC on a cell or expressed on the surface of a cell and mediating an immune response. For example, immune effector cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, immune effector cells are T cells, preferably CD4+ and/or CD8+ T cells, most preferably CD8+ T cells. According to the invention, the term immune effector cell also includes a cell which can mature into an immune cell (such as T cell, in particular T helper cell, or cytolytic T cell) with suitable stimulation. Immune effector cells comprise CD34.sup.+ hematopoietic stem cells, immature and mature T cells and immature and mature B cells. The differentiation ofT cell precursors into a cytolytic T cell, when exposed to an antigen, is similar to clonal selection of the immune system.

(829) A lymphoid cell is a cell which is capable of producing an immune response such as a cellular immune response, or a precursor cell of such cell, and includes lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. A lymphoid cell may be an immune effector cell as described herein. A preferred lymphoid cell is a T cell.

(830) The terms T cell and T lymphocyte are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells. The term antigen-specific T cell or similar terms relate to a T cell which recognizes the antigen to which the T cell is targeted and preferably exerts effector functions of T cells. T cells belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptor (TCR). The thymus is the principal organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with a distinct function.

(831) T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

(832) Cytotoxic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. A majority of T cells have a T cell receptor (TCR) existing as a complex of several proteins. The TCR of a T cell is able to interact with immunogenic peptides (epitopes) bound to major histocompatibility complex (MHC) molecules and presented on the surface of target cells. Specific binding of the TCR triggers a signal cascade inside the T cell leading to proliferation and differentiation into a maturated effector T cell. The actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta (TCR? and TCR?) genes and are called ?- and ?-TCR chains. ?? T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. However, in ?? T cells, the TCR is made up of one ?-chain and one ?-chain. This group of T cells is much less common (2% of total T cells) than the ?? T cells.

(833) Humoral immunity or humoral immune response is the aspect of immunity that is mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides. It contrasts with cell-mediated immunity. Its aspects involving antibodies are often called antibody-mediated immunity. Humoral immunity refers to antibody production and the accessory processes that accompany it, including: Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. It also refers to the effector functions of antibodies, which include pathogen neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

(834) In humoral immune response, first the B cells mature in the bone marrow and gain B-cell receptors (BCR's) which are displayed in large number on the cell surface. These membrane-bound protein complexes have antibodies which are specific for antigen detection. Each B cell has a unique antibody that binds with an antigen. The mature B cells migrate from the bone marrow to the lymph nodes or other lymphatic organs, where they begin to encounter pathogens. When a B cell encounters an antigen, the antigen is bound to the receptor and taken inside the B cell by endocytosis. The antigen is processed and presented on the B cell's surface again by MHC-II proteins. The B cell waits for a helper T cell (TH) to bind to the complex. This binding will activate the TH cell, which then releases cytokines that induce B cells to divide rapidly, making thousands of identical clones of the B cell. These daughter cells either become plasma cells or memory cells. The memory B cells remain inactive here; later when these memory B cells encounter the same antigen due to reinfection, they divide and form plasma cells. On the other hand, the plasma cells produce a large number of antibodies which are released free into the circulatory system. These antibodies will encounter antigens and bind with them. This will either interfere with the chemical interaction between host and foreign cells, or they may form bridges between their antigenic sites hindering their proper functioning, or their presence will attract macrophages or killer cells to attack and phagocytose them.

(835) The term antibody includes an immunoglobulin comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. An antibody binds, preferably specifically binds with an antigen.

(836) Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

(837) An antibody heavy chain, as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.

(838) An antibody light chain, as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, K and A light chains refer to the two major antibody light chain isotypes.

(839) The present disclosure contemplates an immune response that may be protective, preventive, prophylactic and/or therapeutic. As used herein, induces [or inducing] an immune response may indicate that no immune response against a particular antigen was present before induction or it may indicate that there was a basal level of immune response against a particular antigen before induction, which was enhanced after induction. Therefore, induces [or inducing] an immune response includes enhances [or enhancing] an immune response. The term immunotherapy relates to the treatment of a disease or condition by inducing, or enhancing an immune response. The term immunotherapy includes antigen immunization or antigen vaccination.

(840) The terms immunization or vaccination describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.

(841) The term macrophage refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages which are activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens within the macrophage by hydrolytic and oxidative attack resulting in degradation of the pathogen. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, resulting in T and B cell activation and further stimulation of the immune response. Macrophages belong to the class of antigen presenting cells. In one embodiment, the macrophages are splenic macrophages.

(842) The term dendritic cell (DC) refers to another subtype of phagocytic cells belonging to the class of antigen presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample the surrounding environment for pathogens such as viruses and bacteria. Once they have come into contact with a presentable antigen, they become activated into mature dendritic cells and begin to migrate to the spleen or to the lymph node. Immature dendritic cells phagocytose pathogens and degrade their proteins into small pieces and upon maturation present those fragments at their cell surface using MHC molecules. Simultaneously, they upregulate cell-surface receptors that act as co-receptors in T cell activation such as CD80, CD86, and CD40 greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces the dendritic cell to travel through the blood stream to the spleen or through the lymphatic system to a lymph node. Here they act as antigen-presenting cells and activate helper T cells and killer T cells as well as B cells by presenting them antigens, alongside non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce a T cell- or B cell-related immune response. In one embodiment, the dendritic cells are splenic dendritic cells.

(843) The term antigen presenting cell (APC) is a cell of a variety of cells capable of displaying, acquiring, and/or presenting at least one antigen or antigenic fragment on (or at) its cell surface. Antigen-presenting cells can be distinguished in professional antigen presenting cells and non-professional antigen presenting cells.

(844) The term professional antigen presenting cells relates to antigen presenting cells which constitutively express the Major Histocompatibility Complex class II (MHC class II) molecules required for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen presenting cell, the antigen presenting cell produces a co-stimulatory molecule inducing activation of the T cell. Professional antigen presenting cells comprise dendritic cells and macrophages.

(845) The term non-professional antigen presenting cells relates to antigen presenting cells which do not constitutively express MHC class II molecules, but upon stimulation by certain cytokines such as interferon-gamma. Exemplary, non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells or vascular endothelial cells.

(846) Antigen processing refers to the degradation of an antigen into procession products, which are fragments of said antigen (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, such as antigen presenting cells to specific T cells.

(847) The term disease involving an antigen refers to any disease which implicates an antigen, e.g. a disease which is characterized by the presence of an antigen. The disease involving an antigen can be an infectious disease. As mentioned above, the antigen may be a disease-associated antigen, such as a viral antigen. In one embodiment, a disease involving an antigen is a disease involving cells expressing an antigen, preferably on the cell surface.

(848) The term infectious disease refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent (e.g. common cold). Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively. In this regard, the infectious disease can be, for example, hepatitis, sexually transmitted diseases (e.g. chlamydia or gonorrhea), tuberculosis, HIV/acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), the bird flu, and influenza.

(849) Certain Exemplary Embodiments: 1. A method of immunizing against SARS-CoV-2, the method comprising steps of: administering a composition comprising a lipid nanoparticle encapsulated mRNA that encodes at least an epitope of a SARS-CoV-2-encoded polypeptide, according to a regimen established to achieve detectable antibody titer against the epitope in serum within 7 days, which regimen comprises administration of at least one dose of the composition. 2. The method of embodiment 1, wherein the regimen comprises administration of at least two doses of the composition. 3. The method of embodiment 1, wherein the regimen consists of administration of two doses of the composition. 4. The method of embodiment 2 or embodiment 3, wherein the first dose is a different amount that one or more subsequent doses. 5. The method of embodiment 1 or embodiment 4, wherein the first dose is administered a period of time before the subsequent dose, which period of time is at least 1 week, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years or more. 6. The method of any one of embodiments 1-6, wherein the regimen has been established to have an incidence of adverse events below 60% when administered to a relevant population of adults.7. The method of embodiment 6, wherein the regimen has been established not to elicit local injection site reactions above moderate severity with an incidence greater than about 1 in 75. 8. The method of any one of embodiments 1-7, wherein each dose is no more than 60 ug or lower, including, e.g., no more than 50 ug, no more than 40 ug, no more than 30 ug, no more than 20 ug, no more than 10 ug, no more than 5 ug, no more than 2.5 ug, no more than 1 ug. 9. The method of any one of embodiments 1-8, wherein each dose is at least 1 ug or higher, including, e.g., at least 2 ug, at least 5 ug, at least 10 ug, at least 20 ug, at least 30 ug, at least 40 ug, or more. 10. A method comprising administering to a subject a composition comprising a lipid nanoparticle encapsulated mRNA, wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 S protein or a fragment thereof, wherein the composition is administered in an effective amount to induce in the subject a SARS-COV-2 S-protein specific immune response, wherein the effective amount is sufficient to provide sterilizing immunity in the subject at an at least 2-fold (including, e.g., at least 3-fold, at least 4-fold, at least 5-fold) lower dose relative to a reference composition (e.g., a reference RNA vaccine or composition). 11. A method comprising administering to a subject a composition comprising a lipid nanoparticle encapsulated mRNA, wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 S protein or a fragment thereof, wherein the composition is administered in an effective amount to reduce viral load in the subject by at least 80%, relative to a control, at 2 days or more (including, e.g., 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or more) following exposure to SARS-COV2, wherein the control is the viral load in a subject administered a reference composition (e.g., a reference RNA vaccine or composition). 12. A method comprising administering to a subject a composition comprising a lipid nanoparticle encapsulated mRNA, wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 S protein or a fragment thereof, wherein the composition is administered in an effective amount to induce in the subject a SARS-COV-2 S-protein specific immune response, wherein efficacy of the RNA vaccine is at least 80% relative to unvaccinated control subjects. 13. The method of any one of embodiments 10-12, wherein the effective amount is sufficient to produce detectable levels of SARS-COV-2 S protein or a fragment thereof as measured in serum of the subject at 1-72 hours post administration. 14. The method of any one of embodiments 10-12, wherein the effective amount is sufficient to produce a 1,000-10,000 neutralization titer produced by neutralizing antibody against the SARS-COV-2 S protein as measured in serum of the subject at 1-72 hours post administration. 15. The method of any one of embodiments 10-14, wherein an anti-SARS-COV-2 S protein antibody titer produced in the subject is increased by at least 1 log relative to a control, wherein the control is an anti-SARS-COV-2 S protein antibody titer produced in a subject who has not been administered a vaccine against SARS-COV-2. 16. The method of any one of embodiments 10-15, wherein the anti-SARS-COV-2 S protein antibody titer produced in the subject is increased at least 2 times relative to a control, wherein the control is an anti-SARS-COV-2 S protein antibody titer produced in a subject who has not been administered a vaccine against SARS-COV-2. 17. The method of any one of embodiments 1-16, wherein the administering is performed by intramuscular injection. 18. An immunogenic composition comprising a lipid nanoparticle encapsulated RNA (e.g., mRNA) that encodes at least an epitope of a SARS-CoV-2-encoded polypeptide, which vaccine composition has been established to achieve detectable antibody titer against the epitope in serum within 7 days after administration to a population of adult human subjects according to a regimen that includes administration of at least one dose of the vaccine composition. 19. The immunogenic composition of embodiment 18, wherein at least 80% of the uridines in the RNA have a chemical modification. 20. The immunogenic composition of embodiment 18 or 19, wherein 100% of the uridines in the RNA have a chemical modification. 21. The immunogenic composition of any one of embodiments 18-20, wherein the 5 terminal cap is 7mG(5)ppp(5)NlmpNp. 22. The immunogenic composition of any one of embodiments 18-21, wherein the lipid nanoparticles in the composition comprise a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic lipid. 23. The immunogenic composition of any one of embodiments 18-22, wherein lipid nanoparticles in the composition comprise a molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 5-25% non-cationic lipid. 24. The immunogenic composition of embodiment 22 or 23, wherein the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is a cholesterol. 25. The immunogenic composition of any one of embodiments 22 or 23, wherein the cationic lipid is selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate. 26. The immunogenic composition of any one of embodiments 18-25, wherein the RNA comprises a 5 terminal cap and a chemical modification, and the RNA is formulated or is to be formulated as an lipid nanoparticle. 27. The immunogenic composition of any one of embodiments 18-26, wherein the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof is linked to a signal peptide. 28. The immunogenic composition of embodiment 27, wherein the signal peptide is selected from the group consisting of: a HulgGk signal peptide (METPAQLLFLLLLWLPDTTG (SEQ ID NO: 36)); an IgE heavy chain epsilon-1signal peptide (MDWTWILFLVAAATRVHS (SEQ ID NO: 37)); a Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS (SEQ ID NO: 38)) and a VSVg protein signal sequence (MKCLLYLAFLFIGVNCA (SEQ ID NO: 39)). 29. A method for eliciting an immune response in a subject by activating T cells in vivo, comprising administering to the subject a composition comprising a lipid nanoparticle encapsulated modified nucleoside mRNA, wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 S protein or a fragment thereof, thereby activating T cells in vivo against infection by SARS-COV2 in the subject. 30. A method for eliciting an immune response against SARS-COV-2 in a subject, comprising administering to the subject a composition comprising a lipid nanoparticle encapsulated modified nucleoside mRNA, wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 S protein or a fragment thereof, wherein the composition elicits an increased production of the SARS-COV2 polypeptide or fragment thereof production, as compared to a composition comprising a corresponding unmodified mRNA. 31. A method for eliciting an immune response against SARS-COV-2 in a subject, comprising administering to the subject a composition comprising a lipid nanoparticle encapsulated modified nucleoside mRNA, wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 S protein or a fragment thereof, wherein the composition elicits increased antibody titers, as compared to a composition comprising a corresponding unmodified mRNA. 32. A method for eliciting an immune response against SARS-COV-2 in a subject, comprising administering at least one dose to the subject a composition comprising a lipid nanoparticle encapsulated modified nucleoside mRNA, wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 S protein or a fragment thereof, wherein the composition elicits increased antibody titers in the subject at least 7 days after the first dose, as compared to the antibody titers against SARS-COV-2 prior to administration of the composition. 33. A method for eliciting an immune response against SARS-COV-2 in a subject, comprising administering at least one dose to the subject a composition comprising a lipid nanoparticle encapsulated modified nucleoside mRNA, wherein the mRNA encodes an amino acid sequence comprising SARS-COV2 S protein or a fragment thereof, wherein the composition elicits increased antibody titers in the subject at least 7 days after the first dose, as compared to a composition comprising a corresponding unmodified mRNA. 34. The method according to any one of embodiments 29-33, wherein the mRNA is present at a purity of 90% or greater. 35. The method according to any one of embodiments 29-34, wherein the composition does not further comprise a unmodified RNA encoding SARS-COV2 S protein or a fragment thereof. 36. The method according to any one of embodiments 29-35, wherein the composition is administered at least 2 times, the first and second administrations being at least 7 days apart. 37. The method according to any one of embodiments 29-36, wherein the subject is at risk for a SARS-CoV-2 infection. 38. The method according to any one of embodiments 29-37, wherein the subject is undergoing treatment for cardiovascular disease. 39. The method according to any one of embodiments 29-37, wherein the subject is undergoing treatment for diabetes. 40. The method according to any one of embodiments 29-37, wherein the subject is undergoing treatment for chronic cardiopulmonary disease. 41. The method according to any one of embodiments 29-37, wherein the subject is undergoing treatment for chronic renal disease. 42. The method according to any one of embodiments 29-41, wherein the immune response is sustained for at least about 30 days. 43. The method according to any one of embodiments 29-42, wherein the immune response is sustained for at least about 60 days. 44. The method according to any one of embodiments 29-43, wherein the immune response is sustained for at least about 180 days. 45. The method according to any one of embodiments 29-44, wherein the immune response comprises virus neutralizing titer. 46. The method according to any one of embodiments 29-45, wherein the subject is at least 18 years of age. 47. The method according to any one of embodiments 29-46, wherein the dose comprises 100 ug or less of mRNA. 48. The method according to any one of embodiments 29-47, wherein the dose comprises less than 100 ug of mRNA and the composition elicits an immune response that is greater than the immune response elicited by a composition comprising at least 100 ug of mRNA. 49. The method according to any one of embodiments 29-48, wherein the dose comprises about 30 ug of mRNA. 50. The method according to any one of embodiments 29-49, wherein the immune response comprises antibodies against the receptor binding domain of the S protein of SARS-CoV-2. 51. The method according to any one of embodiments 29-50, wherein the immune response comprises RBD-binding IgG. 52. The method according to any one of embodiments 29-50, wherein the SARS-CoV-2 S protein or a fragment thereof comprises a receptor binding domain. 53. A kit comprising a) a composition comprising a lipid nanoparticle encapsulated mRNA; and b) a temperature monitoring system. 54. The kit according to embodiment 53, wherein the temperature monitoring system comprises a temperature sensor and a display, wherein when the temperature monitoring system displays or warns when the temperature of the composition attains a temperature above about ?80? C. 55. The kit according to embodiment 53, wherein the temperature monitoring system comprises a temperature sensor and a display, wherein when the temperature monitoring system displays or warns when the temperature of the composition attains a temperature above about ?60? C. 56. A kit comprising a) a composition comprising a lipid nanoparticle encapsulated mRNA; and b) a light sensor. 57. The kit according to embodiment 56, wherein the light sensor comprises a photosensitive element configured to react to exposure to light, resulting in a change in a material property of the photosensitive element. 58. The method according to any one of embodiments 29-35, wherein the composition is administered at least 2 times, the first and second administrations being at least 14 days apart. 59. The method according to any one of embodiments 29-35, wherein the composition is administered at least 2 times, the first and second administrations being at least 21 days apart. 60. The method according to any one of embodiments 29-48, wherein the dose comprises about 10 ug of mRNA. 61. The method according to any one of embodiments 29-35, wherein the composition is administered at least 2 times, the first and second administrations being at least 28 days apart. 62. The method according to any one of embodiments 29-35, wherein the mRNA encodes any one of the amino acid sequences SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7. 63. An immunogenic composition comprising a messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame encoding a polypeptide that comprises a receptor-binding portion of a SARs-CoV-2 S protein formulated in at least one lipid nanoparticle comprising a cationic lipid, in an effective amount to induce an immune response in a subject administered at least one dose of the immunogenic composition, wherein the isolated mRNA polynucleotide is not self-replicating RNA. 64. The immunogenic composition of embodiment 63, wherein the lipid nanoparticle further comprises any one of a non-cationic lipid, sterol, and PEG-modified lipid. 65. The immunogenic composition of embodiment 63, comprising an isolated messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame encoding a polypeptide that comprises a receptor-binding portion of a SARs-CoV-2 S protein; formulated in at least one lipid nanoparticle that comprises a molar ratio of 20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid, in an effective amount to induce an immune response in a subject administered at least one dose of the immunogenic composition, wherein the isolated mRNA polynucleotide is not self-replicating RNA. 66. The immunogenic composition of embodiment 63, wherein the polypeptide does not comprise the complete S protein. 67. The immunogenic composition of embodiment 63, wherein the polypeptide comprises the receptor binding domain (RBD) of a SARs-CoV-2 S protein. 68. The immunogenic composition of embodiment 63, wherein the polypeptide comprises SEQ ID NO: 5. 69. The immunogenic composition of embodiment 63, wherein the polypeptide comprises SEQ ID NO: 29 or 31. 70. The immunogenic composition of embodiment 63, wherein the polypeptide comprises SEQ ID NO: 3. 71. The immunogenic composition of embodiment 63, wherein the polypeptide comprises SEQ ID NO: 7. 72. The immunogenic composition of any one of embodiments 63-71, wherein the isolated mRNA polynucleotide further comprises a 5 terminal cap, 7mG(5)ppp(5)NlmpNp. 73. The immunogenic composition of any one of embodiments 63-72, wherein at least 80% of the uracil in the open reading frame have a chemical modification selected from N1-methyl-pseudouridine or N1-ethyl-pseudouridine. 74. The immunogenic composition of any one of embodiments 63-73, wherein the chemical modification is in the 5-position of the uracil. 75. The immunogenic composition of any one of embodiments 63-74, wherein the efficacy of the immunogenic composition in vaccinated subjects is at least 60%, relative to unvaccinated subjects, following a single dose of the immunogenic composition. 76. The immunogenic composition of embodiment 75, wherein the efficacy of the immunogenic composition in vaccinated subjects is at least 70%, relative to unvaccinated subjects, following a single dose of the immunogenic composition. 77. The immunogenic composition of embodiment 75, wherein the efficacy of the immunogenic composition in vaccinated subjects is at least 80%, relative to unvaccinated subjects, following a single dose of the immunogenic composition. 78. The immunogenic composition of embodiment 75, wherein the efficacy of the immunogenic composition in vaccinated subjects is at least 90%, relative to unvaccinated subjects, following a single dose of the immunogenic composition. 79. The immunogenic composition of any one of embodiments 63-78, wherein the effective amount is sufficient to produce detectable levels of a polypeptide that comprises a receptor-binding portion of a SARS-CoV-2 S protein as measured in serum of a subject vaccinated with at least one dose of the immunogenic composition at 1-72 hours post administration. 80. The immunogenic composition of any one of embodiments 63-79, wherein the effective amount is sufficient to produce a 1,000-10,000 neutralization titer produced by neutralizing antibody against the antigenic polypeptide that comprises a receptor-binding portion of a SARS-CoV-2 S protein as measured in serum of a subject vaccinated with at least one dose of the immunogenic composition at 1-72 hours post administration. 81. The immunogenic composition of embodiment 80, wherein the 1,000-10,000 neutralization titer is produced in the absence of antibody-dependent enhancement (ADE) of a SARS-CoV-2-associated disease. 82. The immunogenic composition of any one of embodiments 63-81, wherein the effective amount does not induce the immunogenic composition-associated enhanced respiratory disease (ERD). 83. The immunogenic composition of any one of embodiments 63-82, wherein the effective amount reduces the amount of SARS-CoV-2 viral RNA in a lung of the subject after infection with a SARS-CoV-2 virus, as compared to the amount of SARS-CoV-2 viral RNA in a lung of an unvaccinated subject after infection with a SARS-CoV-2 virus. 84. The immunogenic composition of any one of embodiments 63-82, wherein the effective amount reduces the amount of SARS-CoV-2 viral RNA in a lung of the subject at least 3 days after infection with a SARS-CoV-2 virus, as compared to the amount of SARS-CoV-2 viral RNA in a lung of the subject 3 days after infection with a SARS-CoV-2 virus. 85. The immunogenic composition of any one of embodiments 63-82, wherein the effective amount reduces the amount of SARS-CoV-2 viral RNA in a nasal swab sample of the subject after infection with a SARS-CoV-2 virus, as compared to the amount of SARS-CoV-2 viral RNA in a nasal swab sample of an unvaccinated subject after infection with a SARS-CoV-2 virus. 86. The immunogenic composition of any one of embodiments 63-82, wherein the effective amount does not increase the amount of SARS-CoV-2 viral RNA in a nasal swab sample of the subject 3 days after infection with a SARS-CoV-2 virus, as compared to the amount of SARS-CoV-2 viral RNA in a nasal swab sample of the subject 1 day after infection with a SARS-CoV-2 virus. 87. The immunogenic composition of any one of embodiments 63-87, wherein an anti-SARS-CoV-2 antibody titer produced in a subject vaccinated with at least one dose of the immunogenic composition is increased by at least 1 log relative to a control, wherein the control is an anti-SARS-CoV-2 antibody titer produced in a subject who has not been administered an immunogenic composition against SARS-CoV-2. 88. The immunogenic composition of any one of embodiments 63-87, wherein an anti-SARS-CoV-2 antibody titer produced in a subject vaccinated with at least one dose of the immunogenic composition is increased at least 2 times relative to a control, wherein the control is an anti-SARS-CoV-2 antibody titer produced in a subject who has not been administered an immunogenic composition against SARS-CoV-2. 89. The immunogenic composition of any one of embodiments 63-88, wherein the effective amount is a total dose of 2 ?g-100 ?g. 90. The immunogenic composition of embodiment 89, wherein the effective amount is a total dose of 100 ?g. 91. The immunogenic composition of embodiment 89, wherein the effective amount is a total dose of 20 ?g-50 ?g. 92. The immunogenic composition of embodiment 89, wherein the effective amount is a total dose of 10 ?g-30 ?g. 93. The immunogenic composition of embodiment 89, wherein the effective amount is a total dose of 10 ?g. 94. The immunogenic composition of embodiment 89, wherein the effective amount is a total dose of 20 ?g. 95. The immunogenic composition of embodiment 89, wherein the effective amount is a total dose of 30 ?g. 96. The immunogenic composition of any one of embodiments 63-95, wherein the composition is formulated in a single-dose vial. 97. The immunogenic composition of any one of embodiments 63-95, wherein the composition is formulated in a multi-dose vial. 98. The immunogenic composition of any one of embodiments 63-97, wherein intramuscular administration of the effective amount of the immunogenic composition to a subject induces a neutralizing antibody titer in a subject. 99. The immunogenic composition of embodiment 98, wherein the neutralizing antibody titer is sufficient to reduce viral infection of B cells by at least 50% relative to a neutralizing antibody titer of an unvaccinated control subject or relative to a neutralizing antibody titer of a subject vaccinated with a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit viral vaccine. 100. The immunogenic composition of embodiment 98 or 99, wherein the neutralizing antibody titer is induced in the subject following fewer than three doses of the immunogenic composition. 101. The immunogenic composition of any one of embodiments 98-100, wherein the neutralizing antibody titer and/or a T cell immune response is sufficient to reduce the rate of asymptomatic viral infection relative to the neutralizing antibody titer of unvaccinated control subjects. 102. The immunogenic composition of any one of embodiments 98-101, wherein the neutralizing antibody titer and/or a T cell immune response is sufficient to prevent viral latency in the subject. 103. The immunogenic composition of any one of embodiments 98-102, wherein the neutralizing antibody titer is sufficient to block fusion of virus with epithelial cells and/or B cells of the subject. 104. The immunogenic composition of any one of embodiments 63-103, wherein intramuscular administration of the effective amount of the immunogenic composition to a subject induces a T cell immune response in the subject. 105. The immunogenic composition of embodiment 104, wherein the T cell immune response comprises a CD4+ T cell immune response and/or a CD8+ T cell immune response. 106. The immunogenic composition of any one of embodiments 63-105, wherein the encoded polypeptide is presented on the surface of cells of the subject. 107. A method comprising administering to a subject an immunogenic composition of any one of embodiments 63-106, wherein the immunogenic composition is administered to the subject in an effective amount to induce an immune response in the subject. 108. The method of embodiment 107, wherein the immune response is induced against a SARs-CoV-2 virus having a mutation in the RBD, as compared to SEQ ID NO: 5. 109. The method of embodiment 107, wherein the immune response is induced against a SARs-CoV-2 virus having a mutation in the spike protein, as compared to SEQ ID NO: 1. 110. The method of embodiment 108 or 109, wherein the immune response is induced against a SARs-CoV-2 virus having any one of the following mutations in the RBD: Q321L, V3411, A348T, N354D, S359N, V367F, K378R, R408I, Q409E, A435S, N439K, K458R, 1472V, G476S, S477N, V483A, Y508H, and H519P, as compared to SEQ ID NO: 1. 111. The method of embodiment 109, wherein the immune response is induced against a SARs-CoV-2 virus having a D614G mutation in the spike protein, as compared to SEQ ID NO: 1. 112. The method of embodiment 107, wherein the immunogenic composition is administered to the subject annually. 113. An RNA, optionally complexed by a (poly)cationic polymer, polyplex(es), protein(s) or peptide(s), which RNA: (a) comprises an open reading frame encoding a polypeptide that comprises a receptor-binding-portion of a SARS-CoV-2 S protein; and (b) is suitable for intracellular expression of the polypeptide. 114. The RNA of embodiment 113, wherein the polypeptide does not comprise the complete S protein. 115. The RNA of embodiment 113 or 114, wherein the RNA further comprises a 5 terminal cap, 7mG(5)ppp(5)NlmpNp. 116. The RNA of any one of embodiments 113-115, wherein at least 80% of the uracil in the open reading frame have a chemical modification selected from N1-methyl-pseudouridine or N1-ethyl-pseudouridine. 117. The RNA of any one of embodiments 113-116, wherein the chemical modification is in the 5-position of the uracil. 118. The RNA of any one of embodiments 113-117 for use in inducing an immune response in humans or vaccinating humans. 119. The RNA for use of embodiment 118, wherein the humans comprise humans known to have been exposed to SARS-CoV-2. 120. The RNA for use of embodiment 118, wherein the humans comprise humans known to have been infected by SARS-CoV-2. 121. The RNA for use of embodiment 118, wherein the humans comprise humans not known to have been exposed to SARS-CoV-2. 122. Use of the RNA of any one of embodiments 113-117 for vaccinating humans. 123. The use of embodiment 122, wherein the humans comprise humans known to have been exposed to SARS-CoV-2. 124. The use of embodiment 122, wherein the humans comprise humans known to have been infected by SARS-CoV-2. 125. The use of embodiment 122, wherein the humans comprise humans not known to have been exposed to SARS-CoV-2. 126. A single-dose formulation comprising the immunogenic composition of any one of embodiments 63-106. 127. A multi-dose formulation comprising the immunogenic composition of any one of embodiments 63-106 in one vial. 128. The formulation according to embodiment 126, comprising at least 2 doses per vial. 129. The formulation according to embodiment 126, comprising a total of 2-12 doses per vial. 130. The formulation according to any one of embodiments 126-129, wherein each dose is equal in volume. 131. The formulation according to any one of embodiments 126-130, wherein each formulation comprises a total volume of 1-3 mL in the vial. 132. The formulation according to any one of embodiments 126-131, wherein the immunogenic composition is frozen. 133. A pre-filled vaccine delivery device comprising the immunogenic composition of any one of embodiments 63-106.

(850) Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.

(851) The following description is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.

EXAMPLES

Example 1: In Vivo Immunogenicity Using Influenza Hemagglutinin (HA) as a Model Antigen

(852) The potency of the RNA platforms to be used for the coronavirus vaccine described herein has been tested by performing extensive immunogenicity and virus challenge studies using Influenza HA as a model antigen. The studies investigated the induction of antibody responses determined with antigen specific enzyme-linked immunosorbent assay (ELISA) test and functional studies applying virus-neutralization (VNT) assays. One study evaluated the potency of the LNP formulation using a modRNA-LNP vaccine that encodes Influenza HA. Mice were injected IM with 1 ?g on days 0 ad 28 with an LNP-formulated Influenza HA modRNA. On days 14, 28 and 49 blood samples were taken, and tested for immunogenicity. The analysis showed a high antibody immune response, resulting in very high titers of antigen-specific IgG in serum and high viral-neutralization activity (FIG. 3). Moreover, strong Th1 CD4 and CD8 T cell responses (FIG. 4) were induced by the modRNA vaccine.

Example 2: Immunogenicity Studies for Coronavirus Vaccine Candidates

(853) Primary pharmacodynamics studies were performed in BALB/c mice to test the immunogenicity of the vaccine candidates shown in the following table.

(854) TABLE-US-00020 TABLE 2 Vaccine candidates mRNA Vaccine type Vaccine encoded antigen BNT162a1 uRNA RBD (Receptor Binding Domain) of SARS- CoV-2 Spike protein (S protein) BNT162b1 modRNA RBD (Receptor Binding Domain) of SARS- CoV-2 Spike protein (S protein) BNT162b2 modRNA Modified version of SARS-CoV-2 Spike protein (S protein) BNT162c1 saRNA RBD (Receptor Binding Domain) of SARS- CoV-2 Spike protein (S protein)

(855) Thus, as can be seen, embodiments of multiple formats were assessed in parallel. This described approach and system achieved remarkable and efficient success, enabling development of an effective clinical candidate within several months of provision of antigen (e.g., SARS-CoV-2 S1 protein and/or RBD thereof) sequence (as described herein, relevant sequence information (e.g., GenBank: MN908947.3) became available in January 2020)

(856) In the study, four groups of each eight female BALB/c mice were immunized once with the animal trial material at three different doses, or with buffer (control group; see Table 3). While the clinical trial material will be diluted in saline, the animal trial material was diluted in PBS including 300 mM sucrose. As this is the storage buffer of the material itself, the test items are representative for the vaccine that will be used in the planned clinical trials. Immunizations were given IM using a dose volume of 20 ?L.

(857) TABLE-US-00021 TABLE 3 Study design Immuni- Dose Blood End of Group No of Vaccine zation volume collection in-life No animals dose Day [?L]/route Day phase 1 8 buffer 0 20/IM 7, 14, 21 28 2 8 Low 0 20/IM 7, 14, 21 28 3 8 Medium 0 20/IM 7, 14, 21 28 4 8 High 0 20/IM 7, 14, 21 28

(858) Blood of immunized animals was collected on days 7, 14, 21 and 28, and analyzed for the antibody immune response by ELISA and pseudovirus-based neutralization assay (pVNT).

(859) SARS-CoV-2-S specific antibody responses directed against the recombinant S1 subunit or the RBD were detected by ELISA. In brief, high protein-binding 96-well plates (MaxiSorp ELISA plates, VWR International GmbH, Cat. No. 7341284) were coated with 100 ng recombinant S1 subunit (Sino Biological Inc., Cat. No. 40591-V08H) or RBD (Sino Biological Inc., Cat. No. 40592-V02H) per well in 100 ?L coating buffer (50 mM sodium carbonate-bicarbonate buffer, pH9.6) overnight at 4? C. Plates were washed three times with 300 ?L/well 1?phosphate-buffered saline (PBS, VWR International GmbH, Cat. No. 0780-10L) supplemented with 0.01% Tween 20 (Carl Roth GmbH & Co. KG, Cat. No. 9127.1) and blocked with 250 ?L/well 1?Casein Blocking Buffer (Sigma-Aldrich GmbH, Cat No. B6429-500 ml) for 1 hour at 37? C. on a microplate shaker. Plates were again washed three times with 300 ?L/well 1?PBS supplemented with 0.01% Tween 20 and incubated with mouse serum samples diluted in 1?Casein Blocking Buffer for 1 hour at 37? C. on a microplate shaker. Plates were washed three times with 300 ?L/well 1?PBS supplemented with 0.01% Tween 20 and subsequently incubated with Peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Ltd., Cat. No. 115-036-071; diluted 1:7500 in 1? Casein Blocking Buffer) for 45 minutes at 37? C. on a microplate shaker. Plates were washed three times with 300 ?L/well 1?PBS supplemented with 0.01% Tween 20 and 100 ?L/well TMB substrate (Biotrend Chemiekalien GmbH, Cat. No. 4380A) was added. Plates were incubated for 8 min at room temperature and the reaction stopped by addition of 100 ?L 25% sulphuric acid (VWR International GmbH, Cat. No. 1007161000). Plates were read on a microplate reader and the recorded absorbance at 450 nm corrected by subtracting the reference absorbance at 620 nM.

(860) Functional antibody responses to the vaccine candidates were detected by pVNT. The pVNT uses a replication-deficient vesicular stomatitis virus (VSV) that lacks the genetic information for the VSV envelope glycoprotein G but contains an open-reading frame (ORF) for green fluorescent protein (GFP). VSV/SARS-CoV-2 pseudovirus was generated according to a published protocol (Hoffmann et al., Cell, 2020; PMID 32142651). The pseudotype virus bears the SARS-CoV-2 S protein, which mediates cell entry. Therefore, the pseudovirus can be inactivated by neutralizing antibodies that bind SARS-CoV-2 S. This inactivation can be analyzed via in vitro methods.

(861) In brief, 4?10.sup.4 Vero 76 cells (ATCC? CRL-1587?) per well were seeded in a 96-well plate (Greiner Bio-One GmbH, Cat. No. 655160) in 150 ?L/well DMEM (Thermo Fisher Scientific, Cat. No. 61965059) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich GmbH, Cat. No. F7524). Cells were incubated for 4 to 6 hours at 37? C. and 7.5% CO.sub.2. Meanwhile, mouse serum samples were diluted 1:6 up to 1:768 in DMEM/10% FBS in two-fold dilution steps. Diluted serum samples were combined with an equal volume of titrated and pre-diluted VSV/SARS CoV-2 pseudovirus supernatant, resulting in a serum dilution ranging from 1:12 up to 1:1536. The pseudovirus/serum dilution mix was incubated for 5 min at RT on a microplate shaker at 750 rpm with an additional 5 min incubation at RT without agitation. 50 ?L/well pseudovirus/serum dilution mix was added to the seeded Vero-76 cells with the applied pseudovirus volume per well corresponding to 200 infectious units (IU). Each dilution of serum samples was tested in duplicate wells. Cells were incubated for 16 to 24 hours at 37? C. and 7.5% CO.sub.2. Vero 76 cells incubated with pseudovirus in the absence of mouse sera were used as positive controls. Vero 76 cells incubated without pseudovirus were used as negative controls. After the incubation, the cell culture plates were removed from the incubator, placed in an IncuCyte Live Cell Analysis system (Essen Bioscience) and incubated for 30 min prior to the analysis. Whole well scanning for brightfield and GFP fluorescence was performed using a 4? objective. To calculate the neutralizing titer, infected GFP-positive cell number per well was compared with the pseudovirus positive control. Mean values of the pseudovirus positive control multiplied by 0.5 represent the pseudovirus neutralization 50% (pVN50). Serum samples with mean values below this cut-off exhibit >50% virus neutralization activity, respectively.

(862) Immunogenicity Study of BNT162a1 (RBL063.3)

(863) To dissect the potency of the LNP-formulated uRNA vaccine coding for BNT162a1, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

(864) ELISA data 7, 14, 21 and 28 d after the first immunization show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 5).

(865) Immunogenicity Study of BNT162b1 (RBP020.3)

(866) To dissect the potency of the LNP-formulated modRNA vaccine coding for BNT162b1, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

(867) ELISA data 7, 14, 21 and 28 d after the first immunization show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 6). Sera obtained 14, 21, and 28 d after immunization show high SARS-CoV-2 pseudovirus neutralization, especially sera from mice immunized with 1 or 5 ?g BNT162b1 and correlating with the strong increase of IgG antibody titers (FIG. 7).

(868) Immunogenicity Study of BNT162c1 (RBS004.3)

(869) To dissect the potency of the LNP-formulated saRNA vaccine coding for BNT162c1, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

(870) ELISA data 7, 14 and 21 d after the first immunization show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 8). Sera obtained 14, and 21 d after immunization show dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG. 9).

(871) Immunogenicity Study of LNP-Formulated uRNA Encoding the Viral S Protein-V8 (SEQ ID NO: 7, 8) (RBL063.1)

(872) To dissect the potency of the LNP-formulated uRNA vaccine coding for the viral S protein-V8 (RBL063.1), BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response. ELISA data 7, 14, 21 and 28 d after the first immunization are available that show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 10). Sera obtained 14, 21 and 28 d after immunization show dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG. 11).

(873) Immunogenicity Study of BNT162b2 (RBP020.1)

(874) To dissect the potency of the vaccine BNT162b2 (RBP020.1), the immunogenicity of the construct was investigated. For this purpose, a dose titration study in BALB/c mice was initiated where the immune response will be analyzed focusing on the antibody immune response.

(875) ELISA data 7, 14, and 21 d after the first immunization are available that show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 12). Sera obtained 14, and 21 d after immunization show dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG. 13).

(876) Immunogenicity Study of the LNP-Formulated saRNA Encoding the Viral S Protein-V9 (SEQ ID NO: 7, 9) (RBS004.2)

(877) To dissect the potency of the LNP-formulated saRNA vaccine coding for V9, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

(878) ELISA data 7, 14, and 21 d after the first immunization are available that show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 14). Sera obtained 14, and 21 d after immunization show dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG. 15).

(879) The above data demonstrate an immune response for both the RBD with a trimerization domain (V5) and the mutated full-length S protein (V8/V9) in vivo in all tested platforms (including the vaccines BNT162a1, BNT162b1, BNT162b2, and BNT162c1). The antibody immune response was already seen at very early time points by ELISA (i.e., at 7 d post-immunization) Importantly, induced antibodies were able to efficiently neutralize SARS-COV-2 pseudovirus infection in vitro. Also, the induction of an antibody response using a very low immunization dose of 0.2 ?g/mouse when using the modRNA platform (BNT162b1, BNT162b2) as well as the saRNA platform (BNT162c1) indicates a high potency of the vaccine candidates.

(880) In mice, BNT162b2 induced a higher antigen-specific titer compared to BNT162b1 encoded with the identical RNA platform. As expected, the immunogenicity in mice against the antigens differs between the RNA platforms. In mice, the most immunogenic platform based on antigen-specific antibody induction is the modRNA followed by saRNA. The uRNA platform induces the lowest antigen-specific antibody titer.

Example 3: Selection of Formulation

(881) The LNP delivery system was in general developed to effectively and safely deliver therapeutic nucleic acids into the cytosol of various cell types after local administration in vivo. The early formulation work was performed with several promising LNP formulations and surrogate RNA coding for luciferase. The aim of the experiments was to correlate the effect of different ionizable cationic lipids on the efficacy of RNA delivery by LNPs in vivo. Formulations were compared in terms of RNA encapsulation efficiency, apparent pKa, LNP size and polydispersity. Among the screened cationic lipids, ALC-0315 exhibited suitable physical characteristics regarding particle size, homogeneity, and RNA encapsulation efficiency.

(882) Based on this the ALC-0315/DSPC/CHOL/ALC-0159 prototype was submitted for in vivo screening. The results presented in FIG. 16 summarize the in vivo testing of two independent pilot batches using luciferase (Luc) RNA. The results demonstrate improved potency of the ALC-0315 prototype as compared to an internal benchmark (ALC-0218). On the basis of these studies, ALC-0315 was identified as a highly potent cationic lipid and brought forward for further product development studies.

(883) The formulation screening procedure described above involves intravenous administration resulting in delivery primarily to the liver. The mechanism of LNP uptake into hepatocytes is driven by binding of endogenous apolipoproteins to the LNP followed by receptor-mediated endocytosis e.g. through low density lipoprotein receptors. In order to investigate whether the same mechanism is involved for an intramuscular administration, Luc RNA containing LNPs comprising ALC-0315 were injected intravenously (0.3 mg/kg) and intramuscularly (0.2 mg/kg) into ApoE knockout mice in the presence or absence of recombinant human ApoE3. As control, wild-type C57Bl/6 mice were also treated by the different routes of administration. RNA-LNP were pre-incubated with recombinant human ApoE3 (1 mg encapsulated mRNA with 1 mg ApoE3) for 1 hour at room temperature (RT) prior to administration. Luc expression was monitored at 4, 24, 72 and 96 hours post administration (FIG. 17).

(884) When mice were administered intravenously, Luc expression was detected in the wild-type C57Bl/6 mice. In the ApoE knockout mice Luc expression was significantly reduced however when preincubated with exogenous ApoE the expression of Luc was recovered to similar expression levels as wild-type mice (FIG. 18).

(885) In vivo Luc expression experiments using mouse models showed, that similar mechanisms are involved in the uptake of RNA-LNP in case of intramuscular administration as for intravenous administration, and this is not only true for hepatocytes but also for the cells local to the administration site.

(886) In vivo experiments after intramuscular administration of the final ALC-0315/DSPC/CHOL/ALC-0159, confirmed minimal drainage with regards to biodistribution, immunogenicity (vaccine activity) and tolerability.

Example 4: Immunogenicity Studies for Coronavirus Vaccine Candidates

(887) Functional cellular immune responses to the vaccine candidates were detected by ELISpot assay using the IFN-? ELISpot.sup.PLUS kit (Mabtech, Cat. No. 3321-4APT-2). In brief, spleens were removed from animals after sacrifice at day 28 after vaccination. Spleens were mechanically dissociated using the plunger of a syringe and a 70 ?M cell strainer (Greiner Bio-One GmbH, Cat. No. 542070). Splenocytes were washed with an excess volume of DPBS (Thermo Fisher Scientific, Cat. No. 14190-094) followed by centrifugation at 300?g for 6 min at RT and discarding the supernatants. Erythrocytes were then lysed with erythrocyte lysis buffer (154 mM NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM EDTA) for 5 min at RT. The reaction was stopped with an excess volume of DPBS. After another washing step, cells were resuspended in RPMI 1640 medium (Gibco, Cat. no. 61870-010) supplemented with 10% FBS, 1% MEM Non-Essential Amino Acids Solution (Gibco, Cat. No. 11140-035), 1% sodium pyruvate (Gibco, Cat. No. 11360-039), 0.5% penicillin/streptomycin (Gibco, Cat. No. 15140-122), passed through a 70 ?m cell mesh again and counted. CD8+ or CD4+ T cells were isolated from splenocyte cell suspensions using CD8a or CD4 MACS? MicroBeads (Miltenyi Biotec, Cat. No. 130-117-044 and 130-117-043) according to the manufacturer's instructions. In parallel, 96-well ELISpot plates were washed with PBS and blocked with medium (RPMI 1640 medium supplemented with 10% FBS, 1% MEM Non-Essential Amino Acids Solution, 1% sodium pyruvate, 0.5% penicillin/streptomycinmedium) for at least 30 min at 37? C. 1?10.sup.5 CD8+ or CD4+ T cells in 100 ?L medium were subsequently re-stimulated by addition of 50 ?L peptide solution (irrelevant control peptide AH1 (2 ?g/mL; sequence: SPSYVYHQF (SEQ ID NO: 35)), PepMix? SARS-CoV-2 S-RBD (0.025 ?g/mL per peptide; JPT, customized) or PepMix? SARS-CoV-2 Spike Glycoprotein (0.1 ?g/mL per peptide; JPT, Cat. No. PM-WCPV-S-2) and 50 ?L of autologous bone marrow-derived dendritic cells in the IFN-? ELISpot assay. Each condition was tested in duplicate. Plates were incubated overnight in a 37? C. humidified incubator with 5% CO.sub.2 and after approximately 18 h, cells were removed from the plates. IFN-? spots were detected according to the manufacturer's protocol. After plate drying for 2-3 h under the laminar flow, an ELISpot plate reader (ImmunoSpot? S6 Core Analyzer, CTL) was used to count and analyze spot numbers per well.

(888) In addition to ELISpot assay, Luminex analyses were conducted to inform about the T.sub.H1 or T.sub.H2 nature of the detected T cell response. 5?10.sup.5 splenocytes in 100 ?L RPMI 1640 medium supplemented with 10% FBS, 1% MEM Non-Essential Amino Acids Solution, 1% sodium pyruvate, 0.5% penicillin/streptomycinmedium were transferred to a 96-well flat bottom cell culture plate. 100 ?L irrelevant control peptide AH1 (2 ?g/mL; sequence: SPSYVYHQF (SEQ ID NO: 35)), or PepMix? SARS-CoV-2 Spike Glycoprotein (0.1 ?g/mL per peptide; JPT, Cat. No. PM-WCPV-S-2) were added. The plates were incubated for 48 hours and supernatant thereafter was harvested for cytokine profiling. Cytokine concentrations in supernatants of the re-stimulated splenocytes were determined using a bead-based T.sub.H1/T.sub.H2 ProcartaPlex immunoassay (Thermo Fisher Scientific, Cat. No. EPX110-20820-901) according to the manufacturer's instructions. Fluorescence was measured with the Bioplex200 System (Biorad) and analyzed with ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific). The following analytes were measured: IFN-?; IL-12p70; IL-13; IL-1 beta; IL-2; IL-4; IL-5; IL-6; TNF alpha; GM-CSF; IL-18.

(889) For immunophenotyping, flow cytometry analysis was performed. Briefly, erythrocytes from 50 ?L freshly drawn blood were lysed with ACK lysing buffer (Gibco) and cells were stained with fixable viability dye (eBioscience) and anti-CXCR5 (rat IgG2a) antibody in the presence of Fc block (both BD Bioscience) in flow buffer (DPBS (Gibco) supplemented with 2% FCS, 2 mM EDTA (both Sigma) and 0.01% sodium azide (Morphisto) for 20 minutes at room temperature. After staining with anti-rat IgG2a biotin in flow buffer for 20 minutes at 2-8? C., cells were stained extracellularly with antibodies against CD3, CD4, CD8a, CD38, CD44, PD-1, ICOS, CD62L, CXCR5, CD19 and streptavidin in Brilliant Stain Buffer Plus (BD Bioscience) diluted in flow buffer for 20 minutes at 2-8? C. Cells were fixed with 2% RotiHistofix (Roth) for 15 minutes at room temperature. Cells were resuspended in Perm buffer (FoxP3/Transcription Factor Staining Buffer Set, eBioscience) and incubated over night at 2-8? C. Permeabilized cells were intracellularly treated with Fc block for 10 minutes at 2-8? C. and stained with T-bet and GATA (BD Bioscience) antibodies for 30 minutes at 2-8? C. Cells were resuspended in flow buffer and acquired on a BD Symphony A3 flow cytometer (BD Bioscience) and analyzed with FlowJo 10.6.2.

(890) For mouse B cell subtyping in draining lymph nodes, 2.5?10.sup.5 lymph node cells were treated with Fc block for 15 minutes and stained extracellularly with antibodies against CD19, CD45R/B220, IgD, CD138, IgM, CD38, CD95/FAS, IgG1, IgG2a, CD73, GR-1, F4/80, CD4, CD8 in Brilliant Stain Buffer (BD Bioscience) for 20 minutes at 2-8? C. Cells were fixed with 2% RotiHistofix and incubated over night at 2-8? C.

(891) Immunogenicity Study of BNT162b1 (RBP020.3)

(892) To dissect the potency of the LNP-formulated modRNA vaccine coding for BNT162b1, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine was investigated by focusing on the cellular immune response.

(893) After stimulation with an S protein- or RBD-specific peptide pool, but not after stimulation with irrelevant peptide AH1, both CD4+ and CD8+ T cells displayed IFN-? responses in the IFN-? ELISpot assay (FIG. 22). In Luminex analysis, cytokine production after peptide stimulation was confirmed for analytes that indicate a T.sub.H1-driven immune response (FIG. 23).

(894) Immunophenotyping analysis of blood 7 days after immunization (FIG. 24) revealed a significant increase in circulating T follicular helper cells (Tfh) and activated T cells. At day 12 after immunization, draining lymph nodes from immunized BALB/c mice were dissected and B cell subpopulation analysis was performed (FIG. 25). A significant increase in B cells was found in lymph nodes with detectable numbers of plasma cells, class switched B cells and IgG1 or IgG2a positive germinal center B cells. Both in blood and draining lymph node, an activation and maturation of the adaptive immune response was confirmed.

(895) Immunogenicity Study of the LNP-Formulated modRNA Encoding the Viral P2-S Protein V8 (RBP020.1)

(896) To dissect the potency of the LNP-formulated modRNA vaccine coding for RBP020.1, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine was investigated by focusing on the cellular immune response.

(897) After stimulation with an S protein-specific peptide pool, but not after stimulation with irrelevant peptide AH1, both CD4+ and CD8+ T cells displayed IFN-? responses in the IFN-? ELISpot assay (FIG. 26). In Luminex analysis, cytokine production after peptide stimulation was confirmed for analytes that indicate a T.sub.H1-driven immune response (FIG. 27).

(898) Immunogenicity Study of the LNP-Formulated saRNA Encoding the Viral P2-S Protein V9 (RBS004.2)

(899) To dissect the potency of the LNP-formulated saRNA vaccine coding for RBS004.2, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine was investigated by focusing on the cellular immune response.

(900) After stimulation with an S protein-specific peptide pool, but not after stimulation with irrelevant peptide AH1, both CD4+ and CD8+ T cells displayed IFN-? responses in the IFN-? ELISpot assay (FIG. 28). In Luminex analysis, cytokine production after peptide stimulation was confirmed for analytes that indicate a T.sub.H1-driven immune response (FIG. 29).

(901) Immunogenicity Study of BNT162b3 variants BNT162b3c and BNT162b3d

(902) To get an idea about the potential potency of transmembrane-anchored RBD-based vaccine antigens (Schematic in FIG. 30; BNT162b3c (1) and BNT162b3d (2)), BALB/c mice were immunized IM once with 4 ?g LNP-C12 formulated mRNA or with buffer as control. The non-clinical LNP-C12 formulated mRNAs were used as surrogate for the BNT162b3 variants BNT162b3c and BNT162b3d. The immunogenicity of the RNA vaccine was investigated by focusing on the antibody immune response.

(903) ELISA data 6, 14 and 21 d after the first immunization show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 31). Sera obtained 6, 14 and 21 d after immunization show high SARS-CoV-2 pseudovirus neutralization, correlating with the increase of IgG antibody titers (FIG. 32).

Example 5: Immunogenicity Studies for Coronavirus Vaccine Candidates in Non-Human Primates (NHP)

(904) Six Rhesus macaques per group were immunized IM with 30 or 100 ?g of BNT162b1 or with buffer on days 0 and 21. By 14 days after the first dose, antibodies that bound a recombinant S1 were readily detectable, and levels of S1-binding antibodies exceeded the upper limit of quantification of the assay (10,000 U/mL) by day 28. For comparison, S1-binding antibodies of 62 human COVID-19 convalescent sera, obtained after the onset of symptoms were analyzed. All timepoints throughout the two NHP groups significantly exceeded the mean of the human COVID-19 convalescent sera of 422 U/mL (FIG. 33(A)). The VNT geometric mean titers (GMTs) of sera from Rhesus macaques immunized with either dose level of BNT162b1 were detectable by 14 days after a single immunization and reached a geometric mean of 768 (30 ?g dose level) or 1,714 (100 ?g) by day 28 (FIG. 33(B)). The neutralization GMTs were 282 (30 ?g) and 975 (100 ?g) on day 35 (14 days after the boost) (FIG. 33(B)). Flow cytometry analysis of CD4.sup.+?T cells from blood samples stimulated with a S peptide mix on day 42 revealed significant secretion of the T.sub.H1 cytokines IFN?, IL-2 and TNF?. Additionally, IL-21 secretion was significantly increased. IL-21 is known to play a critical role in B cell activation, expansion and plasma cell generation as well as the generation of Tfh. In contrast, no significant amounts of the T.sub.H2 cytokine IL-4 were detected (FIG. 33(C)). In summary and in alignment to the results obtained in mice, BNT162b1 induced a high antibody immune with an early affinity maturation coming with a T.sub.H1 biased immune response.

(905) In summary, we demonstrate that the methyl-nucleoside m1?-modified mRNA encoding the trimeric receptor binding domain from the SARS-CoV-2 S protein is protective in non-human primates.

Example 6: Phase 1/2 Study to Describe the Safety, Tolerability, and Immunogenicity of a COVID-19 RNA Vaccine Candidate (BNT162b1) in Healthy Adults 18 to 55 Years of Age

(906) We report safety, tolerability, and immunogenicity from a placebo-controlled, observer-blinded dose escalation trial among healthy adults randomized to receive 2 doses of 10 ?g, 30 ?g, or 100 ?g of BNT162b1, a lipid nanoparticle (LNP)-formulated, nucleoside-modified, mRNA vaccine candidate that encodes trimerized SARS-CoV-2 spike glycoprotein RBD antigen. Local reactions and systemic events were dose-dependent, generally mild to moderate, and transient. RBD-binding IgG concentrations and SARS-CoV-2 neutralizing titers increased with dose level and after a second dose. Geometric mean neutralizing titers reached 1.8- to 2.8-fold that of a COVID-19 convalescent human serum panel.

(907) The BNT162b1 vaccine candidate now being tested clinically incorporates nucleoside 1-methyl-pseudouridine modified RNA (modRNA) and encodes the receptor binding domain of the SARS-CoV-2 spike, a key target of neutralizing antibodies. The RBD antigen expressed by BNT162b1 is modified by the addition of a T4 fibritin-derived foldon trimerization domain to increase its immunogenicity. This RNA vaccine candidate is being tested in parallel in coordinated studies in Germany and in the US. Here, we present data obtained in the US study.

(908) Methods

(909) Study design and participants: This Phase ?, randomized, placebo-controlled, observer-blinded trial was conducted in the United States to assess the safety, tolerability, and immunogenicity of ascending dose levels of various BNT162 mRNA vaccine candidates. Assessment of three dose levels (10, 30, or 100 ?g) of the BNT162b1 candidate was conducted at two sites in the United States. This study utilized a sentinel cohort design with progression and dose escalation taking place after review of data from the sentinel cohort at each dose level. Healthy men and nonpregnant women 18 to 55 years of age were enrolled. Key exclusion criteria included individuals with known infection with human immunodeficiency virus, hepatitis C virus, or hepatitis B virus; immunocompromised individuals and those with a history of autoimmune disease; those with increased risk for severe COVID-19; previous clinical or microbiological diagnosis of COVID-19; receipt of medications intended to prevent COVID-19; previous vaccination with any coronavirus vaccine; and a SARS-CoV-2 NAAT-positive nasal swab within 24 hours before study vaccination.

(910) The final protocol and informed consent document were approved by institutional review boards for each of the investigational centers participating in this study, and this study was conducted in compliance with all International Council for Harmonisation (ICH) Good Clinical Practice (GCP) guidelines and the ethical principles of the Declaration of Helsinki. A signed and dated informed consent was required before any study-specific activity was performed.

(911) Endpoints: The study's primary endpoints included: the proportion of participants reporting prompted local reactions, systemic events, and use of antipyretic and/or pain medication within 7 days after vaccination, AEs through 1 month after the last dose, and SAEs through 6 months after vaccination, and the proportion of sentinel cohort participants with clinical laboratory abnormalities 1 week after vaccination and grading shifts in laboratory assessments between baseline and 1 and 7 days after Dose 1 and between Dose 2 and 7 days after Dose 2. Secondary endpoints included: SARS-CoV-2 neutralizing geometric mean titers (GMTs); SARS-CoV-2 S1-binding IgG and RBD-binding IgG geometric mean concentrations (GMCs) 7 and 21 days after Dose 1 and 7 and 14 days after Dose 2; geometric mean fold rise (GMFR), ?4-fold rise from baseline and geometric mean ratio (GMR) of SARS-CoV-2 serum neutralizing GMTs to SARS-CoV-2-antigen binding antibody GMCs at each time point.

(912) Procedures: Study participants were randomly assigned to a vaccine group using an interactive web-based response technology system with each group comprising 15 participants (12 active vaccine recipients and 3 placebo recipients). Participants received two 0.5-mL doses of either BNT162b1 or placebo, administered by intramuscular injection into the deltoid muscle.

(913) BNT162b1 incorporates a Good Manufacturing Process (GMP)-grade mRNA drug substance that encodes the trimerized SARS-CoV-2 spike glycoprotein RBD antigen. The mRNA is formulated with lipids as the mRNA-LNP drug product. The vaccine was supplied as a buffered-liquid solution for IM injection and was stored at ?80? C. The placebo was a sterile saline solution for injection (0.9% sodium chloride injection, in a 0.5-mL dose).

(914) Safety assessments for all participants included 4-hour observation after vaccination (for the first 5 participants vaccinated in each sentinel group, and a 30-minute observation (for the remainder of participants) for immediate AEs. The safety assessments also included self-reporting of prompted local reactions (redness, swelling, and pain at the injection site), systemic events (fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, and joint pain), and the use of antipyretic and/or pain medication in an electronic diary (e-diary) for 7 days after vaccination, reporting of unprompted AEs through 1 month after vaccination and SAEs through 6 months after the last vaccination. Hematology and chemistry assessments were conducted at screening, 1 and 7 days after Dose 1, and 7 days after Dose 2.

(915) The protocol specified safety stopping rules for all sentinel-cohort participants. Both an internal review committee (IRC) and an external data monitoring committee (EDMC) reviewed all safety data.

(916) Immunogenicity testing: 50 mL of blood was collected for immunogenicity assessments (SARS-CoV-2 serum neutralization assay, SARS-CoV-2 S1-specific IgG direct Luminex immunoassay, SARS-CoV-2 RBD-specific IgG direct Luminex immunoassay and nonvaccine antigen (NVA) Ig direct Luminex immunoassay) before each study vaccination, at 7 and 21 days after Dose 1 and at 7, 14, 1 month and 6 months after Dose 2.

(917) The SARS-CoV-2 neutralization assay used a previously described strain of SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse genetics and engineered by the insertion of an mNeonGreen (mNG) gene into open reading frame 7 of the viral genome. This reporter virus generates similar plaque morphologies and indistinguishable growth curves from wild-type virus. Serial dilutions of heat inactivated sera were incubated with the reporter virus for 1 hour at 37? C. before inoculating Vero E6 cell monolayers. Infected foci were detected by fluorescence between 16-24 hours after inoculation by the addition of Hoechst 33342 Solution and counted with a Cytation 7 Cell Imaging Multi-Mode Reader.

(918) Statistical analysis: The sample size for the sentinel cohort of the study was not based on statistical hypothesis testing. The primary safety objective was evaluated by descriptive summary statistics for local reactions, systemic events, abnormal hematology and chemistry laboratory parameters, and AEs and SAEs for each vaccine group. A 3-tier approach was used to summarize AEs. The secondary immunogenicity objectives were descriptively summarized at the various time points.

(919) Results

(920) Between 4 May 2020 and 19 Jun. 2020, 76 subjects were screened, and 45 participants were randomized and vaccinated. Twelve participants per dose level (10 ?g, 30 ?g, or 100 ?g) were vaccinated with BNT162b1 on Days 0 and 21, and 9 participants received placebo (FIG. 34). The study population consisted of healthy male and nonpregnant female participants, 18 to 55 years of age with a mean age of 35.4 years (minimum 19 and maximum 54 years). Overall, 51.1% of participants were male and 48.9% were female. Most participants were white (82.2%) and non-Hispanic/non-Latino (93.3%).

(921) Safety and Tolerability

(922) In the 7 days after either vaccination, pain at the injection site was the most frequently local reaction, reported by 58.3% (7/12) in the 10 ag and 100.0% (12/12 each) in the 30 ag and 100 ?g BNT162b1 groups and by 22.2% (2/9) of placebo recipients after Dose 1 and by 83.3% and 100.0% of BNT162b1 recipients at the 10 ?g and 30 ?g dose levels, respectively, after Dose 2. All local reactions were mild or moderate in severity except for one report of severe pain following Dose 1 of 100 ?g BNT162b1.

(923) The most common systemic events reported in the 7 days after vaccination were mild to moderate fatigue and headache in both BNT162b1 and placebo recipients. Systemic events increased with dose level and were reported in a greater number of subjects after the second dose (10 ?g and 30 ?g groups). Following Dose 1, fever ?38.0? C. was reported by 50.0% (6/12) of BNT162b1 recipients in the 100 ?g group and 8.3% (1/12) of participants each in the 10 ?g and 30 ag groups. Following Dose 2, 8.3% (1/12) of participants in the 10 ag group and 75.0% (9/12) of participants in the 30 ag group reported fever ?38.0? C. No Grade 4 systemic events or fever were reported. (FIGS. 35 & 36). Most local reactions and systemic events peaked by Day 2 after vaccination and resolved by Day 7. Based on the reactogenicity profile, participants who received an initial 100 ?g dose did not receive a second vaccination.

(924) Adverse events were reported by 50.0% (6/12) of participants who received 10 ?g or 30 ?g of BNT162b1, by 58.3% (7/12) of those who received 100 ?g of BNT162b1, and by 11.1% (1/9) of placebo recipients. Two participants reported a severe AE, one at the 30 ?g dose level (Grade 3 pyrexia 2 days after vaccination) and one at the 100 ag dose level (sleep disturbance 1 day after vaccination). Related AEs were reported by 25% (3/12) to 50% (6/12) of BNT162b1 recipients and by 11.1% (1/6) of those who received placebo. No serious adverse events were reported.

(925) No Grade 1 or greater change in routine clinical laboratory values or laboratory abnormalities were observed for most subjects after either of the BNT162b1 vaccinations. The most notable changes were decreases in lymphocyte count in 8.3% (1/12), 45.5% (5/11), and 50.0% (6/12) of participants who received 10 ?g, 30 ?g, or 100 ?g, respectively, of BNT162b1. One participant each at the 10 ?g (8.3%) and 30 ?g (9.1%) dose levels and 4 participants at the 100 ?g dose level (33.3%) had Grade 3 decreases in lymphocytes. These hematological changes, which were noted in blood drawn 1-3 days after Dose 1, returned to normal 6-8 days after vaccination. None of the changes in laboratory values after vaccination were associated with clinical findings. In addition, Grade 2 neutropenia was noted 6-9 days after the second dose of 10 ?g or 30 ?g BNT162b1, in 1 participant each. The neutrophil count was not repeated for these two subjects however they continue be followed in the study and no adverse events or clinical manifestation of neutropenia have been reported to date.

(926) Immunogenicity

(927) RBD-binding IgG concentrations and SARS-CoV-2 neutralizing titers were assessed in sera drawn at baseline and at 7 and 21 days after the first dose and 7 days (Day 28) after the second dose of BNT162b1 (FIG. 37(A)). By 21 days after the first dose (for all three dose levels), geometric mean concentrations (GMCs) of RBD-binding IgG were 534-1,778 units/mL, compared to 602 units/mL for a panel of COVID-19 convalescent human sera. By 7 days after the second dose (for the 10 ?g and 30 ?g dose levels) RBD-binding IgG GMCs had increased to 4,813-30,207 units/mL. Because the participants who received a first dose of 100 ?g BNT162b1 did not receive a second dose, the development of the antibody response without a second dose could not be evaluated, and there was no further increase in RBD-binding antibody concentration beyond 21 days after the first dose among participants in this dosing group. Highly elevated RBD-binding antibody concentrations persisted to Day 35 (two weeks after the second dose) in the participants who received 10 ?g and 30 ?g dose levels of BNT162b1.

(928) Modest increases in SARS-CoV-2 neutralizing geometric mean titers (GMTs) were observed 21 days after Dose 1 (FIG. 37(B)). Substantially greater serum neutralizing GMTs were achieved 7 days after participants received a second 10 ?g or 30 ?g dose, reaching 168-267, compared to 94 for the COVID-19 convalescent serum panel.

(929) Discussion

(930) The RNA-based vaccine candidate BNT162b1 was safe and well tolerated. All dose levels exhibited a tolerability and safety profile consistent with those previously observed for mRNA-based vaccines. A clear dose level response was observed after Doses 1 and 2 in adults 18-55 years of age. Reactogenicity was generally higher after the second dose, but symptoms resolved within a few days after presentation. Based on the tolerability profile of the first dose at the 100 ?g dose level, participants randomized to the 100 ?g group did not receive a second vaccination. Transient decreases in lymphocytes (Grades 1-3) were observed within a few days after vaccination; however, lymphocyte counts returned to baseline within 6-8 days in all participants. These laboratory abnormalities were not associated with clinical findings. Lymphopenia following vaccination is most likely explained by transient migration of lymphocytes into the tissues.

(931) Robust immunogenicity was observed after vaccination with BNT162b1. RBD-binding IgG concentrations were detected at Day 21 and substantially increased 7 days after the booster dose given at Day 21. After the first dose, the RBD-binding IgG GMCs in vaccinated participants (10 ?g dose level) were similar to those observed in a panel of 38 sera from COVID-19 convalescing humans obtained 20-40 days after the onset of symptoms and at least 14 days after the start of asymptomatic convalescence. In sera drawn from the 30 ?g and 100 ?g dose level cohorts, GMCs were substantially higher than in the convalescent serum panel. After the booster vaccinations (Dose 2) with 10 ?g or 30 ?g BNT162b1, the RBD-binding IgG GMCs were 8.0-fold to 50-fold higher than the convalescent serum panel GMC.

(932) Sera from vaccinated participants were also tested in the SARS-CoV-2 neutralization assay. Neutralization titers were measurable at Day 21 for all dose levels. At Day 28 (7 days after the booster dose), substantial SARS-CoV-2 neutralization titers were observed. The virus neutralizing GMTs after the 10 ?g and 30 ?g booster vaccinations (Dose 2) were, respectively, 1.8-fold and 2.8-fold higher than the neutralizing GMT of the convalescent serum panel. As the 100 ?g dose level cohort was not boosted, no corresponding data for immunogenicity after a second vaccination are available.

(933) These clinical findings for the BNT162b1 vaccine candidate are very encouraging and provide strong evidence supporting accelerated development and at-risk manufacturing to maximize the opportunity for the soonest availability of a prophylactic vaccine to prevent COVID-19.

Example 7: Concurrent Antibody and, T Cell and Cytokine Responses Elicited by a COVID-19 RNA Vaccine

(934) In this example, we present characterisation of antibody and T cell responses after BNT162b1 vaccination from a non-randomized open-label phase I/II trial in healthy adults, 18-55 years of age. Two doses, of 1 ?g, 10 ?g, 30 ?g and 50 ?g of BNT162b1 administered 21 days apart elicited concomitant antibody, and robust CD4+ and CD8+ T cell responses. All subjects exhibited strong antibody responses with IgG concentrations significantly above those observed in COVID-19 convalescent human sera. Day 43 SARS-CoV-2 serum neutralising geometric mean titers were in the range of 0.7-fold (1 ?g) to 3.3-fold (50 ?g) compared to those of a panel of COVID-19 convalescent human sera, and were broadly active against diverse SARS-CoV-2 spike variants. Interferon (IFN)?, an immune stimulatory cytokine with anti-viral properties, was produced by a high frequency of RBD-antigen specific CD8+ T and numerous CD4+ T cells. IL-12p70, which reinforces a T.sub.H1 immune cell profile, was detected in RBD-stimulated immune cells. The robust RBD-specific antibody, T-cell and favorable cytokine responses by the BNT162b1 mRNA vaccine suggests a potential for multiple beneficial protective mechanisms against COVID-19.

(935) Materials and Methods

(936) Clinical Trial Design

(937) Study BNT162-01 (NCT04380701Germany trial) is an ongoing, first-in-human, Phase I/II, open-label dose-finding clinical trial to assess the safety, tolerability, and immunogenicity of ascending dose levels of various intramuscularly administered BNT162 mRNA vaccine candidates. Healthy men and non-pregnant women 18 to 55 years (amended to add 56-85 of age) of age are eligible. Key exclusion criteria included previous clinical or microbiological diagnosis of COVID-19; receipt of medications to prevent COVID-19; previous vaccination with any coronavirus vaccine; a positive serological test for SARS-CoV-2 IgM and/or IgG at the screening visit; and a SARS-CoV-2 NAAT-positive nasal swab within 24 hours before study vaccination; those with increased risk for severe COVID-19; immunocompromised individuals, those with known infection with HIV, hepatitis C virus, or hepatitis B virus and those with a history of autoimmune disease. The primary endpoints of the study are safety and immunogenicity.

(938) In the part of the study reported here five dose levels (1 ?g, 10 ?g, 30 ?g, 50 ?g or 60 ?g) of the BNT162b1 candidate were assessed at one site in Germany with 12 healthy volunteers per dose level in a dose escalation and de-escalation design. Sentinel dosing was performed in each dose-escalation cohort. Progression in that cohort and dose escalation required data review by a safety review committee. Subjects received a BNT162b1 prime dose on day 1, and a boost dose on day 22?2. Serum for antibody assays was obtained on day 1 (pre-prime), 8?1 (post-prime), 22?2 (pre-boost), 29?3 and 43?4 (post-boost). PBMCs for T cell studies were obtained on day 1 (pre-prime) and 29?3 (post-boost). One subject of the 10 ?g, and one subject of the 50 ?g dose cohort left the study prior to the boost immunisation due to withdrawal of consent and private reasons.

(939) The presented data comprise the BNT162b1-immunised cohorts only and are based on a preliminary analysis with a data extraction date of Jul. 13, 2020, focused on analysis of vaccine-induced immunogenicity (secondary endpoint) descriptively summarised at the various time points. All participants with data available were included in the immunogenicity analyses.

(940) The trial was carried out in Germany in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines and with approval by an independent ethics committee (Ethik-Kommission of the Landesarztekammer Baden-Wurttemberg, Stuttgart, Germany) and the competent regulatory authority (Paul-Ehrlich Institute, Langen, Germany). All subjects provided written informed consent.

(941) Manufacturing of RNA

(942) BNT162b1 incorporates a Good Manufacturing Practice (GMP)-grade mRNA drug substance that encodes the trimerized SARS-CoV-2 spike glycoprotein RBD antigen. The RNA is generated from a DNA template by in vitro transcription in the presence of 1-methylpseudouridine-5-triphosphate (m1YTP; Thermo Fisher Scientific) instead of uridine-5-triphosphate (UTP). Capping is performed co-transcriptionally using a trinucleotide cap 1 analogue ((m.sub.2.sup.7,3-O)Gppp(m.sup.2-O)ApG; TriLink). The antigen-encoding RNA contains sequence elements that increase RNA stability and translation efficiency in human dendritic cells (Holtkamp, S. et al., Blood 108, 4009-4017 (2006); Orlandini von Niessen, A. G. et al., Mol. Ther. 27, 824-836 (2019)). The mRNA is formulated with lipids to obtain the RNA-LNP drug product. The vaccine was transported and supplied as a buffered-liquid solution for IM injection and was stored at ?80? C.

(943) Proteins and Peptides

(944) A pool of 15-mer peptides overlapping by 11 aa and covering the whole sequence of the BNT162b1-encoded SARS-CoV-2 RBD, was used for ex vivo stimulation of PBMCs for flow cytometry, IFN? ELISpot and cytokine profiling. CEF (CMV, EBV, influenza virus; HLA class I epitope peptide pool) and CEFT (CMV, EBV, influenza virus, tetanus toxoid; HLA class II epitope peptide pool) (both JPT Peptide Technologies) were used as controls for general T-cell reactivity.

(945) Human Convalescent Sera and PBMC Panel

(946) Human SARS-CoV-2 infection/COVID-19 convalescent sera (n=38) were drawn from subjects 18-83 years of age at least 14 days after PCR-confirmed diagnosis and at a time when the subjects were asymptomatic. Serum donors had symptomatic infections (n=35), or had been hospitalized (n=1). Sera were obtained from Sanguine Biosciences (Sherman Oaks, CA), the MT Group (Van Nuys, CA) and Pfizer Occupational Health and Wellness (Pearl River, NY). Human SARS-CoV-2 infection/COVID-19 convalescent PBMC samples (n=6) were collected from subjects 41-79 years of age 45-59 days after PCR-confirmed diagnosis when subjects were asymptomatic. PBMC donors had asymptomatic/mild infections (n=4; clinical score 1 and 2) or had been hospitalized (n=2; clinical score 4 and 5). Blood samples were obtained from the Frankfurt University Hospital (Germany).

(947) Cell Culture and Primary Cell Isolation

(948) Vero cells (ATCC CCL-81) and Vero E6 cells (ATCC CRL-1586) were cultured in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX? (Gibco) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich). Cell lines were tested for mycoplasma contamination after receipt and before expansion and cryopreservation. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation and cryopreserved prior to subsequent analysis.

(949) RBD Binding IgG Antibody Assay

(950) A recombinant SARS-CoV-2 RBD containing a C-terminal Avitag? (Acro Biosystems) was bound to streptavidin-coated Luminex microspheres. Heat-inactivated subject sera were diluted 1:500, 1:5,000, and 1:50,000. Following an overnight incubation at 2-8? C. while shaking, plates were washed in a solution containing 0.05% Tween-20. A secondary fluorescently labelled goat anti-human polyclonal antibody (Jackson Labs) was added for 90 minutes at room temperature while shaking, before plates were washed once more in a solution containing 0.05% Tween-20. Data were captured as median fluorescent intensities (MFIs) using a Luminex reader and converted to U/mL antibody concentrations using a reference standard curve with arbitrary assigned concentrations of 100 U/mL and accounting for the serum dilution factor. Three dilutions are used to increase the likelihood that at least one result for any sample will fall within the useable range of the standard curve. Assay results were reported in U/mL of IgG. The final assay results are expressed as the geometric mean concentration of all sample dilutions that produced a valid assay result within the assay range.

(951) SARS-CoV-2 Neutralisation Assay

(952) The neutralisation assay used a previously described strain of SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse genetics and engineered by the insertion of an mNeonGreen (mNG) gene into open reading frame 7 of the viral genome (Xie, X. et al., Cell Host Microbe 27, 841-848.e3 (2020)). This reporter virus generates similar plaque morphologies and indistinguishable growth curves from wild-type virus. Viral master stocks (2?10.sup.7 PFU/mL) were grown in Vero E6 cells as previously described (Xie, X. et al., Cell Host Microbe 27, 841-848.e3 (2020)). Serial dilutions of heat-inactivated sera were incubated with the reporter virus (2?10.sup.4 PFU per well for a final multiplicity of infection (MOI) of 0.5 to yield approximately a 10-30% infection rate of the Vero monolayer) for 1 hour at 37? C. before inoculating Vero CCL81 cell monolayers (targeted to have 8,000 to 15,000 cells per well) in 96-well plates to allow accurate quantification of infected cells. Total cell counts per well were enumerated by nuclear stain (Hoechst 33342) and fluorescent virally infected foci were detected 16-24 hours after inoculation with a Cytation 7 Cell Imaging Multi-Mode Reader (Biotek) with Gen5 Image Prime version 3.09. Titers were calculated in GraphPad Prism version 8.4.2 by generating a 4-parameter (4PL) logistical fit of the percent neutralisation at each serial serum dilution. The 50% neutralisation titre (VNT.sub.50) was reported as the interpolated reciprocal of the dilution yielding a 50% reduction in fluorescent viral foci.

(953) VSV-SARS-CoV-2 Spike Variant Pseudovirus Neutralisation Assay

(954) VSV-SARS-CoV-2-S pseudoparticle generation and neutralisation assays were performed as previously described (Baum, A. et al., Science, eabd0831 (2020). doi:10.1126/science.abd0831). Briefly, human codon optimized SARS-CoV-2 spike (GenBank: MN908947.3) was synthesised (Genscript) and cloned into an expression plasmid. SARS-CoV-2 complete genome sequences were downloaded from GISAID Nucleotide database (www.gisaid.org) (last accessed 24 Aug. 2020). Sequences were curated and genetic diversity of the Spike-encoding gene was assessed across high quality genome sequences using custom pipelines. Amino acid substitutions were cloned into the spike expression plasmid using site-directed mutagenesis. HEK293T cells (ATCC CRL-3216) were seeded (culture medium: DMEM high glucose (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies) and Penicillin/Streptomycin/L-Glutamine (Life Technologies)) and transfected the following day with spike expression plasmid using Lipofectamine LTX (Life Technologies) following the manufacturer's protocol. At 24 hours post-transfection at 37? C., cells were infected with the VSV?G:mNeon/VSV-G virus diluted in Opti-MEM (Life Technologies) at a multiplicity of infection of 1. Cells were incubated 1 hour at 37? C., washed to remove residual input virus and overlaid with infection medium (DMEM high glucose supplemented with 0.7% Low IgG BSA (Sigma), sodium pyruvate (Life Technologies) and 0.5% Gentamicin (Life Technologies)). After 24 hours at 37? C., the supernatant containing VSV-SARS-CoV-2-S pseudoparticles was collected, centrifuged at 3000?g for 5 minutes to clarify and stored at ?80? C. until further use.

(955) For pseudovirus neutralisation assays, Vero cells (ATCC CCL-81) were seeded in 96-well plates in culture medium and allowed to reach approximately 85% confluence before use in the assay (24 hours later). Sera were serially diluted 1:2 in infection medium starting with a 1:40 dilution. VSV-SARS-CoV-2-S pseudoparticles were diluted 1:1 in infection medium for a fluorescent focus unit (ffu) count in the assay of ?1000. Serum dilutions were mixed 1:1 with pseudoparticles for 30 minutes at room temperature prior to addition to Vero cells and incubation at 37? C. for 24 hours. Supernatants were removed and replaced with PBS (Gibco), and fluorescent foci were quantified using the SpectraMax i3 plate reader with MiniMax imaging cytometer (Molecular Devices). Neutralisation titers were calculated in GraphPad Prism version 8.4.2 by generating a 4-parameter logistical (4PL) fit of the percent neutralisation at each serial serum dilution. The 50% pseudovirus neutralisation titre (pVNT.sub.50) was reported as the interpolated reciprocal of the dilution yielding a 50% reduction in fluorescent viral foci.

(956) IFN? ELISpot.

(957) IFN? ELISpot analysis was performed ex vivo (without further in vitro culturing for expansion) using PBMCs depleted of CD4.sup.+ and enriched for CD8.sup.+ T cells (CD8.sup.+ effectors), or depleted of CD8.sup.+ and enriched for CD4.sup.+ T cells (CD4.sup.+ effectors). Tests were performed in duplicate and with a positive control (anti-CD3 monoclonal antibody CD3-2 (1:1,000; Mabtech)). Multiscreen filter plates (Merck Millipore) pre-coated with IFN?-specific antibodies (ELISpotPro kit, Mabtech) were washed with PBS and blocked with X-VIVO 15 medium (Lonza) containing 2% human serum albumin (CSL-Behring) for 1-5 hours. Per well, 3.3?10.sup.5 effector cells were stimulated for 16-20 hours with an overlapping peptide pool representing the vaccine-encoded RBD. Bound IFN? was visualized using a secondary antibody directly conjugated with alkaline phosphatase followed by incubation with BCIP/NBT substrate (ELISpotPro kit, Mabtech). Plates were scanned using an AID Classic Robot ELISPOT Reader and analysed by ImmunoCapture V6.3 (Cellular Technology Limited) or AID ELISPOT 7.0 software (AID Autoimmun Diagnostika). Spot counts were displayed as mean values of each duplicate. T-cell responses stimulated by peptides were compared to effectors incubated with medium only as negative control using an in-house ELISpot data analysis tool (EDA), based on two statistical tests (distribution-free resampling) according to Moodie et al. (Moodie, Z., et al., J. Immunol. Methods 315, 121-32 (2006); Moodie, Z. et al., Cancer Immunol. Immunother. 59, 1489-501 (2010)), to provide sensitivity while maintaining control over false positives.

(958) To account for varying sample quality reflected in the number of spots in response to anti-CD3 antibody stimulation, a normalisation method was applied to enable direct comparison of spot counts/strength of response between individuals. This dependency was modelled in a log-linear fashion with a Bayesian model including a noise component (unpublished). For a robust normalization, each normalisation was sampled 1000 times from the model and the median taken as normalized spot count value. Likelihood of the model: log ?.sub.E=? log ?.sub.P+log ?.sub.j+??, where ?.sub.E is the normalized spot count of the sample, ? is a stable factor (normally distributed) common among all positive controls ?.sub.P, ?.sub.j a sample j specific component (normally distributed) and ?? is the noise component, of which ? is Cauchy distributed and E is Student's-t distributed. ?.sub.j ensures that each sample is treated as a different batch.

(959) Flow Cytometry

(960) Cytokine-producing T cells were identified by intracellular cytokine staining. PBMCs thawed and rested for 4 hours in OpTmizer medium supplemented with 2 ?g/mL DNAsel (Roche), were restimulated with a peptide pool representing the vaccine-encoded SARS-CoV-2 RBD (2 ?g/mL/peptide; JPT Peptide Technologies) in the presence of GolgiPlug (BD) for 18 hours at 37? C. Controls were treated with DMSO-containing medium. Cells were stained for viability and surface markers in flow buffer ((DPBS (Gibco) supplemented with 2% FCS (Biochrom), 2 mM EDTA (Sigma-Aldrich)) for 20 minutes at 4? C. Afterwards, samples were fixed and permeabilized using the Cytofix/Cytoperm kit according to manufacturer's instructions (BD Biosciences). Intracellular staining was performed in Perm/Wash buffer for 30 minutes at 4? C. Samples were acquired on a FACS VERSE instrument (BD Biosciences) and analysed with FlowJo software version 10.5.3 (FlowJo LLC, BD Biosciences). RBD-specific cytokine production was corrected for background by subtraction of values obtained with DMSO-containing medium. Negative values were set to zero. Cytokine production in FIG. 42(B) was calculated by summing up the fractions of all CD4+ T cells positive for either IFN?, IL-2 or IL-4, setting this sum to 100% and calculating the fraction of each specific cytokine-producing subset thereof.

(961) Cytokine Profiling

(962) Human PBMCs were restimulated for 48 hours with SARS-CoV-2 RBD peptide pool (2 ?g/mL final concentration per peptide). Stimulation with DMSO-containing medium served as negative controls. Concentrations of TNF, IL-1? and IL-12p70 in supernatants were determined using a bead-based, 11-plex T.sub.H1/T.sub.H2 human ProcartaPlex immunoassay (Thermo Fisher Scientific) according to the manufacturer's instructions. Fluorescence was measured with a Bioplex200 system (Bio-Rad) and analysed with ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific). RBD-specific cytokine production was corrected for background by subtraction of values obtained with DMSO-containing medium. Negative values were set to zero.

(963) Results

(964) Study Design and Analysis Set

(965) Between Apr. 23 2020 and May 22 2020, 60 subjects were vaccinated with BNT162b1. Twelve participants per 1 ?g, 10 ?g, 30 ?g, and 50 ?g dose levels received a first dose on day 1 and were boosted on day 22, and 12 participants received a 60 ?g prime dose on Day 1 only (FIG. 43). The study population consisted of healthy males and non-pregnant females with a mean age of 41 years (range 19 to 55 years) with equal gender distribution. Most participants were Caucasian (96.7%) with one African American and one Asian subject (1.7% each). Preliminary data analysis was focussed on immunogenicity (Table 4).

(966) TABLE-US-00022 TABLE 4 Subject disposition and analysis sets. T-cell Antibody analysis analysis BNT162b1 Day Day Day Day Day Day Day Cohort Prime Boost 1 8 ? 1 22 ? 2 29 ? 3 43 ? 4 1 29 ? 3 1 ?g 12 12 12 12 12 12 12 8 8 10 ?g 12 11 12 12 12 11 11 (10) 10 (6) 10 (6) 30 ?g 12 12 12 12 12 12 12 (10) 10 (7) 10 (7) 50 ?g 12 11 12 12 12 11 6 8 (5) 8 (5) 60 ?g 12 N/A 12 12 11 12 N/A N/A N/A Antibody analysis: Values indicated number of subjects for which virus neutralisation assay was performed. Values in parentheses indicate number of subjects for which RBD binding IgG antibody assay was performed. T-cell analysis: Values indicated number of subjects for which IFN? ELISpot was performed. Values in parentheses indicate number of subjects for which flow cytometry was performed. N/A: Samples not yet available.

(967) Briefly, no serious adverse events (SAE), no unexpected toxicities, and no withdrawals due to related AEs were observed. Most reported solicited AEs were signs and symptoms of vaccine reactogenicity, typically with onset within the first 24 hours post immunisation, such as systemic, and injection site reactions, chiefly symptoms of pain and tenderness (FIG. 44). Symptomatology was mostly mild or moderate in intensity with occasional severe (Grade 3) AEs such as fever, chills, headache, muscle and joint pain, and injection site reactions. All AEs resolved spontaneously, mostly within 24 hours of onset and could be managed with simple measures (e.g. paracetamol). Based on the reactogenicity reported after the first dose, participants who had received an initial 60 ?g dose did not receive a second 60 ?g dose. Whereas no relevant change in routine clinical laboratory values occurred after BNT162b1 vaccination, a transient increase of the inflammatory marker C-reactive protein (CRP) and temporary reduction of blood lymphocyte counts were observed in a dose-dependent manner in vaccinated subjects (FIG. 45). Based on our previous clinical experience with RNA vaccines, the latter is likely attributable to innate immune stimulation-related transient redistribution of lymphocytes (Kamphuis, E., et al., Blood 108, 3253-61 (2006)).

(968) Vaccine-Induced Antibody Response

(969) RBD-binding IgG concentrations and SARS-CoV-2 neutralising titers were assessed at baseline, 7 and 21 days after the BNT162b1 prime dose (days 8 and 22), and 7 and 21 days after the boost dose (days 29 and 43), except for the 60 ?g cohort, which received prime only (FIG. 39).

(970) All subjects including those who received the 1 ?g dose showed a strong, dose-dependent vaccine-induced antibody response. Twenty-one days after the priming dose (for the four dose levels ranging from 1-50 ?g), geometric mean concentrations (GMCs) of RBD-binding IgG were dose dependently about 265-1,672 U/mL (FIG. 39). Seven days after the boosting dose (day 29) RBD-binding IgG GMCs in subjects treated with 1-50 ?g BNT162b1 strongly increased dose-dependently to about 2,015-25,006 U/mL. At day 43 (21 days after boost), RBD-binding antibody GMCs were in the range of about 3,920-22,700 U/mL in BNT162b1 vaccinated individuals as compared to about 602 U/mL measured in a panel of sera from 38 SARS-CoV-2 infection convalescent patients (18-83 years of age) drawn at least 14 days after PCR-confirmed diagnosis. In the 60 ?g dose cohort with prime dose only, RBD-binding IgG GMCs were about 1,058 U/mL by day 29 indicating the necessity of the second dose for boosting the antibody titer.

(971) SARS-CoV-2 neutralising antibody geometric mean titers (GMTs) increased modestly in a dose-dependent manner 21 days after the priming dose (FIG. 40(A)). Substantially higher serum neutralizing GMTs were achieved 7 days after the boost dose, reaching about 36 (1 ?g dose level), about 158 (10 ?g dose level), about 308 (30 ?g dose level), and about 578 (50 ?g dose level), compared to about 94 for the convalescent serum panel. On day 43 (21 days after the boost), depending on the dose level, the neutralising antibody GMT were further increased to about 62 (1 ?g dose), were relatively stable at about 126 (10 ?g dose), or decreased slightly to about 157 (30 ?g dose), and about 309 (50 ?g dose). Neutralising antibody GMTs were strongly correlated with RBD-binding IgG GMC (FIG. 40(B)). In summary, neutralising antibody titers were largely in the range of those previously reported in the US study with BNT162b1.

(972) Further, by 7 days after the second dose, sera of vaccinated subjects displayed broad neutralising activity across a panel of seventeen SARS-CoV-2 spike variants identified in publicly available SARS-CoV-2 sequences, including sixteen RBD mutants (Baum, A. et al., Science, eabd0831 (2020). doi:10.1126/science.abd0831) and the dominant spike variant D614G (Baum, A. et al., Science, eabd0831 (2020). doi:10.1126/science.abd0831) (FIG. 40(C)).

(973) Vaccine-Induced T Cell Responses

(974) CD4.sup.+ and CD8.sup.+ T cell responses in BNT162b1 immunized subjects were characterized prior to prime vaccination (day 1) and on day 29 after prime (7 days after boost vaccination) using direct ex vivo IFN? ELISPOT with PBMCs from 36 subjects across the 1 ?g to 50 ?g dose cohorts (FIG. 41). In this assay, CD4.sup.+ or CD8.sup.+ T cell effectors were stimulated overnight with overlapping peptides representing the full-length sequence of the vaccine-encoded RBD.

(975) Of 36 subjects, 34 (94.4%, including all subjects treated with ?10 ?g BNT162b1) mounted RBD-specific CD4+ T cell responses. The magnitude varied between individuals with the strongest CD4 T cell responses being more than 10-fold of the memory responses observed against a panel of cytomegalovirus (CMV), Epstein Barrvirus (EBV), influenza virus and tetanus toxoid-derived immuno-dominant peptides in the same subjects (FIG. 41(A)-(C)). No CD4.sup.+ T cell responses were detectable at baseline, except for one subject with a low number of preexisting RBD-reactive CD4.sup.+ T cells, which increased significantly after vaccination (normalized mean spot count from 63 to 1,519, in the 50 ?g dose cohort). The strength of RBD-specific CD4.sup.+ T cell responses correlated positively with both RBD-binding IgG and with SARS-CoV-2 neutralising antibody titers (FIG. 41(D), FIG. 46(A)), in line with the concept of intramolecular help (Sette, A. et al., Immunity 28, 847-58 (2008)). The two subjects lacking CD4.sup.+ response had no detectable VNT.sub.50 titers either (FIG. 41(D)).

(976) Vaccine-induced CD8.sup.+ T cell responses, some strong ones were mounted by the majority of subjects (29/36, 80.6%) (FIG. 41(A)) and were quite comparable with memory responses against CMV, EBV, Influenza virus and tetanus toxoid in the same subjects (FIG. 41 (B), (C)). The strength of RBD-specific CD8.sup.+ T cell responses correlated positively with vaccine-induced CD4.sup.+ T cell responses but did not significantly correlate with SARS-CoV-2 neutralizing antibody titers (FIG. 46(B), (C)).

(977) Of note, although at 1 ?g BNT162b1 the immunogenicity rate was lower (6/8 responding subjects), the magnitude of vaccine-induced CD4.sup.+ and CD8.sup.+ T cells in some subjects was almost as high as with 50 ?g BNT162b1 (FIG. 41(A)). To assess functionality and polarization of RBD-specific T cells, cytokines secreted in response to stimulation with the vaccine antigen were determined by intracellular staining (ICS) with IFN?, IL-2 and IL-4 specific antibodies in pre- and post-vaccination PBMCs of 18 BNT162b1 immunised subjects. RBD-specific CD4.sup.+ T cells secreted IFN?, IL-2, or both, but did not secrete IL-4 (FIG. 42(A)-(C)). Similarly, a fraction of RBD-specific IFN?.sup.+ CD8.sup.+ T cells also secreted IL-2.

(978) The mean fraction of RBD-specific T cells within total circulating T cells obtained by BNT162b1 vaccination was substantially higher than that observed in six subjects recovered from COVID-19. Frequency of RBD-specific IFN?.sup.+ CD8.sup.+ T cells reached up to several percent of total peripheral blood CD8.sup.+ T cells (FIG. 42(C)). Analysis of supernatants of PBMCs stimulated ex vivo with overlapping RBD peptides from a subgroup of five vaccinated subjects showed cognate release of proinflammatory cytokines TNF, IL-13 and IL-12p70 (FIG. 42(D)). In summary, these findings indicate that BNT162b1 induces functional and proinflammatory CD4.sup.+/CD8.sup.+ T cell responses in almost all subjects, with T.sub.H1 polarized helper response.

(979) Discussion

(980) We observed concurrent production of neutralising antibodies, activation of virus-specific CD4.sup.+ and CD8.sup.+ T cells, and robust release of immune-modulatory cytokines such as IFN?, which represents a coordinated immune response to counter a viral intrusion (for review Vabret, N. et al., Immunity 52, 910-941 (2020)). IFN? represents a key cytokine for several anti-viral responses. Indeed, patients with IFN? gene polymorphism related to impaired IFN? activity have been shown to display 5-fold increased susceptibility to SARS (Chong, W. P. et al., BMC Infect. Dis. 6, 82 (2006)). Also, IFN? acts in synergy with type I interferons to inhibit replication of SARS-CoV-2 (Sainz, B., et al., Virology 329, 11-7 (2004)). The robust production of IFN? from CD8.sup.+ T cells indicates a favourable immune response with both anti-viral and immune-augmenting properties.

(981) Importantly, the detection of IFN?, IL-2 and IL-12p70 but not IL-4 indicates a favorable T.sub.H1 profile and the absence of a potentially deleterious T.sub.H2 immune response. CD4.sup.+ and CD8.sup.+ T cells may confer long lasting immunity against corona viruses as indicated in SARS-CoV-1 survivors, where CD8.sup.+ T-cell immunity persisted for 6-11 years (Vabret, N. et al., Immunity 52, 910-941 (2020); Ng, O.-W. et al., Vaccine 34, 2008-14 (2016)).

(982) Some cases of asymptomatic virus exposure have been associated with cellular immune response without seroconversion indicating that SARS-Cov-2 specific T cells could be relevant in disease control even in the absence of neutralising antibodies (Gallais, F. et al. (2020). doi:medRxiv: 10.1101/2020.06.21.20132449). Almost all vaccinated volunteers mounted RBD-specific T cell responses detected with an ex vivo ELISpot assay, which was performed without prior expansion of T cells that captures only high-magnitude T cell responses. Although the strength of the T-cell responses varied considerably between subjects, we observed no clear dose dependency of the T-cell response strength in the dose range of 1 ?g to 50 ?g, indicating that stimulation and robust expansion of T cells might be accomplished at the lowest mRNA-encoded immunogen levels.

(983) The study confirms the dose-dependency of RBD-binding IgG and neutralisation responses, reproduces our previous findings for 10 and 30 ?g dose levels in the US trial, and shows that neutralising antibody titers are further increased by a prime/boost regimen at 50 ?g.

(984) A notable observation is that two injections of BNT162b1 at a dose level as low as 1 ?g are capable of inducing RBD-binding IgG levels higher than those observed in convalescent sera, and serum neutralising antibody titers that are still increasing up to day 43. Considering that the magnitude of a protective neutralising antibody titer is not known, and given the substantial T-cell responses we observed for some subjects in the 1 ?g cohort may hold the promise that a considerable fraction of individuals may benefit even from this lowest tested dose level.

(985) A purely RBD-directed immunity might be considered prone to escape of the virus by single amino acid changes in this small domain. However, neutralisation of 17 pseudo-typed viruses, 16 of which enter cells using a spike with a different RBD variant found in circulating strains and one of which uses the dominant spike variant D614G, alleviates this potential concern.

Example 8: Summary of Safety and Immunogenicity Data from Stage 1 of COVID-19 Vaccine BNT162

(986) This example provides additional safety and immunogenicity data for the BNT162b1 and BNT162b2 vaccine candidates. These safety and tolerability data, as well as immunoglobulin G (IgG) binding and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralization titer data, are coming from US participants in a Stage 1 US Study of these vaccine candidates.

(987) For BNT162b1, the Following is Observed:

(988) For 10 ?g to 30 ?g dose levels, reactogenicity (particularly systemic events) increases with increasing dose level in 18 to 55-year-old and 65 to 85-year-old participants. Reactogenicity (particularly systemic events) increased after Dose 2 compared to Dose 1.

(989) For BNT162b2, the Following is Observed:

(990) Dose level- and dose number-dependent increases in reactogenicity were minimal to modest in either age group. Based on all available data, the reactogenicity profile observed with BNT162b2 (and particularly SEQ ID NO:20) is quite favorable.

(991) The immunogenicity data presented herein, focusing on SARS-CoV-2 neutralizing responses post-dose 2, allow to conclude the following:

(992) For BNT162b1 at Day 28 (7 days post-dose 2):

(993) Neutralizing antibody responses elicited after immunization with 10 ?g and 30 ?g dosages (where data are available in both age groups) are higher in adults 18 to 55 years of age compared to the 65 to 85-year-old group. In the 65 to 85 year old group, neutralizing antibody responses after 20 ?g and 30 ?g dosages were similar, although numerically higher at the 20 ?g dose level.

(994) For BNT162b2 (particularly, SEQ ID NO:20) at Day 28 (7 days post-dose 2):

(995) Neutralizing antibody responses after the 20 ?g dosage (where data are available in both age groups) were higher in the 18 to 55 year old group compared to the 65 to 85-year-old group. In the 18 to 55 year-old group, neutralizing antibody responses were higher after receiving 20 ?g compared to 10 ?g dose levels. The S1 IgG binding antibody data in FIG. 56, and comparisons of post-dose 1 responses across dose levels that are highest at the 30 ?g dose level, suggest that neutralizing antibody levels will likely also be higher for the 30 ?g dose level post-dose 2, as levels of binding antibody correlate well with neutralizing antibody levels. In the 65 to 85 year old group, neutralizing antibody responses after 20 ?g and 30 ?g doses were higher at the 30 ?g dose level.

(996) The data overall show similar neutralizing antibody responses post-dose 2 between BNT162b1 and BNT162b2.

(997) Safety and Tolerability of BNT162b1

(998) 18-55 Years of Age Groups

(999) Safety data are available for this age group through post-dose 2 for all dose levels, except for 20 ?g, which for now has partial data available through post-dose 2 (and 100 ?g where a second dose has not been administered at the recommendation of the Internal Review Committee (IRC)). Local reactions are shown in FIG. 48. Systemic events are shown in FIG. 49.

(1000) Immunogenicity of BNT162b1

(1001) 65-85 Years of Age Groups

(1002) Immunogenicity data are available for this age group through post-dose 2 for all dose levels. RBD-binding IgG geometric mean concentrations (GMCs) are shown in FIG. 50. SARS-CoV-2 neutralizing geometric mean titers (GMTs) are shown in FIG. 51.

(1003) Safety and Tolerability of BNT162b2

(1004) 18-55 Years of Age Groups

(1005) Safety data are available for this age group through post-dose 2 for all dose levels. Local reactions are shown in FIG. 52. Systemic events are shown in FIG. 53.

(1006) 65-85 Years of Age Groups

(1007) Safety data are available for this age group through post-dose 2 for all dose levels, however, the data for the 10 ?g dose level are only partial. Local reactions are shown in FIG. 54. Systemic events are shown in FIG. 55.

(1008) Immunogenicity of BNT162b2

(1009) 18-55 Years of Age Groups

(1010) Immunogenicity data are available for this age group through post-dose 1 for the 30 ?g dose level and post-dose 2 for the 10 ?g and 20 ?g dose levels. S1-binding IgG GMCs are shown in FIG. 56. SARS-CoV-2 neutralizing GMTs are shown in FIG. 57.

(1011) 65-85 Years of Age Groups

(1012) Immunogenicity data are available for this age group through post-dose 2 for the 20 ?g and 30 ?g dose levels. S1-binding IgG GMCs are shown in FIG. 58. SARS-CoV-2 neutralizing GMTs are shown in FIG. 59.

(1013) Conclusions

(1014) The local tolerability profiles of BNT162b1 and BNT162b2 (and particularly SEQ ID NO:20) and the immune response data are similar between the 2 candidates. BNT162b2 (particularly SEQ ID NO:20) may show a favorable systemic reactogenicity profile (particularly in the 65 to 85-year-old group). When selecting the dose level for BNT162b2 (particularly SEQ ID NO:20), the SARS-CoV-2 neutralizing antibody response level in the 65 to 85-year-old group could be of weight to maximize the neutralizing antibody responses in this age group, which is at highest risk of severe disease. Comparing the neutralizing antibody levels in the 20 ?g and 30 ?g older adult cohorts in this study, the 30 ?g dose level showed higher neutralizing antibody levels than those in the 20 ?g cohort (FIG. 59). In comparison to the neutralizing antibody level of a human convalescent serum panel (HCS) with a GMT of 94, the GMT at the 30 ?g dose level was 1.6 times the GMT of the HCS; the GMT at the 20 ?g dose level was 0.9 times the GMT of HCS. Thus, both showed neutralizing antibody titres at least comparable to that of the human convalescent serum panel. The 38 human SARS-CoV-2 infection/COVID-19 convalescent sera were drawn from participants 18 to 83 years of age, at least 14 days after PCR-confirmed diagnosis, and at a time when participants were asymptomatic. The serum donors predominantly had symptomatic infections (35/38), and one had been hospitalized. The sera were obtained from Sanguine Biosciences (Sherman Oaks, CA), the MT Group (Van Nuys, CA), and Pfizer Occupational Health and Wellness (Pearl River, NY). In addition, S1-IgG antibody binding concentrations in both older (FIG. 58, post-dose 2) and younger (FIG. 56, post-dose 1) adult cohorts also favored the selection of the 30 ?g dose level. Preliminary human T cell data that are being generated in a German trial with BNT162b2 are confirming the robust CD4+ and CD8+ expected for the RNA platform. With these considerations, it is proposed to use BNT162b2 (particularly SEQ ID NO:20) at the 30 ?g dose level to proceed into Phase 2b/3 because this dose and construct provides the optimum combination of a favorable reactogenicity profile and a robust immune response, likely to afford protection against COVID-19 in younger and older adults.

Example 9: Immunology of COVID-19 Vaccine BNT162

(1015) To support progression to Phase 2/3 in adults 18-85 years of age, provided herein are nonclinical and clinical data summarizing the T cell response following BNT162b2 immunization in mice and in humans enrolled in a trial with BNT162. The following immunogenicity data are provided: 1. Preliminary and unaudited mouse immunogenicity data: IFN? ELISpot (FIG. 60), intracellular cytokine staining, and Luminex quantification of cytokines produced following BNT162b2 immunization. 2. From a German trial (BNT162-01): IFN? ELISpot (FIG. 61, FIG. 62, FIG. 63) for BNT162b2 at the 10 ?g dose level in 18-55 year old participants before the first dose and 7 days after dose 2.

(1016) T Cell Responses for BNT162b2 in Mice

(1017) Four groups of eight female BALB/c mice were immunized on day 0 with doses of 0.2 ?g, 1 ?g or 5 ?g per animal BNT162b2 (particularly SEQ ID NO:20), or with the buffer alone (control group), by intramuscular (IM) injection. On days 12 and 28, spleens were collected for splenocyte isolation and analysis of T-cell responses using IFN? ELISpot assays. Luminex assays and intracellular cytokine staining (ICS) were performed to assess cytokine responses. A high fraction of splenocytes of both CD4+ and CD8+ T-cell phenotypes isolated from BNT162b2-immunized mice on days 12 and 28 after immunization, when re-stimulated ex vivo with a full-length S peptide mix, exerted a strong antigen-specific IFN?- and IL-2-response in ELISpot and flow cytometry assays (FIG. 60(A) and (B)). Splenocytes harvested on day 28 and stimulated with the full-length S peptide pool produced high levels of the T.sub.H1 cytokines IL-2 and IFN? with correspondingly minimal levels of the T.sub.H2 cytokines IL-4, IL-5, and IL-13 in multiplex immunoassays (FIG. 60(C)).

(1018) T Cell Responses in Humans for BNT162b2 from German Study

(1019) To evaluate the T cell phenotype elicited by immunization of humans with BNT162b2 (particularly SEQ ID NO:20), IFN? ELISpot was performed on peripheral blood mononuclear cells (PBMCs) obtained from participants in a German study.

(1020) IFN? ELISpot

(1021) Vaccine-elicited T cell responses were determined using CD4- or CD8-depleted PBMCs obtained from subjects prior to dose 1 and on day 29 (7 days after dose 2). IFN? ELISpot data were generated for 5 subjects immunized with 10 ?g of BNT162b2 (particularly SEQ ID NO:20) at day 1 and 22. Post-vaccination spike-specific ex vivo CD4+ and CD8+ T cell responses were detected in 5/5 (100%) subjects, respectively. All responses were minimal or undetectable in the prevaccination samples. The responses are considered vaccine induced (FIG. 61, FIG. 62, FIG. 63).

(1022) The BNT162b2 vaccine-elicited, antigen specific CD8+ and CD4+ T cell responses stimulated by S peptide pool 1 (N-terminal portion of the spike, which includes the receptor binding domain [RBD]) and S peptide pool 2 (C-terminal portion of the spike) were comparable to or higher than the memory responses in the same subjects against CMV, EBV, influenza virus, and tetanus toxoid (FIG. 63).

(1023) Conclusions

(1024) These data for the BNT162b2 vaccine candidate confirm prior results obtained in preclinical models and in humans immunized with modRNA (nucleoside-modified) platforms. The data indicate that modRNA elicits substantial Th1-type CD4+ and CD8+ T cell responses.

Example 10: A Trimeric SARS-CoV-2 Receptor-Binding Domain RNA Vaccine is Highly Immunogenic and Protective in Non-Human Primates

(1025) Here, we report the design and non-clinical development of the BNT162b1 vaccine candidate. We demonstrate that nucleoside-modified mRNA encoding a structurally stable, trimerised receptor-binding domain (RBD) of SARS-CoV-2, encapsulated in lipid nanoparticles (LNP) for effective intramuscular delivery, elicits strong antibody and T.sub.H1-dominated cellular immune responses in mice. Immunisation of mice with single doses of BNT162b1 elicited substantial dose level-dependent increases in pseudovirus neutralisation titers and strong IFN?-positive CD4.sup.+ and CD8.sup.+ T-cell responses. Prime-boost vaccination of Rhesus macaques with BNT162b1 elicited authentic SARS-CoV-2 neutralising geometric mean titers, 2.6 to 6.0 times those of a SARS-CoV-2 convalescent human serum panel. Upon SARS-CoV-2 infectious challenge, the immunised macaques had either no or more transient presence of viral RNA in the nose and lungs than did non-immunised control macaques.

(1026) Materials and Methods

(1027) Ethics Statement.

(1028) All mouse studies were performed at BioNTech SE, and protocols were approved by the local authorities (local welfare committee), conducted according to FELASA recommendations and in compliance with the German Animal Welfare Act and Directive 2010/63/EU. Only animals with an unobjectionable health status were selected for testing procedures.

(1029) Immunisations for the non-human primate (NHP) study were performed at the University of Louisiana at Lafayette-New Iberia Research Center (NIRC), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC, Animal Assurance #: 000452). The work was in accordance with USDA Animal Welfare Act and Regulations and the NIH Guidelines for Research Involving Recombinant DNA Molecules, and Biosafety in Microbiological and Biomedical Laboratories. All procedures performed on these animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee orthrough an ethical review process. Infectious SARS-CoV-2 challenge for the NHP study was performed at the Southwest National Primate Research Center. Animal husbandry followed standards recommended by AAALAC International and the NIH Guide for the Care of Use of Laboratory Animals. This study was approved by the Texas Biomedical Research Institute Animal Care and Use Committee.

(1030) Protein and Peptide Reagents.

(1031) A purified recombinant SARS-CoV-2 RBD fusion with a mouse IgG1 constant region was used as a target for Western Blot and tagged with a human Fc-tag (both Sino Biological) was used in ELISA to detect SARS-CoV-2 S-specific IgG. A purified recombinant RBD with a histidine tag (Sino Biological) was used for surface plasmon resonance (SPR) spectroscopy. An overlapping 15-mer peptide pool of the S protein was used for ELISpot, cytokine profiling and intracellular cytokine staining. A peptide control (SPSYVYHQF (SEQ ID NO: 35), derived from gp70 AH-1 (Slansky, J. E. et al., Immunity 13, 529-538, 2000)) was used as control for ELISpot assays. All peptides were obtained from JPT Peptide Technologies.

(1032) Human Convalescent Sera.

(1033) Human COVID-19 convalescent sera (n=38) were drawn from donors 18-83 years of age at least 14 days after PCR-confirmed diagnosis and at a time when the participants were asymptomatic. Serum donors had symptomatic infections (35/38), or had had been hospitalised (1/38). Sera were obtained from Sanguine Biosciences (Sherman Oaks, CA), the MT group (Van Nuys, CA) and Pfizer Occupational Health and Wellness (Pearl River, NY).

(1034) Cell Culture.

(1035) Human embryonic kidney (HEK)293T/17 and Vero-76 cells (both ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX? (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich). Cell lines were tested for mycoplasma contamination after receipt, before expansion and cryopreservation. Vero E6 and Vero CCL81 (both ATCC) cells were cultured in DMEM (Gibco) containing 2% HyClone fetal bovine serum and 100 U/mL penicillium/streptomycin (Gibco). Expi293F? cells were grown in Expi293? media and transiently transfected using ExpiFectamine?293 (all from Thermo Fisher Scientific).

(1036) Manufacturing of In Vitro Transcribed RNA.

(1037) To generate the template for RNA synthesis, a DNA fragment encoding a fusion protein composed of the signal peptide (SP, amino acids 1-16), the SARS-CoV-2 S RBD (GenBank: MN908947) and a T4 fibritintrimerisation motif (foldon), was cloned into a starting plasmid vector with backbone sequence elements for improved RNA stability and translational efficiency (Orlandini von Niessen, A. G. et al., Mol Ther 27, 824-836; 2019; Holtkamp, S. et al., Blood 108, 4009-4017, 2006). Non-coding backbone elements included the regions from the T7 promoter to the 5 and 3 UTR plus a poly(A) tail (100 nucleotides) interrupted by a linker (A30LA70, 10 nucleotides). The DNA was purified, spectrophotometrically quantified, and in vitro transcribed by T7 RNA polymerase in the presence of a trinucleotide cap1 analogue ((m.sub.2.sup.7,3-O)Gppp(m.sup.2-O)ApG; TriLink) and of N1-methylpseudouridine-5-triphosphate (m1?TP; Thermo Fisher Scientific) instead of uridine-5-triphosphate (UTP) (Grudzien-Nogalska, E. et al., Methods in molecular biology (Clifton, N.J.) 969, 55-72, 2013). RNA was purified using magnetic particles (Berensmeier, S., Appl.Microbiol.Biotechnol. 73, 495-504, 2006), integrity assessed by microfluidic capillary electrophoresis (Agilent Fragment Analyser), and concentration, pH, osmolality, endotoxin level and bioburden determined.

(1038) Lipid-Nanoparticle Formulation of the RNA.

(1039) Purified RNA was formulated into LNPs using an ethanolic lipid mixture of ionisable cationic lipid and transferred into an aqueous buffer system via diafiltration to yield an LNP composition similar to one previously described (Maier, M. A. et al., Molecular therapy: the journal of the American Society of Gene Therapy 21, 1570-1578, 2013). BNT162b1 was stored at ?70? C. at a concentration of 0.5 mg/mL.

(1040) mRNA Transfection.

(1041) HEK293T/17 cells were transfected with transfection reagent-mixed BNT162b1 RNA or BNT162b1 by incubation for 18 hours. Non-LNP formulated mRNA (1 ?g for Western blot and flow cytometry, 2.5 ?g for immunofluorescence) was diluted in Opti-MEM medium (Thermo Fisher Scientific) and mixed with the transfection reagents according to the manufacturer's instructions (RiboJuice, Merck Millipore).

(1042) Western Blot Analysis.

(1043) A lysate of BNT162b1 RNA transfected HEK293T/17 cells was analysed by denaturing SDS-PAGE with 10% Mini-Protean TGX precast polyacrylamide gels (Bio-Rad) and Western blot. Transfer to a nitrocellulose membrane (Carl Roth) was performed using a semi-dry transfer system (Trans-Blot Turbo Transfer System, Bio-Rad). Blotted proteins were detected with a primary rabbit polyclonal antibody elicited by a recombinant S1 fragment of the SARS-CoV S (SinoBiological) and a secondary anti-rabbit horse raddish peroxidase (HRP)-conjugated antibody (Sigma Aldrich). Blots were developed with SuperSignal West Femto chemiluminescent substrate (Thermo Fisher Scientific) and imaged with a Bio-Rad ChemiDoc system using the Image Lab software version 5.0.

(1044) Immunofluorescence.

(1045) Transfected HEK293T/17 cells were fixed in 4% paraformaledehyde (PFA) and permeabilised in phosphate-buffered saline (PBS)/0.2% Triton X-100. Free binding sites were blocked and cells incubated with a rabbit polyclonal antibody recognising the S1 Subunit (SinoBiological) and anti-rabbit IgG secondary antibody (Jackson ImmunoResearch), or labelled Concanavalin A (Invitrogen). DNA was stained with Hoechst (Life Technologies). Images were acquired with a Leica SP8 confocal microscope.

(1046) Flow Cytometry.

(1047) Transfected HEK293T/17 cells were stained with Fixable Viability Dye (eBioscience). After fixation (Fixation Buffer, Biolegend), cells were permeabilised (Perm Buffer, eBioscience) and stained with a monoclonal SARS-CoV-2 spike S1 antibody (SinoBiological). Cells were acquired on a FACSCanto II flow cytometer (BD Biosciences) using BD FACSDiva software version 8.0.1 and analysed by FlowJo software version 10.6.2 (FlowJo LLC, BD Biosciences).

(1048) For mouse T-cell analysis in peripheral blood, erythrocytes from 50 ?L freshly drawn blood were lysed (ACK lysing buffer, Gibco), and cells were stained with Fixable Viability Dye (eBioscience) and primary antibodies in the presence of Fc block in flow buffer (DPBS [Gibco] supplemented with 2% FCS, 2 mM EDTA [both Sigma] and 0.01% sodium azide [Morphisto]). After staining with secondary biotin-coupled antibodies in flow buffer, cells were stained extracellularly against surface markers with directly labelled antibodies and streptavidin in Brilliant Stain Buffer Plus (BD Bioscience) diluted in flow buffer. Cells were fixed with 2% RotiHistofix (Carl Roth) and permeabilised (Perm Buffer, FoxP3/Transcription Factor Staining Buffer Set, eBioscience) overnight. Permeabilised cells were intracellularly treated with Fc block and stained with antibodies against transcription factors in Perm Buffer.

(1049) For mouse T-cell analysis in lymphoid tissues, 1?10.sup.6 lymph node and 4?10.sup.6 spleen cells were stained for viability and extracellular antigens with directly labelled antibodies. Cells were washed in 2% RotiHistofix and fixed (Fix/Perm Buffer, FoxP3/Transcription Factor Staining Buffer Set, eBioscience) overnight. Intracellular staining was performed as described for blood T-cell staining. For mouse B-cell subtyping in lymphoid tissues, 2.5?10.sup.5 lymph node and 1?10.sup.6 spleen cells were treated with Fc block, stained for viability and extracellular antigens as described for blood T-cell staining and fixed with 2% RotiHistofix overnight. For mouse intracellular cytokine staining in T cells, 1?10.sup.6 lymph node and 4?10.sup.6 spleen cells were ex vivo restimulated with 0.2 ?g/mL final concentration per peptide of full-length S peptide mix in the presence of GolgiStop and GolgiPlug (both BD Bioscience) for 5 hours. Cells were stained for viability and extracellular antigens as described for lymphoid T-cell staining. Cells were fixed with 2% RotiHistofix and permeabilised overnight. Intracellular staining was performed as described for blood T-cell staining.

(1050) Mouse cells were acquired on a BD Symphony A3 or BD Celesta (B-cell subtyping) flow cytometer (BD Bioscience) using BD FACSDiva software version 9.1 or 8.0.1.1, respectively, and analysed with FlowJo 10.6 (FlowJo LLC, BD Biosciences).

(1051) Protein Expression and Purification.

(1052) To express the RBD-foldon encoded by BNT162b1 for biochemical and structural analysis, DNA corresponding to the RNA coding sequence was cloned into the pMCG1309 vector. A plasmid encoding amino acids 1-615 of human ACE2 with C-terminal His-10 and Avi tags was generated for transient expression of the ACE2 peptidase domain (ACE2 PD) in Expi293F cells. The ACE2/B.sup.0AT1 complex was produced by co-expression of two plasmids in Expi293F cells, one of them encoding ACE2 amino acids 1-17 followed by haemagglutinin and Strep II tags and ACE2 amino acids 18-805, and the other containing a methionine followed by a FLAG tag and amino acids 2-634 of human B.sup.0AT1.

(1053) Secreted ACE2 PD was isolated from conditioned cell culture medium using Nickel Excel resin (GE Healthcare) followed by gel filtration chromatography on a Superdex200 10/30 column (GE Healthcare) in PBS. Approximately 5 mg of purified ACE2 PD was covalently attached per 1 mL of 4% beaded agarose by amine coupling using AminoLink Plus resin (Thermo Fisher Scientific). The RBD-trimerwas purified from conditioned medium by affinity capture with the ACE2 PD crosslinked agarose and was eluted from the resin with 3 M MgCl.sub.2. Following dialysis, the protein was concentrated and purified by gel filtration using a Superdex200 10/300 column in HEPES-buffered saline (HBS) with 10% glycerol. Purification of the ACE2/B.sup.0AT1 complex was based on the procedure described previously (Yan, R. et al., Science (New York, N.Y.) 367, 1444-1448, 2020). To form the ACE2/B.sup.0AT1/RBD-trimer complex, ACE2/B.sup.0AT1 aliquots were combined with purified RBD-foldon diluted in ACE2/B.sup.0AT1 size exclusion chromatography buffer (25 mM Tris pH 8.0, 150 mM NaCl, 0.02% glyco diosgenin) for a 3:1 molar ratio of RBD-trimers to ACE2 protomers. After incubation at 4? C. for 30 minutes, the sample was concentrated and resolved on a Superose 6 Increase 10/300 GL column. Peak fractions containing the complex were pooled and concentrated.

(1054) Surface Plasmon Resonance Spectroscopy.

(1055) Binding kinetics of murine RBD-specific serum IgGs was determined using a Biacore T200 device (Cytiva) with HBS-EP running buffer (BR100669, Cytiva) at 25? C. Carboxyl groups on the CM5 sensor chip matrix were activated with a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form active esters for the reaction with amine groups. Anti-mouse-Fc-antibody (Jackson ImmunoResearch) was diluted in 10 mM sodium acetate buffer pH 5 (30 ?g/mL) for covalent coupling to immobilisation level of ?10,000 response units (RU). Free N-hydroxysuccinimide esters on the sensor surface were deactivated with ethanolamine.

(1056) Mouse serum was diluted 1:50 in HBS-EP buffer and applied at 10 ?L/min for 30 seconds to the active flow cell for capture by immobilised antibody, while the reference flow cell was treated with buffer. Binding analysis of captured murine IgG antibodies to RBD-His (Sino Biological Inc.) was performed using a multi-cycle kinetic method with concentrations ranging from 1.5625 to 50 nM. An association period of 180 seconds was followed by a dissociation period of 600 seconds with a constant flow rate of 40 ?L/min and a final regeneration step. Binding kinetics were calculated using a global kinetic fit model (1:1 Langmuir, Biacore T200 Evaluation Software Version 3.1, Cytiva).

(1057) Biolayer Interferometry.

(1058) Binding of RBD-foldon to the ACE2-PD was measured by biolayer interferometry on an Octet RED384 (Fort?Bio) in a buffer composed of 10 mM HEPES pH 7.5, 150 mM NaCl and 1 mM EDTA at 25? C. Avi-tagged human ACE2 PD was immobilised on streptavidin-coated sensors. Binding data were collected for 10 minutes of association and 15 minutes of dissociation for a concentration series of RBD-trimer. Data were reference-subtracted and fit to a 1:1 binding model with R.sup.2 value greater than 0.96 to determine kinetics and affinity of binding, using Octet Data Analysis Software v10.0 (Fort?Bio). The dissociation rate of interaction (k.sub.d) was slower than the limit of measurement of the instrument, and the binding affinity (K.sub.D) was estimated using an assumed dissociation rate k.sub.d of 1?10.sup.?6 s.sup.?1.

(1059) Electron Microscopy of Negatively Stained Samples.

(1060) Purified RBD-trimer protein in 4 ?L was applied to a glow-discharged copper grid overlaid with formvar and amorphous carbon (Ted Pella). Staining was performed with Nano-W organotungstate stain (Nanoprobes) according to the manufacturer's protocol, and the sample imaged using an FEI TF-20 microscope operating at 200 kV, with a magnification of 62,000? and defocus of ?2.5 ?m. Micrographs were contrast transfer function (CTF)-corrected in RELION using CTFFIND-4.1 (Rohou, A. & Grigorieff, N., Journal of structural biology 192, 216-221, 2015). A small manually picked dataset was used to generate 2D references for auto-picking. The resulting particle set was subjected to 2D classification in RELION 3.0.6 (Zivanov, J. et al., eLife 7; 10.7554/eLife.42166 (2018)).

(1061) Cryo-Electron Microscopy.

(1062) Purified ACE2/B.sup.0AT1/RBD-trimer complex at 6 mg/mL in 4 ?L was applied to gold Quantifoil R1.2/1.3 200 mesh grids glow discharged in residual air for 30 seconds at 20 mA using a Pelco Easiglow. The sample was blotted using a Vitrobot Mark IV for 5 seconds with a force of ?3 before being plunged into liquid ethane cooled by liquid nitrogen. In total, 7,455 micrographs were collected from a single grid on a Titan Krios operating at 300 keV equipped with a Gatan K2 Summit direct electron detector in super-resolution mode at a magnification of 165,000?, for a magnified pixel size of 0.435 ? at the specimen level. Data were collected over a defocus range of ?1.2 to ?3.4 ?m with a total electron dose of 52.06 e.sup.?/?.sup.2 fractionated into 40 frames over a 6-second exposure for 1.30 e.sup.?/?.sup.2/frame. Initial motion correction was performed in Warp (Tegunov, D. & Cramer, P., Nature methods 16, 1146-1152, 2019), during which super-resolution data were binned to give a pixel size of 0.87 ?. Corrected micrographs were imported into RELION 3.1-beta (Zivanov, J. et al., eLife 7; 10.7554/eLife.42166 (2018)) for CTF estimation with CTFFIND-4.1 (Rohou, A. & Grigorieff, N., Journal of structural biology 192, 216-221, 2015). Particles were picked using the LaPlacian-of-Gaussian particle picking algorithm as implemented in RELION and extracted with a box size of 450 pixels. References obtained by 2D classification were used for a second round of reference-based auto-picking, yielding a dataset of 715,356 particles. Particle heterogeneity was filtered out with 2D and 3D classification with a mask size of 280 nm to filter out the non-ACE2-bound RBD copies in each RBD-trimer, yielding a set of 87,487 particles, which refined to 3.73 ? with C2 symmetry. Refinement after subtraction of micelle and B.sup.0AT1 density from the particles yielded an improved map of 3.24 ?. The atomic model from PDB ID 6M17 (Yan, R. et al., Science (New York, N.Y.) 367, 1444-1448, 2020) was rigid-body fitted into the 3.24 ? density, then flexibly fitted to the density using real-space refinement in Phenix (Adams, P. D. et al., Acta crystallographica. Section D, Biological crystallography 66, 213-221, 2010) alternating with manual building in Coot (Emsley, P. et al., Acta crystallographica. Section D, Biological crystallography 66, 486-501, 2010). The microscope was operated for image acquisition using SerialEM software version 3.8.0 beta (Mastronarde, D. N., Journal of structural biology 152, 36-51, 2005). Biolayer interferometry data was collected with Octet Data Acquisition software version 10.0.0.87 and processing was performed using ForteBio Data Analysis software version 10.0.

(1063) Immunisation.

(1064) Mice. Female BALB/c mice (Janvier; 8-12 weeks) were randomly allocated to groups. BNT162b1 was diluted in PBS, 300 mM sucrose or saline (0.9% NaCl) and injected IM into the gastrocnemius muscle at a volume of 20 ?L under isoflurane anaesthesia.

(1065) Rhesus macaques (Macaca mulatta). Male Rhesus macaques (2-4 years) were randomly assigned to receive either BNT162b1 or saline placebo control in 0.5 mL volume administered by IM injection in the left quadriceps muscle on Days 0 and 21. Blood for serum and PBMCs was collected in compliance with animal protocol 2017-8725-023 approved by the NIRC Institutional Animal Care and Use Committee. Animals were anesthetised with ketamine HCl (10 mg/kg; IM) during blood collection and immunisation, and monitored for adequate sedation.

(1066) SARS-CoV-2 Challenge of Rhesus Macaques.

(1067) The SARS-CoV-2 inoculum was obtained from a stock of 2.1?10.sup.6 PFU/mL previously prepared at Texas Biomedical Research Institute (San Antonio, Texas), aliquoted into single use vials, and stored at ?70? C. The working virus stock was generated from two passages of the SARS-CoV-2 USA-WA1/2020 isolate (a 4.sup.th passage seed stock purchased from BEI Resources; NR-52281) in Vero E6 cells. The virus was confirmed to be SARS-CoV-2 by deep sequencing and identical to the published sequence (GenBank accession number MN985325.1).

(1068) BNT162b1-immunised (n=6) and age-matched saline control-immunised (n=6) male Rhesus macaques (control) were challenged with 1?10.sup.6 plaque forming units of SARS-CoV-2 USA-WA1/2020 isolate, split equally between the intranasal (IN; 0.2 mL) and intratracheal (IT; 0.2 mL) routes as previously described (Singh, D. K. et al., SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species, 2020). The challenge was performed 41 to 48 days after the second immunisation. A separate sentinel group of non-immunised age- and sex-matched animals (n=3) received only DMEM supplemented with 10% FCS IN (0.2 mL) and IT (0.2 mL). Approximately two weeks prior to challenge, animals were moved to the Animal Biosafety Level 3 (ABSL-3) facility at Southwest National Primate Research Center (SNPRC; San Antonio, TX). Animals were monitored regularly by a board-certified veterinary clinician for rectal body temperature, weight and physical examination. Specimen collection was performed under tiletamine zolazepam (Telazol) anaesthesia as described (Singh, D. K. et al., SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species, 2020). Nasal swabs were collected from macaques at 0, 1, 3, and 6 days after inoculation to assess viral titers. Bronchoalveolar lavage (BAL) was performed the week before challenge and at Days 3 and 6 post-inoculation by instilling four times 20 mL of saline. These washings were pooled, aliquoted and stored frozen at ?70? C.

(1069) Reverse-Transcription Quantitative Polymerase Chain Reaction.

(1070) To detect and quantify SARS-CoV-2, viral RNA was extracted from nasal swabs and BAL specimens as previously described (Mehra, S. et al., The Journal of infectious diseases 207, 1115-1127, 2013; Gautam, U. S. et al., Proceedings of the National Academy of Sciences of the United States of America 115, E62-E71; 2018; Joosten, S. A. et al., PLoS pathogens 6, e1000782, 2010) and tested by RT-qPCR as previously described (Singh, D. K. et al., SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species, 2020). Briefly, 10 ?g yeast tRNA and 1?10.sup.3 PFU of MS2 phage (Escherichia coli bacteriophage MS2, ATCC) were added to each thawed sample, and RNA extraction performed using the NucleoMag Pathogen kit (Macherey-Nagel). The SARS-CoV-2 RT-qPCR was performed on extracted RNA using a CDC-developed 2019-nCoV_N1 assay on a QuantStudio 3 instrument (Applied Biosystems). The cut-off for positivity (limit of detection, LOD) was established at 10 gene equivalents (GE) per reaction (800 GE/mL). Samples were tested in duplicate. On day 6, one BAL specimen from the control group and one day 1 nasal swab from the BNT162b1-immunised group had, on repeated measurements, viral RNA levels on either side of the LLOD. These specimens were categorised as indeterminate and excluded from the graphs and the analysis.

(1071) Tissue Preparation.

(1072) Mice. Peripheral blood was collected from the retro-orbital venous plexus or vena facialis under isoflurane anaesthesia. Blood was centrifuged for 5 minutes at 16.000?g, and the serum was immediately used for downstream assays or stored at ?20? C. Spleen single-cell suspensions were prepared in PBS by mashing tissue against the surface of a 70 ?m cell strainer (BD Falcon) using the plunger of a 3-mL syringe (BD Biosciences). Erythrocytes were removed by hypotonic lysis. Popliteal, inguinal and iliac lymph nodes were pooled, cut into pieces, digested with collagenase D (1 mg/mL; Roche) and passed through cell strainers. Rhesus macaques (Macaca mulatta). Blood for serum and PBMCs was collected in compliance with animal protocol 2017-8725-023 approved by the NIRC Institutional Animal Care and Use Committee.

(1073) RBD-Binding IgG Antibody Assay.

(1074) For mouse sera, MaxiSorp plates (Thermo Fisher Scientific) were coated with recombinant RBD (100 ng/100 ?L) in sodium carbonate buffer, and bound IgG was detected using an HRP-conjugated secondary antibody and TMB substrate (Biotrend). Data collection was performed using a BioTek Epoch reader and Gen5 software version 3.0.9. For concentration analysis, the signal of the specific samples was correlated to a standard curve of an isotype control. For rhesus macaque and human sera, a recombinant SARS-CoV-2 RBD containing a C-terminal Avitag? (Acro Biosystems) was bound to streptavidin-coated Luminex microspheres. Bound rhesus macaque or human anti-RBD antibodies present in the serum were detected with a fluorescently labelled goat anti-human polyclonal secondary antibody (Jackson ImmunoResearch). Data were captured as median fluorescent intensities (MFIs) using a Bioplex200 system (Bio-Rad) and converted to U/mL antibody concentrations using a reference standard curve consisting of 5 pooled human COVID-19 convalescent serum samples (obtained >14 days PCR diagnosis), diluted in antibody depleted human serum, with arbitrary assigned concentrations of 100 U/mL and accounting for the serum dilution factor.

(1075) VSV-SARS-CoV-2 Spike Variant Pseudovirus Neutralisation.

(1076) A recombinant replication-deficient vesicular stomatitis virus (VSV) vector that encodes GFP instead of VSV-G (VSV?G-GFP) was pseudotyped with SARS-CoV-2 S protein according to published pseudotyping protocols (Berger Rentsch, M. & Zimmer, G., PLoS ONE 6, e25858, 2011; Lester, S. et al., Access Microbiology 1, 20290, 2019). In brief, HEK293T/17 monolayers transfected to express SARS-CoV-2 S truncated of the C-terminal cytoplasmic 19 amino acids (SARS-CoV-2-S-C?19) were inoculated with VSV?G-GFP vector. After incubation for 1 hour at 37? C., the inoculum was removed and cells were washed with PBS before medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) was added to neutralise residual input virus. VSV/SARS-CoV-2 pseudovirus-containing medium was harvested 20 hours after inoculation, 0.2 ?m filtered and stored at ?80? C.

(1077) Vero-76 cells were seeded in 96-well plates. Serial dilutions of mouse serum samples were prepared and pre-incubated for 10 minutes at room temperature with VSV/SARS-CoV-2 pseudovirus suspension (4.8?10.sup.3 infectious units [IU]/mL) before transferring the mix to Vero-76 cells. Inoculated Vero-76 cells were incubated for 20 hours at 37? C. Plates were placed in an IncuCyte Live Cell Analysis system (Sartorius) and incubated for 30 minutes prior to the analysis (IncuCyte 2019B Rev2 software). Whole well scanning for brightfield and GFP fluorescence was performed using a 4? objective. The 50% pseudovirus neutralisation titre (pVNT50) was reported as the reciprocal of the first serum dilution yielding a 50% reduction in GFP-positive infected cell number per well compared to the mean of the no serum pseudovirus positive control. Each serum sample dilution was tested in duplicates.

(1078) SARS-CoV-2 Neutralisation by Human Convalescent and Rhesus Macaque Sera.

(1079) The SARS-CoV-2 neutralisation assay used a previously described strain of SARS-CoV-2 (USA_WA1/2020) that had been rescued by reverse genetics and engineered by the insertion of an mNeonGreen (mNG) gene into open reading frame 7 of the viral genome (Xie, X. et al., Cell host & microbe 27, 841-848.e3, 2020). This reporter virus generates similar plaque morphologies and indistinguishable growth curves from wild-type virus. Viral master stocks were grown in Vero E6 cells as previously described (Lester, S. et al., Access Microbiology 1, 20290, 2019). When testing human convalescent serum specimens, the fluorescent neutralisation assay produced comparable results as the conventional plaque reduction neutralisation assay. Serial dilutions of heat-inactivated sera were incubated with the reporter virus (2?10.sup.4 PFU per well) to yield approximately a 10-30% infection rate of the Vero CCL81 monolayer) for 1 hour at 37? C. before inoculating Vero CCL81 cell monolayers (targeted to have 8,000 to 15,000 cells per well) in 96-well plates to allow accurate quantification of infected cells. Total cell counts per well were enumerated by nuclear stain (Hoechst 33342) and fluorescent virally infected foci were detected 16-24 hours after inoculation with a Cytation 7 Cell Imaging Multi-Mode Reader (Biotek) with Gen5 Image Prime version 3.09. Titers were calculated in GraphPad Prism version 8.4.2 by generating a 4-parameter (4PL) logistical fit of the percent neutralisation at each serial serum dilution. The 50% neutralisation titre (VNT.sub.50) was reported as the interpolated reciprocal of the dilution yielding a 50% reduction in fluorescent viral foci.

(1080) IFN? ELISpot.

(1081) ELISpot assays were performed with mouse IFN? ELISpot.sup.PLUS kits according to the manufacturer's instructions (Mabtech). A total of 5?10.sup.5 splenocytes was ex vivo were restimulated with the full-length S peptide mix (0.1 ?g/mL final concentration per peptide, JPT) or controls (gp70-AH1 [SPSYVYHQF (SEQ ID NO: 35)] (Slansky, J. E. et al., Immunity 13, 529-538, 2000), JPT, 4 ?g/mL; Concanavalin A (ConA), Sigma, 2 ?g/mL). Streptavidin-ALP and BCIP/NBT-plus substrate were added, and spots counted using an ELISpot plate reader (ImmunoSpot? S6 Core Analyzer, CTL). Spot numbers were evaluated using ImmunoCapture Image Aquision Software V7.0 and ImmunoSpot 7.0.17.0 Professional. For T-cell subtyping, CD8.sup.+ T cells were isolated from splenocyte suspensions using MACS MicroBeads (CD8a [Ly-2], Miltenyi Biotec) according to the manufacturer's instructions. The flow-through served as a source of CD4.sup.+ T cells. CD8.sup.+ or CD4.sup.+ T cells were subsequently restimulated with syngeneic bone marrow-derived dendritic cells loaded with full-length S peptide mix (0.1 ?g/mL final concentration per peptide) or medium as control. Purity of isolated T-cell subsets was determined by flow cytometry to calculate spot counts per 1?10.sup.5 CD8.sup.+ or CD4.sup.+ T cells.

(1082) Cytokine Profiling.

(1083) Mouse splenocytes were re-stimulated for 48 hours with full-length S peptide mix (0.2 ?g/mL final concentration per peptide) or medium only. Concentrations of IFN?, IL-2, IL-4 and IL-5 in supernatants were determined using a bead-based, 11-plex T.sub.H1/T.sub.H2 mouse ProcartaPlex multiplex immunoassay (Thermo Fisher Scientific) according to the manufacturer's instructions. Fluorescence was measured with a Bioplex200 system (Bio-Rad) and analysed with ProcartaPlex Analyst 1.0 software (Thermo Fisher Scientific).

(1084) Statistics and Reproducibility.

(1085) No statistical methods were used to predetermine group and samples sizes (n). All experiments were performed once. P-values reported for RT-qPCR analysis were determined by categorical analysis for binomial response (undetectable viral RNA after challenge as success, measurable viral RNA after challenge as failure) with logit link to treatment and day effects using PROC GENMOD from SAS? 9.4. Samples from post challenge days (Days 3 and 6 for BAL; Days 1, 3 and 6 for nasal swab) were included in the analysis. Indeterminate results were excluded from this analysis. All remaining analyses were carried out using GraphPad Prism 8.4.

(1086) Results

(1087) We designed a SARS-CoV-2 vaccine named BNT162b1, which is composed of an LNP-encapsulated N1-methyl-pseudouridine (m1?) nucleoside-modified mRNA that encodes the RBD fused at its C-terminus to the natural trimerisation domain (foldon) of T4 fibritin (Meier, S. et al., Journal of molecular biology 344, 1051-1069, 2004) (FIG. 64a). The SARS-CoV-2 S signal peptide (SP) enables ER translocation and secretion of the trimeric RBD. The m1?-modification of the RNA dampens innate immune sensing and, together with optimized non-coding sequence elements, increases RNA translation in vivo (Orlandini von Niessen, A. G. et al., Mol Ther 27, 824-836, 2019; Karik?, K. et al., Molecular therapy: the journal of the American Society of Gene Therapy 16, 1833-1840, 2008).

(1088) BNT162b1 RNA in vitro transcribed by T7 polymerase from a plasmid DNA template had a single, sharp peak microfluidic capillary electrophoresis profile, consistent with its calculated length of 1262 nucleotides, indicating purity and integrity (data not shown). Western blot analysis of a lysate of BNT162b1 RNA-transfected HEK293T/17 cells demonstrated that the RBD was expressed from the RNA and had an apparent molecular weight consistent with its calculated weight of 29.46 kDa (data not shown). Protein expression and endoplasmic reticulum localisation on the secretory pathway in transfected cells were confirmed by flow cytometry and immunofluorescence microscopy, respectively (data not shown).

(1089) For structural characterization, the trimerised RBD was expressed from a DNA sequence corresponding to the coding sequence of BNT162b1 RNA in Expi293F cells and purified by affinity capture with the ACE2 peptidase domain immobilized on agarose beads. The trimerised RBD bound to the human ACE2 peptidase domain (PD) with high affinity (5 pM K.sub.D), which is approximately 1,000-fold the reported K.sub.D of 5.09 nM for monomeric RBD and consistent with the avidity effect of multimeric binding (data not shown). The trimeric valency of the RBD-foldon and its flexibility were visualized by electron microscopy (EM) of negatively stained specimens, which revealed a range of conformations (FIG. 64b). Although the flexibility of the RBD-foldon precluded direct structural analysis at high resolution, the RBD domains could be immobilized by binding to a complex of ACE2 and the B.sup.0AT1 neutral amino acid transporter, which ACE2 chaperones, when that complex was in the previously reported closed conformation (Yan, R. et al., Science (New York, N.Y.) 367, 1444-1448, 2020). The size and symmetry of the RBD-foldon/ACE2/B.sup.0AT1 ternary complex aided image reconstruction by electron cryomicroscopy (cryoEM), and the structure of the RBD domains in the complex was determined to 3.24 ? resolution (FIG. 64c). One copy of the RBD was resolved for each bound trimer. The binding interface between the resolved RBD and the ACE2 extracellular domain was fitted to a previously reported structure and showed good agreement (He, Y. et al., Biochemical and Biophysical Research Communications 324, 773-781, 2004; Yi, C. et al., Cellular & molecular immunology; 10.1038/s41423-020-0458-z, 2020). The high affinity binding to ACE2 and well-resolved structure in complex with ACE2 demonstrate that the recombinant RBD-foldon authentically presents the ACE2 binding site targeted by many SARS-CoV-2 neutralising antibodies (Brouwer, P. J. M. et al., Science (New York, N.Y.); 10.1126/science.abc5902 (2020); Zost, S. J. et al., Nature medicine; 10.1038/s41591-020-0998-x (2020)).

(1090) BNT162b1-elicited B- and T-cell immune responses were characterised in a series of experiments in BALB/c mice after a single intramuscular (IM) immunisation with 0.2, 1, or 5 ?g BNT162b1 or with buffer alone. RBD-specific serum IgG developed quickly at all dose levels in a dose-dependent manner and plateaued around day 21 (at 1.63?0.13 mg/mL for the 5 ?g dose level; FIG. 65a). Vaccine-elicited IgG had high RBD-binding affinity (geometric mean K.sub.D 48.0 pM) with high on-rate (geometric mean k.sub.on 1.72?10.sup.6/Ms) and low off-rate (geometric mean K.sub.off 8.27?10.sup.?5/s; FIG. 65b). SARS-CoV-2 neutralising activity in mouse serum was measured by a vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudovirus neutralisation assay. Mean 50% pseudovirus neutralisation titers (pVNT.sub.50) increased steadily after immunisation to 102, 192, and 1,056 on day 28 for the 0.2, 1, and 5 ?g dose levels, respectively (FIG. 65c).

(1091) A high fraction of splenocytes of both CD4.sup.+ and CD8.sup.+ T-cell phenotypes isolated from BNT162b1-immunised mice on days 12 and 28 after immunisation, when re-stimulated ex vivo with a full-length S peptide mix, exerted a strong antigen-specific IFN?-response in ELISpot assays (FIG. 65d). Full-length S peptide-stimulated bulk splenocytes and CD4.sup.+ and CD8.sup.+ subsets also show high IFN? production at day 12 and significant IL-2 responses but much lower IL-4 responses in flow cytometric cytokine release analyses, indicating a T.sub.H1 phenotype response (FIG. 65e). The T.sub.H1 phenotype persists, with total splenocytes harvested on day 28 and stimulated with the full-length S peptide pool producing high levels of IL-2 and IFN? but undetectable amounts of the T.sub.H2 cytokines IL-4 and IL-5 in multiplex immunoassays (FIG. 65f). In draining lymph nodes (dLN) and spleens obtained 12 days after immunisation of mice with BNT162b1 or buffer, much higher numbers of B cells (including plasma cells, class switched IgG1- and IgG2a-positive B cells, and germinal center B cells) were observed in the samples from mice that received BNT162b1 (data not shown). In blood obtained 7 days after immunisation, the number of circulating B cells was lower than in buffer-immunised mice, most likely due to B-cell homing to lymphoid compartments (data not shown). dLNs from BNT162b1-immunised mice also displayed an elevation in T-cell counts, particularly numbers of T follicular helper (T.sub.FH) cells, including subsets with ICOS upregulation, which are known to play an essential role in the formation of germinal centers (Hutloff, A., Oncotarget 6, 21785-21786, 2015) (data not shown). BNT162b1-induced elevation of T.sub.FH cells was also detected in the spleen and blood (data not shown). In aggregate, these data indicate a strong and concurrent induction of SARS-CoV-2 S-specific neutralising antibody titers and a T.sub.H1-driven T-cell response by BNT162b1. Intramuscularly administered BNT162b1 appears to be delivered to dLNs as immune-educated sites for proficient vaccine priming, with migration of lymphocytes from the blood to lymphoid tissues to participate in the vaccine response.

(1092) The immunogenicity of BNT162b1 was next tested in 2-4 year old male Rhesus macaques. Groups of six were immunised IM with 30 or 100 ?g of BNT162b1 or with saline control on Days 0 and 21. RBD-binding IgG was readily detectable by Day 14 after a single immunisation, and levels increased further through Day 21, when the boosting dose was given (FIG. 66a). Seven days after the second immunisation (Day 28), the geometric mean RBD-binding IgG concentrations (GMCs) were 20,962 units (U)/mL (30 ?g dose level) and 48,575 U/mL (100 ?g dose level). For comparison, the RBD-binding IgG GMC of a panel of 38 SARS-CoV-2 convalescent human sera was 602 U/mL, substantially lower than the GMC of the immunised Rhesus macaques after one or two doses. Fifty percent neutralisation titers (VNT.sub.50), measured by an authentic SARS-CoV-2 neutralisation assay (Muruato, A. E. et al., bioRxiv: the preprint server for biology; 10.1101/2020.05.21.109546, 2020), were detectable in rhesus sera by Day 14 after a single immunisation and reached geometric mean titers (GMTs) of 768 (30 ?g dose level) or 1,714 (100 ?g dose level) 7 days after the boost (Day 28, FIG. 66b). Robust neutralisation GMTs of 247 for 30 ?g and 564 for 100 ?g dose levels persisted to at least Day 42 (most recent time point tested). For comparison, the 50% neutralisation GMT of the human convalescent serum panel was 93.6.

(1093) The groups of Rhesus macaques (n=6) that had received two immunisations with 100 ?g BNT162b1 or buffer control were challenged 41 to 48 days after the second immunisation with 1?10.sup.6 plaque forming units of SARS-CoV-2 (strain USA-WA1/2020), split equally between the intranasal and intratracheal routes, as previously described (Singh, D. K. et al. SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species, 2020). Three non-immunised, age-matched, male Rhesus macaques (sentinel) were mock-challenged with cell culture medium. At the time of challenge, SARS-CoV-2 neutralising titers ranged from 208 to 1,185 in the BNT162b1-immunised animals and were undetectable in animals from the control-immunised and sentinel groups.

(1094) SARS-CoV-2 RNA was measured in bronchoalveolar lavage (BAL) and nasal swab samples by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). All BAL and nasal swab samples obtained before the infectious challenge and all those obtained from sentinel animals lacked detectable SARS-CoV-2 RNA (FIG. 67). Three days after SARS-CoV-2 challenge, viral RNA was detected in BAL fluid from 5/6 control-immunised and 2/6 BNT162b1-immunised animals (FIG. 67a). By 6 days after challenge, all six BNT162b1-immunised macaques had undetectable viral RNA in their lungs; of the control-immunised macaques, three had a high level of viral RNA in BAL fluid, two had cleared, and one had an indeterminate RT-qPCR result. At the time of necropsy (7-23 days after challenge), no viral RNA was detectable in BAL fluid from any animal. After SARS-CoV-2 challenge, viral RNA was detected in nasal swabs of the control-immunised group at each time point: two animals at Day 1, three animals at Days 3 and 6 after challenge (FIG. 67b), and two animals at the time of necropsy (not shown). In BNT162b1-immunised animals, all nasal swabs were negative or indeterminate at Day 1, and all were negative at Day 3 and at the time of necropsy; at Day 6, swabs from two were positive, indicating a more transient course of viral RNA detection compared to non-immunised Rhesus macaques. The difference in the proportion of animals with detectable viral RNA between BNT162b1-immunised animals and control-immunised animals is statistically significant (p=0.0037 for BAL, and 0.0212 for nasal swab). None of the challenged animals showed clinical or radiographic signs of significant illness, indicating that the 2-4 years old male rhesus challenge model is primarily an infection model for SARS-CoV-2, not a COVID-19 disease model.

(1095) Discussion

(1096) We demonstrate that BNT162b1, an LNP-formulated m1? nucleoside-modified mRNA encoding the trimeric RBD antigen, is highly immunogenic in mice and Rhesus macaques and limits infection in Rhesus macaques challenged with infectious SARS-CoV-2. The RBD-foldon coding sequence directs the expression of a flexible, trimeric protein that binds ACE2 with high affinity and has a structurally intact ACE2 receptor binding site. One key finding is that in mice, a single sub-microgram immunisation rapidly induces high neutralising antibody titers that are in the range or above recently reported SARS-CoV-2 vaccine candidates (van Doremalen, N. et al., bioRxiv: the preprint server for biology; 10.1101/2020.05.13.093195 (2020); Corbett, K. S. et al., bioRxiv: the preprint server for biology; 10.1101/2020.06.11.145920 (2020)). The strong CD4.sup.+ and stronger CD8.sup.+ T-cell responses, both skewing towards a T.sub.H1-bias, and T.sub.FH generation may imply a strong protection capacity induced by the vaccine candidate (Pardi, N. et al., The Journal of Experimental Medicine 215, 1571-1588, 2018). Proliferation of T.sub.FH in germinal centres is integral for generation of an adaptive B-cell response. In humans, T.sub.FH occurring in the circulation after vaccination were correlated with a high frequency of antigen-specific antibodies (Farooq, F. et al., Scientific reports 6, 27944, 2016). Immunisation with BNT162b1 triggered redistribution of B cells and T.sub.FH cells from the blood to lymphoid tissues, where antigen presentation occurs.

(1097) Another significant finding is that in Rhesus macaques two doses of m1? nucleoside-modified mRNA encoding the trimeric SARS-CoV-2 S RBD-foldon elicited SARS-CoV-2 neutralising GMTs 8.2 to 18.2-fold the GMT of a SARS-CoV-2 convalescent human serum panel. Results in nonhuman primates confirm the vaccine's high potency and ability to protect against SARS-CoV-2 challenge in a preclinical model of acute SARS-CoV-2 infection.

Example 11: A RNA Vaccine Encoding the Prefusion-Stable SARS-CoV-2 S is Highly Immunogenic in Mice and Non Human Primates

(1098) Here, we report a SARS-CoV-2 infectious challenge of immunised macaques with BNT162b2 vaccine.

(1099) Materials and Methods

(1100) Manufacturing of In Vitro Transcribed RNA.

(1101) To generate the template for RNA synthesis, a DNA fragment encoding the full-length SARS-CoV-2 S protein (GenBank: MN908947), with amino acid exchanges K986P and V987P, was cloned into a starting plasmid vector with backbone sequence elements for improved RNA stability and translational efficiency (Orlandini von Niessen, A. G. et al., Mol Ther 27, 824-836, 2019; Holtkamp, S. et al., Blood 108, 4009-4017, 2006). Non-coding backbone elements included the regions from the T7 promoter to the 5 and 3 UTR plus a poly(A) tail (100 nucleotides) interrupted by a linker (A30LA70, 10 nucleotides). The DNA was purified, spectrophotometrically quantified, and in vitro transcribed by T7 RNA polymerase in the presence of a trinucleotide cap1 analogue ((m.sub.2.sup.7,3-O)Gppp(m.sup.2-O)ApG; TriLink) and of N.sup.1-methylpseudouridine-5-triphosphate (m1?TP; Thermo Fisher Scientific) instead of uridine-5-triphosphate (UTP) (Grudzien-Nogalska, E. et al., Methods in molecular biology (Clifton, N.J.) 969, 55-72, 2013). RNA was purified using magnetic particles (Berensmeier, S., Appl.Microbiol.Biotechnol. 73, 495-504, 2006), integrity assessed by microfluidic capillary electrophoresis (Agilent Fragment Analyser), and concentration, pH, osmolality, endotoxin level and bioburden determined.

(1102) Lipid-Nanoparticle Formulation of the RNA.

(1103) Purified RNA was formulated into LNPs using an ethanolic lipid mixture of ionisable cationic lipid and transferred into an aqueous buffer system via diafiltration to yield an LNP composition similar to one previously described (Maier, M. A. et al., Molecular therapy: the journal of the American Society of Gene Therapy 21, 1570-1578, 2013). BNT162b2 was stored at ?70? C. at a concentration of 0.5 mg/mL.

(1104) Immunisation.

(1105) Male Rhesus macaques (2-4 years) were randomly assigned to receive either BNT162b2 or saline placebo control in 0.5 mL volume administered by IM injection in the left quadriceps muscle on Days 0 and 21. Blood for serum and PBMCs was collected in compliance with animal protocol 2017-8725-023 approved by the NIRC Institutional Animal Care and Use Committee. Animals were anesthetised with ketamine HCl (10 mg/kg; IM) during blood collection and immunisation, and monitored for adequate sedation.

(1106) SARS-CoV-2 Challenge of Rhesus macaques.

(1107) The SARS-CoV-2 inoculum was obtained from a stock of 2.1?10.sup.6 PFU/mL previously prepared at Texas Biomedical Research Institute (San Antonio, TX), aliquoted into single use vials, and stored at ?70? C. The working virus stock was generated from two passages of the SARS-CoV-2 USA-WA1/2020 isolate (a 4.sup.th passage seed stock purchased from BEI Resources; NR-52281) in Vero E6 cells. The virus was confirmed to be SARS-CoV-2 by deep sequencing and identical to the published sequence (GenBank accession number MN985325.1).

(1108) BNT162b2-immunised (n=6) and age-matched saline control-immunised (n=6) male Rhesus macaques (control) were challenged with 1?10.sup.6 plaque forming units of SARS-CoV-2 USA-WA1/2020 isolate, split equally between the intranasal (IN; 0.2 mL) and intratracheal (IT; 0.2 mL) routes as previously described (Singh, D. K. et al. SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species (2020)). The challenge was performed 41 to 48 days after the second immunisation. A separate sentinel group of non-immunised age- and sex-matched animals (n=3) received only DMEM supplemented with 10% FCS IN (0.2 mL) and IT (0.2 mL). Approximately two weeks prior to challenge, animals were moved to the ABSL-3 facility at Southwest National Primate Research Center (SNPRC; San Antonio, TX). Animals were monitored regularly by a board-certified veterinary clinician for rectal body temperature, weight and physical examination. Specimen collection was performed under tiletamine zolazepam (Telazol) anaesthesia as described (Singh, D. K. et al. SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species (2020)). Nasal swabs were collected from macaques at 0, 1, 3, and 6 days after inoculation to assess viral titers. Bronchoalveolar lavage (BAL) was performed the week before challenge and at Days 3 and 6 post-inoculation by instilling four times 20 mL of saline. These washings were pooled, aliquoted and stored frozen at ?70? C.

(1109) Reverse-Transcription Quantitative Polymerase Chain Reaction.

(1110) SARS-CoV-2 was detected and quantified in NHP essentially as described above in Example 10. Results Results showed COVID-19 mRNA Vaccine BNT162b2 was immunogenic eliciting IgG responses after a single dose which were boosted by a second dose. These also showed a dose-response. At 30 ?g BNT162, the neutralizing geometric mean titre was compared to that seen in covalescent plasma from human patients with SARS CoV-2 and found to be ?8-fold higher with seven days after Dose of the higher dose of 100 ?g giving a higher excess of ?18-fold and remaining 3.3-times higher than this benchmark five weeks after the last immunization. In monkeys the response was also characterised as Th1-dominant with IFN-? and IL-2, but no IL-4 response. CD4 and CD8 positive cellular responses were also observed in monkeys too. Such cellular immune response was characterized as a strongly Th1-biased CD4+ T cell response with a concurrent interferon-? (IFN-?)+CD8+ T cell response.

(1111) The groups of Rhesus macaques (n=6) that had received two immunisations with 100 ?g BNT162b2 or buffer control were challenged 41 to 48 days after the second immunisation with 1?10.sup.6 plaque forming units of SARS-CoV-2 (strain USA-WA1/2020), split equally between the intranasal and intratracheal routes, as previously described (Singh, D. K. et al. SARS-CoV-2 infection leads to acute infection with dynamic cellular and inflammatory flux in the lung that varies across nonhuman primate species, 2020). Three non-immunised, age-matched, male Rhesus macaques (sentinel) were mock-challenged with cell culture medium. At the time of challenge, SARS-CoV-2 neutralising titers ranged from 204 to 938 in the BNT162b2-immunised animals and were undetectable in animals from the control-immunised and sentinel groups.

(1112) SARS-CoV-2 RNA was measured in bronchoalveolar lavage (BAL) and nasal swab samples by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). All BAL and nasal swab samples obtained before the infectious challenge and all those obtained from sentinel animals lacked detectable SARS-CoV-2 RNA (FIG. 68). Three days after SARS-CoV-2 challenge, viral RNA was detected in BAL fluid from 5/6 control-immunised and 2/6 BNT162b2-immunised animals (FIG. 68). By 6 days after challenge, all six BNT162b2-immunised macaques had undetectable viral RNA in their lungs; of the control-immunised macaques, three had a high level of viral RNA in BAL fluid, two had cleared, and one had an indeterminate RT-qPCR result. After SARS-CoV-2 challenge, viral RNA was detected in nasal swabs of the control-immunised group at each time point: two animals at Day 1, three animals at Days 3 and 6 after challenge (FIG. 68). In BNT162b2-immunised animals, all nasal swabs were negative at Day 3 and at Day 6.

(1113) In lung tissues, control monkeys had evidence of pulmonary disease indicated by their increased scores on computed tomography scans with a suggestion of recovery in that scores at day 10 were less than those at day 3; in contrast, the monkeys given COVID-19 mRNA Vaccine BNT162b2 had lower scores. Microscopic analysis of lung tissues showed that lung inflammation was similar between control and BNT162b2-immunized monkeys, and there was no evidence of enhanced respiratory disease.

(1114) Discussion

(1115) Results in nonhuman primates confirm the potency and ability of BNT162b2, an LNP-formulated m1? nucleoside-modified mRNA encoding the S antigen captured in a prefusion conformation, to protect against SARS-CoV-2 challenge in a preclinical model of acute SARS-CoV-2 infection.

Example 12: Storage, Shipping and Dose Preparation

(1116) This example illustrates storage, shipping and dose preparation of a multi-dose vial of BNT162b2 concentrate for injection.

(1117) As shown in FIG. 69, at the stage of primary packing, 2 ml type 1 glass, preservative-free, multi-dose vial (MDV) is used, wherein the MDV has 0.45 ml frozen liquid drug product and there are 5 doses per vial. At the stage of secondary packing, a single tray holds 195 vials, such as 975 doses per tray. The tray (white box) dimensions are 229?229?40 mm. At the stage of teriary packing, a minimum of 1 tray (975 doses) (or up to 5 trays (max 4875 doses)) is stacked in a payload carton. The payload carton is submerged in 23 Kg of dry ice pellets (10 mm-16 mm pellets). The thermal shipper dimensions are as follows: internal dimensions: 245 mm?245 mm?241 mm; external dimensions: 400 mm?400 mm?560 mm. The total weight of the thermal shipper is ?35 Kg.

(1118) Different sizes of Ultra-Low Temperature (ULT) freezers are available in the market. FIG. 70 shows an example for a small volume storage (about 90 litres; about 30K doses (left)) and for a large volume storage (about 500 litres; about 200K doses (right)). Thermal shipper keeps ULT (e.g., ?90? C. to ?60? C.) up to 10 days if stored at 15? C. to 25? C. temperatures without opening and such storage period of time can be extended further by consistently refilling to the top of the container with dry ice. Upon receipt and after opening, the box should be replenished with dry ice within 24 hours (23 Kg of dry ice pellets (10 mm-16 mm pellets). Thermal shipper should be re-iced every 5 days. It is recommended that the thermal shipper is opened not more than twice a day. Thermal shipper should be closed within 1 minute (or less) after opening. The vaccine can be stored at 2? C. to 8? C. up to 2 days or at room temperature for no more than 2 hours after thawing. Post-dilution in use period is 6 hours.

(1119) FIG. 71 shows an exemplary dose preparation for a BNT162b2 5-dose vial which contains a frozen concentrated solution that is preservative-free and must be thawed and diluted prior to administration. The preparation steps are as follows:

(1120) Remove a 5-dose vial of BNT162b2 concentrate for injection from its carton in frozen storage and allow to thaw for approximately 30 minutes at room temperature (e.g., up to 25? C.). In some embodiments, such a multi-dose vial of BNT162b2 may be thawed and stored in a refrigerator (e.g., 2? C.-8? C.), for example, for up to 5 days. Vials thawed at room temperature must be diluted within 2 hours or transferred to a refrigerator. Undiluted vials may be stored for up to 48 hours in the refrigerator. Do not refreeze thawed vials. During storage, minimize exposure to room light, and avoid exposure to direct sunlight and ultraviolet light. Thawed vials can be handled in room light conditions.

(1121) After thawing and prior to use, ensure the vial is equilibrated to room temperature, and invert gently 10 times to mix. Do not shake.

(1122) Using aseptic technique, cleanse the vial stopper with a single-use antiseptic swab, then dilute the thawed vial of BNT162b2 by adding 1.8 mL of 0.9% Sodium Chloride Injection, USP into the vial. Needles 21 gauge or narrower needles are recommended. However, those skill in the art will understand that in some embodiments, wider needeles may be used. For example, in some embodiments, needles 20, 19, 18, 17, 16, 15 or wider needles may be used.

(1123) You may feel some pressure in the vial as you add the diluent. Ensure vial pressure is equalized by withdrawing 1.8 mL air into the empty diluent syringe before removing the needle from the vial.

(1124) Gently invert the diluted vial 10 times to mix. Do not shake.

(1125) Record the date and time of dilution in the appropriate place on the BNT162b2 vial label. Expiry is 6 hours from the time of dilution. Diluted multi-dose vials are stored between 2? C. and 25? C. Do not freeze. Discard if frozen.

(1126) Using aseptic technique, cleanse the vial stopper with a single-use antiseptic swab, and draw up 0.3 mL of the diluted dosing solution into a new sterile dosing syringe with a needle appropriate for intramuscular injection. Adjustments to remove air bubbles should be done with the needle still in the vial to avoid loss of dosing solution. It is recommended to use the same needle to withdraw and administer the dose whenever possible. If a second needle is required for administration, pull back on the syringe plunger until a small amount of air enters the syringe prior to removing the first needle to avoid loss of dosing solution during the needle change. Take care when priming the administration needle to prevent any loss of dose.

(1127) For each additional dose, use a new sterile syringe and needle and ensure the vial stopper is cleansed with antiseptic before each withdrawal. Prepared syringes should be administered immediately. If they cannot be administered immediately, they must be administered within 6 hours of the initial vial dilution. Before administration, ensure a final injection volume in the syringe of 0.3 mL.

Example 13: Vaccine Candidate Against COVID-19 Achieved Success in First Interim Analysis from Phase 3 Study

(1128) The Phase 3 clinical trial of BNT162b2 has enrolled more than 43,000 participants to date, almost 39,000 of whom have received a second dose of the vaccine candidate as of Nov. 8, 2020. Approximately 42% of global participants and 30% of U.S. participants have racially (e.g., including White, Black or African American, American Indian or Alaska native, Asian, native Hawaiian or other Pacific Islander, multiracial) and ethnically (e.g., including Hispanic/Latino and non-Hispanic/non-Latino) diverse backgrounds. The trial is continuing to enroll and is expected to continue through the final analysis when a total of 164 confirmed COVID-19 cases have accrued.

(1129) Vaccine candidate BNT162b2 achieved success in First Interim Analysis from Phase 3 Study. The vaccine candidate was found to be more than 90% effective in preventing COVID-19 in participants without evidence of prior SARS-CoV-2 infection in the first interim efficacy analysis. Analysis evaluated 94 confirmed cases of COVID-19 in trial participants. No serious safety concerns have been observed.

(1130) A set of results from the Phase 3 COVID-19 vaccine trial, assembled by Nov. 4, 2020, provides evidence of the ability of BNT162b2 to prevent COVID-19. The case split between vaccinated individuals and those who received the placebo indicates a vaccine efficacy rate above 90%, at 7 days after the second dose. In particular, early analysis of the results showed that individuals who received two injections of the vaccine three weeks apart experienced more than 90% fewer cases of symptomatic COVID-19 than those who received a placebo. This confirms that protection is achieved 28 days after the initiation of the vaccination, which consists of a 2-dose schedule.

(1131) Preliminary such data includes the following tables:

(1132) TABLE-US-00023 TABLE 5 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days After Dose 2 - Subjects Without Evidence of Infection Prior to 7 Days After Dose 2 - Evaluable Efficacy Population (7 Days) - Interim Analysis 1. Vaccine Group (as Randomized) BNT162b2 (30 ?g) Placebo (N.sup.a = 16061) (N.sup.a = 16218) Pr Efficacy Surveillance Surveillance VE (VE >30% | Endpoint n1.sup.b Time.sup.c (n2.sup.d) n1.sup.b Time.sup.c (n2.sup.d) (%) (95% CI.sup.e) data).sup.f First COVID-19 4 1.72161 90 1.73212 95.5 (88.8, 98.4) >0.9999 occurrence from (15899) (16010) 7 days after Dose 2 Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding; NAAT = nucleic acid amplification test; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; VE = vaccine efficacy. Note: Subjects who had no serological or virological evidence (prior to 7 days after receipt of the last dose) of past SARS-CoV-2 infection (ie, N-binding antibody [serum] negative at Visit 1 and SARS-CoV-2 not detected by NAAT [nasal swab] at Visits 1 and 2), and had negative NAAT at any unscheduled visit prior to 7 days after Dose 2 were included in the analysis. Note: Data from subjects who are not confirmed 7 days post dose 2 cases are included in the analysis to comprehensively show all data reported and/or contribute to the total surveillance time calculation but may be subject to change with additional follow-up. .sup.aN = number of subjects in the specified group. .sup.bn1 = Number of subjects meeting the endpoint definition. .sup.cTotal surveillance time in 1000 person-years for the given endpoint across all subjects within each group at risk for the endpoint. Time period for COVID-19 case accrual is from 7 days after Dose 2 to the end of the surveillance period. .sup.dn2 = Number of subjects at risk for the endpoint. .sup.eCredible interval for VE was calculated using a beta-binomial model adjusted for surveillance time. .sup.fPosterior probability (Pr) was calculated using a beta-binomial model adjusted for surveillance time. This probability must be at least 99.5% at the interim analysis in order to conclude that the vaccine is efficacious.

(1133) TABLE-US-00024 TABLE 6 Severe COVID-19 Occurrence After Dose 1 - Dose 1 All- Available Efficacy Population - Interim Analysis 1. Vaccine Group (as Randomized) BNT162b2 (30 ?g) Placebo (N.sup.a = 21617) (N.sup.a = 21633) Efficacy Endpoint n.sup.b n.sup.b Severe COVID-19 occurrence 0 7 after Dose 1 Note: Data from subjects who are not confirmed 7 days post dose 2 cases are included in the analysis to comprehensively show all data reported and/or contribute to the total surveillance time calculation but may be subject to change with additional follow-up. .sup.aN = number of subjects in the specified group. .sup.bn = Number of subjects meeting the endpoint definition.

Example 14: Efficacy and Immunogenicity Evaluation

(1134) Efficacy Results

(1135) Vaccine efficacy of BNT162b2 against COVID-19 among participants without evidence of past SARS-CoV-2 infection was demonstrated at the first interim analysis conducted after accrual of at least 62 cases following the protocol and SAP. The primary efficacy results presented in this section are from that interim analysis.

(1136) Only the vaccine efficacy of BNT162b2 for the first primary efficacy endpoint (COVID-19 incidence based on central laboratory or locally confirmed NAAT in participants without serological or virological evidence of past SARS-CoV-2 infection prior to 7 days after receipt of the second dose) is analyzed and presented at this interim analysis.

(1137) First Primary Efficacy Endpoint

(1138) Among participants included in the evaluable efficacy population, 32,279 participants (16,061 in BNT162b2 group and 16,218 in placebo group) did not have evidence of infection with SARS-CoV-2 through 7 days after the second dose. There were 4 COVID-19 cases in the BNT162b2 group compared to 90 COVID-19 cases reported in the placebo group. These data give an estimated vaccine efficacy of 95.5% for BNT162b2. The posterior probability of >99.99% met the prespecified interim analysis success criterion of >99.5% (Table 7). The 95% credible interval for the vaccine efficacy was 88.8% to 98.4%, indicating that given the current observed data there is a 95% probability that the true VE lies in this interval. Also, note that the posterior probability that true VE >86.0% is 99.5% and VE >88.8% is 97.5%.

(1139) TABLE-US-00025 TABLE 7 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days After Dose 2 - Subjects Without Evidence of Infection Prior to 7 Days After Dose 2 - Evaluable Efficacy Population (7 Days) - Interim Analysis 1 Vaccine Group (as Randomized) BNT162b2 (30 ?g) Placebo (N.sup.a = 16061) (N.sup.a = 16218) Pr Efficacy Surveillance Surveillance VE (VE >30% | Endpoint n1.sup.b Time.sup.c (n2.sup.d) n1.sup.b Time.sup.c (n2.sup.d) (%) (95% CI.sup.e) data).sup.f First COVID-19 4 1.722 90 1.732 95.5 (88.8, 98.4) >0.9999 occurrence (15899) (16010) from 7 days after Dose 2 Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding; NAAT = nucleic acid amplification test; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; VE = vaccine efficacy. Note: Subjects who had no serological or virological evidence (prior to 7 days after receipt of the last dose) of past SARS-CoV-2 infection (ie, N-binding antibody [serum] negative at Visit 1 and SARS-CoV-2 not detected by NAAT [nasal swab] at Visits 1 and 2), and had negative NAAT at any unscheduled visit prior to 7 days after Dose 2 were included in the analysis. Note: Data from subjects who are not confirmed 7 days post dose 2 cases are included in the analysis to comprehensively show all data reported and/or contribute to the total surveillance time calculation but may be subject to change with additional follow-up. .sup.aN = number of subjects in the specified group. .sup.bn1 = Number of subjects meeting the endpoint definition. .sup.cTotal surveillance time in 1000 person-years for the given endpoint across all subjects within each group at risk for the endpoint. Time period for COVID-19 case accrual is from 7 days after Dose 2 to the end of the surveillance period. .sup.dn2 = Number of subjects at risk for the endpoint. .sup.eCredible interval for VE was calculated using a beta-binomial model adjusted for surveillance time. .sup.fPosterior probability (Pr) was calculated using a beta-binomial model adjusted for surveillance time. This probability must be at least 99.5% at the interim analysis in order to conclude that the vaccine is efficacious.

(1140) The vaccine efficacy of BNT162b2 for the same primary efficacy endpoint based on the all-available efficacy population was 95.7%, with 4 and 93 cases in the BNT162b2 and placebo groups, respectively.

(1141) No clinically meaningful differences in VE by subgroup were observed by age group, country, ethnicity, sex, or race in the in the Dose 2 evaluable efficacy population, with VE estimates that ranged from 91.2% to 100.0% (Table 8).

(1142) TABLE-US-00026 TABLE 8 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days After Dose 2, by Subgroup - Subjects Without Evidence of Infection Prior to 7 Days After Dose 2 - Evaluable Efficacy Population (7 Days) - Interim Analysis 1 Vaccine Group (as Randomized) BNT162b2 (30 ?g) Placebo Efficacy (N.sup.a = 16061) (N.sup.a = 16218) Endpoint Surveillance Surveillance Subgroup n1.sup.b Time.sup.c (n2.sup.d) n1.sup.b Time.sup.c (n2.sup.d) VE (%) (95% CI.sup.e) First COVID-19 occurrence from 7 days after Dose 2 Overall 4 1.722 (15899) 90 1.732 (16010) 95.5 (88.1, 98.8) Age group (years) 16 to 55 2 0.954 (8994) 67 0.959 (9040) 97.0 (88.7, 99.6) >55 2 0.767 (6905) 23 0.773 (6970) 91.2 (64.6, 99.0) Sex Male 2 0.874 (8115) 38 0.865 (8029) 94.8 (79.8, 99.4) Female 2 0.848 (7784) 52 0.867 (7981) 96.1 (85.1, 99.5) Race White 4 1.477 (13399) 85 1.491 (13530) 95.3 (87.4, 98.7) Black or African 0 0.124 (1263) 4 0.124 (1277) 100.0 (?51.8, 100.0) American All others.sup.f 0 0.121 (1237) 1 0.118 (1203) 100.0 (?3690.1, 100.0) Ethnicity Hispanic/Latino 1 0.464 (4389) 34 0.459 (4342) 97.1 (82.7, 99.9) Non-Hispanic/ 3 1.247 (11418) 56 1.262 (11570) 94.6 (83.3, 98.9) non-Latino Country Argentina 0 0.271 (2436) 28 0.266 (2402) 100.0 (86.2, 100.0) Brazil 0 0.087 (878) 2 0.087 (879) 100.0 (?432.5, 100.0) USA 4 1.360 (12384) 60 1.376 (12530) 93.3 (81.8, 98.2) Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding; NAAT = nucleic acid amplification test; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; VE = vaccine efficacy. Note: Subjects who had no serological or virological evidence (prior to 7 days after receipt of the last dose) of past SARS-CoV-2 infection (ie, N-binding antibody [serum] negative at Visit 1 and SARS-CoV-2 not detected by NAAT [nasal swab] at Visits 1 and 2), and had negative NAAT at any unscheduled visit prior to 7 days after Dose 2 were included in the analysis. Note: Data from subjects who are not confirmed 7 days post dose 2 cases are included in the analysis to comprehensively show all data reported and/or contribute to the total surveillance time calculation but may be subject to change with additional follow-up. .sup.aN = number of subjects in the specified group. .sup.bn1 = Number of subjects meeting the endpoint definition. .sup.cTotal surveillance time in 1000 person-years for the given endpoint across all subjects within each group at risk for the endpoint. Time period for COVID-19 case accrual is from 7 days after Dose 2 to the end of the surveillance period. .sup.dn2 = Number of subjects at risk for the endpoint. .sup.eConfidence interval (CI) for VE is derived based on the Clopper and Pearson method adjusted to the surveillance time. .sup.fAmerican Indian or Alaska native, Asian, Native Hawaiian or other Pacific Islander, multiracial, not reported race categories are presented as All others.

(1143) Severe COVID-19 Cases

(1144) Severe COVID-19 cases were reported in a total of 7 participants in Phase 3, all in the placebo group, as of the data cutoff date of 4 Nov. 2020 for the first interim analysis (Table 9). Five of these cases were reported between Dose 1 and Dose 2, none were reported less than 7 days after Dose 2, and 2 cases were reported at least 7 days after Dose 2.

(1145) TABLE-US-00027 TABLE 9 Severe COVID-19 Occurrence After Dose 1 - Dose 1 All- Available Efficacy Population - Interim Analysis 1 Vaccine Group (as Randomized) BNT162b2 (30 ?g) Placebo (N.sup.a = 21617) (N.sup.a = 21633) Efficacy Endpoint n.sup.b n.sup.b Severe COVID-19 occurrence 0 7 after Dose 1 Note: Data from subjects who are not confirmed 7 days post dose 2 cases are included in the analysis to comprehensively show all data reported and/or contribute to the total surveillance time calculation but may be subject to change with additional follow-up. .sup.aN = number of subjects in the specified group. .sup.bn = Number of subjects meeting the endpoint definition.

(1146) Efficacy Conclusions

(1147) The first primary efficacy objective met success criteria. BNT162b2 achieved vaccine efficacy of 95.5% with a 2-sided 95% credible interval of 88.8% to 98.4% among participants without evidence of infection up to 7 days after Dose 2, and a >99.99% posterior probability for the true vaccine efficacy greater than 30% conditioning on available data.

(1148) All 7 severe COVID-19 cases (after Dose 1) were observed in the placebo group, as of the interim analysis cutoff date.

(1149) Immunogenicity Results

(1150) Phase 1

(1151) This Phase 1 interim Clinical Study Report (CSR) presents immunogenicity results for both adult age groups up to 1 month after Dose 2 for the BNT162b1 and BNT162b2 vaccine candidates at the 10-?g, 20-?g, and 30-?g dose levels, and up to 7 weeks after Dose 1 of BNT162b1 at the 100-?g dose level (younger age group only).

(1152) Results for the 7 days after Dose 1 time point are only analyzed and presented in the younger age group (18 to 55 years of age) for 10 ?g and 30 ?g BNT162b1.

(1153) SARS-CoV-2 Neutralizing TitersPhase 1 GMTs

(1154) Overall, for both the BNT162b1 and the BNT162b2 recipients in both age groups, SARS-CoV-2 50% neutralizing GMTs modestly increased by Day 21 after Dose 1 and were substantially increased 7 days after Dose 2. Generally, GMTs in the older age group tended to be somewhat lower than the GMTs in the younger age group at most time points for both BNT162b1 and BNT162b2 recipients.

(1155) BNT162b1

(1156) In the younger age group, SARS-CoV-2 50% neutralizing GMTs modestly increased by Day 21 after Dose 1 and were substantially increased 7 days after Dose 2 (Day 28) of BNT162b1, with higher GMTs observed in the 30-?g dose group compared to the 10-?g and 20-?g dose groups (FIG. 72). GMTs increased at 14 days after Dose 2 (Day 35) for all dose groups, and although GMTs decreased at 1 month after Dose 2 (Day 52), the Day 52 GMTs remained substantially higher than those at the earliertime points after Dose 1.

(1157) In the 100-?g dose group, SARS-CoV-2 50% neutralizing GMTs modestly increased by Day 21 after Dose 1 of BNT162b1 and decreased to a near baseline value by Day 52. Generally similar trends were observed in the older age group, with higher GMTs observed in the 20-?g and 30-?g dose groups of BNT162b1 compared to the 10-?g dose group (FIG. 73).

(1158) Similar trends were observed for the SARS-CoV-2 90% neutralizing GMTs.

(1159) Results for the all-available immunogenicity population in the younger age and older age groups were similar to those observed for the evaluable immunogenicity population.

(1160) RCDCs of SARS-CoV-2 50% and 90% neutralizing titers after BNT162b1 for the younger and older age groups show that the majority of participants responded by 7 days after Dose 2 of BNT162b1.

(1161) BNT162b2

(1162) In the younger age group, SARS-CoV-2 50% neutralizing GMTs increased by Day 21 after Dose 1 and were substantially increased 7 days after Dose 2 (Day 28) of BNT162b2, with higher GMTs observed in the 20-?g and 30-?g dose groups compared to the 10-?g dose group (FIG. 74). The GMTs decreased at 14 days after Dose 2 (Day 35) and 1 month after Dose 2 (Day 52) of BNT162b2; however, the GMTs remained substantially higher than those at the earlier time points after Dose 1.

(1163) Similar trends were generally observed in the older age group, with higher GMTs observed in the 30-?g dose groups compared to the 20-?g and 10-?g dose groups (FIG. 75). SARS-CoV-2 50% neutralizing GMTs were increased 7 days after Dose 2 and were similar in the 10-?g and 20-?g dose groups and higher in the 30-?g dose group. At 1 month after Dose 2, GMTs remained substantially higher than those at the earlier time points after Dose 1. In the older age group, SARS-CoV-2 50% neutralizing GMTs were generally lower than the GMTs in the younger age group. Similar trends were observed for the SARS-CoV-2 90% neutralizing GMTs.

(1164) Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population. RCDCs of SARS-CoV-2 50% and 90% neutralizing titers for the younger and older age groups show that the majority of participants responded by 7 days after Dose 2 of BNT162b2.

(1165) GMFRs

(1166) Overall, for both the BNT162b1 and the BNT162b2 recipients, and in both age groups, GMFRs of SARS-CoV-2 50% neutralizing titers from before vaccination to 7 days after Dose 2 (Day 28) were substantially higher compared to the respective GMFRs after Dose 1. GMFRs in the older age group were generally lower than the those in the younger age group for both BNT162b1 and BNT162b2 recipients.

(1167) BNT162b1

(1168) In the younger age group, GMFRs of SARS-CoV-2 50% neutralizing titers from before vaccination to 7 days after Dose 2 (Day 28) of BNT162b1 were substantially high compared to GMFRs at earlier time points after Dose 1 of BNT162b1 in all dose groups, with GMFRs being highest in the 30-?g dose group. At 1 month after Dose 2, the GMFRs remained higher than those at the earlier time points after Dose 1.

(1169) In the 100-?g dose group, the GMFRs of SARS-CoV-2 50% neutralizing titers were not substantially increased through Day 52 after Dose 1 of BNT162b1.

(1170) In the older age group, GMFRs of SARS-CoV-2 50% neutralizing titers from before vaccination to 7 days after Dose 2 (Day 28) of BNT162b1 were substantially high compared to GMFRs at the earlier time point after Dose 1 of BNT162b1 in the 20-?g and 30-?g dose groups with GMFRs being highest in the 20-?g dose group. The GMFRs remained high in the 20-?g and 30-?g dose groups at 1 month after Dose 2 (Day 52) of BNT162b1 compared to GMFRs at the earlier time point after Dose 1.

(1171) Similar trends were observed for GMFRs of SARS-CoV-2 90% neutralizing titers in the younger age group and older age group.

(1172) Results for the all-available immunogenicity population in the younger age and older age groups were similar to those observed for the evaluable immunogenicity population.

(1173) BNT162b2

(1174) In the younger age group, GMFRs of SARS-CoV-2 50% neutralizing titers from before vaccination to 7 days after Dose 2 (Day 28) of BNT162b2 were substantially high compared to GMFRs at the earlier time point after Dose 1 of BNT162b2 for all dose groups, with GMFRs being similar and highest in the 20-?g and 30-?g dose groups. GMFRs remained high through 1 month after Dose 2 of BNT162b2 compared to GMFRs 21 days after Dose 1 of BNT162b2.

(1175) In the older age group, GMFRs of SARS-CoV-2 50% neutralizing titers from before vaccination to 7 days after Dose 2 (Day 28) of BNT162b2 were substantially high compared to GMFRs at the earlier time point after Dose 1 of BNT162b2 in all dose groups, with GMFRs being highest in the 30-?g dose group. GMFRs remained high through 1 month after Dose 2 of BNT162b2 compared to GMFRs at 21 days after Dose 1 of BNT162b2.

(1176) Similar trends were observed for GMFRs of SARS-CoV-2 90% neutralizing titers in the younger and older age groups.

(1177) Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population.

(1178) Number (%) of Participants Achieving a ?24-Fold Rise

(1179) Overall, for both the BNT162b1 and the BNT162b2 recipients, and in both age groups, most participants achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers from before vaccination to 7 days after Dose 2, except in the older participants in the 10-?g BNT162b1 dose group.

(1180) BNT162b1

(1181) In the younger age group, from before vaccination to 21 days after Dose 1 of BNT162b1, no participants in the 10-?g dose group and 53 participants in the 20-?g and the 30-?g dose groups achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers. From before vaccination to both 7 days and 1 month after Dose 2 of BNT162b1 most or all participants in the 10-?g, 20-?g, and 30-?g dose groups achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers.

(1182) In the older age group, from before vaccination to 21 days after Dose 1 of BNT162b1, only 1 participant in the 30-?g dose group achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers. From before vaccination to both 7 days and 1 month after Dose 2 of BNT162b1, 52 participants in the 10-?g group and 9 to 11 participants in the 20-?g and 30-?g dose groups achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers.

(1183) Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population.

(1184) BNT162b2

(1185) In the younger age group, from before vaccination to 21 days after Dose 1 of BNT162b2, 2 (18.2%) participants in the 10-?g dose group, 3 (25.0%) participants in the 20-?g dose group, and none in the 30-?g group achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers. From before vaccination to 7 days after Dose 2 of BNT162b2, all participants achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers, which was maintained through 1 month after Dose 2 of BNT162b2.

(1186) In the older age group, from before vaccination to 21 days after Dose 1 of BNT162b2, no participants achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers in any dose group. From before vaccination to 7 days after Dose 2 of BNT162b2, 10 (83.3%), 9 (81.8%), and 10 (90.9%) participants achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers in the 10-?g, 20-?g, and 30-?g dose groups, respectively. From before vaccination to 1 month after Dose 2 of BNT162b2, 9 (75.0%), 6 (54.5%), and 11 (100.0%) participants achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers in the 10-?g, 20-?g, and 30-?g dose groups, respectively.

(1187) Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population.

(1188) SARS-CoV-2 Antigen-Specific Binding Antibody LevelsPhase 1

(1189) Vaccine candidate BNT162b1 encodes for the RBD of SARS-CoV-2. RBD-binding IgG responses for each dose level and age group for this candidate are described in this section. RBD-binding IgG levels were also assessed for candidate BNT62b2 which encodes the P2 S of SARS-CoV-2.

(1190) Vaccine candidate BNT162b2 encodes for the P2 S of SARS-CoV-2. S1-binding IgG responses for each dose level and age group for this candidate are described in this section. S1-binding IgG levels were also assessed for candidate BNT62b1 which encodes the RBD of SARS-CoV-2.

(1191) GMCs

(1192) Overall, for both the BNT162b1 and the BNT162b2 recipients, and in both age groups, RBD- and S1-binding GMCs increased substantially by Day 21 after Dose 1 and were further increased 7 days after Dose 2. Responses were maintained through Day 52. GMCs in the older age group were generally lower than the GMCs in the younger age group, with the exception of Day 28 in the 20-?g BNT162b1 dose group for both RBD- and S1-binding IgG levels.

(1193) BNT162b1

(1194) In the younger age group, RBD-binding GMCs increased substantially by Day 21 after Dose 1 of BNT162b1 and further increased 7 days after Dose 2 (Day 28) of BNT162b1, with higher GMCs observed in the 30-?g dose group compared to the 10-?g and 20-?g dose groups (FIG. 76). At 1 month after Dose 2 (Day 52), the GMCs remained substantially higher than at the earlier time points after Dose 1.

(1195) In the 100-?g BNT162b1 group, the RBD-binding GMC increased substantially by 21 days after BNT162b1 and remained higher through Day 52 compared to the Day 7 GMC.

(1196) In the older age group, RBD-binding GMCs increased substantially by Day 21 after Dose 1 of BNT162b1 and further increased 7 days after Dose 2 (Day 28) of BNT162b1, with higher GMCs observed in the 20-?g and 30-?g dose groups compared to the 10-?g group (FIG. 77). At 1 month after Dose 2 (Day 52), the GMCs remained substantially higher than at the earlier time point after Dose 1.

(1197) S1-binding IgG GMC results for BNT162b1 were similar to those observed for RBD-binding IgG GMCs in the younger (FIG. 78) and older age groups (FIG. 79), and in the 100-?g BNT162b1 group.

(1198) Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population. RCDCs of RBD- and S1-binding IgG levels show that the majority of participants responded by 21 days after Dose 1 of BNT162b1.

(1199) BNT162b2

(1200) In the younger age group, S1-binding GMCs increased substantially by Day 21 after Dose 1 of BNT162b2 and were substantially increased by 7 days after Dose 2 (Day 28) of BNT162b2, with higher GMCs observed in the 20-?g and 30-?g dose groups compared to the 10-?g dose group (FIG. 80). At 1 month after Dose 2 (Day 52), the GMCs remained substantially higher than at the earlier time point after Dose 1. Similar trends were observed in the older age group, with higher S1-binding GMCs observed in the 30-?g dose group compared to the 10-?g and 20-?g dose groups (FIG. 81).

(1201) RBD-binding IgG GMC results for BNT162b2 were similar to those observed for S1-binding IgG GMCs in the younger (FIG. 82) and older age groups (FIG. 83). Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population. RCDCs of RBD- and S1-binding IgG levels after BNT162b2 show that the majority of participants responded by 21 days after Dose 1 of BNT162b2.

(1202) GMFRs

(1203) Overall, for the BNT162b1 and the BNT162b2 recipients, and in both age groups, GMFRs of RBD-binding IgG levels and GMFRs of S1-binding IgG levels were substantially high from before vaccination to 21 days after Dose 1, with greater GMFRs observed from before vaccination to 7 days after Dose 2.

(1204) BNT162b1

(1205) GMFRs of RBD-binding IgG levels were substantially high from before vaccination to Day 21 (before Dose 2) after Dose 1 of BNT162b1, with greater GMFRs observed from before vaccination to 7 days after Dose 2 (Day 28) of BNT162b1 in both the younger and older age groups, for the 10-?g, 20-?g, and 30-?g dose groups. GMFRs remained substantially high in the 10-?g, 20-?g, and 30-?g BNT162b1 groups from before vaccination to 1 month after Dose 2 compared to the earlier time points after Dose 1 for both age groups.

(1206) In the 100-?g BNT162b1 group, GMFR of RBD-binding IgG levels was substantially high from before vaccination by 21 days after BNT162b1 and remained higher through Day 52 compared to the Day 7 GMFR.

(1207) Similar trends were observed for GMFRs of S1-binding IgG levels for BNT162b1.

(1208) Results for the all-available immunogenicity population in the younger age and older age groups were similar to those observed for the evaluable immunogenicity population.

(1209) BNT162b2

(1210) GMFRs of S1-binding IgG levels were substantially high from before vaccination to Day 21 (before Dose 2) after Dose 1 of BNT162b2, with greater GMFRs observed from before vaccination to 7 days after Dose 2 (Day 28) of BNT162b2 in both the younger and older age groups, for the 10-?g, 20-?g, and 30-?g dose groups. GMFRs remained substantially high in all BNT162b2 groups from before vaccination to 1 month after Dose 2 compared to the earlier time point after Dose 1 for both age groups.

(1211) Similar trends were observed for GMFRs of RBD-binding IgG levels for BNT162b2. Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population.

(1212) Number (%) of Participants Achieving a ?4-Fold Rise

(1213) Overall, for the BNT162b1 and the BNT162b2 recipients, and in both age groups, all participants achieved a ?4-fold rise in S1- and RBD-binding IgG levels from before vaccination to 7 days after Dose 2, with the exception of 1 participant in the younger 20-?g BNT162b1 group.

(1214) BNT162b1

(1215) In the younger age group, from before vaccination to 21 days following Dose 1 of BNT162b1, all participants (except 1 in the 20-?g dose group) across all dose groups achieved a ?4-fold rise in RBD-binding IgG levels. All participants in the 20-?g dose group achieved a ?4-fold rise in RBD-binding IgG levels from before vaccination to 14 days after Dose 2 (Day 35).

(1216) In the older age group, from before vaccination to 21 days following Dose 1 of BNT162b1, all participants in the 20-?g and 30-?g dose groups and 8 (72.7%) participants in the 10-?g dose group achieved a ?4-fold rise in RBD-binding IgG levels. All participants in the 10-?g dose group achieved a ?4-fold rise in RBD-binding IgG levels from before vaccination to 7 days after Dose 2 (Day 28).

(1217) Similar trends were generally observed for participants achieving a 4-fold rise in S1-binding IgG levels for BNT162b1.

(1218) Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population.

(1219) BNT162b2

(1220) In the younger age group, from before vaccination to 21 days following Dose 1 of BNT162b2, all participants in each dose group achieved a ?4-fold rise in S1-binding IgG levels.

(1221) In the older age group, from before vaccination to 21 days following Dose 1 of BNT162b2, all participants in the 10-?g, and 30-?g dose groups and 11 (91.7%) participants in the 20-?g dose group achieved a ?4-fold rise in S1-binding IgG levels. All participants in the 20-?g dose group achieved a ?4-fold rise in S1-binding IgG levels from before vaccination to 7 days after Dose 2 (Day 28).

(1222) Similar trends were generally observed for participants achieving a 4-fold rise in RBD-binding IgG levels for BNT162b2.

(1223) Results for the all-available immunogenicity population were similar to those observed for the evaluable immunogenicity population in the younger and older age groups.

(1224) GMRs of SARS-CoV-2-Neutralizing Titers to SARS-CoV-2 Antigen-Specific Binding Antibody Levels

(1225) Overall, for BNT162b1 and BNT162b2 recipients, GMRs of SARS-CoV-2 50% neutralizing titers to RBD- or S1-binding IgG levels show a more robust RBD- or S1-binding levels relative to neutralizing titers, which were similar within each age group.

(1226) BNT162b1

(1227) At 21 days after Dose 1 at 10 ?g, 20 ?g, or 30 ?g, GMRs of SARS-CoV-2 50% neutralizing titers to RBD-binding IgG levels were ?0.035 in the younger age group and ?0.183 in the older age group. At 14 days after Dose 2, the GMRs were ?0.032 in the younger age group and ?0.018 in the older age group.

(1228) For the 100-?g dose group, the GMR was 0.018 at 21 days after Dose 1 and 0.014 at 35 days after Dose 1.

(1229) GMRs of SARS-CoV-2 50% neutralizing titers to S1-binding IgG levels were similar to GMRs of SARS-CoV-2 50% neutralizing titers to RBD-binding IgG levels in the younger and older age groups after BNT162b1.

(1230) Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population.

(1231) BNT162b2

(1232) At 21 days after Dose 1, GMRs of SARS-CoV-2 50% neutralizing titers to S1-binding IgG levels were 0.035 in the younger age group and ?0.124 in the older age group. At 14 days after Dose 2, the GMRs were ?0.040 in the younger age group and ?0.037 in the older age group.

(1233) Results for the all-available immunogenicity population in the younger and older age groups were similar to those observed for the evaluable immunogenicity population.

(1234) Evaluating BNT162b1 and BNT162b2 GMRs

(1235) In the younger age group at 21 days after Dose 1, GMRs of SARS-CoV-2 50% neutralizing titers to RBD-binding IgG levels were ?0.035 after BNT162b1 and ?0.054 after BNT162b2. At 14 days after Dose 2, the GMRs were ?0.032 after BNT162b1 and ?0.046 after BNT162b2.

(1236) In the older age group at 21 days after Dose 1, GMRs of SARS-CoV-2 50% neutralizing titers to RBD-binding IgG levels were ?0.183 after BNT162b1 and ?0.196 after BNT162b2. At 14 days after Dose 2, the GMRs were ?0.018 after BNT162b1 and ?0.043 after BNT162b2.

(1237) In the younger age group at 21 days after Dose 1, GMRs of SARS-CoV-2 50% neutralizing titers to S1-binding IgG levels were ?0.061 after BNT162b1 and ?0.035 after BNT162b2. At 14 days after Dose 2, the GMRs were ?0.035 after BNT162b1 and ?0.040 after BNT162b2.

(1238) In the older age group at 21 days after Dose 1, GMRs of SARS-CoV-2 50% neutralizing titers to S1-binding IgG levels were ?0.328 after BNT162b1 and ?0.124 after BNT162b2. At 14 days after Dose 2, the GMRs were ?0.022 after BNT162b1 and ?0.037 after BNT162b2.

(1239) Phase 1 Summary of Immunogenicity Results Evaluating BNT162b1 and BNT162b2

(1240) In general, a modest neutralizing immune response was observed in both the younger and older age groups after the first dose. A much more robust immune response was observed 7 days after the second dose of either BNT162b1 or BNT162b2 at all dose levels in both the younger and older age groups. Antibody levels at the last time point tested were still substantially above those at baseline.

(1241) In the Younger Age Group:

(1242) At 7 days after Dose 2, SARS-CoV-2 50% neutralizing GMTs in the 20-?g and 30-?g dose groups were higher for BNT162b2 recipients than for BNT162b1 recipients. The GMTs were similar in the 10-?g dose group for both recipients. At 1 month after Dose 2 (Day 52), GMTs remained substantially higher than those at the earlier time points after Dose 1 for both BNT162b1 and BNT162b2 recipients.

(1243) From before vaccination to 7 days after Dose 2, GMFRs of SARS-CoV-2 50% neutralizing titers were substantially high for BNT162b1 and BNT162b2 recipients at the 30 g dose level.

(1244) From before vaccination to 7 days after Dose 2, all participants at the 30-?g dose level who received BNT162b1 or BNT162b2 achieved a ?4-fold rise in SARS CoV-2 50% neutralizing titers.

(1245) In the Older Age Group:

(1246) At 7 days after Dose 2, SARS-CoV-2 50% neutralizing GMT in the 30-?g dose group was higher for BNT162b2 recipients than for BNT162b1 recipients. At 1 month after Dose 2 (Day 52), the SARS-CoV-2 50% neutralizing GMTs in the 30-?g dose group were similar for both BNT162b1 and BNT162b2 recipients.

(1247) From before vaccination to 7 days after Dose 2, the GMFR of SARS-CoV-2 50% neutralizing titers were substantially high for BNT162b1 and BNT162b2 recipients at the 30-?g dose level.

(1248) From before vaccination to 7 days after Dose 2, most participants who received BNT162b1 or BNT162b2 at the 30-?g dose level achieved a ?4-fold rise in SARS-CoV-2 50% neutralizing titers.

(1249) Phase 1 Immunogenicity Conclusions

(1250) Both BNT162b1 and BNT162b2 elicited robust SARS-CoV-2 neutralizing antibody response 7 days after Dose 2 in younger and older adults, based on GMTs, GMFRs, proportions of participants achieving a ?4-fold rise in neutralizing titers, and RCDCs. Neutralizing antibody response was maintained through Day 52 and was similar for the candidates within the corresponding age and dose groups.

(1251) Both BNT162b1 and BNT162b2 elicited substantial rises in antigen binding IgG levels 7 days after Dose 2, based on GMCs, GMFRs, and proportions of participants achieving a ?4-fold rise in IgG-antigen specific binding. Responses were maintained through Day 52.

(1252) In the 100-?g dose group, SARS-CoV-2 neutralizing antibody response modestly increased by 3 weeks after Dose 1 of BNT162b1, but neutralizing antibody response returned to levels similar to baseline by 7 weeks after Dose 1.

(1253) These data support the need for a 2-dose vaccination series.

(1254) Phase 2

(1255) Immunogenicity is an exploratory endpoint for the Phase 2 part of the study.

(1256) Phase 3

(1257) Immunogenicity is a secondary (12 to 15 year olds compared with 16 to 25 year olds) and an exploratory endpoint for the Phase 3 part of the study.

Example 15: Safety Evaluation

(1258) In this interim CSR, all participants in Phase 1 and the first 6610 participants in Phase 2/3 (360 participants from Phase 2 included) used an e-diary for reporting local reactions and systemic events. A total of 1125 participants in Phase 2/3 were identified as baseline SARS-CoV-2 positive, defined as having a positive N-binding antibody test result or positive nucleic acid amplification test (NAAT) result on the day of Dose 1; of these, 545 received BNT162b2 and 580 received placebo.

(1259) Phase 1

(1260) Safety data are available up through the date cutoff date (24 Aug. 2020) and are summarized at various time points relative to Dose 1 or Dose 2. Safety results for Phase 1 vaccine candidates BNT162b1 and BNT162b2 for both adult age groups are presented up to 1 month after Dose 2 (or data cutoff date) at the 10-?g, 20-?g, and 30-?g dose levels. Safety results for BNT162b1 at the 100-?g dose level in the younger age group are presented up to 3 weeks after Dose 1 or to before Dose 2 based on the data cutoff date. Note that the group of participants 18 to 55 years of age who received 100 ?g BNT162b1 did not receive a second dose of 100 ?g BNT162b2 per IRC decision.

(1261) Local ReactionsPhase 1

(1262) Overall, for both the BNT162b1 and the BNT162b2 recipients, and in both age groups, pain at the injection site was the most frequent local reaction. Redness and swelling occurred less frequently in the BNT162b2 group and in the BNT162b1 group. In both the BNT162b1 and BNT162b2 groups, the frequency of local reactions was lower in the older age group compared to the younger age group, and there was a trend of a higher frequency of local reactions with increased dose.

(1263) BNT162b1

(1264) In the younger age group, pain at the injection site was the most frequently reported local reaction within 7 days after Dose 1 of BNT162b1. As dose level increased from 10 ?g to 30 ?g, increasing frequencies of pain at the injection site (58.3% to 100.0%, 7 and 12 participants, respectively) were observed compared to none in the placebo group (FIG. 84). Redness was reported in 2 (16.7%) participants in the 30-?g dose group, and swelling was reported in 3 (25.0%) participants in the 20-?g dose group and 2 (16.7%) participants in the 30-?g dose group. In the 100-?g dose group, pain at the injection site (12 [100.0%] participants), swelling (5 [41.7%] participants), and redness (4 [33.3%] participants) were reported, and 1 [8.3%] participant had severe injection site pain (note: per IRC decision, Dose 2 was later administered to participants at the 10-?g dose level).

(1265) Within 7 days after Dose 2 of BNT162b1 in the younger age group, pain at the injection site remained the most frequently reported local reaction reaching 12 (100.0%) participants with the 30-?g dose group compared to the placebo group (2 [22.2%] participants), while the proportions of participants with redness (2 [16.7%]participants) and swelling (3 [25.0%] participants) were highest in the 30-?g dose group (FIG. 84). No redness or swelling was reported in the placebo group.

(1266) In the older age group, pain at the injection site was the most frequently reported local reaction within 7 days after Dose 1 of BNT162b1 in both the 20-?g and 30-?g dose groups (11 [91.7%] participants each) compared to the placebo group (1 [11.1%] participant) (FIG. 85). No redness was reported, and the maximal frequency of swelling (2 [16.7%] participants) was in the 30-?g group. No redness or swelling was reported in the placebo group.

(1267) Within 7 days after Dose 2 of BNT162b1 in the older age group, pain at the injection site was the most frequently reported local reaction in both the 20-?g and 30-?g dose groups (9 [75.0%] participants each). The frequency of swelling (3 [25.0%] participants) was maximal at 30 ?g, while redness (1 [8.3%] participant each) was reported in the 20-?g and 30-?g dose groups. No redness or swelling was reported in the placebo group.

(1268) After the first and second dose and in both age groups, the majority of local reactions were mild or moderate in severity, and no Grade 4 local reactions were reported.

(1269) Overall, for BNT162b1 recipients and in both age groups, pain at the injection site was the most frequent local reaction (58.3% to 100.0%), and redness (0% to 16.7%) and swelling (0% to 25.0%) occurred at a lower frequency. Notably, the frequency of local reactions was lower in the older age group compared to the younger age group, and there was a trend of a higher frequency of local reactions with increased dose.

(1270) In the younger age group, pain at the injection site had median onset day of Day 1.0 (day of vaccination) after either dose of BNT162b1 across doses 10 ?g to 30 ?g and after Dose 1 of BNT162b1 100 ?g. Median onset day for redness and swelling was between Day 1.0 and Day 3.0 in all dose groups.

(1271) In the older age group, pain at the injection site had median onset day of Day 1.0 (day of vaccination) after Dose 1 of BNT162b1 across all dose groups and after Dose 2 for the 20-?g and 30-?g dose groups (median onset day was on Day 1.5 in the 10-?g dose group after Dose 2). With the exception of redness on Day 4 (20-?g dose group) and Day 5 (30-?g dose group) in 1 participant each after Dose 2, all other local reactions of redness or swelling reported had median onset day between Day 1.0 and Day 3.0 for all dose groups.

(1272) Local reactions resolved with median durations between 1.0 and 4.0 days in the younger age group and older age group across dose levels.

(1273) BNT162b2

(1274) In the younger age group, pain at the injection site was the most frequently reported local reaction within 7 days after Dose 1, which was maximal in the 30-?g dose group (11 [91.7%] participants) (FIG. 86). One [8.3%] participant had severe injection site pain after Dose 1 of 30 ?g. Most participants did not report swelling and redness. After Dose 2, pain at the injection site remained the most frequently reported local reaction (83.3%, 10 participants in each) in the 20-?g and 30-?g dose groups compared to the placebo group (2 [22.2%] participants). No participants reported redness and swelling for any dose group including placebo.

(1275) In the older age group, pain at the injection site was reported within 7 days after Dose 1 of BNT162b2 in all dose groups and was maximal in the 30-?g dose group (75.0%, 9 participants), while no redness and swelling was reported in any group (FIG. 87). Local reactions were not reported in the placebo group. After Dose 2, pain at the injection site (8 [66.7%] participants) was reported in the 30-?g group compared to the placebo group (9 [11.1%] participants); no participants who received BNT162b2 or placebo reported redness and swelling.

(1276) After the first and second dose and in both age groups, the majority of local reactions were mild or moderate in severity, and no Grade 4 local reactions were reported.

(1277) Overall, for BNT162b2 recipients and in both age groups, pain at the injection site was the most frequent local reaction (33.3% to 91.7%), and redness (0% to 8.3%) and swelling (0% to 16.7%) were infrequent. The frequency of local reactions was lower in the older age group compared to the younger age group, and there was a trend of a higher frequency of local reactions with increased dose.

(1278) In the younger age group, median onset day for local reactions occurred between Day 1.0 (day of vaccination) to Day 2.0 after any dose of BNT162b2 across any dose level. In the older age group, median onset day for local reactions occurred between Day 1.0 (day of vaccination) to Day 2.0 after any dose of BNT162b2 across any dose level. Local reactions generally resolved with median durations between 1.0 to 2.0 days in the younger and older age groups across dose levels.

(1279) Systemic EventsPhase 1

(1280) Overall, within 7 days after Dose 1, fatigue was generally the most frequently reported systemic event in the both the younger and older BNT162b1 groups and in the older BNT162b2 group; while headache and fatigue were most frequently reported in the younger BNT162b2 dose group. Overall, within 7 days after Dose 2, headache was the most frequently reported systemic event in the both the younger and older BNT162b1 groups and fatigue was the most frequently reported systemic event in the both the younger and older BNT162b2 groups. Chills was generally reported at a higher frequency after Dose 2 and at a higher frequency in the BNT162b1 group than in the BNT162b2 group. Fever was reported more frequently in the younger BNT162b1 group after Dose 2 than in the older BNT162b2 group. For both the BNT162b1 and the BNT162b2 recipients, after the first and second dose and in both age groups, the majority of systemic events were mild or moderate in severity, and no Grade 4 systemic events were reported.

(1281) BNT162b1

(1282) In the younger age group, fatigue was the most frequently reported systemic event within 7 days after Dose 1 of BNT162b1, reported by 4 (33.3%), 8 (66.7%), and 6 (50.0%) participants in the 10-?g, 20-?g, and 30-?g dose groups, respectively (FIG. 88), compared to the placebo group (2 [22.2%] participants). Headache (6 [50.0%] participants) and chills (7 [58.3%] participants) were reported in the 30-?g dose group, and 51 (8.1%) participant reported fever in each group through 30 ?g. In the placebo group, headache (1 [11.1%]) participant) was reported and none reported fever or chills. In the 100-?g dose group, higher frequencies were reported compared to the 30-?g dose group: fatigue (10 [83.3%] participants), headache (9 [75.0%] participants), chills (10 [83.3%] participants), and fever (6 [50.0%] participants).

(1283) Within 7 days after Dose 2 of BNT162b1 in the younger age group, headache was the most frequently reported systemic event, reported by all 12 (100.0%) participants in the 30-?g dose group compared to none in the placebo group, while fatigue and chills were reported by 10 (83.3% participants) and 8 (66.7%) participants in the 30-?g dose group, respectively. Fever was reported in 17% and 75% of participants in the 20-?g and 30-?g dose groups, respectively. In the placebo group, 2 (22.2%) participants reported fatigue, and none reported fever and chills.

(1284) In the older age group, fatigue was the most frequently reported systemic event within 7 days after Dose 1 of BNT162b1, with 7 (58.3%) and 6 (50.0)% of participants reporting fatigue in the 20-?g and 30-?g dose groups, respectively (FIG. 89), compared to 4 (44.4%) participants in the placebo group. Headache (6 [50.0%] participants) and chills (2 [16.7%] participants) were reported in the 30-?g dose group, and fever (3 [25.0%] participants) was reported only in the 30-?g dose group. In the placebo group, chills (2 [22.2%] participants) was reported and none reported headache or fever. One participant each reported severe muscle pain (20-?g dose group) and severe fatigue (30-?g dose group) (the former was pain related to onset of herpes zoster).

(1285) Within 7 days after Dose 2 of BNT162b1 in the older age group, headache was the most frequent systemic event reported in both the 20-?g and 30-?g dose groups (9 [75.0%] participants each) compared to the placebo group (1 [11.1%] participant). Chills was reported in 7 (58.3%) and 4 (33.3%) participants at the 20-?g and 30-?g dose groups, respectively. Fever was reported in 6 (50.0%) participants in the 20-?g dose group and in 4 (33.3%) participants in the 30-?g dose group, with 1 participant reporting fever >38.9? C. to 40.0? C. In the placebo group, fatigue (2 [22.2%] participants) was reported and none reported fever and chills.

(1286) After the first and second dose and in both age groups, the majority of systemic events were mild or moderate in severity, and no Grade 4 systemic events were reported. In the older age group, prompted systemic events after each dose were milder and less frequent than those observed in the younger age group.

(1287) Systemic events had the highest frequency and/or severity with the 100-?g dose group after Dose 1. Use of antipyretic/pain medication also increased with increasing dose level and number of doses in both age groups. For these reasons, the IRC decided that the younger age group participants should not receive a second dose of 100 ?g of BNT162b1.

(1288) In the younger age group, median onset day for most systemic events after either dose of BNT162b1 across doses 10 ?g to 30 ?g and after Dose 1 of BNT162b1 100 ?g was between Day 1.0 and Day 2.0. Most systemic events generally resolved with median durations between 1.0 to 2.0 days. For fatigue, median duration after Dose 1 was 4.0 days in the 10-?g dose group compared with 2.0 days in the 30-?g dose group. In the older age group, median onset day for most systemic events after either dose of BNT162b1, and across any dose group, was between Day 1.0 and Day 3.5. Most systemic events generally resolved with median durations between 1.0 to 3.0 days.

(1289) BNT162b2

(1290) In the younger age group, headache (4 [33.3%] to 6 [50.0%] participants) and fatigue (3 [25.0%] to 5 [41.7%] participants) were the most frequently reported systemic events within 7 days after Dose 1 of BNT162b2 compared to the placebo group (3 [33.3%] participants each) (FIG. 90). Fever (2 [16.7%] participants) and chills (4 [33.3%] participants) were reported only in the 30-?g dose group. One participant in the 30-?g group with a prior history of migraine reported a severe migraine headache on Day 7 after Dose 1.

(1291) Within 7 days after Dose 2 of BNT162b2 in the younger age group, fatigue was the most frequently reported systemic event in the 20-?g and 30-?g dose groups (7 [58.3%] and 9 [75.0%] participants, respectively) compared to the placebo group (5 [55.6%] participants). Headache (8 [66.7%] participants), chills (7 [58.3%] participants), and muscle pain (7 [58.3%] participants), and fever (2 [16.7%] participants) were reported in the 30-?g dose group. Of these events, fatigue (5 [55.6%] participants), headache (1 [11.1%] participant), and chills (1 [11.1%] participant) were reported in the placebo group, and none were reported for muscle pain.

(1292) In the older age group, the most frequently reported systemic event within 7 days after Dose 1 of BNT162b2 was fatigue in the 20-?g and at 30-?g dose groups (4 [33.3%] and 3 [25.0%] participants, respectively) compared to the placebo group (2 [22.2%] participants) (FIG. 91). Headache (3 [25.0%] participants), chills (2 [16.7%] participants), and muscle pain (1 [8.3%] participant) were maximal in the 20-?g dose group. Of these events, only headache (1 [11.1%] participant) and muscle pain (2 [22.2%] participants) were reported in the placebo group. Fever was not reported. Within 7 days after Dose 2 of BNT162b2 in the older age group, fatigue remained the most frequent systemic event in the 20-?g and 30-?g dose groups (6 [50.0%] and 5 [41.7%] participants, respectively), compared to the placebo group (1 [11.1%] participant). Headache was reported in the 20-?g and 30-?g dose groups (4 [33.3%] and 3 [25.0%] participants, respectively), while muscle pain and chills were reported in the 30-?g dose group (3 [25.0%] and 2 [16.7%] participants, respectively). Fever (1 [8.3%] participant) was reported in the 30-?g dose group. Of these events, headache and muscle pain were reported in the placebo group (1 [11.1%] participant each).

(1293) After the first and second dose and in both age groups, the majority of systemic events were mild or moderate in severity, and no Grade 4 systemic events were reported. In the younger age group, median onset day for most systemic events after either dose of BNT162b2, and across any dose group, was between Day 1.0 and Day 4.0. Most systemic events generally resolved with median durations between 1.0 to 2.5 days. In the older age group, median onset day for any systemic event after either dose of BNT162b2, and across any dose level, was between Day 1.5 and Day 2.0, except for systemic events in the 10-?g dose group after Dose 1, which had a median onset day of Day 5.5. Most systemic events generally resolved with median durations between 1.0 to 3.0 days.

(1294) Adverse EventsPhase 1

(1295) Summary of Adverse EventsPhase 1

(1296) All AEs from Dose 1 through the data cutoff date of 24 Aug. 2020 were included in the summary for all dose levels for each vaccine candidate and age group other than BNT162b1 100-?g group for which AEs from Dose 1 to before Dose 2 were summarized.

(1297) Overall, fewer participants reported at least 1 AE after Dose 1 in the older BNT162b2 group (8.3% to 25.0%) compared to the younger (41.7% to 50.0%) and older (25.0% to 58.3%) BNT162b1 groups and the younger BNT162b2 group (33.3% to 41.7%).

(1298) BNT162b1

(1299) In the younger age group, 5 (41.7%) to 6 (50%) participants reported at least 1 AE after Dose 1 of BNT162b1 up to 30 ?g, compared to 2 (22.2%) participants in the placebo group. Related AEs increased with increasing BNT162b1 dose level (25.0% to 50.0%); six (50%) participants reported at least 1 related AE in the 30-?g dose group. One (8.3%) participant reported a severe AE (pyrexia) in the 30-?g dose group.

(1300) In the 100-?g dose group, 8 (66.7%) participants reported at least 1 AE after Dose 1 to before Dose 2 of BNT162b1, compared to 1 (33.3%) participant in the placebo group. Six (50.0%) participants had at least 1 related AE, and 1 (8.3%) participant reported a severe AE (sleep disorder).

(1301) In the older group, 3 (25.0%) participants (30-?g dose group) and 7 (58.3%) participants each (10-?g and 20-?g dose groups) reported at least 1 AE after Dose 1 of BNT162b1, compared to 4 (44.4%) participants in the placebo group. Two (16.7%) to 4 (33.3%) participants reported at least 1 related AE, with the highest frequency in the 20-?g dose group. One participant each reported a severe AE in the 20-?g (herpes zoster) and 30-?g (fatigue) dose groups.

(1302) No SAEs, AEs leading to withdrawals, or deaths were reported in either age group.

(1303) BNT162b2

(1304) In the younger age group, 4 (33.3%) to 5 (41.7%) participants reported at least 1 AE after Dose 1 of BNT162b2, compared to 2 (22.2%) participants in the placebo group. Two (16.7%) to 4 (33.3%) participants reported at least 1 related AE, with the highest frequency in the 20-?g dose group. One participant reported a severe AE (migraine) in the 30-?g dose group.

(1305) In the older group, 1 (8.3%) to 3 (25.0%) participants reported at least 1 AE after Dose 1 of BNT162b2, compared to 2 (22.2%) participants in the placebo group. Only 1 (8.3%) participant reported at least 1 related AE (20-?g dose group). One participant each reported a severe AE in the 30-?g dose group (muscle spasms) and placebo group (radiculopathy).

(1306) No SAEs, AEs leading to withdrawals, or deaths were reported in either age group.

(1307) Analysis of Adverse EventsPhase 1

(1308) Adverse Events by System Organ Class and Preferred TermPhase 1

(1309) AE by SOC and PT summaries in this section included AEs from Dose 1 to 1 month after Dose 2 for all groups other than BNT162b1 100-ug group for which AEs from Dose 1 to 3 weeks after Dose 1 or from Dose 1 to before Dose 2 were summarized.

(1310) General disorders and administration site conditions was the most commonly reported SOC in the older BNT162b1 group and the younger BNT162b2 group. The most commonly reported SOC was gastrointestinal disorders in the younger BNT162b1 group and nervous system disorders in the older BNT162b2 group. Generally, most PTs were reported by ?2 participants per dose group.

(1311) BNT162b1

(1312) In the younger age group, from Dose 1 to 1 month after Dose 2 of BNT162b1, gastrointestinal disorders was the most commonly reported SOC (2 [16.7%] participants each dose group) in the BNT162b1 groups up to 30 ?g. In the 20-?g dose group only, paraesthesia (3 [25.0%]) was the most common AE by PT. All other AEs were reported by ?2 participants perdose group, including those in the placebo group. In the 100-?g dose group, from Dose 1 to 3 weeks after Dose 1 of BNT162b1, psychiatric disorders was the most commonly reported SOC (3 [25.0%] participants), and sleep disorder (3 [25%] participants) was the most common AE by PT. All other AEs were reported by ?2 participants, including those in the placebo group.

(1313) In the older age group, from Dose 1 to 1 month after Dose 2 of BNT162b1, general disorders and administration site conditions was the most commonly reported SOC in the BNT162b1 groups, reported in a total of 6 participants: 1 (8.3%) participant in the 10-?g dose group, 2 (16.7%) participants in the 20-?g dose group, and 3 (25.0%) participants in the 30-?g dose group. Any AEs by PT were reported by no more than 1 participant per dose group.

(1314) BNT162b2

(1315) In the younger age group, general disorders and administration site conditions was the most commonly reported SOC. These events included injection site pain and injection site erythema. Any AEs by PT were reported by no more than 1 participant per dose group.

(1316) In the older age group, nervous system disorders was the most commonly reported SOC, reported in 1 participant each in the 30-?g group (sciatica) and the placebo group (radiculopathy). Any AEs by PT were reported by no more than 1 participant per dose group.

(1317) Related Adverse EventsPhase 1

(1318) Overall, general disorders and administration site conditions was the most commonly reported SOC for the younger and older BNT162b1 groups and the younger BNT162b2 group. In the older BNT162b2 group, nausea, reported in 1 (8.3%) participant, was the only related AE.

(1319) BNT162b1

(1320) In the younger age group, general disorders and administration site conditions was the most commonly reported SOC (injection site pain, pyrexia, chills, fatigue, and injection site swelling). Two (16.7%) participants each in the 30-?g dose group reported related AEs of tachycardia and pyrexia. All other related AEs were reported by ?2 participants per dose group.

(1321) In the 100-?g BNT162b1 group, psychiatric disorders were the most commonly reported SOC. Three (25.0%) participants reported sleep disorder as their psychiatric disorder. All other related AEs were reported by ?2 participants each.

(1322) In the older age group, general disorders and administration site conditions was the most commonly reported SOC (fatigue, injection site bruising, injection site pain, and peripheral swelling). Any related AEs by PT were reported by no more than 1 participant per dose group.

(1323) BNT162b2

(1324) In the younger age group, general disorders and administration site conditions was the most commonly reported SOC (injection site pain and injection site erythema). Any related AEs by PT were reported by no more than 1 participant per dose group, including those in the placebo group.

(1325) In the older age group, only 1 (8.3%) participant reported a related AE of nausea in the 20-?g dose group.

(1326) Immediate Adverse EventsPhase 1

(1327) BNT162b1

(1328) In the younger age group, 1 participant reported an immediate AE of paraesthesia after Dose 1 of 20 ?g BNT162b1. In the 100-?g group, no participants reported an immediate AE after Dose 1.

(1329) In the older age group, 1 participant reported an immediate AE of eye paraesthesia after Dose 1 of 10 ?g BNT162b1.

(1330) There were no participants in either age group who reported any immediate AEs after Dose 2 of BNT162b1.

(1331) BNT162b2

(1332) In the younger age group, after Dose 1 of BNT162b2, there were 3 participants who reported an immediate AE: injection site erythema (10-?g dose group), ageusia (20-?g dose group), and injection site pain (30-?g dose group). After Dose 2 of BNT162b2, there was 1 participant who reported an immediate AE of taste disorder (20-?g dose group).

(1333) There were no participants in the older age group who reported any immediate AE after any dose of BNT162b2.

(1334) Severe Adverse EventsPhase 1

(1335) BNT162b1

(1336) In the younger age group, there was 1 participant who reported a severe AE of pyrexia (102.4? F.) 2 days after Dose 2 (30-?g dose group) and 1 participant who reported a severe AE of sleep disorder 1 day after Dose 1 (100-?g dose group). Both AEs were determined by the investigator to be related to study intervention.

(1337) In the older age group, 2 participants reported a severe AE: herpes zoster which occurred 2 days after Dose 1 (20-?g dose group, considered unrelated to BNT162b1) and fatigue 1 day after Dose 2 (30-?g dose group, considered related to BNT162b1).

(1338) BNT162b2

(1339) In the younger age group, 1 participant with a history of migraines reported a severe migraine 7 days after Dose 1 (30-?g dose group, considered unrelated). In the older age group, 2 participants reported a severe AE: muscle spasms 2 days after Dose 2 (30-?g dose group, considered unrelated to BNT162b2) and radiculopathy 3 days after Dose 1 (placebo), considered unrelated to study intervention.

(1340) Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals, and Other Significant Adverse EventsPhase 1

(1341) DeathsPhase 1

(1342) There were no Phase 1 participants who died through the data cutoff date of 24 Aug. 2020 in this interim CSR.

(1343) Serious Adverse EventsPhase 1

(1344) There were no Phase 1 participants who reported any SAEs during the period covered in this interim CSR.

(1345) Safety-Related Participant WithdrawalsPhase 1

(1346) There were no Phase 1 participants with any AEs leading to withdrawal from the study through the data cutoff date of 24 Aug. 2020 in this interim CSR.

(1347) Other Significant Adverse EventsPhase 1

(1348) AEs of special interest were not defined for Phase 1 of this study.

(1349) Other Safety AssessmentsPhase 1

(1350) Severe COVID-19 IllnessPhase 1

(1351) There were no COVID-19 cases reported in the Phase 1 participants through the data cutoff date of 24 Aug. 2020.

(1352) PregnancyPhase 1

(1353) Pregnancy was not reported in any Phase 1 participants through the data cutoff date of 24 Aug. 2020.

(1354) Analysis and Discussion of Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals, and Other Significant Adverse EventsPhase 1

(1355) During the period covered in this interim CSR, there were no SAEs, AEs leading to withdrawals, or deaths reported in either age group.

(1356) Clinical Laboratory EvaluationPhase 1

(1357) Overall, 1 to 3 days after Dose 1, there were transient decreases in lymphocytes (<0.8?LLN), which returned to normal by 6 to 8 days after Dose 1, in the younger and older BNT162b1 and BNT162b2 groups. Most shifts were from normal or Grade 1 to Grade 1, 2, or 3 decrease in lymphocyte counts, which returned to normal by 6 to 8 days after Dose 1, and were observed in all age and dose groups. Shifts from normal to Grade 1 (younger BNT162b1 group) or Grade 2 (older BNT162b2 group) neutrophil decrease were also observed but were infrequent.

(1358) Overall, other clinical chemistry abnormalities reported or shifts of laboratory results were infrequent. The incidence of decreased lymphocyte counts was lower for BNT162b2 recipients compared with BNT162b1 recipients. None of the laboratory abnormalities were associated with clinical findings.

(1359) BNT162b1

(1360) In the younger age group, laboratory abnormalities of transient decreases in lymphocytes (<0.8?LLN) were observed in 1 (8.3%), 4 (33.3%), and 6 (54.5%) of participants 1 to 3 days after Dose 1 of BNT162b1 10 ?g, 20 ?g, or 30 ?g, respectively, which returned to normal by 6 to 8 days after Dose 1. A shift from normal to Grade 3 decrease in lymphocyte counts was observed in 1 participant each in the 10-?g and 30-?g dose groups and 2 (16.7%) participants the 20-?g dose group. No Grade 3 decrease in lymphocyte counts was observed by 6 to 8 days after Dose 1. After Dose 1, a shift from normal to Grade 2 neutrophil decrease was observed in 1 (11.1%) participant in the placebo group, which was not observed by 19 to 23 days after Dose 1. At 6 to 8 days after Dose 2, a shift in neutrophil decrease was observed in 1 participant each in the 10-?g dose group (Grade 1 to Grade 2) and in the 30-?g dose group (normal to Grade 2). Both participants had a shift to Grade 1 at the unplanned visit approximately 1 month after Dose 2.

(1361) In the 100-?g BNT162b1 group, laboratory abnormalities of transient decreases in lymphocytes (<0.8?LLN) were observed in 9 (75.0%) participants 1 to 3 days after Dose 1, which returned to normal by 6 to 8 days after Dose 1. A shift from normal to Grade 3 decrease in lymphocyte counts was observed in 4 (33.3%) participants 1 to 3 days after Dose 1, which returned to normal by 6 to 8 days after Dose 1. A shift from normal to Grade 1 neutrophil decrease was observed in 3 (25.0%) participants at 6 to 8 days after Dose 1, which returned to normal by 19 to 23 days after Dose 1.

(1362) In the older age group, laboratory abnormalities of transient decreases in lymphocytes (<0.8?LLN) were also observed in 1 (8.3%), 3 (25.0%), and 2 (16.7%) participants 1 to 3 days after Dose 1 of BNT162b1 10 ?g, 20 ?g, or 30 ?g, respectively, which returned to normal by 6 to 8 days after Dose 1. At 1 to 3 days after Dose 1 of BNT162b1, shifts from normal to Grade 3 or Grade 4 decrease in lymphocyte counts were observed in 1 (8.3%) participant each in the 30-?g and 10-?g dose groups, respectively, and both returned to normal by 6 to 8 days after Dose 1.

(1363) Overall, other clinical chemistry abnormalities reported or shifts of laboratory results were infrequent. None of the abnormalities were associated with clinical findings.

(1364) BNT162b2

(1365) In the younger age group, laboratory abnormalities of transient decreases in lymphocytes (<0.8?LLN) were observed in 1 (8.3%) participant each 1 to 3 days after Dose 1 of BNT162b2 in the 20-?g and 30-?g dose groups, which returned to normal by 6 to 8 days after Dose 1. At 1 to 3 days after Dose 1 of BNT162b2, shifts from normal to Grade 1 decrease in lymphocyte counts were observed in 3 (25.0%), 2 (16.7%), and 4 (33.3%) participants in the 10-?g, 20-?g, and 30-?g dose groups, respectively, and shifts from normal to Grade 2 decrease in lymphocyte counts were observed in 1 (8.3%) participant each in the 20-?g and 30-?g dose groups. By 6 to 8 days after Dose 1, no Grade 2 or Grade 3 decrease in lymphocyte counts were observed.

(1366) In the older age group, laboratory abnormalities of transient decreases in lymphocytes (<0.8?LLN) were also observed in 1 (8.3%) participant each 1 to 3 days after Dose 1 of BNT162b2 across all dose levels, which returned to normal by 6 to 8 days after Dose 1. A shift from normal to Grade 3 (10-?g dose group) and a Grade 1 to Grade 3 (30-?g dose group) decrease in lymphocyte counts was observed in 1 (8.3%) participant each after Dose 1. A shift from normal to Grade 2 neutrophil decrease was observed in 2 (16.7%) participants in the 20-?g dose group at 1 to 3 days after Dose 1, and no shifts to Grade 2 were observed by 6 to 8 days after Dose 1. A shift from normal to Grade 2 neutrophil decrease was observed in 1 (8.3%) participant in the 10-?g dose group at 6 to 8 days after Dose 1. By 19 to 23 days after Dose 1, no shifts to Grade 2 neutrophil decrease were observed for any dose group.

(1367) Overall, other clinical chemistry abnormalities reported or shifts of laboratory results were infrequent. The incidence of decreased lymphocyte counts was lower for BNT162b2 recipients compared with BNT162b1 recipients. None of the laboratory abnormalities were associated with clinical findings.

(1368) Physical Examination FindingsPhase 1

(1369) Overall, there were fewer abnormalities noted during physical examinations after BNT162b2 than after BNT162b1 in both age groups. Abnormalities were generally observed 1 to 3 days after Dose 1 and most were of the extremities, musculoskeletal system, or skin.

(1370) BNT162b1

(1371) In the younger age group, there were no abnormalities noted during baseline physical examinations. Overall, after randomization, most abnormalities were observed 1 to 3 days after Dose 1 of 10 ?g, 20 ?g, or 30 ?g BNT162b1 (9 [20.0%] participants) and 6 to 8 days after Dose 2 (7 [15.6%] participants). In the 30-?g dose group, a maximum of 6 (50.0%) participants had abnormalities 1 to 3 days after Dose 1, and most abnormalities were of the extremities.

(1372) In the 100-?g dose group, only 1 (8.3%) participant had an abnormality at baseline. From Dose 1 to 3 weeks after Dose 1, 9 (75.0%) participants had abnormalities 1 to 3 days after BNT162b1, and most abnormalities were of the extremities.

(1373) In the older age group, there were 5 (11.1%) participants with abnormalities noted during baseline physical examinations, with ?2 participants in any dose group. Overall, after randomization, most abnormalities were observed 1 to 3 days after Dose 1 of BNT162b1 (15 [33.3%] participants). In the 20-?g and 30-?g dose groups, 6 (50.0%) and 4 (33.3%) participants had abnormalities 1 to 3 days after Dose 1, and most abnormalities involved either the musculoskeletal system or extremities.

(1374) There were no clinically important findings from physical examinations.

(1375) BNT162b2

(1376) In the younger age group, there were 5 (11.1%) participants with abnormalities noted during baseline physical examinations, with ?2 participants in any dose group. Overall, after randomization, most abnormalities were observed 1 to 3 days after Dose 1 of 10 ?g, 20 ?g, or 30 ?g BNT162b2 (5 [11.1%] participants) and 6 to 8 days after Dose 2 (4 [8.9%] participants), with most being abnormalities of the extremities or skin.

(1377) In the older age group, there was 1 (8.3%) participant in the 30-?g dose group with an abnormality noted during the baseline physical examination. After randomization, ?2 participants in any dose group overall had an abnormality in physical examination during any visit window.

(1378) There were no clinically important findings from physical examinations at baseline.

(1379) Phase 1 Summary of Safety Results Evaluating BNT162b1 and BNT162b2

(1380) Overall, reactogenicity events were well tolerated and short-lived (median durations 1.0 to 4.0 days). All participants returned to receive their second dose. All AEs as a result of reactogenicity events resolved without sequelae.

(1381) For local reactions in both age groups, pain at the injection site (58.3% to 100.0%), redness (0% to 16.7%), and swelling (0% to 25.0%) were reported for BNT162b1 recipients, which were more frequent than for BNT162b2 recipients: pain at the injection site (33.3% to 91.7%), redness (0% to 8.3%), and swelling (0% to 16.7%). In general, frequencies of local reactions were observed to be higher with increased dose level.

(1382) The frequency of local reactions was lower in the older age group compared to the younger age group. The frequency of pain at the injection site, the most frequently reported local reaction, was lower in the older age groups after 30 ?g BNT162b1 (91.7% and 75.0%) and after 30 ?g of BNT162b2 (75.0% and 66.7% for Dose 1 and Dose 2, respectively), compared to the younger age groups after 30 ?g of BNT162b1 (100.0% for both Dose 1 and Dose 2) and 30 ?g of BNT162b2 (91.7% and 83.3% for Dose 1 and Dose 2, respectively).

(1383) BNT162b2 recipients in the older age group reported lower frequencies of local reactions compared with BNT162b1 recipients in the older age group. In the older 30-?g BNT162b2 group, pain at the injection site was lower after Dose 1 (75.0%) and Dose 2 (66.7%) than in the older 30-?g BNT162b1 group after Dose 1 (91.7%) and Dose 2 (75.0%).

(1384) Common systemic events in both age groups after either Dose 1 or Dose 2 included fatigue (16.7% to 83.3%), headache (25.0% to 100%), chills (8.3% to 66.7%), fever (0% to 75.0%), and muscle pain (8.3% to 75.0%) for BNT162b1 recipients up to 30 ?g, which were more frequent than BNT162b2 recipients up to 30 ag: fatigue (8.3% to 75.0%), headache (0% to 66.7%), chills (0% to 58.3%), fever (0% to 16.7%), and muscle pain (0% to 58.3%). In general, frequencies of systemic events were observed to be higher with increased dose level.

(1385) The frequency of systemic events was lower in the older age group compared to the younger age group. The frequency of fatigue was lower in the older age groups after 30 ?g of BNT162b1 (50.0% and 66.7%) and after 30 ?g of BNT162b2 (25.0% and 41.7% for Dose 1 and Dose 2, respectively), compared to the younger age groups after 30 ?g of BNT162b1 (50.0% and 83.3%) and after 30 ?g of BNT162b2 (41.7% and 75.0%) for Dose 1 and Dose 2, respectively.

(1386) BNT162b2 recipients in the older age group reported lower frequencies of systemic events compared with BNT162b1 recipients in the older age group. The frequency of fatigue was lower in the older 30-?g BNT162b2 group (25.0% and 41.7% for Dose 1 and Dose 2, respectively) than in the older 30-?g BNT162b1 group (50.0% and 66.7% for Dose 1 and Dose 2, respectively).

(1387) Most AEs were mild or moderate in severity. Most related AEs were similar to the solicited reactogenicity events reported in the e-diary. Few severe AE were reported but were considered not related to study intervention.

(1388) There were no SAEs, deaths, or discontinuations because of AEs.

(1389) Transient decrease in lymphocytes were observed in all age and dose groups 1 to 3 days after Dose 1, which resolved by 6 to 8 days after Dose 1.

(1390) There were no clinically important findings from physical examinations.

(1391) BNT162b2 demonstrated a favorable reactogenicity and safety profile compared with BNT162b1, contributing to the selection of BNT162b2 for Phase 2/3 development.

(1392) Phase 1 Safety Conclusions

(1393) All doses tested for BNT162b1 and BNT162b2 (10 ?g, 20 ?g, and 30 ?g) were safe and well tolerated except for BNT162b1 at 100 ?g, which was discontinued after the first dose due to the reactogenicity profile.

(1394) Reactogenicity was generally higher after Dose 2 than Dose 1.

(1395) The frequency of local and systemic reactogenicity was generally lower for BNT162b2 compared to BNT162b1 especially after the second dose.

(1396) Reactogenicity events after each dose for both BNT162b1 and BNT162b2 in older adults were milder and less frequent than those observed in younger adults. The majority of reactogenicity events were mild or moderate in severity.

(1397) Most AEs were mild or moderate. There were no SAEs or discontinuations because of AEs.

(1398) Overall, fewer AEs were experienced by participants who received BNT162b2 compared with those who received BNT162b1, with the least number of participants experiencing AEs in the BNT162b2 older age group. Few severe AEs in the older age group after BNT162b2 were observed, and all were considered unrelated to study intervention.

(1399) Clinical laboratory evaluations showed a transient decrease in lymphocytes that was observed in all age and dose groups after Dose 1, which resolved within a few days, were not associated with any other clinical sequelae, and were not considered clinically relevant.

(1400) BNT162b2 at 30 ?g was selected to proceed into the Phase 2/3 portion of the study because this dose and construct provided the optimum combination of a favorable reactogenicity profile and a robust immune response.

(1401) Phase 2

(1402) Safety data are available up to the data cutoff date (2 Sep. 2020) and are summarized up to the data cutoff date for the 360 participants in Phase 2. All participants in Phase 2 used an e-diary for reporting local reactions and systemic events.

(1403) Local ReactionsPhase 2

(1404) After the first and second dose of BNT162b2 and in both age groups, the majority of local reactions were mild or moderate in severity, and no Grade 4 (potentially life-threatening) local reactions were reported.

(1405) In the BNT162b2 group, pain at the injection site was reported more frequently in the younger age group (FIG. 92) than in the older age group (FIG. 93), and frequency was similar after Dose 1 compared with Dose 2 of BNT162b2 in the younger age group (85.2% vs. 80.2%, respectively) and in the older age group (70.7% vs. 72.5%, respectively). In the placebo group, pain at the injection site was reported at similar frequencies (7.8% to 10.2%) in the younger and older age groups after Dose 1 and Dose 2.

(1406) In the BNT162b2 group, redness and swelling were similar in the younger and older age group after Dose 1. After Dose 2, the frequency of redness and swelling was slightly higher in the older age group (7.7% and 12.1%, respectively) than in the younger age group (3.5% and 3.5%, respectively). In the placebo group, only 1 participant in the older age group reported redness after Dose 1, and no swelling was reported.

(1407) One participant in the BNT162b2 group (older age group) reported severe injection site pain after Dose 1, and 1 participant in the younger age group reported severe injection site pain after Dose 2. One participant in the BNT162b2 group (older age group) reported severe redness after Dose 2.

(1408) Overall, across age groups, pain at the injection site was the most frequent local reaction and did not increase after Dose 2, and redness and swelling were generally similar in frequency after Dose 1 and Dose 2.

(1409) Across age groups, local reactions for the BNT162b2 group after either dose had a median onset day between Day 1.0 and Day 3.0 (Day 1.0 was the day of vaccination), and ranges were generally similar in the younger and older age groups. Across age groups, after either dose of BNT162b2, local reactions resolved after a median duration of 1.0 to 3.0 days, which was generally similar in the younger and older age groups.

(1410) Systemic EventsPhase 2

(1411) In the BNT162b2 group, systemic events were generally reported more frequently and were of higher severity in the younger group (FIG. 94) compared with the older group (FIG. 95), with frequencies and severity increasing with number of doses (Dose 1 vs Dose 2). Vomiting and diarrhea were exceptions with vomiting infrequent and similar in both age groups and vomiting and diarrhea similar after each dose. Frequencies of systemic events in the younger and older BNT162b2 groups (Dose 1 vs Dose 2) are listed below: fatigue: younger group (50.0% vs 59.3%) compared to older group (35.9% vs 52.7%) headache: younger group (31.8% vs 51.2%) compared to older group (27.2% vs 36.3%) muscle pain: younger group (23.9% vs 45.3%) compared to older group (14.1% vs 28.6%) chills: younger group (9.1% vs 40.7%) compared to older group (7.6% vs 20.9%) joint pain: younger group (9.1% vs 17.4%) compared to older group (4.3% vs 16.5%) fever: younger group (3.4% vs 17.4%) compared to older group (0.0% vs 11.0%). vomiting: similar in both age groups and after either dose. diarrhea: reported less frequently in the older group and was similar after each dose. Systemic events were generally reported less frequently in the placebo group than in the BNT162b2 group, for both age groups and doses, with some exceptions. In the younger age group, fever, headache, chills, vomiting, and diarrhea after Dose 1, and vomiting after Dose 2 were reported at similar frequencies in both the placebo and BNT162b2 groups (FIG. 94). In the older age group, vomiting, diarrhea, muscle pain, and joint pain after Dose 1, and vomiting and diarrhea after Dose 2 were reported at similar frequencies in the placebo and BNT162b2 groups (FIG. 95).

(1412) Use of antipyretic/pain medication was slightly less frequent in the older age group after both doses but increased in both age groups overall after Dose 2 as compared with after Dose 1. Use of antipyretic/pain medication was less frequent in the placebo group than in the BNT162b2 group.

(1413) After the first and second dose and in both age groups, the majority of systemic events were mild or moderate in severity, and no Grade 4 (potentially life-threatening) systemic events were reported. Across age groups, severe systemic events were only reported after Dose 2 of BNT162b2 overall and included fever (1.1%), fatigue (4.0%), headache (2.8%), chills (2.3%), and muscle pain (1.7%).

(1414) Across age groups, systemic events after both doses of BNT162b2 had a median onset day between Day 2.0 to Day 3.0 (Day 1.0 was the day of vaccination), and ranges were similar in the younger and older age groups. Across age groups, systemic events for this group after either dose resolved with a median duration of 1 day, which was similar in the younger and older age groups. There was no clear difference in the durations of systemic events that occurred after Dose 1 compared to those that occurred after Dose 2.

(1415) Adverse EventsPhase 2

(1416) Summary of Adverse EventsPhase 2

(1417) The number of participants who reported at least 1 AE was similar in the BNT162b2 group compared with the placebo group, which was generally similar in the 2 vaccine groups in the younger and older age groups (Table 10 and Table 11, respectively).

(1418) Two severe events were reported for 2 participants in the BNT162b2 younger age group: myalgia (AE) and gastric adenocarcinoma (SAE) The SAE of gastric adenocarcinoma occurred 23 days after receiving Dose 1. Both events were assessed by the investigator as not related to study intervention.

(1419) From 7 days after Dose 2 to the data cutoff date (2 Sep. 2020), no additional participants reported any AE.

(1420) TABLE-US-00028 TABLE 10 Number (%) of Subjects Reporting at Least 1 Adverse Event From Dose 1 to 7 Days After Dose 2, by Age Group - Phase 2 - Safety Population Age Group: 18-55 Years Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo (N.sup.a = 88) (N.sup.a = 90) Adverse Event n.sup.b (%) n.sup.b (%) Any event 8 (9.1) 10 (11.1) Related.sup.c 3 (3.4) 6 (6.7) Severe 2 (2.3) 0 Life-threatening 0 0 Any serious adverse event 1 (1.1) 0 Related.sup.c 0 0 Severe 1 (1.1) 0 Life-threatening 0 0 Any adverse event leading 1 (1.1) 0 to withdrawal Related.sup.c 0 0 Severe 1 (1.1) 0 Life-threatening 0 0 Death 0 0 .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified adverse event category. For any event, n = the number of subjects reporting at least 1 occurrence of any adverse event. .sup.cAssessed by the investigator as related to investigational product.

(1421) TABLE-US-00029 TABLE 11 Number (%) of Subjects Reporting at Least 1 Adverse Event From Dose 1 to 7 Days After Dose 2, by Age Group - Phase 2 - Safety Population Age Group: 56-85 Years Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo (N.sup.a = 92) (N.sup.a = 90) Adverse Event n.sup.b (%) n.sup.b (%) Any event 4 (4.3) 8 (8.9) Related.sup.c 2 (2.2) 2 (2.2) Severe 0 0 Life-threatening 0 0 Any serious adverse event 0 0 Related.sup.c 0 0 Severe 0 0 Life-threatening 0 0 Any adverse event leading 0 0 to withdrawal Related.sup.c 0 0 Severe 0 0 Life-threatening 0 0 Death 0 0 .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified adverse event category. For any event, n = the number of subjects reporting at least 1 occurrence of any adverse event. .sup.cAssessed by the investigator as related to investigational product.

(1422) Analysis of Adverse EventsPhase 2

(1423) Adverse Events by System Organ Class and Preferred TermPhase 2

(1424) Table 12 presents the number of participants who reported at least 1 AE from Dose 1 to 7 days after Dose 2 by SOC and PT.

(1425) The number of participants who reported at least 1 AE was similar in the BNT162b2 group compared to the placebo group from Dose 1 to 7 days after Dose 2.

(1426) In the younger age group, 8 (9.1%) and 10 (11.1%) participants reported at least 1 AE in the BNT162b2 group and the placebo group, respectively. In the older age group, 4 (4.3%) and 8 (8.9%) participants reported at least 1 AE in the BNT162b2 group and the placebo group, respectively.

(1427) Overall, most AEs reported up to 7 days after Dose 2 were in the SOCs of gastrointestinal disorders (3 [1.7%] in the BNT162b2 group and 2 [1.1%] in the placebo group), general disorders and administration site conditions (3 [1.7%] in the BNT162b2 group and 7 [3.9%] in the placebo group), and musculoskeletal and connective tissue disorders (3 [1.7%] in the BNT162b2 group and 1 [0.6%] in the placebo group). The most frequently reported AE by PT was injection site pain (3 [3.4%]) in the younger BNT162b2 group, which all occurred on the day of vaccination with Dose 1 during the reporting period for local reactions. Two events resolved within 3 days, and 1 event resolved 11 days later. All other AEs by PT were reported in 52 participants in each vaccine group.

(1428) One participant in the older BNT162b2 group had an AE of contusion in the upper left arm deltoid region, which was assessed by the investigator as related to study intervention.

(1429) TABLE-US-00030 TABLE 12 Number (%) of Subjects Reporting at Least 1 Adverse Event From Dose 1 to 7 Days After Dose 2, by System Organ Class and Preferred Term - Phase 2 - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo 18-55 Years 56-85 Years 18-85 Years 18-85 Years System Organ Class (N.sup.a = 88) (N.sup.a = 92) (N.sup.a = 180) (N.sup.a = 180) Preferred Term n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) Any event 8 (9.1) (4.0, 17.1) 4 (4.3) (1.2, 10.8) 12 (6.7) (3.5, 11.4) 18 (10.0) (6.0, 15.3) BLOOD AND LYMPHATIC 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) SYSTEM DISORDERS Lymphadenopathy 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) GASTROINTESTINAL 1 (1.1) (0.0, 6.2) 2 (2.2) (0.3. 7.6) 3 (1.7) (0.3, 4.8) 2 (1.1) (0.1, 4.0) DISORDERS Diarrhoea 1 (1.1) (0.0, 6.2) 1 (1.1) (0.0, 5.9) 2 (1.1) (0.1, 4.0) 1 (0.6) (0.0, 3.1) Odynophagia 0 (0.0, 4.1) 1 (1.1) (0.0, 5.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) Tongue discomfort 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) GENERAL DISORDERS 3 (3.4) (0.7, 9.6) 0 (0.0, 3.9) 3 (1.7) (0.3, 4.8) 7 (3.9) (1.6, 7.8) AND ADMINISTRATION SITE CONDITIONS Injection site erythema 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 2 (1.1) (0.1, 4.0) Injection site pain 3 (3.4) (0.7, 9.6) 0 (0.0, 3.9) 3 (1.7) (0.3, 4.8) 0 (0.0, 2.0) Fatigue 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 2 (1.1) (0.1, 4.0) Chills 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Injection site discolouration 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Injection site swelling 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) INFECTIONS AND 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) INFESTATIONS Vulvovaginal mycotic 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) infection INJURY, POISONING 0 (0.0, 4.1) 1 (1.1) (0.0, 5.9) 1 (0.6) (0.0, 3.1) 3 (1.7) (0.3, 4.8) AND PROCEDURAL COMPLICATIONS Contusion 0 (0.0, 4.1) 1 (1.1) (0.0, 5.9) 1 (0.6) (0.0, 3.1) 1 (0.6) (0.0, 3.1) Fall 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Muscle rupture 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Tendon rupture 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) INVESTIGATIONS 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) White blood cell count 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) increased MUSCULOSKELETAL AND 2 (2.3) (0.3, 8.0) 1 (1.1) (0.0, 5.9) 3 (1.7) (0.3, 4.8) 1 (0.6) (0.0, 3.1) CONNECTIVE TISSUE DISORDERS Myalgia 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 1 (0.6) (0.0, 3.1) Arthralgia 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) Neck pain 0 (0.0, 4.1) 1 (1.1) (0.0, 5.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) NEOPLASMS BENIGN, 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) Adenocarcinoma gastric 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) NERVOUS SYSTEM 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) DISORDERS Headache 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) RESPIRATORY, THORACIC 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 2 (1.1) (0.1, 4.0) AND MEDIASTINAL DISORDERS Oropharyngeal pain 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Productive cough 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Rhinorrhoea 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) SKIN AND SUBCUTANEOUS 1 (1.1) (0.0, 6.2) 1 (1.1) (0.0, 5.9) 2 (1.1) (0.1, 4.0) 1 (0.6) (0.0, 3.1) TISSUE DISORDERS Dermatitis 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Hangnail 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) Macule 0 (0.0, 4.1) 1 (1.1) (0.0, 5.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) Rash macular 0 (0.0, 4.1) 0 (0.0, 3.9) 0 (0.0, 2.0) 1 (0.6) (0.0, 3.1) Note: MedDRA (v23.0) coding dictionary applied. .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified event. For any event, n = number of subjects reporting at least 1 occurrence of any event. .sup.cExact 2-sided CI based on the Clopper and Pearson method.

(1430) Related Adverse Events by System Organ Class and Preferred TermPhase 2

(1431) The number of participants with AEs assessed by the investigator as related to study intervention from Dose 1 to 7 days after Dose 2 were low in frequency and similar in the BNT162b2 group and placebo group. Within the BNT162b2 group, a similar proportion of participants in the young and old age groups reported related AEs. Most investigator-assessed related AEs were reactogenicity events in the SOC of general disorders and administration site conditions, and they were reported by a similar proportion of participants in the BNT162b2 group overall compared with the placebo group, with injection site pain being the PT reported most frequently and exclusively in the BNT162b2 younger age group.

(1432) Immediate Adverse EventsPhase 2

(1433) There were no immediate AEs after any dose of BNT162b2 30 ?g or placebo.

(1434) Severe or Life-Threatening Adverse EventsPhase 2

(1435) Two participants (both in the BNT162b2 younger age group) reported severe events of myalgia (AE) and gastric adenocarcinoma (SAE). The participant who reported myalgia had scapular muscle pain, which began 2 days after Dose 2 and was ongoing at the time of the data cutoff. Both events were assessed by the investigator as not related to study intervention.

(1436) Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals, and Other Significant Adverse EventsPhase 2

(1437) DeathsPhase 2

(1438) There were no Phase 2 participants who died through the data cutoff date of 2 Sep. 2020 in this interim CSR.

(1439) Serious Adverse EventsPhase 2

(1440) One participant had an SAE from Dose 1 to 7 days after Dose 2 (Table 13). One participant, who was in the BNT162b2 younger age group, had an SAE of gastric adenocarcinoma 23 days after Dose 1, which was assessed by the investigator as not related to study intervention (Table 13). The SAE was ongoing at the time of the data cutoff, and the participant was withdrawn from the study because of the SAE. From 7 days after Dose 2 to the data cutoff date (2 Sep. 2020), no additional participants reported any SAE.

(1441) TABLE-US-00031 TABLE 13 Number (%) of Subjects Reporting at Least 1 Serious Adverse Event From Dose 1 to 7 Days After Dose 2, by System Organ Class and Preferred Term - Phase 2 - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo 18-55 56-85 18-85 18-85 System Organ Years Years Years Years Class (N.sup.a = 88) (N.sup.a = 92) (N.sup.a = 180) (N.sup.a = 180) Preferred Term n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) Any event 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) NEOPLASMS BENIGN, 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) Adenocarcinoma 1 (1.1) (0.0, 6.2) 0 (0.0, 3.9) 1 (0.6) (0.0, 3.1) 0 (0.0, 2.0) gastric Note: MedDRA (v23.0) coding dictionary applied. .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified adverse event. For any event, n = number of subjects reporting at least 1 occurrence of any adverse event. .sup.cExact 2-sided CI based on the Clopper and Pearson method.

(1442) Safety-Related Participant WithdrawalsPhase 2

(1443) The participant in the BNT162b2 younger age group who reported an SAE of gastric adenocarcinoma was discontinued from the study on Day 23 after Dose 1 of BNT162b2.

(1444) Narratives of Safety-Related Participant WithdrawalsPhase 2

(1445) A narrative for the Phase 2 participant who was withdrawn from the study because of an SAE through the data cutoff date (2 Sep. 2020) was provided.

(1446) Other Significant Adverse EventsPhase 2

(1447) AEs of special interest were not defined for Phase 2 of this study; however, targeted medical events were monitored throughout the study.

(1448) Analysis and Discussion of Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals, and Other Significant Adverse EventsPhase 2

(1449) Up to the data cutoff date of 2 Sep. 2020, there was 1 participant in the younger age group (BNT162b2 group) withdrawn from the study because of an SAE of gastric adenocarcinoma, which was assessed by the investigator as not related to study intervention.

(1450) Phase 2 Safety Conclusions

(1451) Across age groups, local reactions were generally similar in frequency after each dose, and systemic events generally increased in frequency and severity after Dose 2 compared to Dose 1. Local and systemic reactogenicity events were well-tolerated and short-lived.

(1452) Reactogenicity events after each dose of BNT162b2 in older adults were generally milder and less frequent than those observed in younger adults. The majority of reactogenicity events were mild or moderate in severity. No Grade 4 events were reported.

(1453) AEs in participants were low in frequency, and most AEs were mild or moderate in severity. There were no SAEs or discontinuations because of AEs that were assessed as related by the investigator.

(1454) The reactogenicity and AE profile after BNT162b2 30 ?g evaluated in 360 participants was consistent with the safety profile observed after BNT162b2 30 ?g in Phase 1. BNT162b2 at 30 ?g was safe and well tolerated up to 7 days after Dose 2.

(1455) Phase 2/3

(1456) In this interim CSR, safety results for Phase 3 included 36,855 older adolescent and adult participants (16 to 91 years of age) up through the safety data cutoff date of 6 Oct. 2020. AE summaries included any AE reported, regardless of whether participants completed the visit at 1 month after Dose 2. The first 6610 adult participants (18 to 85 years of age, which included the 360 participants in Phase 2) used an e-diary for reporting local reactions and systemic events and had safety data summarized through at least 1 month after Dose 2.

(1457) During the Phase 2/3 portion of the study, a stopping rule for the theoretical concern of vaccine enhanced disease was to be triggered if the 1-sided probability of observing the same or more extreme adverse severe case split was 5% or less, given the same true incidence for vaccine and placebo recipients, and alert criteria were to be triggered if this probability was less than 11%. It is also noted that, with ?18,000 per arm, the study has >83% probability of detecting at least 1 adverse event.

(1458) Local ReactionsPhase 2/3

(1459) In the BNT162b2 group, pain at the injection site was reported more frequently in the younger age group (FIG. 96) than in the older age group (FIG. 97), and frequency was similar after Dose 1 compared with Dose 2 of BNT162b2 in the younger age group (85.3% vs. 79.5%, respectively) and in the older age group (71.7% vs. 66.6%, respectively). In the placebo group, pain at the injection site after Doses 1 and 2 was reported at slightly higher frequencies in the younger age group (13.8% and 11.9%, respectively) than in the older age group (8.8% and 7.7%, respectively).

(1460) In the BNT162b2 group, frequencies of redness and swelling were similar in the younger and older age group after Doses 1 and 2. Frequencies of redness were similar after Dose 1 compared with Dose 2 of BNT162b2 in the younger age group (4.3% vs 5.4%, respectively) and in the older age group (4.5% vs 6.6%, respectively). Frequencies of swelling were similar after Dose 1 compared with Dose 2 of BNT162b2 in the younger age group (5.5% vs 5.9%, respectively) and in the older age group (6.5% vs 7.0%, respectively). In the placebo group, redness and swelling were reported infrequently in the younger (50.8%) and older (51.3%) age groups after Doses 1 and 2. Overall, across age groups, pain at the injection site did not increase after Dose 2, and redness and swelling were generally similar in frequency after Dose 1 and Dose 2. Severe local reactions (50.8%) were reported infrequently in the BNT162b2 group after either dose overall but occurred more frequently in the younger group. After the first and second dose and in both age groups, the majority of local reactions were mild or moderate in severity, and no Grade 4 local reactions were reported.

(1461) Subgroup Analyses

(1462) No clinically meaningful differences in local reactions were observed by country, sex, race, or ethnicity.

(1463) Across age groups, local reactions for the BNT162b2 group after either dose had a median onset day between Day 1.0 and Day 3.0 (Day 1.0 was the day of vaccination), and ranges were similar in the younger and older age groups. Across age groups, local reactions for this group after either dose resolved with median durations between 1.0 to 2.0 days, which were similar in the younger and older age groups.

(1464) Systemic EventsPhase 2/3

(1465) Systemic events were generally increased in frequency and severity in the younger group (FIG. 98) compared with the older group (FIG. 99), with frequencies and severity increasing with number of doses (Dose 1 vs Dose 2) Vomiting and diarrhea were exceptions with vomiting reported similarly infrequently in both age groups and both vomiting and diarrhea similar after each dose. Frequencies of systemic events in the younger and older BNT162b2 groups (Dose 1 vs Dose 2) are listed below: fatigue: younger group (49.0% vs 61.6%) compared to older group (34.3% vs 51.2%) headache: younger group (42.9% vs 53.1%) compared to older group (25.4% vs 39.5%) muscle pain: younger group (22.0% vs. 38.6%) compared to older group (14.0% vs 28.5%) chills: younger group (14.4% vs 36.5%) compared to older group (6.2% vs 22.8%) joint pain: younger group (10.9% vs 22.4%) compared to older group (8.3% vs 18.9%) fever: younger group (3.7% vs 16.6%) compared to older group (1.4% vs 11.5%). vomiting: similar in both age groups and after either dose. diarrhea: reported less frequently in the older group and was similar after each dose.

(1466) Systemic events were generally reported less frequently in the placebo group than in the BNT162b2 group, for both age groups and doses, with some exceptions. In the younger age group, fever and joint pain (after Dose 1) and vomiting and diarrhea (after Dose 1 and Dose 2) were reported at similar frequencies in the placebo group and the BNT162b2 group (FIG. 98). In the older age group, fever and joint pain (after Dose 1) and vomiting and diarrhea (after Dose 1 and Dose 2) were reported at similar frequencies in the placebo group and the BNT162b2 group (FIG. 99).

(1467) Use of antipyretic/pain medication was slightly less frequent in the older age group (20.1% to 37.4%) than in the younger age group (28.1% to 45.8%) after both doses, and medication use increased in both age groups after Dose 2 as compared with after Dose 1. Use of antipyretic/pain medication was less frequent in the placebo group than in the BNT162b2 group and was similar after Dose 1 and Dose 2 in the younger and older placebo groups (9.8% to 13.7%).

(1468) Severe systemic events across age groups after Dose 1 of BNT162b2 were generally lower in frequency than after Dose 2: fever (0.1% vs 0.8%), fatigue (0.8% vs 3.7%), headache (0.5% vs 1.9%), chills (0.2% vs 1.7%), muscle pain (0.3% vs. 1.6%), and joint pain (0.1% vs 0.6%). Diarrhea and vomiting frequencies were generally similar.

(1469) In the placebo group, severe fever was reported at a similar frequency (0.1%) after Dose 1 and Dose 2. One participant in the younger BNT162b2 group reported fever of 41.2? C. only on Day 2 after Dose 2 and was nonfebrile for all other days of the reporting period.

(1470) After the first and second dose and in both age groups, the majority of systemic events were mild or moderate in severity, and no Grade 4 (potentially life-threatening) systemic events were reported other than fever occurring only 1 day in 1 participant (41.2? C.) in the BNT162b2 group.

(1471) Subgroup Analyses

(1472) No clinically meaningful differences in systemic events were observed by country, ethnicity, sex, or race.

(1473) Across age groups, median onset day for most systemic events after either dose of BNT162b2 was Day 2.0 (Day 1.0 was the day of vaccination), and ranges were similar in the younger and older age groups. Across age groups, all systemic events resolved with median duration of 1.0 day, which was similar in the younger and older age groups.

(1474) Adverse EventsPhase 2/3

(1475) In this interim CSR, the first 6610 adult participants (which included the 360 participants in Phase 2) had safety data summarized through at least 1 month after Dose 2. AE summaries for all 36,855 participants up to the cutoff date (6 Oct. 2020) included any event reported, regardless of whether participants completed the visit at 1 month after Dose 2. At the time of the data cutoff date, there was a small percentage (?0.7%) of participants with at least 1 uncoded term.

(1476) Summary of Adverse EventsPhase 2/3

(1477) First 6610 ParticipantsPhase 2/3

(1478) Table 14 presents a summary of the first 6610 participants reporting at least 1 AE from Dose 1 to 1 month after Dose 2.

(1479) The number of participants who reported at least 1 AE was similar in the BNT162b2 group as compared with the placebo group. Severe AEs, SAEs, and AEs leading to withdrawal were reported by ?1.1%, 0.5%, and 0.2%, respectively, in both groups. In the younger and older age groups, the numbers of participants who reported at least 1 AE from Dose 1 to 1 month after Dose 2 were similar in the BNT162b2 group and the corresponding placebo group. Rates of related AEs, severe AEs, SAEs, and AEs leading to withdrawal in the younger and older age groups were also similar to the corresponding placebo group.

(1480) The first 6610 participants who reported at least 1 AE from Dose 1 to the data cutoff date in the BNT162b2 group and the placebo group were similar to those in the corresponding groups at 1 month after Dose 2 (Table 14). From 1 month after Dose 2 to the data cutoff date, 4 additional participants in the younger age group (3 in BNT162b2 and 1 in placebo) and 10 additional participants in the older age group (3 in BNT162b2 and 7 in placebo) reported at least 1 AE. There were no additional related AEs, severe AEs, SAEs, or AEs leading to withdrawal reported in either group.

(1481) TABLE-US-00032 TABLE 14 Number (%) of Subjects Reporting at Least 1 Adverse Event From Dose 1 to 1 Month After Dose 2 - ~6000 Subjects for Phase 2/3 Analysis - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo (N.sup.a = 3314) (N.sup.a = 3296) Adverse Event n.sup.b (%) n.sup.b (%) Any event 374 (11.3) 316 (9.6) Related.sup.c 135 (4.1) 68 (2.1) Severe 35 (1.1) 19 (0.6) Life-threatening 4 (0.1) 7 (0.2) Any serious adverse event 18 (0.5) 17 (0.5) Related.sup.c 0 0 Severe 9 (0.3) 8 (0.2) Life-threatening 4 (0.1) 7 (0.2) Any adverse event leading 6 (0.2) 5 (0.2) to withdrawal Related.sup.c 2 (0.1) 1 (0.0) Severe 2 (0.1) 1 (0.0) Life-threatening 1 (0.0) 2 (0.1) Death 0 0 .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified adverse event category. For any event, n = the number of subjects reporting at least 1 occurrence of any adverse event. .sup.cAssessed by the investigator as related to investigational product.
All ParticipantsPhase 2/3

(1482) From Dose 1 to the data cutoff date, the number of overall participants who reported at least 1 AE was higher in the BNT162b2 group as compared with the placebo group. Severe AEs, SAEs, and AEs leading to withdrawal were reported by ?0.8%, 0.3%, and 0.1%, respectively, in both groups. Discontinuations due to related AEs were reported in 6 participants in the BNT162b2 group and 4 participants in the placebo group. Three Phase 3 participants died: 1 participant in the BNT162b2 group and 2 participants in the placebo group. The participant in the BNT162b2 group who died experienced an SAE of arteriosclerosis which was assessed by the investigator as not related to study intervention.

(1483) In the younger age group, the number of participants who reported at least 1 AE was 1920 (18.1%) and 880 (8.3%) in the BNT162b2 and placebo groups, respectively. In the older age group, the number of participants who reported at least 1 AE was 1166 (14.9%) and 582 (7.4%) in the BNT162b2 and placebo groups, respectively.

(1484) Analysis of Adverse EventsPhase 2/3

(1485) Adverse Events by System Organ Class and Preferred TermPhase 2/3

(1486) First 6610 ParticipantsPhase 2/3

(1487) There are no Tier 1 AEs identified for this program.

(1488) There were no Tier 2 AEs (defined as an event rate ?1.0% in any vaccine group [PT level]) reported from Dose 1 to 1 month after Dose 2.

(1489) Most AEs reported up to 1 month after Dose 2 overall were reactogenicity and in the SOCs of general disorders and administration site conditions (81 [2.4%] in the BNT162b2 group and 57 [1.7%] in the placebo group), musculoskeletal and connective tissue disorders (81 [2.4%] in the BNT162b2 group and 56 [1.7%] in the placebo group), infections and infestations (56 [1.7%] in the BNT162b2 group and 48 [1.5%] in the placebo group), and gastrointestinal disorders (54 [1.6%] in the BNT162b2 group and 41 [1.2%] in the placebo group) (Table 15). In the younger BNT162b2 group, rates of AEs in these SOCs were: general disorders and administration site conditions (54 [3.0%]), musculoskeletal and connective tissue disorders (53 [3.0%]), infections and infestations (31 [1.7%]), and gastrointestinal disorders (32 [1.8%]). In the older BNT162b2 group, rates of AEs in these SOCs were: general disorders and administration site conditions (27 [1.8%]), musculoskeletal and connective tissue disorders (28 [1.8%]), infections and infestations (25 [1.6%]), and gastrointestinal disorders (22 [1.4%]).

(1490) In the BNT162b2 group, the most frequently reported AEs by PT overall were injection site pain (30 [0.9%]), headache (30 [0.9%]), and fatigue (27 [0.8%]) (Table 15), and during this time period (from Dose 1 to 1 month after Dose 2) most of these AEs were reported during the e-diary 1 week reporting period. The majority of these PTs were reported in the younger age group: headache (21[1.2%]), and fatigue (17 [1.0%]). Injection site pain was reported at a similar frequency in the younger (16 [0.9%]) and older (14 [0.9%]) age groups.

(1491) In the BNT162b2 group, there were 10 (0.3%) participants who reported an AE of lymphadenopathy: 6 in the younger age group and 4 in the older age group compared to none in the placebo group; 1 (0.1%) was male and 9 (0.5%) were females. AEs of lymphadenopathy occurred in the arm and neck region (in axillary, left axillary, left para clavicular, left supra clavicular, bilateral cervical, or unspecified lymph nodes). Most lymphadenopathy events were reported within 2 to 4 days after vaccination (2 events were reported 8 days after vaccination). Five of the events lasted 54 days, 3 events lasted between 12 to 16 days, and 2 events were ongoing at the time of the data cutoff date.

(1492) In the younger age group, an AE of angioedema 13 days after Dose 1 (both eyes) and hypersensitivity (allergy attack [no additional information available at the time of this report], unrelated to study intervention) were reported in 1 participant each (BNT162b2 group), and an AE of drug hypersensitivity (oral penicillin reaction) was reported in 1 participant (placebo). None of these events were assessed by the investigator as related to study intervention. Three participants in the younger BNT162b2 group reported appendicitis compared to 1 participant in the older placebo group with perforated appendicitis; all were assessed by the investigator as unrelated to study intervention.

(1493) TABLE-US-00033 TABLE 15 Number (%) of Subjects Reporting at Least 1 Adverse Event From Dose 1 to 1 Month After Dose 2, by System Organ Class and Preferred Term - ~6000 Subjects for Phase 2/3 Analysis - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo System Organ Class (N.sup.a = 3314) (N.sup.a = 3296) Preferred Term n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) Any event 374 (11.3) (10.2, 12.4) 316 (9.6) (8.6, 10.6) BLOOD AND LYMPHATIC SYSTEM 14 (0.4) (0.2, 0.7) 0 (0.0, 0.1) DISORDERS Lymphadenopathy 10 (0.3) (0.1, 0.6) 0 (0.0, 0.1) Anaemia 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Iron deficiency anaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Lymph node pain 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) CARDIAC DISORDERS 6 (0.2) (0.1, 0.4) 4 (0.1) (0.0, 0.3) Atrial fibrillation 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Mitral valve incompetence 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Palpitations 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Acute coronary syndrome 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Acute myocardial infarction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Angina pectoris 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Angina unstable 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Atrial flutter 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Cardiac failure congestive 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery disease 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery dissection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery occlusion 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Left atrial enlargement 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Left ventricular hypertrophy 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Mitral valve prolapse 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tachycardia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) CONGENITAL, FAMILIAL AND 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) GENETIC DISORDERS Congenital cystic kidney 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) disease EAR AND LABYRINTH 7 (0.2) (0.1, 0.4) 6 (0.2) (0.1, 0.4) DISORDERS Vertigo 2 (0.1) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Vertigo positional 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Ear discomfort 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Deafness unilateral 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Ear pain 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tinnitus 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Tympanic membrane 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) perforation ENDOCRINE DISORDERS 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Goitre 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypogonadism 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) EYE DISORDERS 8 (0.2) (0.1, 0.5) 6 (0.2) (0.1, 0.4) Cataract 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Chalazion 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Vision blurred 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Blepharitis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Conjunctival hyperaemia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Dacryostenosis acquired 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Diplopia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Eye pain 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Lacrimation increased 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Retinal detachment 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vitreous detachment 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) GASTROINTESTINAL 54 (1.6) (1.2, 2.1) 41 (1.2) (0.9, 1.7) DISORDERS Diarrhoea 17 (0.5) (0.3, 0.8) 15 (0.5) (0.3, 0.7) Nausea 12 (0.4) (0.2, 0.6) 5 (0.2) (0.0, 0.4) Toothache 5 (0.2) (0.0, 0.4) 2 (0.1) (0.0, 0.2) Vomiting 4 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Abdominal pain 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Constipation 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Dyspepsia 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Gastrooesophageal reflux 0 (0.0, 0.1) 3 (0.1) (0.0, 0.3) disease Odynophagia 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Dental caries 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Inguinal hernia 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Small intestinal 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) obstruction Abdominal adhesions 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Abdominal pain upper 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Abdominal rigidity 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Angular cheilitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Colitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Diverticular perforation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Faeces soft 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Gastric ulcer haemorrhage 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Gastrointestinal disorder 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Gingival discomfort 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Haematochezia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Hiatus hernia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypoaesthesia oral 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Intestinal obstruction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Irritable bowel syndrome 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Mouth ulceration 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Parotid duct obstruction 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Rectal haemorrhage 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Salivary gland calculus 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tongue discomfort 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tongue ulceration 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) GENERAL DISORDERS AND 81 (2.4) (1.9, 3.0) 57 (1.7) (1.3, 2.2) ADMINISTRATION SITE CONDITIONS Fatigue 27 (0.8) (0.5, 1.2) 17 (0.5) (0.3, 0.8) Injection site pain 30 (0.9) (0.6, 1.3) 14 (0.4) (0.2, 0.7) Chills 15 (0.5) (0.3, 0.7) 7 (0.2) (0.1, 0.4) Injection site erythema 10 (0.3) (0.1, 0.6) 6 (0.2) (0.1, 0.4) Pyrexia 13 (0.4) (0.2, 0.7) 2 (0.1) (0.0, 0.2) Injection site swelling 4 (0.1) (0.0, 0.3) 4 (0.1) (0.0, 0.3) Pain 4 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Malaise 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Injection site bruising 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Injection site reaction 3 (0.1) (0.0, 0.3) 0 (0.0, 0.1) Asthenia 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Injection site pruritus 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Chest discomfort 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Chest pain 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Face oedema 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Inflammation 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Injection site discolouration 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Injection site discomfort 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Injection site hyperaesthesia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Non-cardiac chest pain 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Swelling 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Unevaluable event 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vaccination site nodule 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Vascular stent occlusion 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) HEPATOBILIARY DISORDERS 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Cholelithiasis 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Cholecystitis acute 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) IMMUNE SYSTEM DISORDERS 4 (0.1) (0.0, 0.3) 6 (0.2) (0.1, 0.4) Seasonal allergy 1 (0.0) (0.0, 0.2) 3 (0.1) (0.0, 0.3) Food allergy 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Allergy to vaccine 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Anaphylactic reaction 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Drug hypersensitivity 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Hypersensitivity 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Milk allergy 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) INFECTIONS AND 56 (1.7) (1.3, 2.2) 48 (1.5) (1.1, 1.9) INFESTATIONS Urinary tract infection 10 (0.3) (0.1, 0.6) 7 (0.2) (0.1, 0.4) Sinusitis 8 (0.2) (0.1, 0.5) 1 (0.0) (0.0, 0.2) Diverticulitis 3 (0.1) (0.0, 0.3) 4 (0.1) (0.0, 0.3) Tooth infection 2 (0.1) (0.0, 0.2) 3 (0.1) (0.0, 0.3) Otitis media 0 (0.0, 0.1) 4 (0.1) (0.0, 0.3) Upper respiratory tract infection 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Appendicitis 3 (0.1) (0.0, 0.3) 0 (0.0, 0.1) Cellulitis 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Ear infection 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Herpes zoster 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Pneumonia 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Tonsillitis 0 (0.0, 0.1) 3 (0.1) (0.0, 0.3) Conjunctivitis 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Gastroenteritis 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Infected bite 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Otitis externa 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Otitis media acute 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Skin infection 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Tooth abscess 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Acute sinusitis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Appendicitis perforated 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Cystitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Dermatitis infected 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Eye infection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Folliculitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Fungal infection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Fungal skin infection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Genital herpes 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Genital herpes simplex 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Gingivitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Hordeolum 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Kidney infection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Lower respiratory tract infection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Oral candidiasis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Oral fungal infection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Parotitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Peritoneal abscess 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Peritonitis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Pharyngitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Pharyngitis streptococcal 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Respiratory tract infection viral 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Rhinitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Sepsis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Skin bacterial infection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Soft tissue infection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Subcutaneous abscess 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vulvovaginal candidiasis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Vulvovaginal mycotic infection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) INJURY, POISONING AND 28 (0.8) (0.6, 1.2) 42 (1.3) (0.9, 1.7) PROCEDURAL COMPLICATIONS Fall 8 (0.2) (0.1, 0.5) 16 (0.5) (0.3, 0.8) Arthropod bite 5 (0.2) (0.0, 0.4) 3 (0.1) (0.0, 0.3) Muscle strain 4 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Contusion 2 (0.1) (0.0, 0.2) 4 (0.1) (0.0, 0.3) Skin abrasion 1 (0.0) (0.0, 0.2) 4 (0.1) (0.0, 0.3) Skin laceration 0 (0.0, 0.1) 4 (0.1) (0.0, 0.3) Ankle fracture 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Joint dislocation 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Tooth fracture 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Fibula fracture 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Foot fracture 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Joint injury 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Ligament sprain 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Limb injury 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Meniscus injury 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Muscle rupture 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Rib fracture 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Bone contusion 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Chest injury 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Corneal abrasion 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Craniocerebral injury 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Forearm fracture 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Hand fracture 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Humerus fracture 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Lumbar vertebral fracture 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Muscle injury 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Postoperative ileus 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Procedural pain 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Radius fracture 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Road traffic accident 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Sunburn 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Tendon rupture 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Thermal burn 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) INVESTIGATIONS 9 (0.3) (0.1, 0.5) 4 (0.1) (0.0, 0.3) Body temperature increased 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Blood glucose increased 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Blood cholesterol increased 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Blood pressure increased 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Blood triglycerides increased 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Cardiac stress test abnormal 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Electrocardiogram QT prolonged 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Heart rate increased 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Weight decreased 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) White blood cell count increased 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) METABOLISM AND NUTRITION 12 (0.4) (0.2, 0.6) 7 (0.2) (0.1, 0.4) DISORDERS Decreased appetite 3 (0.1) (0.0, 0.3) 0 (0.0, 0.1) Hypercholesterolaemia 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Type 2 diabetes mellitus 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Dehydration 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Diabetes mellitus inadequate control 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Dyslipidaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Glucose tolerance impaired 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Hyperlipidaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypertriglyceridaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypoglycaemia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Hypokalaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Iron deficiency 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Obesity 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Vitamin D deficiency 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) MUSCULOSKELETAL AND 81 (2.4) (1.9, 3.0) 56 (1.7) (1.3, 2.2) CONNECTIVE TISSUE DISORDERS Arthralgia 20 (0.6) (0.4, 0.9) 15 (0.5) (0.3, 0.7) Myalgia 20 (0.6) (0.4, 0.9) 12 (0.4) (0.2, 0.6) Back pain 8 (0.2) (0.1, 0.5) 8 (0.2) (0.1, 0.5) Pain in extremity 10 (0.3) (0.1, 0.6) 4 (0.1) (0.0, 0.3) Neck pain 5 (0.2) (0.0, 0.4) 5 (0.2) (0.0, 0.4) Muscle spasms 4 (0.1) (0.0, 0.3) 2 (0.1) (0.0, 0.2) Musculoskeletal pain 2 (0.1) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Osteoarthritis 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Tendonitis 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Arthritis 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Flank pain 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Joint effusion 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Plantar fasciitis 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Bursitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Exostosis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Limb discomfort 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Muscle twitching 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Muscular weakness 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Musculoskeletal discomfort 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Musculoskeletal stiffness 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Osteitis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Osteopenia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Pain in jaw 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Rotator cuff syndrome 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tenosynovitis stenosans 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Torticollis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NEOPLASMS BENIGN, MALIGNANT 4 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) AND UNSPECIFIED (INCL CYSTS AND POLYPS) Basal cell carcinoma 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Adenocarcinoma gastric 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Breast cancer 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Seborrhoeic keratosis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Uterine leiomyoma 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vaginal neoplasm 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NERVOUS SYSTEM DISORDERS 44 (1.3) (1.0, 1.8) 32 (1.0) (0.7, 1.4) Headache 30 (0.9) (0.6, 1.3) 24 (0.7) (0.5, 1.1) Dizziness 3 (0.1) (0.0, 0.3) 2 (0.1) (0.0, 0.2) Migraine 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Paraesthesia 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Syncope 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Burning sensation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Cervical radiculopathy 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Dysgeusia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Hypoaesthesia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Radiculopathy 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Sciatica 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Transient ischaemic attack 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Tremor 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) PSYCHIATRIC DISORDERS 13 (0.4) (0.2, 0.7) 13 (0.4) (0.2, 0.7) Anxiety 4 (0.1) (0.0, 0.3) 4 (0.1) (0.0, 0.3) Depression 2 (0.1) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Anxiety disorder 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Irritability 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Mental status changes 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Bipolar disorder 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Depressed mood 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Disorientation 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Gastrointestinal somatic 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) symptom disorder Insomnia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Mental disorder 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Mood swings 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Panic reaction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Sleep disorder 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Suicidal ideation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) RENAL AND URINARY 3 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) DISORDERS Acute kidney injury 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Chronic kidney disease 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Dysuria 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Haematuria 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Nephrolithiasis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Urinary retention 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) REPRODUCTIVE SYSTEM AND 4 (0.1) (0.0, 0.3) 2 (0.1) (0.0, 0.2) BREAST DISORDERS Dysmenorrhoea 3 (0.1) (0.0, 0.3) 0 (0.0, 0.1) Cervical dysplasia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Menorrhagia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Prostatitis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) RESPIRATORY, THORACIC AND 21 (0.6) (0.4, 1.0) 26 (0.8) (0.5, 1.2) MEDIASTINAL DISORDERS Oropharyngeal pain 4 (0.1) (0.0, 0.3) 7 (0.2) (0.1, 0.4) Cough 5 (0.2) (0.0, 0.4) 5 (0.2) (0.0, 0.4) Rhinitis allergic 3 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Rhinorrhoea 1 (0.0) (0.0, 0.2) 4 (0.1) (0.0, 0.3) Nasal congestion 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Dyspnoea 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Dyspnoea exertional 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Upper-airway cough syndrome 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Acute respiratory failure 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Allergic respiratory disease 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Asthma 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Bronchospasm 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Chronic obstructive pulmonary 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) disease Productive cough 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Pulmonary embolism 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Sinus congestion 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Throat irritation 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) SKIN AND SUBCUTANEOUS 32 (1.0) (0.7, 1.4) 19 (0.6) (0.3, 0.9) TISSUE DISORDERS Dermatitis contact 10 (0.3) (0.1, 0.6) 3 (0.1) (0.0, 0.3) Rash 7 (0.2) (0.1, 0.4) 2 (0.1) (0.0, 0.2) Erythema 3 (0.1) (0.0, 0.3) 1 (0.0) (0.0, 0.2) Urticaria 2 (0.1) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Dermatitis 1 (0.0) (0.0. 0.2) 2 (0.1) (0.0, 0.2) Pruritus 3 (0.1) (0.0. 0.3) 0 (0.0, 0.1) Hyperhidrosis 0 (0.0, 0.1) 2 (0.1) (0.0. 0.2) Macule 1 (0.0) (0.0, 0.2) 1 (0.0) (0.0. 0.2) Angioedema 1 (0.0) (0.0, 0.2) 0 (0.0. 0.1) Dermatitis atopic 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Dermatitis bullous 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Drug eruption 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Ecchymosis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Hangnail 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Papule 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Rash erythematous 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Rash maculo-papular 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Rosacea 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Skin discolouration 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Skin ulcer 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) SURGICAL AND MEDICAL 5 (0.2) (0.0, 0.4) 4 (0.1) (0.0, 0.3) PROCEDURES Dental care 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Dental operation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Gingival operation 1 (0.0) (0.0. 0.2) 0 (0.0, 0.1) Hip surgery 1 (0.0) (0.0. 0.2) 0 (0.0, 0.1) Inguinal hernia repair 1 (0.0) (0.0. 0.2) 0 (0.0, 0.1) Laryngeal operation 0 (0.0. 0.1) 1 (0.0) (0.0, 0.2) Postoperative care 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Sclerotherapy 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Tooth extraction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) VASCULAR DISORDERS 8 (0.2) (0.1, 0.5) 15 (0.5) (0.3, 0.7) Hypertension 5 (0.2) (0.0, 0.4) 6 (0.2) (0.1, 0.4) Haematoma 1 (0.0) (0.0, 0.2) 3 (0.1) (0.0, 0.3) Aortic aneurysm 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Deep vein thrombosis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Essential hypertension 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Hypotension 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) LYMPHOEDEMA 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Lymphorrhoea 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Orthostatic hypotension 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Raynaud's phenomenon 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Note: MedDRA (v23.0) coding dictionary applied. .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified event. For any event, n = number of subjects reporting at least 1 occurrence of any event. .sup.cExact 2-sided CI based on the Clopper and Pearson method.

(1494) All ParticipantsPhase 2/3

(1495) For all 36,855 participants up to the data cutoff date, there were a total of 121 (0.7%) participants in the BNT162b2 group and 51 (0.3%) participants in the placebo group with at least 1 uncoded term. As a result, uncoded terms are also present in other AE tables summarized by SOC and PT.

(1496) From Dose 1 to the data cutoff date, the number of overall participants who reported at least 1 AE was higher in the BNT162b2 group (3086 [16.8%]) as compared with the placebo group (1462 [7.9%]). Most AEs reported in all participants from Dose 1 to the data cutoff date were reactogenicity and in the SOCs of general disorders and administration site conditions (1941 [10.5%] in the BNT162b2 group and 438 [2.4%] in the placebo group), musculoskeletal and connective tissue disorders (742 [4.0%] in the BNT162b2 group and 227 [1.2%] in the placebo group), and nervous system disorders (567 [3.1%] in the BNT162b2 group and 251 [1.4%] in the placebo group). In the BNT162b2 group, the most frequently reported AEs by PT were injection site pain (1222 [6.6%]), pyrexia (504 [2.7%]), fatigue (481 [2.6%]), headache (470 [2.6%]), chills (458 [2.5%]), and myalgia (454 [2.5%]). The majority of these PTs were reported in the younger age group: injection site pain (787 [7.4%]), pyrexia (351 [3.3%]), fatigue (309 [2.9%]), headache (303 [2.9%]), chills (316 [3.0%]), and myalgia (304 [2.9%]).

(1497) Beyond the first 6610 participants, events related to reactogenicity are no longer reported using an e-diary but are instead reported as AEs. Therefore, a post hoc analysis was conducted to evaluate if the imbalance in AEs observed in the overall participants from Dose 1 to the data cutoff date but not observed in the first 6610 participants from Dose 1 to 1 month after Dose 2 was attributed to reactogenicity events. The analysis examined the AEs reported within 7 days after each dose, which represented the reactogenicity reporting period. The time period was chosen because many AEs were reported in the SOCs of general disorders and administration site conditions, musculoskeletal and connective tissue disorders, and nervous system disorders, which contains AEs consistent with reactogenicity events, and could only be attributed to reactogenicity if they occurred during this time period as opposed to occurring up to a month from each dose.

(1498) From Dose 1 to 7 days after Dose 1 (as of the data cutoff date), 1494 (8.1%) participants reported at least 1 AE in the BNT162b2 group, which represented approximately half of the total number of the 3086 [16.8%] participants who reported at least 1 AE up to the data cutoff date. In the placebo group, 555 (3.0%) participants reported at least 1 AE from Dose 1 to 7 days after Dose 1, compared with the total number of 1462 (7.9%) participants who reported at least 1 AE up to the data cutoff date.

(1499) From Dose 2 to 7 days after Dose 2 (as of the data cutoff date), 1165 (6.3%) participants reported at least 1 AE in the BNT162b2 group, which represented approximately 38% of the total number of the 3086 [16.8%] participants who reported at least 1 AE up to the data cutoff date. From Dose 2 to 7 days after Dose 2, fewer participants reported AEs in the placebo group than the BNT162b2 group. In the placebo group, 268 (1.5%) participants reported at least 1 AE from Dose 2 to 7 days after Dose 2, compared with the total number of 1462 (7.9%) participants who reported at least 1 AE up to the data cutoff date.

(1500) AEs were reported from Dose 1 to 7 days after Dose 1 in the SOC of general disorders and administration site conditions (1127 [6.1%] in the BNT162b2 group and 251 [1.4%] in the placebo group), which represented more than half of the total number of participants reporting at least 1 AE in this SOC (1941 [10.5%] in the BNT162b2 group and 438 [2.4%] in the placebo group) up to the data cutoff date. Musculoskeletal and connective tissue disorders (252 [1.4%] in the BNT162b2 group and 76 [0.4%] in the placebo group) and nervous system disorders (220 [1.2%] in the BNT162b2 group and 115 [0.6%] in the placebo group) were also commonly reported, representing a smaller proportion of the total number of participants reporting AEs for these SOCs.

(1501) In the BNT162b2 group, the most frequently reported AEs from Dose 1 to 7 days after Dose 1 by PT were injection site pain (881 [4.8%]), fatigue (231 [1.3%]), headache (181 [1.0%]), myalgia (147 [0.8%]), pyrexia (110 [0.6%]), and chills (100 [0.5%]). The majority of these PTs were reported in the younger age group: injection site pain (566 [5.3%]), fatigue (153 [1.4%]), headache (118 [1.1%]), myalgia (99 [0.9%]), pyrexia (82 [0.8%]), and chills (75 [0.7%]). Injection site pain reported from Dose 1 to 7 days after Dose 1 (881 [4.8%]) represented a large proportion of the total participants who reported AEs for this PT (1222 [6.6%]).

(1502) AEs were reported from Dose 2 to 7 days after Dose 2 in the SOCs of general disorders and administration site conditions (828 [4.5%] in the BNT162b2 group and 93 [0.5%] in the placebo group), musculoskeletal and connective tissue disorders (377 [2.0%] in the BNT162b2 group and 38 [0.2%] in the placebo group), and nervous system disorders (294 [1.6%] in the BNT162b2 group and 40 [0.2%] in the placebo group). Musculoskeletal and connective tissue disorders and nervous system disorders reported from Dose 2 to 7 days after Dose 2 represented at least half of the total number of participants who reported at least 1 AE in these SOCs.

(1503) In the BNT162b2 group, the most frequently reported AEs from Dose 2 to 7 days after Dose 2 by PT were pyrexia (375 [2.0%]), chills (327 [1.8%]), injection site pain (313 [1.7%]), myalgia (282 [1.5%]), headache (258 [1.4%]), and fatigue (227 [1.2%]). The majority of these PTs were reported in the younger age group: pyrexia (251 [2.4%]), chills (216 [2.0%]), myalgia (185 [1.7%]), injection site pain (183 [1.7%]), headache (154 [1.5%]), and fatigue (134 [1.3%]). AEs for most of these PTs reported from Dose 2 to 7 days after Dose 2 represented at least half of the total number of participants who reported an AE for these PTs: pyrexia (504 [2.7%]), chills (458 [2.5%]), myalgia (454 [2.5%]), headache (470 [2.6%]), and fatigue (481 [2.6%]).

(1504) Overall, AEs reported from Dose 1 to 7 days after Dose 1 and from Dose 2 to 7 days after Dose 2 were largely attributable to reactogenicity events. This observation provides a reasonable explanation for the greater rates of AEs observed overall in the BNT162b2 group compared with the placebo group.

(1505) From Dose 1 to the data cutoff date, there were a total of 44 (0.2%) participants in the BNT162b2 group who reported an AE of lymphadenopathy, inclusive of those reported in the first 6610 participants (10 [0.3%]). Up to the data cutoff date, 34 additional participants in the BNT162b2 group and 4 additional participants in the placebo group reported an AE of lymphadenopathy. In the BNT162b2 group, lymphadenopathy was reported in 34 (0.3%) participants in the younger age group and 10 (0.1%) participants in the older age group compared to 4 (0.0%) in the placebo group (3 in the younger age group and 1 in the older age group). Lymphadenopathy occurred predominantly in the arm and neck region with most events reported in left axillary lymph node(s). Most lymphadenopathy events occurred after Dose 2, 53 days after Dose 1 or Dose 2, were Grade 1 or Grade 2 in severity, and 32 of 48 events were resolved by the data cutoff date. In 1 participant in the younger BNT162b2 age group, Grade 1 lymphadenopathy (swollen right axillary lymph nodes) was an immediate AE, which occurred after Dose 1 and was continuing at the data cutoff date.

(1506) In the younger age group, there was 1 participant each with an AE of suspected COVID-19 in the BNT162b2 (SAE) and placebo groups.

(1507) In the BNT162b2 group, 6 participants reported immunization reactions (vaccine reaction or systemic vaccine reaction [no additional information currently available at the time of this report]) assessed as related to study intervention. Three participants reported drug hypersensitivity in the BNT162b2 group in addition to the drug hypersensitivity in a participant in the placebo group. Drug hypersensitivity (allergic reaction) was assessed as related in 1 participant in the BNT162b2 group and drug hypersensitivity (drug allergy or allergic reaction to dipyrone) was assessed as unrelated to study intervention in 2 participants in the BNT162b2 group.

(1508) Nineteen (0.1%) participants in the BNT162b2 group (14 in the younger age group and 5 in the older age group reported at least 1 vaccine complication (most were descriptive of reactogenicity events) compared to none in the placebo group. All were assessed as related to the study intervention and included: post vaccination myalgia, fever, body aches, headache, chills, nausea, adverse reaction, arthralgia, fatigue, aches, muscle aches, malaise, and sore left shoulder. Most events were Grade 1, started within 3 days of vaccination, and lasted from 1 to 3 days.

(1509) In addition to the 4 participants with appendicitis (including 1 appendicitis perforated in the placebo group) in the first 6610 participants, there were an additional 3 participants with appendicitis (including 1 participant with appendicitis perforated) reported in the BNT162b2 group from Dose 1 through the data cutoff date for all participants. Therefore, a total of 6 participants in the BNT162b2 group reported appendicitis (including 1 appendicitis perforated) with 4 in the younger age group and 2 in the older age group, and 1 participant in the placebo group (older age group) reported appendicitis (perforated). All events were severe or life-threatening and none were assessed as related to study intervention.

(1510) Related Adverse Events by System Organ Class and Preferred TermPhase 2/3 First 6610 ParticipantsPhase 2/3

(1511) From Dose 1 to 1 month after Dose 2, 135 (4.1%) participants reported at least 1 AE assessed as related by the investigator in the BNT162b2 group, and 68 (2.1%) participants reported at least 1 related AE in the placebo group. Most related AEs were reactogenicity events and in the SOC of general disorders and administration site conditions (69 [2.1%] in the BNT162b2 group and 40 [1.2%] in the placebo group). The AEs of lymphadenopathy reported in 8 of 10 participants were assessed by the investigator as related to study intervention.

(1512) All ParticipantsPhase 2/3

(1513) From Dose 1 to the data cutoff date, 2303 (12.5%) participants in the BNT162b2 group and 593 (3.2%) participants in the placebo group reported at least 1 AE assessed as related by the investigator, inclusive of the related AEs for the first 6610 participants. Most related AEs were reactogenicity events and in the SOC of general disorders and administration site conditions (1869 [10.1%] in the BNT162b2 group and 365 [2.0%] in the placebo group).

(1514) The AEs of lymphadenopathy reported in 30 of 44 participants in the BNT162b2 group and 2 of 4 participants in the placebo group were assessed by the investigator as related to study intervention.

(1515) In the BNT162b2 group, based on all information currently available at the time of this report:

(1516) Six participants reported immunization reaction (vaccine reaction or systemic vaccine reaction) assessed as related to the study intervention. In most participants, immunization reactions occurred 1 or 2 days after Dose 2, lasted 2 or 3 days (1 participant was recovering at data cutoff date), and were Grade 1 or Grade 2 in severity. In 1 participant, immunization reactions (systemic vaccine reactions) occurred 2 days after Dose 1 (Grade 1) and lasted 2 days, and 1 day after Dose 2 (Grade 3) and lasted 4 days.

(1517) One participant reported an AE each of drug hypersensitivity (allergic reaction), urticaria (allergic reaction), and headache, which were all Grade 2 and assessed by the investigator as related to study intervention. The AEs of drug hypersensitivity and urticaria both occurred within 1 day after Dose 1 and resolved that same day. The AE of headache occurred the following day after vaccination and lasted 4 days.

(1518) Immediate Adverse EventsPhase 2/3

(1519) First 6610 ParticipantsPhase 2/3

(1520) After Dose 1, ?0.3% of participants reported immediate AEs. Most immediate AEs were in the SOC of general disorders and administration site conditions and were events related to injection site reactions (injection site pain, injection site erythema and injection site swelling).

(1521) After Dose 2, 0.1% of participants in each group reported immediate AEs. Most immediate AEs were in the SOC of general disorders and administration site conditions and were events related to injection site reactions (injection site pain, injection site hyperaesthesia, and injection site pruritus).

(1522) After either dose of BNT162b2, no participant reported an immediate allergic reaction to the vaccine.

(1523) All ParticipantsPhase 2/3

(1524) After Dose 1, 0.3% of participants in each group reported immediate AEs. Most immediate AEs were in the SOC of general disorders and administration site conditions and most events were related to injection site reactions with injection site pain most frequently reported (40 [0.2%] participants in the BNT162b2 group and 27 (0.1%) participants in the placebo group). One participant had an immediate AE of lymphadenopathy after Dose 1. All other immediate AEs were reported by ?3 participants each in the BNT162b2 group.

(1525) After Dose 2, 0.1% of participants in each group reported immediate AEs. Most immediate AEs were in the SOC of general disorders and administration site conditions and most events were injection site reactions with injection site pain most frequently reported (10 [0.1%] participants in the BNT162b2 group and 7 [0.0%] participants in the placebo group). All other immediate AEs were reported by ?2 participants each. After either dose of BNT162b2, no participant reported an immediate allergic reaction to the vaccine.

(1526) Severe or Life-Threatening Adverse EventsPhase 2/3

(1527) First 6610 ParticipantsPhase 2/3

(1528) From Dose 1 to 1 month after Dose 2, severe AEs reported were reported by 35 (1.1%) participants in the BNT162b2 group and 19 (0.6%) in the placebo group.

(1529) Four (0.1%) participants in the BNT162b2 group and 7 (0.2%) participants in the placebo group had at least 1 life-threatening AE from Dose 1 to 1 month after Dose 2. None of these events were assessed by the investigator as related to study intervention.

(1530) In the BNT162b2 Group:

(1531) One participant from Phase 2 had a severe event of gastric adenocarcinoma (SAE), which is discussed in a previous section.

(1532) Two participants had severe events of appendicitis: 1 event began 9 days after Dose 1 and the other event began 15 days after Dose 2 (SAEs) which were assessed by the investigator as not related to study intervention.

(1533) One participant had 2 life-threatening AEs of appendicitis and peritoneal abscess 7 days after Dose 1 (both SAEs); both events were assessed by the investigator as not related to study intervention.

(1534) One participant had 8 severe events: anemia, cardiac failure congestive, abdominal adhesions, sepsis, hypokalaemia, mental status changes, acute kidney injury, and acute respiratory failure (all SAEs). None of the events were assessed by the investigator as related to study intervention.

(1535) All ParticipantsPhase 2/3

(1536) Severe AEs reported up to the data cutoff date, inclusive of those discussed for the first 6610 participants, were reported by 142 [0.8%] participants in the BNT162b2 group and 70 (0.4%) in the placebo group. Additional events included:

(1537) Two participants in the BNT162b2 group had a severe event each of appendicitis: 1 event began 17 days after Dose 1 and the other event began 11 days after Dose 1 (SAE) which were assessed by the investigator as not related to study intervention. One participant in the BNT162b2 group had a severe event of perforated appendicitis on the same day after Dose 1 (SAE) which was assessed by the investigator as not related to study intervention.

(1538) Nine participants (0.0%) in the BNT162b2 group and 12 (0.1%) participants in the placebo group had at least 1 life-threatening AE from Dose 1 to the data cutoff date, inclusive of those discussed for the first 6610 participants. None of these events were assessed by the investigator as related to study intervention.

(1539) Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals, and Other Significant Adverse EventsPhase 2/3

(1540) DeathsPhase 2/3

(1541) There were 3 Phase 3 participants (1 in the BNT162b2 group and 2 in the placebo group) who died through the data cutoff date of 6 Oct. 2020. None of these deaths were among the first 6610 participants (Table 14) and none were assessed by the investigator as related to study intervention.

(1542) One participant in the older BNT162b2 group experienced a Grade 4 SAE of arteriosclerosis 4 days after Dose 1 and died 15 days after Dose 1.

(1543) One participant in the younger placebo group experienced a Grade 4 SAE of unevaluable event (unknown of unknown origin [no additional information currently available at the time of this report) 8 days after Dose 1 and died the same day.

(1544) One participant in the older placebo group experienced a Grade 4 SAE of hemorrhagic stroke 15 days after Dose 2 and died 35 days after Dose 2.

(1545) Death Narratives

(1546) Narratives for the participants who died through the data cutoff date (6 Oct. 2020) were provided.

(1547) Serious Adverse EventsPhase 2/3

(1548) First 6610 ParticipantsPhase 2/3

(1549) From Dose 1 to 1 month after Dose 2, the number of participants who reported at least 1 SAE was similar in the BNT162b2 group (18 (0.5%]) and in the placebo group (17 [0.5%]) (Table 16). None of the SAEs were assessed by the investigator as related to study intervention. Most PTs for SAEs were reported by only 1 participant (3 participants reported an SAE of appendicitis).

(1550) From Dose 1 to 1 month after Dose 2, the number of participants who reported at least 1 SAE in the younger and older age groups was similar.

(1551) In the BNT162b2 Group:

(1552) Two participants had an SAE each of appendicitis: 1 event began 9 days after Dose 1 and the other event began 15 days after Dose 2.

(1553) One participant had an SAE each of appendicitis and peritoneal abscess 7 days after Dose 1, which was considered life-threatening. Both events lasted for 17 days.

(1554) One participant had 8 SAEs 17 days after Dose 1: anemia, cardiac failure congestive, abdominal adhesions, sepsis, hypokalaemia, mental status changes, acute kidney injury, and acute respiratory failure (all were severe). The SAEs of abdominal adhesions and acute respiratory failure lasted for 2 and 14 days, respectively. All other SAEs lasted for 19 days.

(1555) One participant had an SAE of anaphylactic reaction 9 days after Dose 2 as a result of a bee sting which was considered life threatening. The event resolved on the same day.

(1556) In the placebo group, 1 participant had an SAE each of appendicitis perforated and peritonitis 13 and 15 days after Dose 2, respectively (both severe). Both events lasted 4 and 5 days, respectively.

(1557) From 1 month after Dose 2 to the data cutoff date, no additional SAEs were reported for these first 6610 participants.

(1558) TABLE-US-00034 TABLE 16 Number (%) of Subjects Reporting at Least 1 Serious Adverse Event From Dose 1 to 1 Month After Dose 2, by System Organ Class and Preferred Term - ~6000 Subjects for Phase 2/3 Analysis - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo System Organ Class (N.sup.a = 3314) (N.sup.a = 3296) Preferred Term n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) Any event 18 (0.5) (0.3, 0.9) 17 (0.5) (0.3, 0.8) BLOOD AND LYMPHATIC SYSTEM 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) DISORDERS Anaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) CARDIAC DISORDERS 3 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Acute coronary syndrome 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Atrial fibrillation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Cardiac failure congestive 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery disease 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery dissection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery occlusion 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) EAR AND LABYRINTH DISORDERS 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Vertigo 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) EYE DISORDERS 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Diplopia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) GASTROINTESTINAL DISORDERS 3 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Abdominal adhesions 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Colitis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Diarrhoea 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Diverticular perforation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Intestinal obstruction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Small intestinal obstruction 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) GENERAL DISORDERS AND 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) ADMINISTRATION SITE CONDITIONS Chest pain 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Non-cardiac chest pain 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Unevaluable event 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vascular stent occlusion 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) HEPATOBILIARY DISORDERS 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) Cholelithiasis 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) Cholecystitis acute 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) IMMUNE SYSTEM DISORDERS 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Anaphylactic reaction 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) INFECTIONS AND INFESTATIONS 4 (0.1) (0.0, 0.3) 3 (0.1) (0.0, 0.3) Appendicitis 3 (0.1) (0.0, 0.3) 0 (0.0, 0.1) Appendicitis perforated 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Peritoneal abscess 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Peritonitis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Pneumonia 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Sepsis 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Urinary tract infection 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) INJURY, POISONING AND 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) PROCEDURAL COMPLICATIONS Forearm fracture 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Skin laceration 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) INVESTIGATIONS 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Cardiac stress test abnormal 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) METABOLISM AND NUTRITION 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) DISORDERS Hypokalaemia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NEOPLASMS BENIGN, 2 (0.1) (0.0, 0.2) 1 (0.0) (0.0, 0.2) MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) Adenocarcinoma gastric 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Breast cancer 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Vaginal neoplasm 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NERVOUS SYSTEM DISORDERS 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Syncope 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) PSYCHIATRIC DISORDERS 2 (0.1) (0.0, 0.2) 2 (0.1) (0.0, 0.2) Bipolar disorder 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Mental disorder 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Mental status changes 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Suicidal ideation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) RENAL AND URINARY 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) DISORDERS Acute kidney injury 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) RESPIRATORY, THORACIC 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) AND MEDIASTINAL DISORDERS Acute respiratory failure 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) VASCULAR DISORDERS 0 (0.0, 0.1) 2 (0.1) (0.0, 0.2) Deep vein thrombosis 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Orthostatic hypotension 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Note: MedDRA (v23.0) coding dictionary applied. .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified event. For any event, n = number of subjects reporting at least 1 occurrence of any event. .sup.cExact 2-sided CI based on the Clopper and Pearson method.

(1559) All ParticipantsPhase 2/3

(1560) From Dose 1 to the data cutoff date, inclusive of those discussed for the first 6610 participants, the number of participants who reported at least 1 SAE was similar in the BNT162b2 group (63 [0.3%]) and in the placebo group (49 [0.3%]) (Table 17).

(1561) Additional Events Included:

(1562) In the BNT162b2 group, there were 2 participants in the younger age group with an SAE each assessed by the investigator as related to study intervention: One participant had an SAE of lymphadenopathy (right axilla) 13 days after Dose 1, which was not resolved at the time of the data cutoff. The participant was a 48-year-old woman with a relevant medical history of eczema and topical crisaborole use who was administered BNT162b2 vaccine in the left deltoid and had right axillary pain and lymphadenopathy. She had no injuries to the right arm, no fever, and no history of a similar incident. Her WBC was normal with a normal lymphocyte count and a right axilla ultrasound showed 4 enlarged lymph nodes (largest 2.5?1.1?2.4 cm). A biopsy was performed and was reported to be normal and without markers for lymphoma or other cancer. A follow-up visit with oncology (and possible repeat ultrasound) was planned for 3 months time.

(1563) One participant had an SAE of shoulder injury related to vaccine administration (SIRVA, erroneously administered into or near the shoulder joint capsule) after Dose 2, which was recovering at the time of the data cutoff.

(1564) From Dose 1 to the data cutoff date, a total of 6 participants in the BNT162b2 group reported an SAE of appendicitis. Three of these SAEs of appendicitis occurred in the first 6610 participants. The 3 additional SAEs of appendicitis are described below along with other specified SAEs that were assessed as not related to study intervention in the BNT162b2 group:

(1565) Two participants had an SAE each of appendicitis: 1 event began 17 days after Dose 1 which lasted for 3 days (younger age group), and the other event began 11 days after Dose 1 which lasted 5 days (older age group).

(1566) One participant in the older age group had an SAE of appendicitis perforated on the same day after Dose 1, which was resolving at the time of the data cutoff.

(1567) One participant in the younger age group had an SAE of suspected COVID-19 on the same day after Dose 2, which lasted for 6 days. The nasal swab result was negative.

(1568) TABLE-US-00035 TABLE 17 Number (%) of Subjects Reporting at Least 1 Serious Adverse Event From Dose 1 to Data Cutoff Date (6 OCT. 2020), by System Organ Class and Preferred Term - Phase 2/3 (All Participants) - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo System Organ Class (N.sup.a = 18419) (N.sup.a = 18436) Preferred Term n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) Any event 63 (0.3) (0.3, 0.4) 49 (0.3) (0.2, 0.4) BLOOD AND LYMPHATIC 2 (0.0) (0.0, 0.0) 2 (0.0) (0.0, 0.0) SYSTEM DISORDERS Anaemia 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Leukocytosis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Lymphadenopathy 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Neutropenia 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Thrombocytosis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) CARDIAC DISORDERS 10 (0.1) (0.0, 0.1) 6 (0.0) (0.0, 0.1) Cardiac failure congestive 2 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Acute coronary syndrome 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Atrial fibrillation 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Acute myocardial infarction 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Angina pectoris 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Arrhythmia 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Arrhythmia supraventricular 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Arteriospasm coronary 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Coronary artery disease 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Coronary artery dissection 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Coronary artery occlusion 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Myocardial infarction 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) EAR AND LABYRINTH DISORDERS 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Vertigo 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) EYE DISORDERS 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Diplopia 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Retinal artery occlusion 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) GASTROINTESTINAL DISORDERS 7 (0.0) (0.0. 0.1) 5 (0.0) (0.0, 0.1) Small intestinal obstruction 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Abdominal adhesions 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Colitis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Diarrhoea 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Diverticular perforation 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Gastrointestinal haemorrhage 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Intestinal obstruction 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Obstructive pancreatitis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Oesophageal food impaction 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Pancreatitis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Salivary gland calculus 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) GENERAL DISORDERS AND 3 (0.0) (0.0, 0.0) 3 (0.0) (0.0, 0.0) ADMINISTRATION SITE CONDITIONS Unevaluable event 0 (0.0, 0.0) 2 (0.0) (0.0, 0.0) Chest pain 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Influenza like illness 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Non-cardiac chest pain 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Shoulder injury related to vaccine 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) administration Vascular stent occlusion 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) HEPATOBILIARY DISORDERS 3 (0.0) (0.0, 0.0) 2 (0.0) (0.0, 0.0) Cholecystitis acute 0 (0.0, 0.0) 2 (0.0) (0.0, 0.0) Cholelithiasis 2 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Bile duct stone 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) IMMUNE SYSTEM DISORDERS 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Anaphylactic reaction 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) INFECTIONS AND INFESTATIONS 15 (0.1) (0.0, 0.1) 9 (0.0) (0.0, 0.1) Appendicitis 5 (0.0) (0.0, 0.1) 0 (0.0, 0.0) Pneumonia 1 (0.0) (0.0, 0.0) 4 (0.0) (0.0, 0.1) Appendicitis perforated 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Cellulitis 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Diverticulitis 2 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Pyelonephritis 2 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Osteomyelitis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Peritoneal abscess 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Peritonitis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Pharyngitis streptococcal 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Sepsis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Suspected COVID-19 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Urinary tract infection 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Urosepsis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) INJURY, POISONING AND PROCEDURAL 2 (0.0) (0.0, 0.0) 2 (0.0) (0.0, 0.0) COMPLICATIONS Forearm fracture 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Head injury 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hip fracture 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Skin laceration 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) INVESTIGATIONS 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Cardiac stress test abnormal 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hepatic enzyme increased 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) METABOLISM AND NUTRITION 3 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) DISORDERS Fluid retention 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hyperglycaemia 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hypoglycaemia 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Hypokalaemia 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) MUSCULOSKELETAL AND 1 (0.0) (0.0, 0.0) 2 (0.0) (0.0, 0.0) CONNECTIVE TISSUE DISORDERS Musculoskeletal chest pain 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Osteoarthritis 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Osteochondritis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) NEOPLASMS BENIGN, MALIGNANT 3 (0.0) (0.0, 0.0) 2 (0.0) (0.0, 0.0) AND UNSPECIFIED (INCL CYSTS AND POLYPS) Adenocarcinoma gastric 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Breast cancer 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Metastases to central nervous 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) system Uterine leiomyoma 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Vaginal neoplasm 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) NERVOUS SYSTEM DISORDERS 5 (0.0) (0.0, 0.1) 7 (0.0) (0.0, 0.1) Syncope 0 (0.0, 0.0) 3 (0.0) (0.0, 0.0) Subarachnoid haemorrhage 2 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Cerebrovascular accident 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Diplegia 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Haemorrhagic stroke 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Ischaemic stroke 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Loss of consciousness 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Paraesthesia 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Transient ischaemic attack 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) PREGNANCY, PUERPERIUM AND 0 (0.0, 0.0) 3 (0.0) (0.0, 0.0) PERINATAL CONDITIONS Pregnancy 0 (0.0, 0.0) 2 (0.0) (0.0, 0.0) Abortion spontaneous incomplete 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) PSYCHIATRIC DISORDERS 3 (0.0) (0.0, 0.0) 3 (0.0) (0.0, 0.0) Suicidal ideation 0 (0.0, 0.0) 2 (0.0) (0.0, 0.0) Bipolar disorder 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Mental disorder 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Mental status changes 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Psychotic disorder 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) RENAL AND URINARY DISORDERS 4 (0.0) (0.0, 0.1) 0 (0.0, 0.0) Nephrolithiasis 2 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Acute kidney injury 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Renal colic 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) RESPIRATORY, THORACIC AND 2 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) MEDIASTINAL DISORDERS Acute respiratory failure 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Pneumonitis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Pulmonary embolism 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) SURGICAL AND MEDICAL 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) PROCEDURES Breast conserving surgery 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) UNCODED TERM 3 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) INVASIVE DUCTAL CARCINOMA 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) STAGE 1B, LEFT BREAST@@ LEFT OVARIAN CYST, BENIGN 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) TUMOR@@ MRSA INFECTION RIGHT 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) STUMP@@ PROLAPSED UTERUS@@ 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) STEMI: ST ELEVATION 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) MYOCARDIAL INFARCTION@@ VASCULAR DISORDERS 4 (0.0) (0.0, 0.1) 2 (0.0) (0.0, 0.0) Deep vein thrombosis 1 (0.0) (0.0, 0.0) 1 (0.0) (0.0, 0.0) Arteriosclerosis 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hypertension 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Hypertensive urgency 1 (0.0) (0.0, 0.0) 0 (0.0, 0.0) Orthostatic hypotension 0 (0.0, 0.0) 1 (0.0) (0.0, 0.0) Note: MedDRA (v23.0) coding dictionary applied. .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified event. For any event, n = number of subjects reporting at least 1 occurrence of any event. .sup.cExact 2-sided CI based on the Clopper and Pearson method.

(1569) Serious Adverse Event NarrativesPhase 2/3

(1570) Narratives for the Phase 3 participants who reported SAEs assessed as related to study intervention by the investigator who completed their visit at 1 month after Dose 2 and through the data cutoff date (6 Oct. 2020) were provided.

(1571) Safety-Related Participant WithdrawalsPhase 2/3

(1572) First 6610 ParticipantsPhase 2/3

(1573) From Dose 1 to 1 month after Dose 2, 6 (0.2%) participants in the BNT162b2 group and 5 (0.2%) participants in the placebo group were withdrawn because of AEs (Table 18), and no additional withdrawals were reported for these participants from 1 month after Dose 2 until the data cutoff date.

(1574) Withdrawals of Interest in the BNT162b2 Group:

(1575) Two participants were withdrawn because of AEs that were assessed by the investigator as related to study intervention. One participant in the younger age group had an AE of myalgia 8 days after Dose 1 which was recovering at the time of the data cutoff. One participant in the older age group had an AE of pruritus and an AE of tachycardia 2 days after Dose 1; both events had a duration of 1 day and both were severe.

(1576) Three participants each had an SAE and were withdrawn from the study: younger age group (gastric adenocarcinoma), and in the older age group (coronary artery disease and coronary artery dissection).

(1577) Withdrawals of Interest in the Placebo Group:

(1578) One participant (younger age group) was withdrawn because of an AE of allergy to vaccine (study intervention) and an AE of erythematous rash 2 days after Dose 1; both AEs resolved 18 days later, and both were assessed by the investigator as related to study intervention.

(1579) One participant in the older group had an SAE (coronary artery occlusion) assessed by the investigator as not related and was withdrawn from the study.

(1580) One participant in the older group was withdrawn from the study because of an AE of urticaria 10 days after Dose 1. The event resolved on the same day and was assessed by the investigator as not related to study intervention.

(1581) TABLE-US-00036 TABLE 18 Number (%) of Subjects Withdrawn Because of Adverse Events From Dose 1 to 1 Month After Dose 2, by System Organ Class and Preferred Term - ~6000 Subjects for Phase 2/3 Analysis - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo System Organ Class (N.sup.a = 3314) (N.sup.a = 3296) Preferred Term n.sup.b (%) (95% CI.sup.c) n.sup.b (%) (95% CI.sup.c) Any event 6 (0.2) (0.1, 0.4) 5 (0.2) (0.0, 0.4) CARDIAC DISORDERS 3 (0.1) (0.0, 0.3) 2 (0.1) (0.0, 0.2) Atrial fibrillation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Coronary artery disease 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery dissection 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Coronary artery occlusion 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Left ventricular hypertrophy 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Tachycardia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) GASTROINTESTINAL DISORDERS 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Diverticular perforation 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) IMMUNE SYSTEM DISORDERS 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Allergy to vaccine 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) INJURY, POISONING AND 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) PROCEDURAL COMPLICATIONS Ankle fracture 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Fall 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) MUSCULOSKELETAL AND 2 (0.1) (0.0, 0.2) 0 (0.0, 0.1) CONNECTIVE TISSUE DISORDERS Muscular weakness 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Myalgia 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NEOPLASMS BENIGN, 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) Adenocarcinoma gastric 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) NERVOUS SYSTEM DISORDERS 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Transient ischaemic attack 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) SKIN AND SUBCUTANEOUS 1 (0.0) (0.0, 0.2) 2 (0.1) (0.0, 0.2) TISSUE DISORDERS Pruritus 1 (0.0) (0.0, 0.2) 0 (0.0, 0.1) Rash erythematous 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Urticaria 0 (0.0, 0.1) 1 (0.0) (0.0, 0.2) Note: MedDRA (v23.0) coding dictionary applied. .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects reporting at least 1 occurrence of the specified event. For any event, n = number of subjects reporting at least 1 occurrence of any event. .sup.cExact 2-sided CI based on the Clopper and Pearson method.

(1582) All ParticipantsPhase 2/3

(1583) From Dose 1 to the data cutoff date, 18 (0.1%) participants in the BNT162b2 group and 14 (0.1%) participants in the placebo group were withdrawn because of AEs. In addition to withdrawals discussed for the first 6610 participants, other withdrawals included:

(1584) One participant in the younger BNT162b2 group had an SAE of lymphadenopathy (right axilla) 13 days after Dose 1 assessed by the investigator as related to study intervention and was withdrawn, which was not resolved at the time of the data cutoff.

(1585) Three participants in the younger age group (1 BNT162b2 and 2 placebo) became pregnant after Dose 1 and were withdrawn.

(1586) One participant in the younger placebo group had a positive pregnancy test (exposure during pregnancy) 39 days after Dose 1 and was withdrawn.

(1587) Narratives of Safety-Related Participant WithdrawalsPhase 2/3

(1588) Narratives for the Phase 2/3 participants with any AEs leading to withdrawal from the study through the data cutoff date (6 Oct. 2020) were provided.

(1589) Other Significant Adverse EventsPhase 2/3

(1590) AEs of special interest were not defined for Phase 2/3 of this study; however, targeted medical events were monitored throughout the study.

(1591) Other Safety AssessmentsPhase 2/3

(1592) Severe COVID-19 IllnessPhase 2/3

(1593) At the time of the efficacy interim analysis cutoff date of 4 Nov. 2020, all 7 severe COVID-19 cases were reported in the placebo group.

(1594) PregnancyPhase 2/3

(1595) Pregnancy was reported in 5 Phase 3 participants through the data cutoff date of 6 Oct. 2020: in 1 participant in the BNT162b2 group and 4 participants in the placebo group. Incomplete spontaneous abortion occurred in 1 participant in the placebo group.

(1596) Narratives for pregnancy were provided.

(1597) Analysis and Discussion of Deaths, Serious Adverse Events, Safety-Related Participant Withdrawals, and Other Significant Adverse EventsPhase 2/3

(1598) Up to the data cutoff date of 6 Oct. 2020, the numbers of SAEs were similar in the BNT162b2 group (63 [0.3%]) and in the placebo group (49 [0.3%]). Two participants in the BNT162b2 group reported SAEs that were assessed by the investigator as related to study intervention.

(1599) Few participants in the BNT162b2 group (18 [0.1%]) and in the placebo group (14 [0.1%]) were withdrawn because of AEs.

(1600) There were 3 deaths (1 in the BNT162b2 group and 2 in the placebo group); none of the deaths were assessed by the investigator as related to study intervention.

(1601) Phase 2/3 Safety Conclusions

(1602) Across age groups, local reactions were generally similar in frequency after each dose, and systemic events generally increased in frequency and severity after Dose 2 compared to Dose 1. Local and systemic reactogenicity events were well-tolerated and short-lived (median durations of 1.0 to 2.0 days).

(1603) Reactogenicity events after each dose of BNT162b2 in older adults were generally milder and less frequent than those observed in younger adults. The majority of reactogenicity events were mild or moderate in severity. No Grade 4 events were reported other than fever in 1 participant in the BNT162b2 group that began 1 day after Dose 2 and lasted 1 day.

(1604) The reactogenicity and AE profile after BNT162b2 30 ?g evaluated in 6610 participants was consistent with the safety profile observed after BNT162b2 30 ?g in Phase 1 and Phase 2.

(1605) AEs were reported in 16.8% of participants in the BNT162b2 group, and most AEs were mild or moderate in severity. At the time of the data cutoff date, the number of participants with AEs in the BNT162b2 group was greater as compared with the placebo group (7.9%), which upon analysis, was attributed to reactogenicity events reported as AEs within 7 days after each dose.

(1606) At the time of the data cutoff date, there were 2 related SAEs in the BNT162b2 group (lymphadenopathy and shoulder injury related to vaccine administration (SIRVA, erroneously administered into or near the shoulder joint capsule), and there were 6 discontinuations because of related AEs. There was 1 death in the BNT162b2 group (arteriosclerosis) and 2 deaths in the placebo group that were assessed as not related to study intervention.

(1607) Overall, BNT162b2 at 30 ?g was well tolerated when measured up to 1 month after Dose 2 following dosing regimen.

Example 16: Conclusion of Phase 3 Study of COVID-19 Vaccine, Meeting all Primary Efficacy Endpoints

(1608) After conducting the final efficacy analysis in the ongoing Phase 3 study, the mRNA-based COVID-19 vaccine, BNT162b2, met all of the study's primary efficacy endpoints. Analysis of the data indicated a vaccine efficacy rate of 95% (p<0.0001) in participants without prior SARS-CoV-2 infection (first primary objective) and also in participants with and without prior SARS-CoV-2 infection (second primary objective), in each case measured from 28 days after the first dose, 7 days after the second dose. The first primary objective analysis is based on 170 cases of COVID-19 of which 162 cases of COVID-19 were observed in the placebo group versus 8 cases in the BNT162b2 group. Efficacy was consistent across age, gender, race and ethnicity demographics. The observed efficacy in adults over 65 years of age was over 94%.

(1609) There were 10 severe cases of COVID-19 observed in the trial, with nine of the cases occurring in the placebo group and one in the BNT162b2 vaccinated group. No serious safety concerns related to the vaccine were reported. A review of unblinded reactogenicity data from the final analysis which consisted of a randomized subset of at least 8,000 participants 18 years and older in the Phase 2/3 study demonstrated that the vaccine was well tolerated, with most solicited adverse events resolving shortly after vaccination. The only Grade 3 (severe) solicited adverse events greater than or equal to 2% in frequency after the first or second dose were fatigue at 3.8% and headache at 2.0% following dose 2. Consistent with earlier shared results, older adults tended to report fewer and milder solicited adverse events following vaccination. The local reactogenicity profile among SARS-CoV-2 positive participants was consistent with that of the overall reactogenicity subset; similarly, on comparison of AE data with that of the All subjects, there is no indication of a worse safety profile in baseline positive participants. Indeed, there is no indication of a worse safety profile in baseline positive participants; therefore, BNT162b2 can be used irrespective of COVID-19 history or SARS-CoV-2 serological status.

(1610) In addition, the safety milestone required by the U.S. Food and Drug Administration (FDA) for Emergency Use Authorization (EUA) has been achieved.

(1611) The results of this first global trial to reach the final efficacy analysis mark indicated that a high rate of protection against COVID-19 can be achieved very fast after the first 30 ?g dose, underscoring the potential of BNT162 to provide early protection.

(1612) In summary: Primary efficacy analysis demonstrated BNT162b2 to be 95% effective against COVID-19 beginning 28 days after the first dose; 170 confirmed cases of COVID-19 were evaluated, with 162 observed in the placebo group versus 8 in the vaccine group Efficacy was consistent across age, gender, race and ethnicity demographics; observed efficacy in adults over 65 years of age was over 94% Safety data milestone required by U.S. Food and Drug Administration (FDA) for Emergency Use Authorization (EUA) has been achieved Data demonstrates vaccine was well tolerated across all populations with over 43,000 participants enrolled; no serious safety concerns observed; the only Grade 3 adverse event greater than 2% in frequency was fatigue at 3.8% and headache at 2.0%

Example 17: All Confirmed Cases of COVID-19 After Dose 1

(1613) A number of confirmed cases of COVID-19 are not captured in the analyses of the first primary endpoint for the evaluable efficacy population because they occurred less than 7 days after Dose 2, or because they occurred in participants who were excluded from the evaluable efficacy population or who had evidence of infection before or during the vaccination regimen.

(1614) All reports of COVID-19 with onset at any time after Dose 1 are accounted for in Table 19, which provides a summary of cases for all participants in the Dose 1 all-available efficacy (modified intention-to-treat) population, regardless of evidence of infection before or during the vaccination regimen. Among these participants, 50 cases of COVID-19 occurred after Dose 1 in the BNT162b2 group compared to 275 cases in the placebo group (Table 19). Notably, in the BNT162b2 group, most cases occurred before Dose 2. The estimated VE against confirmed COVID-19 occurring after Dose 1 was 82% (2-sided 95% CI: 75.6%, 86.9%), with an estimated VE of 52.4% (2-sided 95% CI: 29.5%, 68.4%) against confirmed COVID-19 occurring after Dose 1 but before Dose 2.

(1615) TABLE-US-00037 TABLE 19 COVID-19 Occurrence After Dose 1 - Dose 1 All-Available Efficacy Population BNT162b2 (30 ?g) Placebo (N.sup.a = 21669) (N.sup.a = 21686) Efficacy Endpoint n.sup.b n.sup.b COVID-19 occurrence after Dose 1 50 275 After Dose 1 to before Dose 2 39 82 Dose 2 to 7 days after Dose 2 2 21 ?7 days after Dose 2 9 172 .sup.aN = number of subjects in the specified group. .sup.bn1 = Number of subjects meeting the endpoint definition.

(1616) The early onset of protection is readily apparent in FIG. 100, which displays cumulative incidence for the first COVID-19 occurrence after Dose 1 among all vaccinated participants based on Dose 1 all-available efficacy (modified intention-to-treat) population. Disease onset appears to track together for BNT162b2 and placebo until approximately 14 days after Dose 1, at which point the curves diverge, with cases steadily accumulating in the placebo group, while remaining virtually flat in the BNT162b2 group.

(1617) The cumulative incidence of COVID-19 cases over time among placebo and vaccine recipients begins to diverge by 14 days after Dose 1, approximately 9 days after the estimated median incubation period of 5 days, indicating the early onset of a partially protective effect of immunization. In the interval between Dose 1 and Dose 2, the observed vaccine efficacy was 52%, and in the first 7 days after Dose 2, it was 91%, reaching full efficacy for COVID-19 with onset at least 7 days after Dose 2.

Example 18: Secondary Efficacy ResultsFinal Analysis

(1618) Vaccine Efficacy for COVID-19 Occurring Within 14 Days After Dose 2Final Analysis Participants Without Evidence of Infection Before Vaccination

(1619) For this efficacy endpoint, participants with positive or unknown NAAT results at any illness visit prior to 14 days after Dose 2 were not included in the evaluation for efficacy.

(1620) Among participants without evidence of SARS-CoV-2 infection before and during vaccination regimen, VE against confirmed COVID-19 occurring at least 14 days after Dose 2 was 94.2%, with 8 and 139 cases in the BNT162b2 and placebo groups respectively. The posterior probability of >99.99% for the true VE greater than 30% met the prespecified success criterion of >98.6% for this endpoint. The 95% credible interval for the vaccine efficacy was 88.7% to 97.2%, indicating that the true VE is at least 88.7% with a 97.5% probability given the available data.

(1621) Participants With or Without Evidence of Infection Before Vaccination

(1622) Among participants with or without evidence of SARS-CoV-2 infection before and during vaccination regimen, VE against confirmed COVID-19 occurring at least 14 days after Dose 2 was 94.4%, with 8 and 144 cases in the BNT162b2 and placebo groups respectively. The posterior probability of >99.99% for the true VE greater than 30% met the prespecified success criterion of >98.6% for this endpoint. The 95% credible interval for the vaccine efficacy was 89.1% to 97.3%, indicating that the true VE is at least 89.1% with a 97.5% probability given the available data.

(1623) Vaccine Efficacy for Severe COVID-19 CasesFinal Analysis

(1624) Efficacy Against Severe COVID-19 (?7 Days After Dose 2)

(1625) Participants Without Evidence of Infection Before and During Vaccination Regimen

(1626) For this efficacy endpoint, participants with positive or unknown NAAT results at any illness visit prior to 7 days after Dose 2 were not included in the evaluation for efficacy. Among participants without evidence of severe SARS-CoV-2 infection before and during vaccination regimen, the estimated VE against severe COVID-19 occurring at least 7 days after Dose 2 was 66.4%, with 1 and 3 cases in the BNT162b2 and placebo groups respectively. The posterior probability for the true vaccine efficacy greater than 30% is 74.29%, which did not meet the prespecified success criterion of >98.6% for this endpoint due to the small number of severe cases observed after Dose 2 in the study.

(1627) Participants With and Without Evidence of Infection Before and During Vaccination Regimen

(1628) Among participants with or without evidence of severe SARS-CoV-2 infection prior to 7 days after Dose 2, VE against severe COVID-19 occurring at least 7 days after Dose 2 was 66.3%, with 1 and 3 cases in the BNT162b2 and placebo groups respectively. The posterior probability for the true vaccine efficacy greater than 30% is 74.19%.

(1629) All Confirmed Cases of Severe COVID-19 After Dose 1All-Available Population

(1630) Among participants in the all-available efficacy population, 1 case of COVID-19 occurred after Dose 1 in the BNT162b2 group compared to 9 cases in the placebo group. The estimated VE against severe COVID-19 occurring after Dose 1 was 88.9% (2-sided 95% CI: 20.1%, 99.7%), with an estimated VE of 75.0% against severe COVID-19 occurring at least 7 days after Dose 2.

(1631) Efficacy Against Severe COVID-19 (214 Days After Dose 2)

(1632) Participants Without Evidence of Infection Before and During Vaccination Regimen (14 Days)Severe

(1633) Among participants without evidence of severe SARS-CoV-2 infection before and during vaccination regimen, the estimated VE against severe COVID-19 occurring at least 14 days after Dose 2 was 66.4%, with 1 and 3 cases in the BNT162b2 and placebo groups respectively. The posterior probability for the true vaccine efficacy greater than 30% is 74.32%.

(1634) Participants With or Without Evidence of Infection Before and During Vaccination Regimen (14 Days)Severe

(1635) Among participants with or without evidence of severe SARS-CoV-2 infection before and during vaccination phase, VE against severe COVID-19 occurring at least 14 days after Dose 2 was 66.3%, with 1 and 3 cases in the BNT162b2 and placebo groups respectively. The posterior probability for the true vaccine efficacy greater than 30% is 74.18%.

(1636) Vaccine Efficacy for COVID-19 Cases per CDC DefinitionFinal Analysis

(1637) Efficacy Against COVID-19 Based on CDC-Defined Symptoms (?7 Days After Dose 2) Participants Without Evidence of Infection Before and During Vaccination RegimenCDC Defined7 Days

(1638) Among participants without evidence of SARS-CoV-2 infection before and during vaccination regimen, VE against CDC-defined COVID-19 occurring at least 7 days after Dose 2 was 95.1% (2-sided 95% CI: 90.2%, 97.9%), with 8 and 165 cases in the BNT162b2 and placebo groups, respectively.

(1639) Participants With and Without Evidence of Infection Before and During Vaccination RegimenCDC Defined7 Days

(1640) Among participants with and without evidence of SARS-CoV-2 infection before and during vaccination regimen, VE against CDC-defined COVID-19 occurring at least 7 days after Dose 2 was 94.7% (2-sided 95% CI: 89.8%-97.6%), with 9 and 172 cases in the BNT162b2 and placebo groups, respectively.

(1641) Efficacy Against COVID-19 Based on CDC-Defined Symptoms (?14 Days After Dose 2)

(1642) Among participants without and with or without evidence of SARS-CoV-2 infection before and during vaccination regimen, VE against CDC-defined COVID-19 occurring at least 14 days after Dose 2 were similar to those occurring at least 7 days after Dose 2.

Example 19: Efficacy ConclusionsFinal Analysis

(1643) In the final efficacy analysis, among participants without evidence of SARS-CoV-2 infection before and during vaccination regimen, VE against confirmed COVID-19 occurring at least 7 days after Dose 2 was 95.0%, with 8 COVID-19 cases in the BNT162b2 group compared to 162 COVID-19 cases in the placebo group. The 95% credible interval for the vaccine efficacy was 90.3% to 97.6%. For the second primary endpoint, VE against confirmed COVID-19 occurring at least 7 days after Dose 2 in participants with and without evidence of SARS-CoV-2 infection before and during vaccination regimen was 94.6%, with 9 and 169 cases in the BNT162b2 and placebo groups respectively. The posterior probability of >99.99% for the true VE greater than 30% met the prespecified success criterion of >98.6% for this endpoint. The 95% credible interval for the vaccine efficacy was 89.9% to 97.3%, indicating that the true VE is at least 89.9% with a 97.5% probability given the available data

(1644) Observed VE was very high for the first primary efficacy endpoint across subgroups of age, sex, race/ethnicity, and country, as VE was >93% in all subgroups, with the exception of all others race group (89.3% VE) and Brazil (87.7% VE).

(1645) A total of 10 cases of severe COVID-19 occurred after Dose 1, 1 in the BNT162b2 group, compared with 9 cases in the placebo group.

(1646) Among all participants (regardless of evidence of infection before or during the vaccination regimen) 50 cases of COVID-19 occurred after Dose 1 in the BNT162b2 group compared with 275 cases in the placebo group, indicating an estimated VE of 82% (95% CI: 75.6%, 86.9%) against confirmed COVID-19 occurring after Dose 1.

(1647) The early onset of protection is readily apparent from cumulative incidence curves, which show that disease onset tracks conjointly for BNT162b2 and placebo until approximately 14 days after Dose 1, at which point the curves diverge, with cases steadily accumulating in the placebo group, while remaining virtually flat after BNT162b2.

(1648) In conclusion, the final efficacy results show that BNT162b2 at 30 ?g provided protection against COVID-19 in participants who had no evidence of prior infection with SARS-CoV-2, including across demographic subgroups, with severe cases observed predominantly in the placebo group.

(1649) Details of demographic populations assessed are presented below in Tables 20 and 21.

(1650) TABLE-US-00038 TABLE 20 Demographic Characteristics - ~38000 Subjects for Phase 2/3 Analysis - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo Total (N.sup.a = 18860) (N.sup.a = 18846) (N.sup.a = 37706) n.sup.b (%) n.sup.b (%) n.sup.b (%) Sex Male 9639 (51.1) 9436 (50.1) 19075 (50.6) Female 9221 (48.9) 9410 (49.9) 18631 (49.4) Race White 15636 (82.9) 15630 (82.9) 31266 (82.9) Black or African American 1729 (9.2) 1763 (9.4) 3492 (9.3) American Indian or Alaska 102 (0.5) 99 (0.5) 201 (0.5) native Asian 801 (4.2) 807 (4.3) 1608 (4.3) Native Hawaiian or other 50 (0.3) 26 (0.1) 76 (0.2) Pacific Islander Multiracial 449 (2.4) 406 (2.2) 855 (2.3) Not reported 93 (0.5) 115 (0.6) 208 (0.6) Ethnicity Hispanic/Latino 5266 (27.9) 5277 (28.0) 10543 (28.0) Non-Hispanic/non-Latino 13482 (71.5) 13459 (71.4) 26941 (71.5) Not reported 112 (0.6) 110 (0.6) 222 (0.6) Country Argentina 2883 (15.3) 2881 (15.3) 5764 (15.3) Brazil 1145 (6.1) 1139 (6.0) 2284 (6.1) South Africa 372 (2.0) 372 (2.0) 744 (2.0) USA 14460 (76.7) 14454 (76.7) 28914 (76.7) Age group 16-55 Years 10889 (57.7) 10896 (57.8) 21785 (57.8) >55 Years 7971 (42.3) 7950 (42.2) 15921 (42.2) Age at vaccination (years) Mean (SD) 50.5 (15.65) 50.3 (15.72) 50.4 (15.68) Median 52.0 52.0 52.0 Min, max (16, 89) (16, 91) (16, 91) Body mass index (BMI) Underweight (<18.5 kg/m.sup.2) 201 (1.1) 235 (1.2) 436 (1.2) Normal weight (?18.5 5517 (29.3) 5460 (29.0) 10977 (29.1) kg/m.sup.2-24.9 kg/m.sup.2) Overweight (?25.0 kg/m.sup.2- 6578 (34.9) 6481 (34.4) 13059 (34.6) 29.9 kg/m.sup.2) Obese (?30.0 kg/m.sup.2) 6556 (34.8) 6662 (35.3) 13218 (35.1) Missing 8 (0.0) 8 (0.0) 16 (0.0) Note: HIV-positive subjects are included in this summary but not included in the analyses of the overall study objectives. .sup.aN = number of subjects in the specified group, or the total sample. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects with the specified characteristic.

(1651) TABLE-US-00039 TABLE 21 Baseline Charlson Comorbidities - ~38000 Subjects for Phase 2/3 Analysis - Safety Population Vaccine Group (as Administered) BNT162b2 (30 ?g) Placebo Total Charlson Comorbidity (N.sup.a = 18860) (N.sup.a = 18846) (N.sup.a = 37706) Index Category n.sup.b (%) n.sup.b (%) n.sup.b (%) Subjects with any 3934 (20.9) 3809 (20.2) 7743 (20.5) Charlson comorbidity AIDS/HIV 59 (0.3) 62 (0.3) 121 (0.3) Any Malignancy 733 (3.9) 662 (3.5) 1395 (3.7) Cerebrovascular Disease 195 (1.0) 166 (0.9) 361 (1.0) Chronic Pulmonary Disease 1478 (7.8) 1453 (7.7) 2931 (7.8) Congestive Heart Failure 88 (0.5) 83 (0.4) 171 (0.5) Dementia 7 (0.0) 11 (0.1) 18 (0.0) Diabetes With Chronic 99 (0.5) 113 (0.6) 212 (0.6) Complication Diabetes Without Chronic 1473 (7.8) 1478 (7.8) 2951 (7.8) Complication Hemiplegia or Paraplegia 13 (0.1) 21 (0.1) 34 (0.1) Leukemia 12 (0.1) 10 (0.1) 22 (0.1) Lymphoma 22 (0.1) 32 (0.2) 54 (0.1) Metastatic Solid Tumor 4 (0.0) 3 (0.0) 7 (0.0) Mild Liver Disease 125 (0.7) 89 (0.5) 214 (0.6) Moderate or Severe Liver 1 (0.0) 2 (0.0) 3 (0.0) Disease Myocardial Infarction 194 (1.0) 188 (1.0) 382 (1.0) Peptic Ulcer Disease 52 (0.3) 71 (0.4) 123 (0.3) Peripheral Vascular 124 (0.7) 117 (0.6) 241 (0.6) Disease Renal Disease 123 (0.7) 133 (0.7) 256 (0.7) Rheumatic Disease 62 (0.3) 56 (0.3) 118 (0.3) Note: MedDRA (v23.1) coding dictionary applied. Note: HIV-positive subjects are included in this summary but not included in the analyses of the overall study objectives. .sup.aN = number of subjects in the specified group. This value is the denominator for the percentage calculations. .sup.bn = Number of subjects with the specified characteristic. Subjects with multiple occurrences within each category are counted only once. For Subjects with any Charlson comorbidity, n = number of subjects reporting at least 1 occurrence of any Charlson comorbidity.

Example 20: Certain Observations Regarding Response of Young Adolescent Populations to Immunization with BNT162b2

(1652) In clinical trials described in Examples 13-19, the following were observed in younger adolescent populations.

(1653) Local Reactions in Younger Adolescents

(1654) Younger adolescents 12 to 15 years of age (N=100; 49 in the BNT162b2 group and 51 in the placebo group) contributed preliminary data to the reactogenicity subset and were analyzed separately. In this age group, pain at the injection site was the most frequently prompted local reaction in the BNT162b2 group, reported in 71.4% of participants compared to 17.6% in the placebo group after Dose 1. The incidence of pain was reduced in the BNT162b2 group and placebo group after Dose 2 (down to 58.7% vs 8.7%). Redness was reported in 1 participant in the BNT162b2 group after Dose 1 and in 2 participants after Dose 2, and in none in the placebo group after either dose. Swelling was reported in 2 participants in the BNT162b2 group after Dose 1 and in 3 participants after Dose 2, and in 1 in the placebo group after Dose 1 and none after Dose 2. Most local reactions were mild to moderate in severity. Two severe reactions were reported, both in the BNT162b2 group: severe redness and severe pain at the injection site.

(1655) Systemic Reactions in Younger Adolescents

(1656) Younger adolescents 12 to 15 years of age (N=100; 49 in the BNT162b2 group and 51 in the placebo group) contributed preliminary data to the reactogenicity subset and were analyzed separately. Most systemic events (other than vomiting and diarrhea, which had low incidences across groups) were reported at higher incidence in the BNT162b2 group than in the placebo group. However, there was no clear trend for increasing incidence or severity after Dose 1 compared to after Dose 2. In this age group, the most frequent prompted systemic events after Dose 1 compared to Dose 2 were (Dose 1 vs Dose 2): fatigue: BNT162b2 (49.0% vs 50.0%) compared to placebo (25.5% vs 6.5%) headache: BNT162b2 (42.9% vs 45.7%) compared to placebo (35.3% vs 21.7%) muscle pain: BNT162b2 (22.4% vs 30.4%) compared to placebo (13.7% vs 4.3%) chills: BNT162b2 (30.6% vs 28.3%) compared to placebo (7.8% vs 8.7%) joint pain: BNT162b2 (12.2% vs 17.4%) compared to placebo (9.8% vs 6.5%) fever: BNT162b2 (14.3% vs 19.6%) compared to placebo (0% vs 0%) vomiting: reported at similar frequencies in both groups and similar after each dose diarrhea: reported at similar frequencies in both groups and similar after each dose.

(1657) Most systemic events in younger adolescents were mild to moderate in severity. Severe events were relatively infrequent in both groups, occurring in no more than 1 or 2 participants after either dose.

(1658) Antipyretic/pain medication use in the younger adolescent group was modestly increased after Dose 2 compared to Dose 1 (30.6% vs 41.3%) and was greater than use in the placebo group (9.8% vs 13%).

(1659) In summary, as observed in older age groups (e.g., greater than 16 years of age such as 16-85 years of age), reactogenicity was mostly mild to moderate and short-lived after dosing for younger adolescents 12 to 15 years of age, and the adverse event profile did not suggest any serious safety concerns.

(1660) Examples 21-24 below further confirm that neutralizing antibody responses and/or cell-mediated immune responses can be achieved with mRNA compositions described herein (including, e.g., BNT162b1 and BNT162b2) administered according to various dosing regimens described herein, including for example dosing regimens that involve administration of one or more doses lower than 30 ug, including, e.g., 20 ug, 10 ug, 3 ug, etc. Among other things, data provided in these Examples 21-24 further confirm induction of an immune response (e.g., as described herein) against SARS-CoV-2 upon administration of certain mRNA compositions described herein (including, e.g., BNT162b1 and BNT162b2) with one or more doses of 3 ug or above.

(1661) Those of ordinary skill in the art, reading the present disclosure, will appreciate that it demonstrates among other things, that administration of various mRNA compositions described herein can induce immune responses that include neutralizing antibodies against SARS-CoV-2; it furthermore confirms that certain such compositions (i.e., that induce neutralizing antibodies and/or that induce cell-mediated immune response such as T cell response) can induce protective immune responses that reduce SARS-CoV-2 infection and/or incidence of COVID19 sickness in organisms, specifically including primate organisms in which they have induced such neutralizing antibodies and/or cell-mediated immune response and furthermore including humans. In some embodiments, it also confirms that certain such compositions (e.g., described herein) do not significantly induce vaccine-mediated disease enhancement, for example, as evidenced by only one of the 10 cases of severe COVID-19 that were observed after a first dose. Indeed, the present disclosure documents that such compositions can effectively vaccinate humans (see, for example, clinical trial results included in Examples 13-19), for example, against severe COVID-19 disease.

Example 21: Immunogenicity Studies for Functional Antibody Responses

(1662) In clinical trials described in Example 7, the following were observed in healthy younger adults (18-55 years of age) and older adults (56-85 years of age) after BNT162b1 or BNT162b2 vaccination. Two doses, of 1 ?g, 3 ?g, 10 ?g, 20 ?g, or 30 ?g were administered 21 days apart in younger adults. Two doses of 20 ?g was administered 21 days apart in older adults. Functional antibody data for younger adult cohorts was determined up until Day 50 after an initial dose was administered for dose groups 1 ?g and 3 ?g, and up until Day 85 for dose groups 10, 20, and 30 ?g. For BNT162b2-dosed older adults, data is available until Day 29 after an initial dose was administered.

(1663) For virus neutralizing antibody GMTs (neutralizing GMTs) and 95% confidence intervals for participants aged 18 to 55 years after dosing with BNT162b1, see FIG. 40.

(1664) For virus neutralizing antibody GMTs (neutralizing GMTs) and 95% confidence intervals for younger participants aged 18 to 55 yrs and older participants aged 56 to 85 yrs after dosing with BNT162b2, see FIG. 101 (50% neutralizing titer).

(1665) Geometric means fold increase (GMFI) from baseline in functional antibody titer data are shown in FIG. 102 (BNT162b1) and FIG. 103 (BNT162b2).

(1666) Participants dosed with BNT162b1 showed a strong dose-dependent antibody response. On Day 22, at 21 days after dose 1, virus neutralising antibody GMTs had increased in a dose-dependent manner for the 1, 10, 30 and 50 ag dose groups. At Day 29 (7 days after Dose 2), neutralising GMTs showed a strong, dose level dependent booster response. In the single, 60 ag dose group, neutralising GMTs remained at a lower level, indicating a booster dose is necessary to increase functional antibody titers.

(1667) On Day 43 (21 days after Dose 2 of BNT162b1), neutralising GMTs decreased (with the exception of the 1 ?g dose level). Day 43 virus neutralising GMTs were 0.7-fold (1 ?g) to 3.6-fold (50 ?g) those of a COVID-19 HCS panel.

(1668) The COVID-19 HCS panel is comprised of 38 human COVID-19 HCS sera drawn from individuals aged 18 to 85 yrs, at least 14 d after confirmed diagnosis, and at a time when the individuals were asymptomatic. The serum donors predominantly had symptomatic infections (35/38), and one had been hospitalized. The sera were obtained from Sanguine Biosciences (Sherman Oaks, CA), the MT Group (Van Nuys, CA), and Pfizer Occupational Health and Wellness (Pearl River, NY).

(1669) Participants dosed with BNT162b2 showed a strong antibody response induced by BNT162b2. Virus neutralizing GMTs were detected at 21 days after Dose 1 (Day 22) and had increased substantially in younger participants (aged 18 to 55 years) immunized with ?3 ag of BNT162b2, and older participants (aged 56-85 years) immunized with 20 ag BNT162b2 by 7 days after Dose 2 (Day 29). Day 29 virus neutralizing GMTs were comparable between the younger and older adult 20 ?g dose level cohorts. The lowest treated dose of 1 ag BNT162b2 elicited a minimal neutralising response in participants aged 18 to 55 years.

(1670) On Day 43 (21 days after Dose two of BNT162b2), virus neutralising GMTs in the younger adult cohorts decreased for the 3, 20, and 30 ag dose levels. Thereafter, neutralising GMTs in between Days 29 and 43, neutralizing GMTs remained stable up to Day 85 (63 days after Dose two) for younger adult dose groups 10, 20 and 30 ?g and were 1.3-fold to 1.9-fold those of a COVID-19 HCS panel.

(1671) Seroconversion in this context is defined as a minimum of a 4-fold increase of antibody GMT as compared to baseline. The frequency of participants with seroconversion is shown in FIGS. 104 (BNT162b1) and 105 (BNT162b2).

(1672) All participants dosed with Dose 1 at 30 ?g BNT162b1 or BNT162b2 seroconverted either by 7 days or 21 days after Dose 2 (Day 29 or Day 43). All participants dosed with 30 ?g BNT162b2 remained seropositive throughout the follow-up until Day 85.

Example 22: Immunogenicity Studies for Binding Antibody Concentrations

(1673) In clinical trials described in Example 7, the following were observed in healthy younger adults (18-55 years of age) and older adults (56-85 years of age) after BNT162b1 or BNT162b2 vaccination. Binding antibody concentration data is available up until Day 43 for BNT162b1-dosed younger participants aged 18 to 55 yrs dosed with 1, 10, 30, 50, or 60 ?g on Days 1 (all dose levels) and 22 (all dose levels except 60 ?g) (n=12 per group).

(1674) For BNT162b2-dosed participants, data is available for younger participants aged 18 to 55 yrs dosed with 1, 3, 10, 20, or 30 ?g, and older participants aged 56 to 85 yrs dosed with 20 ?g on Days 1 and 22 (n=12 per group). Binding antibody concentration data for younger participant dose groups is available up until Day 50 for dose groups 1 ?g and 3 ?g, and up until Day 85 for dose groups 10, 20, and 30 ?g. For the BNT162b2-dosed older participants, data is available up until Day 29.

(1675) The fold increase from baseline in binding antibody concentrations after dosing with BNT162b1 and BNT162b2 are shown in FIG. 106 and FIG. 107, respectively. Participants dosed with BNT162b1 showed a strong dose-dependent antibody response against the SARS-CoV-2 spike (S) protein S1 subunit at Day 21 after Dose 1 (Day 22). At 7 days after Dose 2 (Day 29), S1-binding immunoglobulin (IgG) GMCs showed a strong, dose-dependent booster response. In the 60 ag dose group, which was only dosed once, S1-binding IgG GMCs remained at a lower level, indicating that a booster dose is necessary to increase antibody concentrations.

(1676) At 21 days after Dose 2 of BNT162b1 (Day 43), S1-binding IgG GMCs decreased (with exception of the 1 ag dose group), but were clearly above those of a COVID-19 HSC panel for all doses tested.

(1677) BNT162b2 dosed participants showed a strong BNT162b2-induced S1-binding IgG response at 21 days after Dose 1 (Day 22) with evidence of a dose-dependent response only between the 1 ag and 10 ag dose levels. S1-binding IgG GMCs showed a substantial boster response by 7 days after Dose 2 (Day 29). Day 29 S1-binding IgG GMCs were comparable between the younger and older participants at the 20 ?g dose level.

(1678) Across all dose-level cohorts antibody levels decreased over time, but with S1-binding antibody GMCs well above that observed in a COVID-19 HCS panel at Day 85 (63 days after Dose 2; 10 to 30 ?g dose level) (FIG. 107).

(1679) Almost all BNT162b1- and BNT162b2-immunized participants seroconverted with regard to the S1-binding antibody response as early as 21 days after Dose 1 (Day 22). Frequency of participants with seroconversion after dosing with BNT162b1 is shown in FIG. 108 and with BNT162b2 is shown in FIG. 109. Similar observations were made using only the RBD domain as the target antigen.

Example 23: Exemplary Cell-Mediated Immune Responses: SARS-CoV-2-specific CD4.SUP.+ and CD8.SUP.+ T-cell Responses

(1680) In clinical trials described in Example 7, the following were observed in healthy younger adults (18-55 years of age) and older adults (56-85 years of age) after BNT162b1 or BNT162b2 vaccination. CD4+ and CD8+ T-cell response data were available from 97 study participants receiving BNT162b1, 70 younger participants at dose levels of 1, 3, 10, 20, 30, 50, or 60 ?g (note: Dose 2 was not given in the 60 ?g dose group), and 27 older participants at dose levels of 10, 20, or 30 ?g, as well as 76 participants receiving BNT162b2 at dose levels of 1, 3, 10, 20, or 30 ?g (47 younger participants), or 10, 20, or 30 ?g (older participants).

(1681) BNT162b1 induced strong RBD-specific CD4.sup.+ T-cell responses in the majority of participants given both dose one and dose two (86 of 88 [97.7%]), including all older participants (27 of 27 [100%]); CD8.sup.+ responses were induced in 47 of 61 (77.0%) younger participants and in 21 of 27 (77.7%) of older participants. In contrast, T-cell responses were detected less often and were lower in magnitude in 9 younger participants who received only Dose 1 in the 60 ?g dose group, indicating the importance of a booster dose BNT162b2 induced strong SARS-CoV-2 S protein-specific CD4.sup.+ T-cell responses in all of the dosed younger or older participants (76 of 76 [100%]); CD8.sup.+ T-cell responses were induced in 45/47 (95.7%) of younger participants and 24/29 (82.8%) older participants. Despite the slightly lower CD8.sup.+ immunogenicity rate in older participants, the magnitude of the BNT162b2-induced responses was comparable to those induced in younger participants receiving 30 ?g of BNT162b2. These T-cell responses were directed against different parts of the antigen including non-RBD sequences, indicating the induction of multi-epitopic responses by BNT162b2 in both age groups.

(1682) Dosing twice with BNT162b1 or BNT162b2 led to a substantial increase in incidence and magnitude of T-cell responses in both age groups, and across all dose levels for BNT162b1. While the magnitude of CD4.sup.+ T-cell responses induced by BNT162b2 was also similar across different dose levels, the magnitude of CD8.sup.+ T-cell responses was highest at the 30 ?g dose level. The participants with the strongest CD4.sup.+ T-cell responses had more than 10-fold of the memory responses observed in the same participants against immunodominant peptides from cytomegalovirus, Epstein Barr virus, influenza virus, and tetanus toxoid in the same participants. The same participants also had strong CD8.sup.+ T-cell responses that were comparable to memory responses against the above mentioned viral antigens.

(1683) RBD- and S protein-specific CD4.sup.+ T-cell responses observed after vaccination were induced de novo by BNT162b1 in 97.5% of participants and by BNT162b2 in 100% of participants. RBD- and S protein-specific CD8.sup.+ T-cell responses observed after vaccination were induced de novo by BNT162b1 in 95.5% of participants and by BNT162b2 in 96.6% of participants.

Example 24: Exemplary Cell-Mediated Immune Responses: Functional and Pro-Inflammatory CD4.SUP.+./CD8.SUP.+ T-Cell Responses

(1684) In clinical trials described in Example 7, the following were observed in healthy younger adults (18-55 years of age) and older adults (56-85 years of age) after BNT162b1 or BNT162b2 vaccination. De novo induction of SARS-CoV-2 S protein or RBD protein directed T-cells was confirmed using intracellular cytokine staining (ICS). As described in Example 7 for BNT162b1, similar cell-mediated immune responses were also observed with BNT162b2 as described below.

(1685) For example, IFN?-producing CD4+ and CD8+ T-cells against SARS-CoV-2 S protein or RBD were induced robustly by both BNT162b1 and BNT162b2. No clear dose dependency was observed for both BNT162b1 and BNT162b2. The cytokine responses elicited after dosing with either BNT162b1 or BNT162b2 in older participants was mostly identical in response pattern and intensity with that in younger participants.

(1686) BNT162b1 and BNT162 induced poly-functional and pro-inflammatory CD4+/CD8+ T-cell responses in almost all participants. The detection of interferon (IFN)?, interleukin (IL)-2 but not IL-4 indicates a favorable Th1 profile and the absence of a potentially deleterious Th2 immune response.

(1687) Regarding BNT162b2, peripheral blood mononuclear cell (PBMC) fractions isolated from blood of study participants collected at baseline (pre-Dose 1) and 29?3 d after Dose 1 BNT162b2 were analyzed. This includes data for a total of 74 study participants: Younger participants aged 18 to 55 yrs per dose group: 1 ?g (n=8), 3 ?g (n=9), 10 ?g (n=10), 20 ?g (n=9), 30 ?g (n=10). Older participants aged 56 to 85 yrs per dose group: 10 ?g (n=11), 20 ?g (n=8), 30 ?g (n=9).

(1688) The functionality and polarization of vaccine-induced SARS-CoV-2 S-specific T cells were assessed by intracellular accumulation of cytokines IFN-gamma, IL-2, and IL-4 in response to stimulation with overlapping peptides representing the full-length sequence of the vaccine-encoded RBD and the wild-type SARS CoV-2 protein, respectively. As a control, PMBCs from 18 COVID-19 convalescent virologically confirmed patients were used.

(1689) Two doses of BNT162b2 (dose range 1 to 30 ?g), induced vaccine-specific T-cell responses in both age groups analyzed (FIGS. 110 and 111). Testing for SARS-CoV-2 S protein specific T-cell responses was performed with two different peptide pools, S pool 1 comprising overlapping peptides from the N-terminal region of the S protein (which is not equilvalent to structural domains) and S pool 2 comprising C-terminal regions of the S protein. S-specific CD4.sup.+ T-cell responses analyzed in 74 participants dosed with BNT162b2 were characterized by a Th1 cytokine profile secreting IFN-gamma, or IL-2, or both.

(1690) Almost no Th2 cytokine IL-4 secreting T cells were detectable in response to S peptide sub-pool stimulations (mean fractions: 0.01% and 0.02% of antigen-specific circulating CD4.sup.+ T cells in the 20 and 30 ?g adult cohort, respectively; separate stimulation with S protein sub-pool 1 and sub-pool 2). S-specific CD8.sup.+ T cells secreted IFN? in 61 of the 74 analyzed participants (adults: 40 of 46 participants and older adults: 21 of 28 participants) and also IL-2 secreting CD8.sup.+ T cells were detectable. Fractions of S-specific IFN?.sup.+ CD8.sup.+ T cells targeting the N-terminal domain of the S protein reached up to 1% of total peripheral blood CD8.sup.+ T cells in the 20 and 30 ?g younger participant dose groups and up to 2.4% in the 30 ag older participant dose group. Pre-existing CD8.sup.+ T-cell responses against the C-terminal region of the S protein were detected in 17 of 74 dosed participants (range: 0.07 to 5.59% IFN?-producing CD8.sup.+ T cells). In 6 of 17 participants, these pre-existing responses were slightly amplified upon BNT162b2 dosing.

(1691) Overall, the mean fractions of S-specific CD4.sup.+ and CD8.sup.+ T cells were substantially higher (e.g., the S protein pool 1 IFN? CD8.sup.+ response of 30 ?g dosed participants was 12.5-fold higher) than that observed in 18 patients who recovered from COVID-19. Importantly, for the clinically targeted 30 ag dose group, the cytokine responses elicited after vaccination with BNT162b2 in older participants was mostly identical in response pattern and intensity with that of the younger participants.

(1692) BNT162b2-induced T-cell responses, especially for CD8.sup.+ T cells, were not limited to the RBD, and pronounced and strong T cell recognition of non-RBD regions of the S protein were observed.

(1693) BNT162b2 induced poly-functional and pro-inflammatory CD4.sup.+/CD8.sup.+ T-cell responses in almost all participants. The Th1 polarization of the helper response was characterized by a robust IFN?/IL-2 and only minor IL-4 production upon antigen-specific (wild-type SARS-CoV-2 S protein peptide pools) re-stimulation.

Example 25: Certain T Cell Responses Induced by BNT162b2

(1694) In addition to Examples 23 and 24, which describe certain T cell responses induced by immunization with BNT162b2 as observed in the German trial (Study BNT162-01; NCT04380701), the present Example further demonstrates immunogenicity of prime-boost vaccination with 1, 10, 20 and 30 ?g BNT162b2 in participants 19-55 years of age, including detailed characterisation of T cell responses, e.g., the first identification of epitopes recognised by CD8+ T cells induced by a COVID-19 vaccine described herein. Without wishing to be bound by any particular theory, it is noted that identity of epitopes to which a response is raised in a subject, and/or extent of response to particular epitope or combination of epitopes may impact one or more features (e.g., effectiveness and/or duration) of an immune response and/or of immune protection provided by an administered vaccine. In some embodiments, an administration regimen may involve one or more steps of monitoring one or more features of an immune response, including, for example, presence and/or level of response (e.g., of T cells and/or antibodies) that recognize one or more particular epitopes. In some embodiments, need for, timing of, and/or magnitude of a subsequent dose may be determined in light of such monitoring.

(1695) As further described below, the present Example demonstrates, in part, that the epitopes recognised by several BNT162b2-induced CD8.sup.+ T cells when presented on frequent MHC alleles were identified using peptide MHC multimers; and that CD8.sup.+ T cells were shown to be of the early-differentiated effector-memory phenotype, with single specificities reaching 0.01-3% of circulating CD8.sup.+ T cells. Without wishing to be bound by any particular theory, it is noted that cells that exhibit effector-memory phenotype may provide longer term protection.

(1696) The present Example also documents that certain participants receiving BNT162b2 had pre-existing T cell responses. Thus, among other things, this example confirms that compositions as described herein, and particularly BNT162b2 may well be useful even in subjects who have already been exposed to one or more related viruses, including potentially the same virusi.e., SARS-CoV-2 and/or to an antigen thereof or another antigen that shares one or more epitopes with SARS-CoV2 spike protein.

(1697) Prevalence and Magnitude of Vaccine-Induced T Cell Responses

(1698) T cell responses of 37 BNT162b2 immunised participants from whom sufficient peripheral blood mononuclear cells (PBMCs) were available were analysed pre-vaccination (day 1) and seven days after the booster dose (day 29) by direct ex vivo IFN? enzyme-linked immunosorbent spot (ELISpot) assay (FIG. 112 and FIG. 113). One of ordinary skill in the art will understand that SARS-CoV-2 S protein is composed of a signal peptide (aa 1-13), the N-terminal S1 protease fragment (aa 14-685), and the C-terminal S2 protease fragment (aa 686-1273); and that S1 contains the RBD (aa 319-541), which binds to the host receptor, and that S2 mediates fusion between the viral envelope and cell membrane. To deconvolute reactivity against S protein, CD4.sup.+ or CD8.sup.+ T cell effectors were stimulated overnight with overlapping peptides representing different portions of the wild-type sequence of SARS-CoV-2 S, namely N-terminal pools S pool 1 (aa 1-643) and RBD (aa 1-16 fused to aa 327-528 of S), and the C-terminal S pool 2 (aa 633-1273).

(1699) Seven days after the boost with BNT162b2 at any of the indicated doses, robustly expanded SARS-CoV-2 S-specific CD4.sup.+ T-cells were detectable in all 37 participants (FIG. 112(A), FIG. 113(A)). In 34 of these participants, comparison to pre-vaccination PBMCs was possible. Thirty of the 34 subjects (88.2%) had de novo (not existent at baseline) CD4.sup.+ T cell responses against both S pool 1 and S pool 2 of SARS-CoV-2. One participant had de novo response only against pool 2. The remaining three participants had de novo responses against S pool 1 and low numbers of pre-existing S pool 2-reactive CD4.sup.+ T cells. In two of these three participants, the pre-existing responses against S pool 2 were amplified by vaccination (from 91 and 188 spots/10.sup.6 cells pre-vaccination to 1391 and 965 spots after vaccination, respectively), whereas in one of the three participants, the pre-existing responses against S pool 2 remained stable (53 to 140 spots/10.sup.6 cells). These data demonstrate that in 94.1% (32/34) of participants, two doses of BNT162b2 induce poly-epitopic CD4.sup.+ T cell responses (de novo or amplified) directed against both N- and C-terminal portions of S and thus against epitopes outside the RBD (FIG. 113(B)).

(1700) Although for dose levels ?10 ?g the magnitude of CD4.sup.+ T cell responses did not appear to be dose-dependent, it varied between individuals. In the strongest responders, the S-specific CD4.sup.+ T cell responses were more than 10-fold of the individual memory responses to common viruses and recall antigens (those from cytomegalovirus, Epstein Barr virus, influenza virus and tetanus toxoid) (FIG. 112 (B), (C)).

(1701) Vaccine-induced S-specific CD8.sup.+ T cell responses were detected in 34 of 37 vaccinated participants (91.9%). The majority were strong responses (FIG. 112(A), FIG. 113(A)) comparable to individual memory responses against cytomegalovirus (CMV), Epstein Barr virus (EBV) and influenza virus (FIG. 112(B), (C)). De novo S-specific CD8.sup.+ T cell responses were induced in 33 participants, these were either directed against both (22 participants), or one of the S pools (S pool 1 in ten participants, and S pool 2 in two participants), indicating a preponderance of a poly-epitopic response including non-RBD S-specific T cells (FIG. 113(B)). In seven participants, pre-existing CD8.sup.+ T cell responses to S pool 2 were detected that were not further augmented by vaccination. Six out of these seven participants had a concurrent de novo response to pool 1 of S, which in strength did not differ significantly from those observed in individuals without pre-existing responses to S pool 2 (FIG. 113(C)). Of note, the strongest responses (higher than third quartile) against S pool 1 among the 34 participants with detectable CD8.sup.+ T cell responses were observed in those without pre-existing S pool 2-specific responses.

(1702) The magnitude of S-specific CD4.sup.+ T cell responses correlated positively with S1-binding IgG (FIG. 114(A)), and, in line with the concept of intramolecular help (e.g., a CD4 response to one eptiope in an antigen can support development of a CD8 response to an epitope in the same antigen), also with the strength of S-specific CD8.sup.+ T cell responses (FIG. 114(B)). S-specific CD8.sup.+ T cell responses also correlated positively with S1-binding IgG (FIG. 114(C)), indicating a convergent development of the humoral and cellular adaptive immunity.

(1703) Polarisation of Vaccine-Induced T Cell Responses

(1704) To assess functionality and polarisation of S-specific T cells, cytokines secreted in response to stimulation with S pool 1, S pool 2 and RBD pool were determined by intracellular staining (ICS) for IFN?, IL-2 and IL-4 specific responses in pre- and post-vaccination PBMCs of 37 BNT162b2-immunised participants receiving different doses. A considerable fraction of vaccine-induced, S-specific CD4.sup.+ T cells secreted IFN?, IL-2, or both, while T cells secreting the T.sub.H2 cytokine IL-4 were barely detectable (FIG. 115(A)-(C), FIG. 113(D)-(E)). Vaccine-induced S-specific CD8.sup.+ T cells secreted predominantly IFN? and lower levels of IL-2 in response to S pool 1 and S pool 2 stimulation. Fractions of IFN?.sup.+ CD8.sup.+ T cells specific to S pool 1 constituted up to about 1% of total peripheral blood CD8.sup.+ T cells (FIG. 115(D)). Of note, several of the analysed participants (n=3 in the 20 ?g dose cohort and n=3 in the 30 ?g dose cohort) displayed pre-existing S pool 2 specific CD8.sup.+ T cell responses, which in 5 out of the 6 participants were not further amplified after vaccination. A strong pre-existing S pool 2 specific IFN?.sup.+ CD4.sup.+ T cell response was detectable in one participant (20 ?g dose cohort) (FIG. 115(C)).

(1705) In both assay systems, cytokine production of CD4.sup.+ as well as CD8.sup.+ T cells in response to peptide pools comprising the full SARS-CoV-2 S exceeded the responses against the RBD peptide pool, further confirming the poly-epitopic nature of T cell responses elicited by BNT162b2. The mean fraction of BNT162b2-induced S-specific IFN?.sup.+ or IL-2.sup.+ CD4.sup.+ and CD8.sup.+ T cells within total circulating T cells was higher than that detected in eighteen control subjects who had recovered from COVID-19 (HCS) (FIG. 115(C), (D)).

(1706) Epitope Specificity and Phenotype of CD8.sup.+ T Cells Observed in Immunization with BNT162b2

(1707) CD8.sup.+ T cell responses were characterised on the epitope level in three participants vaccinated with a 2-dose regimen with two doses (e.g., 10 ?g/dose or 30 ?g/dose) given 21 days apart.

(1708) Pre- and post-vaccination peripheral blood mononuclear cells (PBMCs) collected from the participants were stained with individualised peptide/MHC multimer staining cocktails for flow cytometry analysis. Twenty-three (4 for HLA-B*0702, 19 for HLA-A*2402), 14 (HLA-B*3501) and twenty-three (7 for HLA-B*4401, 16 for HLA-A*0201) diverse peptide/MHC allele pairs were used for participant 1, 2 and 3, respectively, thus probing a selected set of potential reactivities rather than comprehensively capturing the poly-epitopic T cell response. For each participant, de novo induced CD8.sup.+ T cell reactivities against multiple epitopes were identified adding up to a total of eight different epitope/MHC pairs spread across the full length of S protein (FIG. 116 (A), (C)). The magnitude of epitope-specific T cell responses ranged between 0.01-3.09% of peripheral CD8.sup.+ T cells and the most profound expansion was observed for HLA-A*0201 YLQPRTFLL (SEQ ID NO: 40) (3.09% multimer.sup.+ of CD8.sup.+), HLA-A*2402 QYIKWPWYI (SEQ ID NO: 42) (1.27% multimer.sup.+ of CD8.sup.+) and HLA-B*3501 QPTESIVRF (SEQ ID NO: 45) (0.17% multimer.sup.+ of CD8.sup.+). Comparison with the bulk IFN?.sup.+ CD8.sup.+ T cell response against full S protein in these individuals determined by ELISpot and intracellular staining (ICS) indicated that pMHC technology may be more useful to assess the true extent of the cellular immune response (FIG. 113(F)).

(1709) Phenotyping of the identified pMHC multimer.sup.+ S antigen-experienced CD8.sup.+ T cell specificities revealed an early differentiated effector memory phenotype characterized by low expression of CCR7 and CD45RA and high expression of the costimulatory molecules CD28 and CD27. CD8.sup.+ T cells also expressed markers associated with cognate activation, such as CD38, HLA-DR and PD-1 (FIG. 116(B)).

(1710) Discussion

(1711) Effectors of the adaptive immune system have complementary roles in the defense of viral infections. While neutralising antibodies are the first line of defense, CD8.sup.+ cytotoxic T lymphocytes (CTLs) contribute to virus clearance from intracellular compartments that are inaccessible to neutralising antibodies. Antigen-specific CD4.sup.+ T cells have immune orchestrating functions, including provision of cognate help to B cells and CD8.sup.+ T cells, support of memory generation, as well as indirect (e.g. via IFN?) or direct (against MHC class II-expressing target cells) cytotoxic activity.

(1712) This Example shows that vaccination with BNT162b2 induces a coordinated immune response with SARS-CoV-2 S-specific neutralising antibodies (as described in other Examples), CD4+ T cells, CD8+ T cells, and immune-modulatory cytokines such as IFN?. All participants vaccinated with BNT162b2 mounted de novo S-specific CD4+ T cell responses and almost 92% of participants mounted CD8+ T cell responses, as detected with an ex vivo ELISpot assay. The magnitude of the T cell responses varied inter-individually and showed no clear dose dependency. Even with the lowest dose of 1 ?g BNT162b2, most of the vaccinated participants demonstrated robust expansion of CD4+ and CD8+ T cells. T cell responses were directed against RBD, S1 and S2 regions of S protein, indicating immune recognition of multiple independent MHC I and II epitopes.

(1713) Expression of IFN? and IL-2 but only low levels of IL-4 in BNT162b2-induced CD4+ T cells indicated a TH1 profile and the absence of a potentially deleterious TH2 immune response.

(1714) While all CD8+ T cell responses against the S1 subunit of S protein were de novo and not detected at baseline, pre-existing immune responses against the S2 subunit were identified in several individuals. The S1 fragment has less sequence similarity to the corresponding seasonal coronavirus sequences than the S2 fragment does; without wishing to be bound by theory, it is proposed that this finding indicates that pre-existing cross-reactive CD8+ T cells may have been detected.

(1715) Peptide MHC (pMHC) multimer technology enabled the identification of S protein epitopes recognised by vaccine-induced CD8.sup.+ T cells as well as direct quantification of the respective epitope-specific T cells. The cumulative T cell frequencies in each participant exceeded the overall T cell response measured in ELISpot and ICS assays, indicating that those assays underestimate the true magnitude of the poly-epitopic response. One of skill in the art will appreciate that single peptide analyses are known to yield higher T cell frequencies as compared to functional T cell assays that stimulate with peptide pools, with a multitude of immunogenic epitopes competing. A high proportion of induced CD8.sup.+ T cells were early differentiated effector memory cells. This favourable phenotype has the potential to respond rapidly, but has a limited capacity to produce IFN?, and thus is less likely to be detected in functional T cell assays. While epitopes in SARS-CoV-2 S against which infected individuals raise CD8+ T cells were identified and known in the art (see, e.g., Shomuradova et al., Immunity (2020) doi:10.1016/j.immuni.2020.11.004; and Peng et al. Nat. Immunol. 21, 1336-1345 (2020)), the data presented herein is the first demonstration of epitopes recognised by COVID-19 vaccine-induced T cells. Of note, the immunodominant HLA-A*02:01 restricted peptide YLQPRTFLL (SEQ ID NO: 40) identified in this stidy has previously been described in convalescent COVID-19 patients. Id.

(1716) Materials and Methods

(1717) Proteins and Peptides.

(1718) Two pools of 15-mer peptides overlapping by 11 amino acids (aa) and together covering the whole sequence of wild-type SARS-CoV-2 S (S pool 1 featuring aa 1-643, S pool 2 featuring aa 633-1273) and one pool covering the SARS-CoV-2 RBD (aa 327-528) with the signal peptide of S (aa 1-16) fused to its N-terminus were used for ex vivo stimulation of PBMCs for flow cytometry and IFN? ELISpot. CEF (CMV, EBV, influenza virus; human leukocyte antigen [HLA] class I epitope peptide pool) and CEFT (CMV, EBV, influenza virus, tetanus toxoid; HLA class II epitope peptide pool) were used as controls for general T-cell reactivity and to benchmark the magnitude of memory T cell responses. All peptides were obtained from JPT Peptide Technologies.

(1719) Human Convalescent Serum and PBMC Panel.

(1720) Human SARS-CoV-2 infection/COVID-19 convalescent sera (n=38) were drawn from donors 18-83 years of age at least 14 days after PCR-confirmed diagnosis and at a time when the participants were asymptomatic. The mean age of the donors was 45 years. Neutralising GMTs in subgroups of the donors were as follows: symptomatic infections, 90 (n=35); asymptomatic infections, 156 (n=3); hospitalized, 618 (n=1). Sera were obtained from Sanguine Biosciences (Sherman Oaks, CA), the MT Group (Van Nuys, CA) and Pfizer Occupational Health and Wellness (Pearl River, NY). Human SARS-CoV-2 infection/COVID-19 convalescent PBMC samples (n=18) were collected from donors 22-79 years of age 30-62 days after PCR-confirmed diagnosis, when donors were asymptomatic. PBMC donors had asymptomatic or mild infections (n=16, clinical score 1 and 2) or had been hospitalized (n=2, clinical score 4 and 5). Blood samples were obtained from the Frankfurt University Hospital.

(1721) Primary Cell Isolation.

(1722) PBMCs were isolated by Ficoll-Paque? PLUS (Cytiva) density gradient centrifugation and cryopreserved prior to analysis.

(1723) IFN? ELISpot.

(1724) IFN? ELISpot analysis was performed ex vivo (without further in vitro culturing for expansion) using PBMCs depleted of CD4.sup.+ and enriched for CD8.sup.+ T cells (CD8.sup.+ effectors) or depleted of CD8.sup.+ and enriched for CD4.sup.+ T cells (CD4.sup.+ effectors). Tests were performed in duplicate and with a positive control (anti-CD3 monoclonal antibody CD3-2 [1:1,000; Mabtech]). Multiscreen filter plates (Merck Millipore) pre-coated with IFN?-specific antibodies (ELISpotPro kit, Mabtech) were washed with PBS and blocked with X-VIVO 15 medium (Lonza) containing 2% human serum albumin (CSL-Behring) for 1-5 hours. Per well, 3.3?10.sup.5 effector cells were stimulated for 16-20 hours with three overlapping peptide pools representing different portions of the wild-type sequence of SARS-CoV-2 S (N-terminal pools S pool 1 [aa 1-643] and RBD [aa1-16 fused to aa 327-528], and the C-terminal S pool 2 [aa 633-1273]). Bound IFN? was visualised using a secondary antibody directly conjugated with alkaline phosphatase followed by incubation with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) substrate (ELISpotPro kit, Mabtech). Plates were scanned using an AID Classic Robot ELISPOT Reader and analysed by AID ELISPOT 7.0 software (AID Autoimmun Diagnostika). Spot counts were displayed as mean values of each duplicate. T-cell responses stimulated by peptides were compared to effectors incubated with medium only as a negative control using an in-house ELISpot data analysis tool (EDA), based on two statistical tests (distribution-free resampling), to provide sensitivity while maintaining control over false positives.

(1725) To account for varying sample quality reflected in the number of spots in response to anti-CD3 antibody stimulation, a normalisation method was applied, enabling direct comparison of spot counts and strength of response between individuals. This dependency was modelled in a log-linear fashion with a Bayesian model including a noise component (unpublished). For a robust normalization, each normalisation was sampled 1000 times from the model and the median taken as normalized spot count value. Likelihood of the model: log ?.sub.E=? log ?.sub.P+log ?.sub.j+??, where ?.sub.E is the normalized spot count of the sample; ? is a stable factor (normally distributed) common among all positive controls ?.sub.P; ?.sub.j is a sample j specific component (normally distributed); and ?? is the noise component, of which ? is Cauchy distributed, and ? is Student's-t distributed. ?.sub.j ensures that each sample is treated as a different batch.

(1726) Flow Cytometry.

(1727) Cytokine-producing T cells were identified by intracellular cytokine staining. PBMCs thawed and rested for 4 hours in OpTmizer medium supplemented with 2 ?g/mL DNase I (Roche), were restimulated with different portions of the wild-type sequence of SARS-CoV-2 S in peptide pools described in the ELISpot section (2 ?g/mL/peptide; JPT Peptide Technologies) in the presence of GolgiPlug (BD) for 18 hours at 37? C. Controls were treated with DMSO-containing medium. Cells were stained for viability and surface markers (CD3 BV421, 1:250; CD4 BV480, 1:50; CD8 BB515, 1:100; all BD Biosciences) in flow buffer (DPBS [Gibco] supplemented with 2% FBS [Biochrom], 2 mM ethylenediaminetetraacetic acid [EDTA; Sigma-Aldrich]) for 20 minutes at 4? C. Afterwards, samples were fixed and permeabilised using the Cytofix/Cytoperm kit according to manufacturer's instructions (BD Biosciences). Intracellular staining (CD3 BV421, 1:250; CD4 BV480, 1:50; CD8 BB515, 1:100; IFN? PE-Cy7, 1:50 [for HCS]; IFN? BB700, 1:250 [for participants]; IL-2 PE, 1:10; IL-4 APC, 1:500; all BD Biosciences) was performed in Perm/Wash buffer for 30 minutes at 4? C. Samples were acquired on a fluorescence-activated cell sorter (FACS) VERSE instrument (BD Biosciences) and analysed with FlowJo software version 10.6.2 (FlowJo LLC, BD Biosciences). S- and RBD-specific cytokine production was corrected for background by subtraction of values obtained with dimethyl sulfoxide (DMSO)-containing medium. Negative values were set to zero. Cytokine production in FIG. 116(B) was calculated by summing up the fractions of all CD4.sup.+ T cells positive for either IFN?, IL-2 or IL-4, setting this sum to 100% and calculating the fraction of each specific cytokine-producing subset thereof. Pseudocolor plot axes are in log 10 scale.

(1728) Peptide/MHC Multimer Staining.

(1729) In order to select MHC-class I epitopes for multimer analysis, a mass spectrometry-based binding and presentation predictor (e.g., as described in Abelin et al., Immunity 46, 315-326 (2017); and Poran et al., Genome Med. 12, 70 (2020)) was applied to 8-12 amino acid long peptide sequences from the Spike glycoprotein derived from the GenBank reference sequence for SARS-CoV-2 (accession: NC_045512.2, www.ncbi.nlm.nih.gov/nuccore/NC_045512) and paired with 18 MHC-class-I alleles with >5% frequency in the European population. Top predicted epitopes were identified by setting thresholds to the binding percent-rank (?1%) and presentation scores (?10.sup.?2.2) and considered for synthesis of peptides of >90% purity. pMHC complexes were refolded with the easYmer technology (easYmer? kit, ImmuneAware Aps), and complex formation was validated in a bead-based flow cytometry assay according to the manufacturer's instructions. Combinatorial labeling was used for dissecting the antigen specificity of T cells utilizing two-color combinations of five different fluorescent labels to enable detection of up to ten different T cell populations per sample. For tetramerisation, streptavidin (SA)-fluorochrome conjugates were added: SA BV421, SA BV711, SA PE, SA PE-Cy7, SA APC (all BD Biosciences). For three BNT162b2 vaccinated participants, individualized pMHC multimer staining cocktails contained up to ten pMHC complexes, with each pMHC complex encoded by a unique two-color combination. PBMCs (2?10.sup.6) were stained ex vivo for 20 minutes at room temperature with each pMHC multimer cocktail at a final concentration of 4 nM in Brilliant Staining Buffer Plus (BSB Plus [BD Horizon?]). Surface and viability staining was carried out in flow buffer (DPBS [Gibco] with 2% FBS [Biochrom], 2 mM EDTA [Sigma-Aldrich]) supplemented with BSB Plus for 30 minutes at 4? C. (CD3 BUV395, 1:50; CD45RA BUV563, 1:200; CD27 BUV737, 1:200; CD8 BV480, 1:200; CD279 BV650, 1:20; CD197 BV786, 1:15; CD4 BB515, 1:50; CD28 BB700, 1:100; CD38 PE-CF594, 1:600; HLA-DR APC-R700, 1:150; all BD Biosciences; DUMP channel: CD14 APC-eFluor780, 1:100; CD16 APC-eFluor780, 1:100; CD19 APC-eFluor780, 1:100; fixable viability dye eFluor780, 1:1,667; all ThermoFisher Scientific). Cells were fixed for 15 minutes at 4? C. in 1?Stabilization Fixative (BD), acquired on a FACSymphony? A3 flow cytometer (BD Biosciences) and analysed with FlowJo software version 10.6.2 (FlowJo LLC, BD Biosciences). CD8.sup.+ T cell reactivities were considered positive, when a clustered population was observed that was labelled with only two pMHC multimer colors.

Example 26: Evidence Suggesting Possibility of Re-Infection

(1730) The primary endpoint was evaluated in individuals without prior evidence of COVID-19 disease, and very few cases of confirmed COVID-19 occurred among participants with evidence of infection prior to vaccination (although more cases occurred in the placebo group compared with the vaccine group). However, available data, while limited, as shown in Tables 22-23 suggest that previously infected individuals can be at risk of COVID-19 (i.e., reinfection) and could benefit from vaccination.

(1731) TABLE-US-00040 TABLE 22 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days After Dose 2, by Test Status - Subjects With or Without Evidence of Infection Prior to 7 Days After Dose 2 - Evaluable Efficacy (7 Days) Population Vaccine Group Control Group N.sup.a = 19965 N.sup.a = 20172 Cases Cases n1.sup.b n1.sup.b Vaccine RT-PCR NP Swab Results and surveillance surveillance Efficacy % Serostatus: Time Points time.sup.c (n2.sup.d) time.sup.c (n2.sup.d) (95% CI.sup.e) Pre-dose 1 SARS-CoV-2 RT-PCR (NP swab) Positive 0 0 NE (NE, 0.013 (119) 0.015 (137) NE) Negative 9 166 94.5 (89.4, 2.301 (18259) 2.314 (18410) 97.6) Unknown 0 3 100.0 (?126.2, 0.017 (181) 0.016 (161) 100.0) Pre-dose 2 SARS-CoV-2 RT-PCR (NP swab) Positive 0 1 100 (?4916.4, 0.009 (83) 0.012 (106) 100.0) Negative 9 167 94.6 (89.5, 2.301 (18263) 2.315 (18408) 97.6) Unknown 0 1 100.0 (?3173.8, 0.022 (213) 0.018 (194) 100.0) Subjects with negative RT-PCR pre-dose 1 and positive RT-PCR pre-dose 2 Subjects with documented 0 0 COVID-19 symptoms 0.000 (0) 0.000 (1) between dose 1 and 2 Subjects with no documented 0 1 COVID-19 symptoms 0.004 (44) 0.006 (52) between dose 1 and 2 Pre-dose 1 serostatus.sup.f Positive 1 1 ?8.0 (?8378.1, 0.052 (488) 0.056 (525) 98.6) Negative 8 167 95.2 (90.3, 2.255 (17823) 2.260 (17894) 98.0) Unknown 0 1 100.0 (?4527.0, 0.025 (248) 0.030 (289) 100.0) 1-month post-dose 2 (Visit 3) No data shown serostatus* Positive Negative Subjects who seroconverted** No data shown between dose 1 and 1-month post-dose 2 Subjects with documented COVID-19 symptoms during time period Subjects with no documented COVID-19 symptoms during time period (protocol- defined vaccine efficacy against asymptomatic infection) Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding; RT-PCR = reverse transcription-polymerase chain reaction; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; VE = vaccine efficacy; NP = nasopharyngeal; NE = Not estimable. .sup.aN = number of subjects in the specified group. .sup.bn1 = Number of subjects meeting the endpoint definition. .sup.cTotal surveillance time in 1000 person-years for the given endpoint across all subjects within each group at risk for the endpoint. Time period for COVID-19 case accrual is from 7 days after Dose 2 to the end of the surveillance period. .sup.dn2 = Number of subjects at risk for the endpoint. .sup.eConfidence interval (CI) for VE is derived based on the Clopper and Pearson method adjusted for surveillance time. .sup.fBased on N-binding test result.

(1732) TABLE-US-00041 TABLE 23 Vaccine Efficacy - First COVID-19 Occurrence From 7 Days After Dose 2, by Test Status - Subjects With or Without Evidence of Infection Prior to 7 Days After Dose 2 - All Available Efficacy Population Vaccine Group Control Group N.sup.a = 20488 N.sup.a = 20459 Cases Cases n1.sup.b n1.sup.b Vaccine RT-PCR NP Swab Results and surveillance surveillance Efficacy % Serostatus: Time Points time.sup.c (n2.sup.d) time.sup.c (n2.sup.d) (95% CI.sup.e) Pre-dose 1 SARS-CoV-2 RT-PCR (NP swab) Positive 0 0 NE (NE, 0.014 (122) 0.015 (138) NE) Negative 9 169 94.7 (89.7, 2.358 (18740) 2.338 (18668) 97.6) Unknown 0 3 100.0 (?125.3, 0.018 (187) 0.016 (165) 100.0) Pre-dose 2 SARS-CoV-2 RT-PCR (NP swab) Positive 0 1 100.0 (?4739.5, 0.010 (86) 0.012 (107) 100.0) Negative 9 170 94.7 (89.8, 2.357 (18743) 2.339 (18660) 97.6) Unknown 0 1 100.0 (?3290.7, 0.022 (220) 0.019 (204) 100.0) Subjects with negative RT-PCR pre-dose 1 and positive RT-PCR pre-dose 2 Subjects with documented 0 0 COVID-19 symptoms 0.000 (0) 0.000 (1) between dose 1 and 2 Subjects with no documented 0 1 COVID-19 symptoms 0.005 (47) 0.006 (53) between dose 1 and 2 Pre-dose 1 serostatus.sup.f Positive 1 1 ?7.5 (?8335.1, 0.053 (500) 0.057 (537) 98.6) Negative 8 170 95.3 (90.6, 2.308 (18278) 2.283 (18144) 98.0) Unknown 0 1 100.0 (?4081.4, 0.028 (271) 0.030 (290) 100.0) 1-month post-dose 2 (Visit 3) No data shown serostatus* Positive Negative Subjects who seroconverted** No data shown between dose 1 and 1-month post-dose 2 Subjects with documented COVID-19 symptoms during time period Subjects with no documented COVID-19 symptoms during time period (protocol- defined vaccine efficacy against asymptomatic infection) Abbreviations: N-binding = SARS-CoV-2 nucleoprotein-binding; RT-PCR = reverse transcription-polymerase chain reaction; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2; VE = vaccine efficacy; NP = nasopharyngeal; NE = Not estimable. .sup.aN = number of subjects in the specified group. .sup.bn1 = Number of subjects meeting the endpoint definition. .sup.cTotal surveillance time in 1000 person-years for the given endpoint across all subjects within each group at risk for the endpoint. Time period for COVID-19 case accrual is from 7 days after Dose 2 to the end of the surveillance period. .sup.dn2 = Number of subjects at risk for the endpoint. .sup.eConfidence interval (CI) for VE is derived based on the Clopper and Pearson method adjusted for surveillance time. .sup.fBased on N-binding test result.

Example 27: Pharmacokinetics (PK) and Absorption, Distribution, Metabolism, and Excretion (ADME) Analysis of Certain Lipid Excipients

(1733) The present Example describes various assessed characteristics (e.g., PK/ADME characteristics) of lipids used in a vaccine composition as described herein. Without wishing to be bound by any particular theory it is noted that such characteristics of lipid components may contribute to relevant features (e.g., distribution, expression, etc) of administered vaccines, including to efficacy generally and/or in particular circumstances (e.g., when administered according to particular regimens and/or to particular populations, etc).

(1734) Absorption

(1735) A single dose PK study of ALC-0315 and ALC-0319 following intravenous (IV) bolus injection of a nanparticle formulation in rats was conducted to assess the PK and metabolism of lipid excipients ALC-0315 and ALC-0159. This study used LNPs containing surrogate luciferase RNA, with the lipid composition being identical to BNT162b2, to investigate the in vivo disposition of ALC-0159 and ALC-0315.

(1736) Concentrations of ALC-0159 dropped approximately 8000- and >250-fold in plasma and liver, respectively, during this 2-week study. For ALC-0315, the elimination of the molecule from plasma and liver was slower, but concentrations fell approximately 7000- and 4-fold in two weeks for plasma and liver, respectively. Overall, the apparent terminal t % in plasma and liver were similar in both tissues and were 2-3 and 6-8 days for ALC-0159 and ALC-0315, respectively. The apparent terminal t % in plasma likely represents the re-distribution of the respective lipids from the tissues into which they have distributed as the LNP, back to plasma where they are eliminated.

(1737) Metabolism

(1738) In vitro metabolism of ALC-0315 and ALC-0159 was evaluated in blood, liver microsomes, S9 fractions, and hepatocytes from mice, rats, monkeys, and humans. In vivo metabolism was examined in rat plasma, urine, faeces, and liver samples from the PK study. Metabolism of ALC-0315 and ALC-0159 appears to occur relatively slowly in vitro and in vivo. ALC-0315 and ALC-0159 are apparently metabolised by hydrolytic metabolism of the ester and amide functionalities, respectively, and this hydrolytic metabolism is observed across the species evaluated.

(1739) Excretion

(1740) Excretion studies appeared to demonstrate that 50% of ALC-0159 was eliminated unchanged in faeces, and that metabolism played a role in the elimination of ALC-0315, as little to no unchanged material was detected in either urine or faeces. Investigations of urine, faeces and plasma from the rat PK study identified a series of ester cleavage products of ALC-0315. Without wishing to be bound by any particular theory, it is proposed that this likely represents the primary clearance mechanism acting on this molecule in vivo. In vitro, ALC-0159 was metabolized slowly by hydrolytic metabolism of the amide functionality.

Example 28: Distribution Analysis of Administered Vaccine Composition

(1741) In vivo biodistribution of COVID-19 mRNA Vaccine BNT162b2 was evaluated using mice as a model system and assessing luciferase expression as a surrogate reporter. Protein expression was demonstrated at the site of injection and to a lesser extent, and more transiently, in the liver after mice received an IM injection of RNA encoding luciferase in an LNP formulation like BNT162b2. Luciferase expression was identified at the injection site at 6 hours after injection and diminsihed to near near baseline levels by day 9. Expression in the liver was also present at 6 hours after injection and was not detected by 48 hours after injection. All other tissues than liver evaluated contain equal to or less than 1% of the dose.

Example 29: Repeat-Dose Toxicity Study of Various Dosing Regimens

(1742) A GLP-compliant repeat-dose study performed in rats to evaluate immunogenicity and toxicity of COVID-19 mRNA vaccines, including BNT162b2.

(1743) In certain studies, male and female Wistar Han rats were given a vaccine composition as described herein; compositions based on various RNA platforms (e.g., BNT162b2) were tested as IM injection(s) into the hind limb on three occasions each a week apart (dosing days 1, 8 and 15). Different doses (10, 30 and 100 ?g) were tested; the lower doses were given as a single injection of 20-70 ?l while the highest doses (100 ?g) and controls were given as two injections (one in each hind limb) of 100 ?l each. The control was phosphate buffered saline/300 mM sucrose, corresponding to the storage buffer of the vaccine product. Each group had 18 male and 18 female rats, assigned as 10 to the main study, 5 for recovery groups and 3 as additional animals for cytokine analyses. The recovery period was 3 weeks after the last dose. Necropsy was performed on study day 17, ?48 hours after the last dose and after the 3-week recovery period.

(1744) No unscheduled deaths were observed.

(1745) Dosing was considered well tolerated and did not present any signs of systemic toxicity; there was a slight increase in body temperature in the hours after dosing and some loss in body weight over the same period but these were not of a magnitude to be considered adverse.

(1746) Local inflammatory reactions were observed at the intramuscular injection site. Injection site changes noted were oedema, erythema, and induration, more severe and more frequent after the second and/or third doses compared to the first; however, these resolved prior to subsequent dosing and were fully recovered at the end of the 3-week recovery period.

(1747) Macroscopic findings at the injection sites included induration or thickening, occasionally accompanied by encrustation, which was noted for nearly all rats. This correlated microscopically with inflammation and variable fibrosis, oedema, and myofibre degeneration. Inflammation at the injection site was accompanied by elevations in circulating white blood cells and acute phase proteins (fibrinogen, alpha-2 macroglobulin, and alpha-1 acid glycoprotein).

(1748) Inflammation was occasionally evident extending into tissues adjacent to the injection site. There was enlargement of the draining (iliac) lymph nodes evident at the end of dosing. This correlated with increased cellularity of germinal centres and increased plasma cells in the draining (iliac) lymph node and is an anticipated immune response to the administered vaccine.

(1749) Enlargement of spleen and increased spleen weights correlated microscopically to increased haematopoiesis and increased haematopoiesis was also evident in the bone marrow. These findings are likely secondary to the immune/inflammatory responses to the vaccine.

(1750) At the end of the recovery period, injection sites were normal, clinical pathology findings and macroscopic observations had resolved and there was evidence of recovery of the injection site inflammation on microscopy.

(1751) Microscopic vacuolation of portal hepatocytes was present after the dosing phase. This observation was absent after the recovery period. There were no elevations in alanine aminotransferase (ALAT). There were elevations in gamma-glutamyltransferase (GGT) in all vaccinated rats, but there were no macroscopic or microscopic findings consistent with cholestasis or hepatobiliary injury to explain the increased gamma-GT activity which was completely resolved at the end of the 3-week recovery period. The vacuolation may be related to hepatic distribution of the pegylated lipids in the LNP. No changes were seen in serum cytokine concentrations. There were no effects noted on ophthalmological and auditory assessments, nor on external appearance or behaviour; in particular, gait was normal meaning that the changes seen did not affect the rats' mobility. No vaccine-related changes were seen in serum cytokine concentrations.

(1752) Testing for immunogenicity showed that COVID-19 mRNA Vaccines (including, e.g., BNT162b2 such as BNT162b2 v8) elicited a specific IgG antibody response to SARS CoV-2 spike protein directed against the S1 fragment and the receptor binding domain. A neutralizing antibody response was also observed with the vaccine in a pseudovirus neutralization assay.

(1753) Results from ELISA assays are shown in FIGS. 117 and 118 (from day 17 or day 10 as noted in the figures), in which the very top traces are those for COVID-19 mRNA Vaccine BNT162b2 and other traces are those for other COVID-19 mRNA vaccines using different constructs as described herein: similar results were shown for day 38 (not shown here). These translated into neutralising activity as seen in the VSV/SARS-CoV2-S pseudovirus neutralisation test using Vero 76 cells (FIG. 119): similar results were presented for day 38 (not shown here). Across the vaccines tested in this study those with a higher antigen-specific antibody titre also had a more pronounced virus neutralisation effect.

(1754) COVID-19 mRNA vaccines (e.g., BNT162b2) were well tolerated, and produced inflammatory changes at the injection sites and the draining lymph nodes, increased haematopoiesis in the bone marrow and spleen, and clinical pathology changes consistent with an immune response or inflammation in the injection sites.

(1755) Those skilled in the art, reading the present disclosure, will appreciate that the findings in this Example can be considered typical of those expected with dosing of various mRNA constructs and/or lipid nanoparticles as described herein.

Example 30: Toxicity and Immunogenicity Study of Three-Dose Regimen

(1756) A study was performed to assess toxicity in rats given COVID-19 mRNA Vaccine (e.g., BNT162b2). This study was in compliance with Good Laboratory Practice.

(1757) Male and female Wistar Han rats were given BNT162b2 as an IM injection into the hind limb on three occasions, each a week apart (dosing days 1, 8 and 15). Necropsy was performed on study day 17, ?48 hours after the last dose, and after the 3-week recovery period. COVID-19 mRNA Vaccine BNT162b2 was supplied at 0.5 mg/ml and the dose volume was 60 l, to give 30 ?g per dose. Control rats received saline.

(1758) Blood was taken at various points during the assessment, prior to and during dosing, and also during recovery, and antibody responses to vaccine components were assessed.

(1759) All rats given COVID-19 mRNA Vaccine (e.g., BNT162b2) survived to their scheduled necropsy: there were no changes noted in clinical signs or body weight changes noted. A reduction in food intake was noted on days 4 and 11 (to 0.83? controls) and there was an increase in mean body temperature post-dose on day 1 (up to 0.54? C.), day 8 (up to 0.98? C.), and day 15 (up to 1.03? C.) compared to controls.

(1760) At injection sites, there were instances of oedema and erythema on days 1 (maximum of slight oedema and very slight erythema), 8 (maximum of moderate oedema and very slight erythema) and 15 (maximum of moderate oedema and very slight erythema) which fully resolved and were not noted prior to dosing on days 8 and 15. Haematological tests showed higher white blood cells (up to 2.95? controls), primarily involving neutrophils (up to 6.80? controls), monocytes (up to 3.30? controls), and large unstained cells, LUC, (up to 13.2? controls) and slightly higher eosinophils and basophils on days 4 and 17. White blood cells were higher on day 17 as compared with day 4. There were transiently lower reticulocytes on day 4 (to 0.27? controls) in both sexes and higher reticulocytes on day 17 (up to 1.31? controls) in females only. Lower red blood cell mass parameters (to 0.90? controls) were present on days 4 and 17. There were lower A:G ratios (to 0.82?) on days 4 and 17. Higher fibrinogen was noted on day 17 (up to 2.49?) compared to controls, consistent with an acute phase response. The acute phase proteins alpha-1-acid glycoprotein (up to 39? on day 17) and alpha-2 macroglobulin (up to 71? on Day 17) were elevated on days 4 and 17 with higher concentrations in males. There were no changes urinalysis parameters.

(1761) At post-mortem there were higher absolute and relative spleen weights in vaccinated rats (up to 1.42? in males and to 1.62? in females). There were no other changes in organ weights. Macroscopic findings included enlarged draining lymph nodes and pale/dark firm injection sites in a minority of vaccinated rats. The dosing was tolerated without inducing any systemic toxicity with all changes consistent with an inflammatory response and immune activation: findings are consistent with those typically associated with dosing of lipid nanoparticle-encapsulated mRNA vaccines.

Example 31: Reproduction Toxicity

(1762) A study was performed to assess reproduction toxicity in female rats given COVID-19 mRNA vaccines, including BNT162b2. Female rats were given a COVID-19 mRNA vaccine (e.g., BNT162b2) twice before the start of mating and twice during gestation at a human clinical dose (e.g., 30 ?g RNA/dosing day). The COVID-19 mRNA vaccine was administered intramuscularly (IM) to FO female Wistar rats 21 and 14 days before the start of mating (M-21 and M-14, respectively) and then on Gestation Day (GD) 9 and GD20, for a total of 4 doses. A subgroup was terminated at GD21 and another (litter) group was terminated at PostNatal Day (PND) 21. SARS-CoV-2 neutralizing antibody titers were found in the majority of females just prior to mating (M-14), in most females and foetuses at the end of gestation (GD21), and in most offspring at the end of lactation (PND21). There was transient reduced body weight gain and food consumption after each dose. No effects on the estrous cycle or fertility index were observed. While there was an increase (?2?) of pre-implantation loss (as compared to control), the pre-implantation loss percent observed in the vaccinated group was within historical control data range (5.1%-11.5%). Among foetuses (from a total of n=21 dams/litters), there was a very low incidence of gastroschisis, mouth/jaw malformations, right sided aortic arch, and/or cervical vertebrae abnormalities. Regarding skeletal findings, the exposed group had comparable to control group levels of presacral vertebral arches supernumerary lumbar ribs, supernumerary lumbar short ribs, caudal vertebrae number <5). There were no signs of adverse effects on the postnatal pups (terminated at PND21). This study shows that there is no significant adverse effects on fertility and early embryogenesis.

Example 32: Safety and Immunogenicity of the SARS-COV-2 BNT162b1 mRNA Vaccine in Younger and Older Chinese Adults: A Randomized, Placebo-Controlled, Observer-Blind Phase I Study

(1763) The present Example reports initial results from a phase I trial testing BNT162b1 in 144 healthy Chinese participants. BNT162b1 encodes the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) and is one of several RNA-based SARS-CoV-2 vaccine candidates described herein.

(1764) The present Example specifically reports on the observed safety profile, in whichfever >39? C. was the only Grade 3 adverse event reported. Prime-boost vaccination with 10 ?g or 30 ?g BNT162b1 induced robust antibody and T-cell responses in both young (18 to 59 years of age) and older (65 to 85) Chinese adults. Both dose levels induced seroconversion after 41 d: Geometric mean titres of SARS-CoV-2 serum-neutralizing antibodies in younger participants in the 10 ?g and 30 ?g dose groups were 1.9 and 2.1 times that of convalescent sera from recovered COVID-19 patients; and in older participants 0.7 and 1.3 times. Interferon-? T cell responses to RBD antigen challenge were significantly higher in participants receiving BNT162b1 than those in placebo group.

(1765) Increased reactogenicity as well as a more favorable vaccine-elicited virus-neutralizing response were associated with the 30 ?g dose of BNT162b1 in both younger and older Chinese adults.

(1766) The safety and immunogenicity data provided in the present Example for BNT162b1, specifically with respect to vaccination of healthy, young and elderly Chinese participants, suggests that prime-boost vaccination with 10 ?g and 30 ?g dose levels of the BNT162b1 vaccine induces a strong humoral and cellular immunogenic response in both younger adults of 18 to 55 years of age and older adults of 65 to 85 years of age, with robust RBD-specific antibody and T-cell responses seen in in both younger and older participants, at least within 28 days following the prime-boost vaccination. Certain findings in this study further confirm aspects of the tolerability profile for BNT162b1, for example as also observed in American and German populations.

(1767) Methods

(1768) A randomized, placebo-controlled, observer-blind phase 1 trial was conducted in 144 healthy young adults, 18 to 59 years of age, and older adults 65 to 85 years of age in Taizhou, Jiangsu Province, China. Eligible participants were randomized to receive two doses, administered 21 days apart, of either BNT162b1 at a dose of 10 ?g or 30 ?g or placebo, administered as an intramuscular injection. Study participants, investigators, and laboratory staff were blinded to treatment administration. The primary safety endpoints were local reactions at injection site or systemic adverse reactions within 14 days after vaccination, and adverse events occurring up to 28 days after receiving the boost vaccination. The immunogenic endpoints of virus-neutralizing antibody, and antigen-specific binding antibodies and cellular immune responses elicited by vaccine were measured at predefined timepoints.

(1769) Results

(1770) Study Design and Analysis Set

(1771) A total of 296 adults aged between 18-55 years or 65-85 years were screened at Taizhou vaccine clinical research center in Jiangsu Province, in China. 144 eligible participants consented to participate in the trial and were randomized 1:1:1 to receive prime and boost doses of BNT162b1 at 10 ?g or 30 ?g, or two placebo doses 21 days apart. Following priming doses, two participants between the ages of 65 and 85 years had withdrawn from boost dose administration (one at 10 ?g, one at 30 ?g). The demographic characteristics of the participants are shown in Table 24. The mean age among the younger participants ranged from 37.9 to 42.0 years, and the mean age among the older participants ranged from 68.5 to 70.7 years in the treatment groups, with equal gender distribution across treatment groups. The medical history or existing underlying disorders of the participants were similar across treatment groups, with the exception of hypertension, which was noted in older participants at baseline.

(1772) Observed Safety and Tolerability Data

(1773) Within 14 days after completion of dosing, 21 (88%) of the younger participants in the 10 ?g BNT162b1 dose group and 24 (100%) of the younger participants in the 30 ?g BNT162b1 dose group reported at least one solicited adverse reaction, versus 17% of the younger participants in the placebo group (Tables 25A-25B). Reactogenicity was dose level dependent and most evident in the 30 ag BNT162b1 dose group. The most common solicited adverse reactions reported were injection site pain, fever, headache, fatigue, malaise, joint pain, muscle pain chills. The adverse events were transient and either managed with simple standard of care management, or resolved spontaneously. Most of the reported adverse reactions were mild or moderate in severity, and resolved within the first seven days after each BNT162b1 dose. No injection site reactions were graded as severe (grade 3). All of the grade 3 systemic adverse reactions associated with the vaccination were fever, predominantly observed in the younger participants. One male participant in the older age group experienced an episodic Grade 3 fever accompanied by pain and pruritus at the injection site after administration of the prime BNT162b1 dose at 30 ?g, and electively withdrew from boost vaccination administration.

(1774) No pre-specified trial-halting rules were met during the study. Only one serious adverse event was reported by a participant of 67 years of age (a humerus fracture caused by a car accident, preventing the participant from receiving the boost dose) which was considered as not related to the vaccine or study procedure. The overall frequencies of injection site adverse reactions post-vaccination were comparable after the BNT162b1 prime and boost doses. Some systematic adverse reactions such as fever, headache, fatigue, and malaise occurred more commonly after the BNT162b1 boost dose than after the prime dose in younger adults. In contrast to the younger participants, elderly participants did not present with increased reactogenicity after the BNT162b1 boost dose.

(1775) There were no changes reported in blood pressure and respiratory rates among the participants across different treatment groups before and after BNT162b1 administration. Transient increases of temperature and pulse rate 24 hours post-vaccination were noted in both younger and older participants, especially in the 30 ?g dose group. The most common abnormalities in laboratory values from baseline were transient decreases in lymphocyte and platelet counts and increases in C-reaction protein. All laboratory abnormalities were self-limited and resolved in a short period of time without clinical manifestations. These data are consistent with certain findings reported in other populations (e.g., as described elsewhere herein).

(1776) Vaccine-Induced Antibody Responses

(1777) All participants were seronegative at baseline (day 1, pre-vaccination), and were monitored for seroconversion at days 8, 22, 29 and 43 by analyses of SARS-CoV-2 neutralizing antibodies and RBD and S1 protein binding antibodies. The BNT162b1 induced antibody responses in vaccinated participants were compared with a panel of human COVID-19 convalescent serum obtained at least 14 days after PCR-confirmed diagnosis from 28 COVID-19 patients. The highest neutralization titers were observed on day 43 (i.e., 21 days after the BNT162b1 boost dose), indicating a continuous uptrend in this group of Asian participants after day 29, which seems to further increase in this Asian population at day 43 among older participants as compared with reports in other populations, showing peak titres occurring earlier and subsequently subsiding in this subject population. On day 43, both 10 ?g and 30 ?g BNT162b1 dose levels induced significant virus-neutralizing antibody responses after the BNT162b1 prime dose which was boosted by the second BNT162b1 dose, with geometric mean titers (GMTs) of 232.9 (95% CI 151.3 to 358.5) and 254.0 (184.6 to 349.4) in the younger participants, and 80.0 (49.2 to 130.2) and 160.0 (96.7 to 264.6) in the older participants in the 10 ?g and 30 ?g dose groups, respectively (FIG. 120). The virus-neutralizing responses of younger participants in the 10 ?g and 30 ?g dose groups were 1.9 and 2.1 times the GMT of a panel of the convalescent sera (GMT, 119.9; 95% CI, 70.4 to 203.9). In the older participants, the corresponding ratios were 0.7 and 1.3 times in the 10 ?g and 30 ?g dose groups, respectively. All the younger recipients showed positive seroconversion on Day 43, and the seroconversion rate was 91% at the 10 ?g dose and 96% at the 30 ?g dose in the older recipients on Day 43, respectively. Participants who received the 30 ?g dose appeared to have somewhat higher virus-neutralizing antibody responses than those received the 10 ?g dose. However, the older participants between the ages of 65 and 85 generally showed a slower virus-neutralizing response and lower peak response than the younger participants between the ages 18 and 55.

(1778) Similarly, both doses of BNT162b1 induced high levels of S1- and RBD-binding IgG in the participants after the prime-boost regimen. The S1- and RBD-binding IgG levels after vaccination across all timepoints in the vaccine recipients were highly correlated with the neutralizing titers regardless of the age and dose groups, with a correlation coefficient of 0.85, and 0.79 (p<0.0001), respectively.

(1779) Vaccine-Induced T-Cell Responses

(1780) Vaccine-induced CD8+ T cell responses in individuals immunized with BNT162b1 were characterized before the priming vaccination (day 1), on day 29 (7 days after the boost vaccination) and on day 43 (21 days after the boost vaccination), using a direct ex vivo IFN? enzyme-linked immunosorbent spot (ELISpot) assay with peripheral blood mononuclear cells (PBMCs). At day 29, specific IFN-? ELISpot responses against the Sp1 peptide pool (covering RBD) were significantly higher in participants receiving BNT162b1 than those in placebo group (FIG. 121). Younger participants aged 18 to 55 years had average spot-forming cells of 227.5 (95% CI, 146.5 to 308.5) in those who had received 10 ?g vaccinations, and 223.5 (181.2 to 265.9) in those who had received the 30 ?g vaccinations per 105 PBMCs. In older participants aged 65 to 85 years, a slightly lower spot-forming cells with averages of 156.5 (84.1 to 229.0) and 171.9 (113.4 to 230.3) were noted post-vaccination across the two dose groups. At day 43, younger participants receiving the prime-boost BNT162b1 regimen tended to show a mild decrease in their S1-specific IFN-? ELISpot response compared to that seen on day 29; no blood samples were collected at this time point from the older participants, as so this data is not available. No differences between the BNT162b1 and the placebo groups were observed for IFN-? ELISpot responses to the Sp2 peptide pool (which does not include peptides of the RBD encoded by BNT162b1) and minor non-specific responses to CD8+ T cells were observed in both dose groups.

(1781) Discussion

(1782) The trial described in this Example was conducted in China in parallel with other BNT162 vaccine candidates in multiple regions.sup.14. One focus of the study was to establish data with respect to safety and immunogenicity of mRNA vaccines in Asian populations. This Example reports a first evaluation of both the safety and immunogenicity profiles of such an mRNA vaccine in a Chinese population, and furthermore of younger and older Chinese populations.

(1783) This is a preliminary report for the clinical trial of the modified-RNA-based SARS-CoV-2 vaccine candidate BNT162b1, which encodes the SARS-CoV-2 RBD, administrated to a healthy adult Chinese population. BNT162b1, like BNT162b2 (modRNA encoding the S protein derived from the same nucleoside-modified platform) induces strong vaccine-induced antibody responses and strong T cell responses. Clinical safety and immunogenicity for both BNT162b1 and BNT162b2 candidates have been evaluated in healthy adults in both German (younger adults; BNT162-01) and American (younger adults and elderly adults aged 65 to 85 years; BNT162-02) populations. In younger adult groups, severe local reactogenicity AEs within 7 days were fewer in American study (BNT162-02) and the present study (BNT162-03) as compared with the German study (BNT162-01). Systemic reactogenicity AEs within 7 days were broadly similar across studies. Systemic AEs (independent of relatedness) within 28 days post Dose 2 were slightly higher in the BNT162-02 and BNT162-03 studies, as compared to the BNT162-01 study.

(1784) In older adult groups, severe local reactogenicity AEs within 7 days were similar across studies. Systemic reactogenicity AEs within 7 days were slightly lower in the BNT162-03 study, as compared to the BNT162-01 and the BNT162-02 studies. Systemic AEs (independent of relatedness) within 28 days post Dose 2 were slightly higher in the BNT162-03 study as compared to the BNT162-02 study, however the severe AEs were lower. In summary, comparative analyses of the BNT162b1 safety profile between the BNT162-01, BNT162-02 and BNT162-03 studies at 30 ?g showed a generally comparable profile, and in the systemic reactogenicity/older population even a better safety profile in the Asian population vs non-Asian. Thus, findings reported here further complements and expands reporting of BNT162b1 and other RNA-based vaccine candidates from clinical trials conducted in Germany and the United States.sup.7,8,15.

(1785) The rationale for this study was to evaluate whether intrinsic and extrinsic differences between German and Chinese population have any impact on tolerability or immune responses to this novel type of vaccine. The safety profile of the vaccine candidate BNT162b1 in healthy Chinese adults observed in our study appear to be better than that reported in other populations, in term of severe reactogenicity by local and systemic reactions.sup.7,15. Body habitus, endogenous antibody repertoire may have an influence. The reactogenicity of BNT162b1 was dose-dependent. Increased frequencies of adverse events were observed after administration of the boost vaccination compared with those after the prime vaccination, especially in the younger participants. Older adults had lower incidences of adverse reactions than the younger participants. Grade 3 fever was reported by 17% of the younger participants and 8% of the older participants receiving 30 ?g dose, respectively. Nearly all severe fever reactions were transient and self-limiting. One participant had withdrawn from the boost vaccination due to the reactions after the prime dose administration, having episodic fever or cold intolerance with or without temperature record accompanied by the injection site pain, itching and pruritus, which lasted over two weeks and resolved after taking Hydrocortisone Butyrate ointment. Transient decreases in lymphocyte counts as pharmacodynamic markers were predominantly observed in the younger recipients at 30 ?g dose level of BNT162b1, which was associated with the redistribution of lymphocytes into lymphoid tissues by innate immune stimulation.sup.16.

(1786) Both doses of the vaccine candidate BNT162b1 were effective at eliciting specific humoral and cellular immune responses, with a clear boost effect of the second vaccination on antibody titers found in both younger and older adults. BNT162b1 administered at a 30 ?g dose following a prime-boost regimen induced an optimal level of immune responses in terms of virus-neutralizing antibody to SARS-CoV-2, which was higher than those in a panel of human convalescent serum samples, regardless of age. The humoral response in the Chinese participants showed a unique temporal pattern and peaked at day 43 in both age groups. Although the number of participants was small, and methodological differences in measurements that may occur can influence observed results, the findings reported here suggest that there may be a populational difference in response to the vaccine.

(1787) Since the vaccine candidate BNT162b1 is a modified RNA vaccine encoding a trimeric version of the RBD, the vaccine recipients in the study reported in the present Example demonstrated significant T-cell responses specific to S1 peptide pool (containing 166 15-mer S1 peptides of from the human SARS-CoV-2 virus), but not to the S2 peptide pool. The results indicated the cellular responses elicited by BNT162b1 was antigen specific. By contrast, the vaccine candidate BNT162b2 spectrum was different from other RNA-based SARS-CoV-2 vaccine, inducing T-cell responses could recognize both S1 and S2 peptide pools.sup.15. Nevertheless, data showed BNT162b1 at the 30 ?g dose was highly immunogenic capable of eliciting strong humoral and cell-mediated responses in healthy Chinese adults.

(1788) Those skilled in the art appreciate that small sample size and age restriction of 18 years and older may limit the conclusive rigor of findings observed in the present Example. Regardless, given that prophylactic RNA vaccines as described herein represent a novel approach to vaccination, safety assessments, including in particular populations (e.g., in children and adolescent populations) are particularly valuable. Also, although comparison of serum neutralizing responses elicited by the vaccine candidates described herein with that in human convalescent serum panels provides meaningful assessment of the vaccines, the level of serological immunity needed to protect against COVID-19 has not yet been rigorously established.sup.17. Those skilled in the art also recognize that the human convalescent serum panels that have been used in different trials are not standardized among laboratories, and thus may have a different distribution of patient characteristics and timepoints of collection, so that direct comparison of results (e.g., characterizing different vaccine candidates and/or characterizing vaccine candidates relative to different convalescent serum) may not be informative.

(1789) In summary, results described in the present Example confirmed the dose-dependency safety and good immunogenicity profile of the RNA-based SARS-CoV-2 vaccine candidate BNT162b1 and further expand the previous findings for BNT162b1 in the Germany and the United States trials.sup.7,8,15. Increased reactogenicity as well as a more favorable vaccine-elicited virus-neutralizing response were found associated with the 30 ?g of the BNT162b1 in both younger and older adults. In contrast, another vaccine candidate BNT162b2 manufactured from same platform, showed a more favorable safety profile.sup.8. BNT162b1 encodes a relatively small RBD immunogen, which might induce a narrower spectrum of neutralizing antibodies that are less robust to potential antigenic drift of SARS-CoV-2, compared with BNT162b2, which encodes a full-length spike immunogen.sup.18. It is worth noting that the candidate BNT162b2 has been found to be more than 95% effective in preventing COVID-19 in participants, with no decreasing efficacy in those over 65 years of age.sup.19.

(1790) Methods

(1791) Study Design and Participants

(1792) This randomized, placebo-controlled, observer-blind phase I trial was performed in healthy young adults between 18 and 59 years old, and older adults between 65 and 85 years of age, in Taizhou, Jiangsu Province, China. Participants were in overall good health established by medical history, physical examination, and laboratory tests at the screening visit. Both males and females were included and agreed to use contraception during the trial. We excluded participants that were pregnant or breast-feeding. Participants that tested positive for SARS-CoV-2 via a commercial rapid diagnostic kit for IgM/IgG antibody to SARS-CoV-2 (manufactured by Livzon diagnostics inc., Zhuhai, China), or via testing with a pharyngeal swab nucleic acid diagnostic test (manufactured by Fosun pharma, Shanghai, China) were excluded. Imaging features of COVID-19 present in a chest CT scan was a further exclusion criteria. Participants with serious cardiovascular disease or chronic conditions such as uncontrolled diabetes and hypertension, human immunodeficiency virus, hepatitis B and hepatitis C were excluded. Written informed consent was obtained from each participant before the start of the study.

(1793) The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. The trial protocol was reviewed and approved by the National Medical Products Administration, China, and the institutional review board of the Jiangsu Provincial Center of Disease Control and Prevention.

(1794) Randomization and Blinding

(1795) Eligible participants between 18 and 55 years of age were enrolled in the younger age group, and older participants aged greater than or equal to 65 years and less than or equal to 85 years were enrolled in the older age group. Participants were randomized in a ratio of 1:1:1 to receive the low-dose BNT126b1 or high-dose BNT126b1 or placebo. Participants were stratified by gender, using a Web-based interactive response technology (IRT) system. The blocked randomization list was generated by an independent statistician using SAS software (version 9.4).

(1796) Authorized unblinded pharmacists prepared the vaccines or placebo according to the allocation of participants through the IRT system, and nurses administrated the investigational products to participants. The unblinded staff had no further involvement in the trial, and were forbidden to disclosure allocation information to others. All other investigators, participants, laboratory staff and the sponsor remained blinded throughout the trial.

(1797) Vaccine and Vaccination

(1798) BNT162b1 as administered consisted of a Good Manufacturing Practice (GMP)-grade mRNA drug substance encoding the trimerized SARS-CoV-2 spike glycoprotein RBD antigen, formulated with lipids to obtain the RNA-LNP drug product. Vaccine was transported and supplied as a buffered-liquid solution for intramuscular injection, and stored at ?80? C., e.g., as described herein.

(1799) The low-dose and high-dose BNT126b1 contained 10 ?g and 30 ?g active ingredient, respectively, and the placebo was a commercial saline solution. Each participant received a prime-boost dosing regimen of vaccine candidate BNT162b1 at either 10 ?g/0.5 ml or at 30 ?g/0.5 ml or placebo of 0.5 ml administered into the deltoid, 21 days apart.

(1800) Monitoring of Safety and Immunogenicity

(1801) Each participant was asked to remain at the study site for at least six hours post vaccine administration to donate blood samples prior to and 24 hours post prime vaccination and again prior to and 8 days post boost vaccination for laboratory testing. Vital signs including temperature, blood pressure, pulse, and respiratory rate were measured at baseline, and one hour, three hours and six hours post-vaccination. Any adverse events following the vaccination were documented by participants using diaries until day 28 post-administration of the boost dose. Younger group participants were enrolled and received the vaccination first. Enrollment of the older age group was launched following evaluation of the preliminary safety data of the younger age group for the first 14 days post-prime vaccination. Severity of adverse events and laboratory abnormal changes are graded with both the scale issued by the China State Food and Drug Administration.sup.20 and the U.S. Food and Drug Administration (FDA).sup.21. Serum and PBMCs were collected before the vaccination, at day 8 and/or day 22 after the boost dose, to facilitate measurement of specific IgG antibody responses to RBD and spike glycoprotein S1, neutralizing antibody to SARS-CoV-2, and T-cell responses. All reported adverse events were reviewed by investigators. Adverse events were categorized as either possibly, probably, or definitely related to the vaccine candidate.

(1802) Human Convalescent Sera

(1803) The neutralizing titer is the reciprocal of the highest sample dilution that protects at least 50% of cells from cytopathic effects. A panel of 24 convalescent human serum samples were obtained from donors 18 to 70 years of age (mean age, 45.8 years) who had recovered from SARS-CoV-2 infection; samples were obtained at least 14 days after a polymerase chain reaction-confirmed diagnosis and after symptom resolution. The disease severities of these patients varied from non-symptomatic (n=3, 13%), mild (n=8, 33%), moderate (n=10, 42%), or severe (n=3, 13%).

(1804) Neutralizing geometric mean titers (GMTs) in subgroups of the donors were as follows: 40 for the 3 donors with non-symptomatic infections; 91.9 for the 8 donors with mild infection; 160 for those with moderate infection; and 226.3 in the 3 donors with severe infection. Each serum sample in the panel was from a different donor. Thus, most of the serum samples were obtained from persons with moderate Covid-19. The convalescent serum samples were tested side by side as comparators with the serum samples obtained from participants in this trial.

(1805) ELISA

(1806) We assessed binding antibody responses against the SARS-CoV-2 RBD and S1 by using the enzyme-linked immunosorbent assay (ELISA).

(1807) Microneutralization Assay

(1808) We detected the SARS-CoV-2 specific neutralizing antibody in serum by microneutralization assay based on cytopathy observed in a biosafety level 3 laboratory (BSL-3) with SARS-CoV-2 virus strain BetaCoV/JS02/human/2020 (EPI_ISL_411952).

(1809) ELISpot

(1810) Specific T-cell responses against the peptides were assessed by using a commercial ex-vivo interferon-? (INF-?) enzyme-linked Immunospot (ELISpot) kit manufactured by Mabtech (Nacka Strand, Stockholm, Sweden).sup.22. PBMCs were isolated from fresh blood samples, and stimulated with different overlapping peptide pools before the measurement. The S1 peptide pool, which covers the N-terminal half of SARS-CoV-2 spike, including the RBD, and the S2 peptide pool, which covers the C-terminal of SARS-CoV-2 spike, which does not include the RBD were used in this study.sup.23. A peptide pool consisting of 32 MHC class I restricted viral peptides from human Cytomegalovirus, Epstein-Barr virus and Influenza virus (CEF peptide pool), was used to stimulate CD8+ T cells to assess general T-cell reactivity (not specific to SARS-CoV-2.sup.24.)

(1811) Outcomes

(1812) The primary and secondary objectives of this trial were to evaluate safety and immunogenicity of the candidate vaccine BNT162b1 in healthy Chinese adults. The primary endpoints for safety evaluation were the incidence of solicited local reactions at the injection site or systemic adverse reactions within 14 days post vaccination, and adverse events following the immunization until 28 days after receiving the boost dose. Any clinical laboratory abnormalities from baseline to 24 hours or 7 days after vaccination, and any serious adverse event (SAE) that occurred were also recorded.

(1813) The secondary endpoints for immunogenicity were geometric mean titer (GMT), seroconversion rates, and fold increase of virus-neutralizing antibody, and ELISA IgG antibodies binding to S1 or RBD measured at days 8, 22 after each vaccination. Seroconversion is defined as an increase by a factor of four or more in antibody titer over the baseline, or the lower limit value if the baseline titer is below the limit of detection. The serum dilution for ELISA started at 1:100, while that for microneutralization assay started at 1:10.

(1814) Cellular immune responses in terms of the number of positive cells with interferon gamma (IFN-?) secretion among PBMCs at a concentration of 1?10.sup.5/well at day 8 and 22 after the boost dose were explored as an exploratory endpoint.

(1815) Statistical Analysis

(1816) The total sample size in this study was 144 participants, 24 participants of each age group was included in each treatment group. Based on an assumption of 8% of the adverse reaction occurrence post-vaccination, the probability of observation of at least one event in 24 participants in each dose group was 86.5%.

(1817) All randomized participants who received at least one dose of the investigational vaccine were included in the safety analysis. Safety endpoints were described as frequencies (%) with 95% confidence interval (CI) of the adverse reactions or events during the observation period. We compared the proportions of the participants with adverse reactions or events across the groups using Chi-square or Fisher exact. All participants who received at least one vaccination and had results of serologic measurements before or after vaccination were included in the immunogenicity analysis. The immunological endpoints were descriptively summarized at the specified time points, and compared across the groups, using ANOVA for log-transformed antibody titres, or Wilcoxon rank-sum test for non-normal data. The neutralising antibody responses of the participants in each dose group were compared with those of patients who had PCR-confirmed SARS-CoV-2 infection. Any serologic values below the lower limit of detection were set to half of the value (1:50 for ELISA and 1:5 for microneutralization assay), while the values above the highest dilution titer were assigned values of the highest dilution for calculation. Pearson correlation analysis of the RBD or S1 specific ELISA antibody and neutralising antibody was performed to assess the relationship between responses on different assays.

REFERENCES

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(1819) TABLE-US-00042 TABLE 24 Baseline characteristics of the participants, by age groups. Younger participants aged 18-55 years Older participants aged 65-85 years Characteristic 10 ?g 30 ?g Placebo 10 ?g 30 ?g Placebo No. of participants 24 24 24 24 24 24 Age, mean (SD), years 37.9 (9.6) 39.7 (9.0) 42.0 (8.7) 70.5 (5.0) 68.5 (3.0) 70.7 (4.4) Sex (female) 12 (50%) 12 (50%) 12 (50%) 12 (50%) 12 (50%) 12 (50%) Body-mass index, 24.7 (3.2) 23.0 (2.7) 24.3 (3.4) 24.0 (3.0) 24.8 (2.9) 23.5 (2.5) kg/m.sup.2 Medical history or existing disorder Cardiac ischemia 2 (8%) 2 (8%) 2 (8%) 0 0 0 Sinus bradycardia 0 2 (8%) 1 (4%) 0 0 0 Hyperuricemia 3 (13%) 1 (4%) 1 (4%) 3 (13%) 2 (8%) 2 (8%) Nasopharyngitis 2 (8%) 0 0 0 0 0 Blood uric acid 2 (8%) 1 (4%) 1 (4%) 0 0 0 increased Hypertension 3 (13%) 0 1 (4%) 12 (50%) 9 (38%) 7 (29%) Diabetes 0 0 0 1 (4%) 2 (8%) 1 (4%) Gastric inflammation 0 0 0 0 0 2 (8%) Others* 3 (13%) 5 (21%) 1 (4%) 3 (13%) 3 (13%) 4 (17%) Data are mean (SD) or n (%). *Others includes tonsillitis, helicobacter infection, human papilloma virus infection, periodontitis, electrocardiogram high voltage, lymphadenopathy, anemia, hepatic cyst, oropharyngeal discomfort, hyperthyroidism, noninfective gingivitis, hyperlipaemia, benign prostatic hyperplasia, prostatitis, blindness unilateral, cerebral infarct, limb injury, deformity of spine, calculus urinary and lymphadenopathy.

(1820) TABLE-US-00043 TABLE 25A Solicited adverse reactions within 14 days post-vaccination, and unsolicited adverse reactions till day 43, by age groups. Younger participants Older participants aged 18-55 years aged 65-85 years Adverse 10 ?g 30 ?g Placebo 10 ?g 30 ?g Placebo reactions (n = 24) (n = 24) (n = 24) P value (n = 24) (n = 24) (n = 24) P value Solicited adverse reactions within 14 days Any 21 (88%) 24 (100%) 4 (17%) <0.0001 21 (88%) 23 (96%) 2 (8%) <0.0001 Grade 3 3 (13%) 9 (38%) 0 0.0015 0 2 (8%) 0 0.3239 Injection-site adverse reactions Any 21 (88%) 24 (100%) 2 (8%) <0.0001 18 (75%) 21 (88%) 0 <0.0001 Grade 3 0 0 0 0 0 0 Pain 21 (88%) 23 (96%) 2 (8%) <0.0001 16 (67%) 21 (88%) 0 <0.0001 Redness 6 (25%) 8 (33%) 0 0.0059 3 (13%) 4 (17%) 0 0.1492 Swelling 5 (21%) 7 (29%) 0 0.0137 0 5 (21%) 0 0.0091 Induration 0 3 (13%) 0 0.1018 0 1 (4%) 0 1.0000 Systemic adverse reactions Any 17 (71%) 22 (92%) 3 (13%) <0.0001 9 (38%) 19 (79%) 2 (8%) <0.0001 Grade 3 3 (13%) 9 (38%) 0 0.0015 0 2 (8%) 0 0.3239 Fever* 14 (58%) 21 (88%) 1 (4%) <0.0001 7 (29%) 19 (79%) 1 (4%) <0.0001 Grade 3 3 (13%) 9 (38%) 0 0.0015 0 2 (8%) 0 0.3239 Headache 11 (46%) 19 (79%) 3 (13%) <0.0001 1 (4%) 2 (8%) 0 0.7682 Fatigue 12 (50%) 16 (67%) 0 <0.0001 3 (13%) 8 (33%) 0 0.0045 Malaise 8 (33%) 9 (38%) 0 0.0013 2 (8%) 4 (17%) 1 (4%) 0.4858 Joint pain 4 (17%) 10 (42%) 1 (4%) 0.0067 0 1 (4%) 0 1.0000 Muscle pain 2 (8%) 10 (42%) 0 <0.0001 0 1 (4%) 0 1.0000 Chills 4 (17%) 7 (29%) 0 0.0118 1 (4%) 4 (17%) 0 0.1185 Nausea 3 (13%) 3 (13%) 0 0.2330 0 0 0 Anorexia 1 (4%) 4 (17%) 0 0.1185 0 3 (13%) 1 (4%) 0.3143 Diarrhea 2 (8%) 1 (4%) 1 (4%) 1.0000 0 0 0 1.0000 Vomiting 0 2 (8%) 0 0.3239 0 0 0

(1821) TABLE-US-00044 TABLE 25B (modified) Solicited adverse reactions within 14 days post-vaccination, and unsolicited adverse reactions until day 43, by age groups including placebo-corrected AE rates. Younger participants aged 18-55 years 10 ?g 30 ?g (minus (minus Adverse 10 ?g placebo- 30 ?g placebo- Placebo reactions (n = 24) AEs) (n = 24) AEs) (n = 24) P value Solicited adverse reactions within 14 days Any 21 (88%) 17 (71%) 24 (100%) 20 (83%) 4 (17%) <0.0001 Grade 3 3 (13%) 3 (13%) 9 (38%) 9 (38%) 0 0.0015 Injection site adverse reactions Any 21 (88%) 19 (79%) 24 (100%) 22 (92%) 2 (8%) <0.0001 Grade 3 0 0 0 0 0 Pain 21 (88%) 19 (79%) 23 (96%) 21 (88%) 2 (8%) <0.0001 Redness 6 (25%) 6 (25%) 8 (33%) 8 (33%) 0 0.0059 Swelling 5 (21%) 5 (21%) 7 (29%) 7 (29%) 0 0.0137 Induration 0 0 3 (13%) 3 (13%) 0 0.1018 Systemic adverse reactions Any 17 (71%) 14 (58%) 22 (92%) 19 (79%) 3 (13%) <0.0001 Grade 3 1 (4%) 1 (4%) 4 (17%) 4 (17%) 0 0.0015 Fever* 14 (58%) 13 (54%) 21 (88%) 20 (83%) 1 (4%) <0.0001 Grade 3 1 (4%) 1 (4%) 4 (17%) 4 (17%) 0 0.0015 Grade 3 by 3 (13%) 3 (13%) 9 (38%) 9 (38%) 0 0.0015 NMPA criteria Headache 11 (46%) 8 (33%) 19 (79%) 16 (67%) 3 (13%) <0.0001 Fatigue 12 (50%) 12 (50%) 16 (67%) 16 (67%) 0 <0.0001 Malaise 8 (33%) 8 (33%) 9 (38%) 9 (38%) 0 0.0013 Joint pain 4 (17%) 3 (13%) 10 (42%) 9 (38%) 1 (4%) 0.0067 Muscle pain 2 (8%) 2 (8%) 10 (42%) 10 (42%) 0 <0.0001 Chills 4 (17%) 4 (17%) 7 (29%) 7 (29%) 0 0.0118 Nausea 3 (13%) 3 (13%) 3 (13%) 3 (13%) 0 0.2330 Anorexia 1 (4%) 1 (4%) 4 (17%) 4 (17%) 0 0.1185 Diarrhea 2 (8%) 1 (4%) 1 (4%) 0 1 (4%) 1.0000 Vomiting 0 0 2 (8%) 2 (8%) 0 0.3239 Unsolicited adverse reactions within 28 days Any 9 (38%) 8 (33%) 10 (42%) 9 (38%) 1 (4%) 0.0046 Fever.sup. 0 0 0 0 0 Temperature 2 (8%) 2 (8%) 6 (25%) 6 (25%) 0 0.0230 intolerance Injection site 3 (13%) 3 (13%) 4 (17%) 4 (17%) 0 0.1492 discomfort Injection site 2 (8%) 2 (8%) 3 (13%) 3 (13%) 0 0.3580 pruritus Pain not at 1 (4%) 1 (4%) 1 (4%) 1 (4%) 0 1.0000 injection site Dizziness 3 (13%) 3 (13%) 1 (4%) 1 (4%) 0 0.3142 Blood uric 1 (4%) 1 (4%) 1 (4%) 1 (4%) 0 1.0000 acid increased Older participants aged 65-85 years 10 ?g 30 ?g (minus (minus Adverse 10 ?g placebo- 30 ?g placebo- Placebo reactions (n = 24) AEs) (n = 24) AEs) (n = 24) P value Solicited adverse reactions within 14 days Any 21 (88%) 19 (79%) 23 (96%) 21 (88%) 2 (8%) <0.0001 Grade 3 0 0 2 (8%) 2 (8%) 0 0.3239 Injection site adverse reactions Any 18 (75%) 18 (75%) 21 (88%) 21 (88%) 0 <0.0001 Grade 3 0 0 0 0 0 Pain 16 (67%) 16 (67%) 21 (88%) 21 (88%) 0 <0.0001 Redness 3 (13%) 3 (13%) 4 (17%) 4 (17%) 0 0.1492 Swelling 0 0 5 (21%) 5 (21%) 0 0.0091 Induration 0 0 1 (4%) 1 (4%) 0 1.0000 Systemic adverse reactions Any 9 (38%) 7 (29%) 19 (79%) 17 (71%) 2 (8%) <0.0001 Grade 3 0 0 2 (8%) 2 (8%) 0 0.3239 Fever* 7 (29%) 6 (25%) 19 (79%) 18 (75%) 1 (4%) <0.0001 Grade 3 0 0 2 (8%) 2 (8%) 0 0.3239 Grade 3 by 0 0 2 (8%) 2 (8%) 0 0.3239 NMPA criteria Headache 1 (4%) 1 (4%) 2 (8%) 2 (8%) 0 0.7682 Fatigue 3 (13%) 3 (13%) 8 (33%) 8 (33%) 0 0.0045 Malaise 2 (8%) 1 (4%) 4 (17%) 3 (13%) 1 (4%) 0.4858 Joint pain 0 0 1 (4%) 1 (4%) 0 1.0000 Muscle pain 0 0 1 (4%) 1 (4%) 0 1.0000 Chills 1 (4%) 1 (4%) 4 (17%) 4 (17%) 0 0.1185 Nausea 0 0 0 0 0 Anorexia 0 ?1 3 (13%) 2 (8%) 1 (4%) 0.3143 Diarrhea 0 0 0 0 0 1.0000 Vomiting 0 0 0 0 0 Unsolicited adverse reactions within 28 days Any 4 (17%) 2 (8%) 9 (38%) 7 (29%) 2 (8%) 0.0590 Fever.sup. 0 0 1 (4%) 1 (4%) 0 1.0000 Temperature 0 0 4 (17%) 4 (17%) 0 intolerance Injection site 2 (8%) 2 (8%) 3 (13%) 3 (13%) 0 0.3580 discomfort Injection site 0 0 1 (4%) 1 (4%) 0 1.0000 pruritus Pain not at 0 0 0 0 0 injection site Dizziness 0 0 3 (13%) 3 (13%) 0 0.1018 Blood uric 2 (8%) 0 1 (4%) ?1 2 (8%) 1.0000 acid increased Data are shown as number of participants with event (%). Grade 3 was severe reaction (i.e., prevented activity). SAEs = Serious adverse events. A participant was only counted once in the specific reaction category, also with more than one episode of the adverse reaction. Only unsolicited adverse reactions reported by two or more participants were listed. *Those febrile participants were graded according to the guidelines of Food and Drug Administration (FDA), the United States. Fever was also graded according to the grading guidelines for adverse events in vaccine clinical trials, issued by the National Medical Products Administration (NMPA), China, which defines grade 3 fever as axillary temperature ?38.5? C. .sup.One participant experienced grade 3 fever accompanied with pain, itching and pruritus at the injection site after the prime dose, and electively withdrew from the boost vaccination.

Example 33: Neutralization of SARS-CoV-2 Lineage B.1.1.7 Pseudovirus by BNT162b2 Vaccine-Elicited Sera

(1822) In September 2020, the SARS-CoV-2 variant B.1.1.7 was detected in the United Kingdom, and it subsequently increased in prevalence, showed enhanced transmissibility, and spread to other countries and continents. B1.1.7 has a series of mutations in its spike protein: ?H69/V70, ?Y144, N501Y, A570D, D614G, P681H, T7161, S982A, and D1118H. One of these, N501Y, was of particular concern because it is located in the receptor binding site; the spike with this mutation binds more tightly to its cellular receptor, ACE-2; and virus with this mutation has increased host range that includes mice. 19 pseudoviruses, each bearing a SARS-CoV-2 S with a different mutation found in circulating strains, were neutralized as efficiently as non-mutant pseudoviruses by BNT162b2-immune sera. The following study demonstrates that a virus with the full set of mutations in the UK variant spike is also neutralized efficiently by BNT162b2-immune sera.

(1823) We generated VSV-SARS-CoV-2-S pseudoviruses bearing the Wuhan reference strain or lineage B.1.1.7 strain spike protein. Sera of 16 participants in the previously reported trial (Sahin U. et al., medRxiv 2020.12.09.20245175; doi: doi.org/10.1101/2020.12.09.20245175), drawn from eight younger (18-55 yrs) and eight older adults (56-85 yrs) 21 days after the booster immunization with 30 ?g BNT162b2, were tested for neutralization of SARS-CoV-2 Wuhan and lineage B.1.1.7 spike-pseudotyped VSV by a 50% pseudovirus neutralization assay (pVNT.sub.50; FIG. 122). The ratio of the 50% neutralization GMT of the sera against the SARS-CoV-2 lineage B.1.1.7 spike-pseudotyped VSV to that against the Wuhan reference spike-pseudotyped VSV was 0.79, indicating no biologically significant difference in neutralization activity against the two pseudoviruses.

(1824) The preserved neutralization of pseudoviruses bearing the B.1.1.7 spike by BNT162b2-immune sera suggests that the UK variant viruses will not escape BNT162b2-mediated protection. Furthermore, since there is good concordance between pseudotype neutralization and SARS-CoV-2 neutralization assays, the use of a non-replicating pseudovirus system is not expected to be a potential limitation of the work.

(1825) Materials and Methods

(1826) VSV-SARS-CoV-2 S Variant Pseudovirus Generation

(1827) A recombinant replication-deficient vesicular stomatitis virus (VSV) vector that encodes green fluorescent protein (GFP) and luciferase instead of the VSV-glycoprotein (VSV-G) was pseudotyped with SARS-CoV-2 spike (S) derived from either the Wuhan reference strain (NCBI Ref: 43740568) or the variant of concern (VOC)-202012/01 (also known as SARS-CoV-2 lineage B.1.1.7) according to published pseudotyping protocols (FIG. 123) (PMID: 21998709). In brief, HEK293T/17 monolayers transfected to express SARS-CoV-2 S were inoculated with VSV-G complemented VSV?G vector. After incubation for 1 h at 37? C., the inoculum was removed. Cells were washed with PBS before medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) was added to neutralize residual VSV-G complemented input virus. VSV-SARS-CoV-2-S pseudotype-containing medium was harvested 20 h after inoculation, 0.2 m filtered and stored at ?80? C. Prior to use in the neutralization test, the pseudovirus batches were titrated on Vero 76 cells, and the percent infected cells determined by flow cytometry (FIG. 124). Individual titers were calculated in transducing units (TU) per mL. Production of the VSV-SARS-CoV-2-S pseudoviruses bearing the Wuhan reference strain or lineage B.1.1.7 strain spike protein yielded similar titers (Table 26).

(1828) TABLE-US-00045 TABLE 26 Titers of SARS-CoV-2 Wuhan reference strain and lineage B.1.1.7 spike-pseudotyped VSV in transducing units (TU) per mL. VSV pseudovirus bearing Titer [TU/mL] Wuhan strain SARS-CoV-2 S 1.59 ? 10.sup.5 Lineage B.1.1.7 SARS-CoV-2 S 1.30 ? 10.sup.5

(1829) Serum Specimens and Neutralization Assay

(1830) The immunization and serum collection regimen is illustrated schematically in FIG. 125. For measuring neutralization titers, each serum was 2-fold serially diluted in culture medium with the first dilution of 1:20 (dilution range of 1:20 to 1:2560). VSV-SARS-CoV-2-S particles were diluted in culture medium to obtain 100 TU in the assay. Serum dilutions were mixed 1:1 with pseudovirus for 30 minutes at room temperature prior to addition to Vero 76 cell monolayers in 96-well plates and incubation at 37? C. for 24 hours. Supernatants were removed, and the cells were lysed with luciferase reagent (Promega). Luminescence was recorded, and neutralization titers were calculated in GraphPad Prism version 9 by generating a 4-parameter logistical (4PL) fit of the percent neutralization at each serial serum dilution. The 50% pseudovirus neutralisation titre (pVNT.sub.50) was reported as the interpolated reciprocal of the dilution yielding a 50% reduction in luminescence. A table of the neutralization titers is provided (Table 27). The ratio for each serum of the pVNT.sub.50 against SARS-CoV-2 lineage B.1.1.7 and the Wuhan reference strain spike-pseudotyped VSV is plotted in FIG. 126.

(1831) TABLE-US-00046 TABLE 27 pVNT.sub.50 values of 16 BNT162b2 post-immunization sera against SARS-CoV-2 Wuhan reference strain spike-pseudotype and lineage B.1.1.7 spike-pseudotyped VSV. pVNT.sub.50 pVNT.sub.50 ratio Serum ID Wuhan ref. B.1.1.7 (B.1.1.7/Wuhan ref.) 1 160 161.2 1.01 2 114.1 85.8 0.75 3 223.2 128.6 0.58 4 193 268.4 1.39 5 111.9 64.3 0.57 6 128 99.1 0.77 7 278.1 226.8 0.82 8 203.6 185 0.91 9 94.9 58.4 0.62 10 209.7 126.8 0.60 11 50.8 41.7 0.82 12 241.3 486.1 2.01 13 174 84.8 0.49 14 292.5 136.7 0.47 15 186.7 121.6 0.65 16 86.3 116.2 1.35

Example 34: Exemplary Regimen for Administration of a SARS-CoV-2 RNA Vaccine in Pregnant Women

(1832) The present Example describes an exemplary regimen for administration of a SARS-CoV-2 RNA vaccine described herein (specifically, in this Example, BNT162b2) in pregnant women (e.g., in healthy pregnant women 18 years of age and older).

(1833) Pregnant women are at risk for acquiring SARS-CoV-2 infection and COVID-19. Pregnancy may confer increased risk of severe COVID-19 because of physiological changes during pregnancy that can increase susceptibility to respiratory infections and subsequent rapid progression to respiratory failure. Additionally, pregnant women with COVID-19 have been reported to have higher rates of preterm birth, cesarean delivery, fetal distress, and infants requiring neonatal intensive care.

(1834) The present Example describes certain protocols in accordance with which BNT162b2 may be administered to pregnant women and/orto infants born from such pregnant women, and also describes certain assessments that may be performed and/or results that may be achieved. For example, this Example describes a study that will assess safety of BNT162b2 in pregnant women and their infants; it will also assess the immunogenicity of BNT162b2 in pregnant women, the transfer of antibody to their infants, and the kinetics of antibody transfer in the infant.

(1835) Among other things, the present Example describes a study that will assess the safety and tolerability of prophylactic BNT162b2 when administered to maternal participants 18 years of age or older vaccinated at 24 to 34 weeks' gestation. Without wishing to be bound by any particular theory, the present Example proposes that vaccination beginning within this time period may provide particular advantages. Recognizing that proposals ranging from vaccination at any time during pregnancy (see, for example, Israel Recommends COVID Vaccination in All Stages of Pregnancy, Updating Guidelines Haaretz Feb. 1, 2021) and others have proposed refraining from vaccinating during pregnancy (see, for example, WHO Strategic Advisory Group recommendation), the present Example describes a particular regimen in which pregnant mothers receive a first dose of vaccine between about 24 to about 34, or in some embodiments between about 27 to about 34 weeks of gestation, and a second dose about 21 days later, ideally prior to delivery of the baby.

(1836) Without wishing to be bound by any particular theory, the present Example proposes that vaccination according to this regimen may, for example, reduce risk to the fetus as may result, for example, from exposure to an immunized maternal immune response early in pregnancy. Furthermore, still without wishing to be bound by any particular theory, the present Example proposes that the provided vaccination schedule may provide particular benefits when at least two doses are administered prior to delivery of the baby. Among other things, the present Example proposes that a provided regimen may provide a particularly beneficial risk/benefit balance. Among other things, the present disclosure teaches that benefits that may be provided by immunization of pregnant mothers, and particularly by such immunization in accordance with a regimen described in the present Example, may impart immunity to the baby that, in some embodiments, may carry past delivery, this reducing need for immunization of the baby, at least for a period of days weeks, months, or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks or more, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more months, or 1, 2, 3, 4, or 5 years) post-delivery. Thus, in some embodiments, as noted herein, infants born of mothers vaccinated during pregnancy, e.g, according to a particular regimen as described herein, may not need further vaccination, or may need reduced vaccination (e.g., lower doses and/or smaller numbers of administrationse.g., boosters, and/or lower overall exposure over a given period of time), for a period of time (e.g., as noted herein) after birth.

(1837) For example, in maternal participants receiving at least 1 dose of study intervention from each vaccine group, the percentage of maternal participants reporting: (i) Local reactions for up to 7 days following each dose; (ii) Systemic events for up to 7 days following each dose (iii) AEs from Dose 1 through 1 month after Dose 2 (iv) SAEs from Dose 1 through 1 month after delivery will be assessed. Alternatively or additionally, in maternal participants complying with certain key protocol criteria (evaluable maternal participants) and no serological or virological evidence (up to 1 month after receipt of the second dose) of past SARS-CoV-2 infection: (v) GMR, estimated by the ratio of the geometric mean of SARS-CoV-2 neutralizing titers in pregnant women to those in nonpregnant women 1 month after Dose 2 may be assessed.

(1838) Still further alternatively or additionally, in maternal participants complying with the key protocol criteria (evaluable participants) and/or with without or without (e.g., separately for those with and those without, or independent of) serological or virological evidence (prior to 7 days after receipt of Dose 2) of past SARS-CoV-2 infection: (vi) 100?(1?IRR) [ratio of active vaccine to placebo] may be assessed.

(1839) Yet further alternatively or additionally, one or more of the following may be assessed: In maternal participants complying with the key protocol criteria (evaluable maternal participants) from each vaccine group: (a) GMCs/GMTs, at baseline (before Dose 1), 2 weeks after Dose 2, 1 month after Dose 2, and 6 months after delivery (b) GMFRs from baseline through 2 weeks after Dose 2, 1 month after Dose 2, and 6 months after delivery; In infants born to maternal participants receiving at least 1 dose of study intervention from each vaccine group, the percentage of infants with: (a) Specific birth outcomes (b) AEs from birth through 1 month of age (c) SAEs and AESIs (major congenital anomalies, developmental delay) through 6 months of age; In infants born to evaluable maternal participants from each vaccine group: (a) GMCs and GMFRs, at birth and 6 months after delivery; In maternal participants who received BNT162b2 (at initial randomization and at 1 month after delivery): (a) Incidence per 1000 person-years of follow-up; In maternal participants who received BNT162b2 at initial randomization and without evidence of prior SARS-CoV-2 infection: (a) Incidence per 1000 person-years of follow-up In each subset of evaluable maternal participants from each vaccine group with: (a) Confirmed COVID-19 (b) Confirmed severe COVID-19 (c) SARS-CoV-2 infection but no confirmed COVID-19 (d) GMCs/GMTs and GMFRs at baseline, 1 month after Dose 2, and 6 months after delivery; In evaluable maternal participants: (a) GMCs/GMTs at baseline and before Dose 2 (b) GMFRs from baseline to before Dose 2; In infants born to maternal participants from each vaccine group, based on the breastfeeding status: (a) GMCs and GMFRs, at birth and 6 months after delivery; In infants born to maternal participants receiving at least 1 dose of study intervention from each vaccine group, based on the breastfeeding status, the percentage of infants with: (a) AEs from birth through 1 month of age (b) SAEs and AESIs (major congenital anomalies, developmental delay) through 6 months of age; In infants born to maternal participants from each vaccine group: (a) Incidence rate of infant participants with confirmed COVID-19; In infants born to maternal participants from each vaccine group: (a) Incidence rate of MIS-C.

(1840) In some embodiments, a first dose will be administered to pregnant women during their 27 to 34 weeks of gestation, followed by a second dose approximately 21 days later. In some embodiments, a first dose will be administered to pregnant women during their 24 to 34 weeks of gestation, followed by a second dose approximately 21 days later. In some embodiments, participant mothers are assessed for a period of time up to about 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18 months or more (e.g., after initiation of the study, after administration of the first dose, after administration of the second dose, and/or after birth of the infant).

(1841) Is some embodiments, an infant born to a mother to whom one or more (e.g., two) vaccine doses have been administered (e.g., to whom two doses were administered during gestation) is assessed for a period of time up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months or more (e.g., after initiation of the study, after administration of the first dose, after administration of the second dose, and/or after birth of the infant).

(1842) In some embodiments, a dose will be 30 ug of BNT162b2 as described herein.

(1843) In some embodiments, assessment of vaccine performance are made in populations of pregnant women of any age, or within a particular age range (e.g., equal to or above 18 years of age). In some embodiments, assessment of vaccine performance are made in populations of women carrying singleton pregnancies.

(1844) In some embodiments, gestational age is assessed by one or more of last menstrual period, ultrasound examiniation, physical examination, and/or combinations thereof. In some embodiments, gestational age is determined by ultrasound. In some embodiments, gestational age is determined by consideration of two or more assessments (e.g., two or more ultrasounds performed at different times, such as in different trimesters, of the pregnancy).

(1845) In some embodiments, assessment of vaccine performance is made in populations characterized by one or more of: ultrasound examination performed at at least 18 weeks of pregnancy with no significant fetal abnormalities observed (e.g., upon assessment by a licensed investigator); documented negative HIV, syphilis, and/or HBV tests or combinations thereof; prepregnancy BMI of ?40 kg/m.sup.2.

(1846) In some embodiments, assessment of vaccine performance is made in populations that do not include subjects characterized by one or more of: suffering from a medical or psychiatric condition that may increase the risk of vaccination or otherwise, in the reasonable judgement of a licensed investigator make the subject inappropriate for receipt of the vaccine; prevopis clinical or micobiological diagnosis of COVID-19; history of severe adverse reaction associated with a vaccine and/or sever allergic reaction (e.g., anaphylaxis) to any component of the vaccine; known or suspected immunodeficiency; bleeding diathesis or condition associated with prolonged bleeding, gestational hypertension or preeclampsia-eclampsia, placental abnormality, polyhydramnios or oligohydramnios, significant bleeding or blood clotting disorder, gestational diabetes, one or more signs of premature labor with the current pregnancy or having ongoing intervention (medical/surgical) in the current pregnancy to prevent preterm birth, prior stillbirth or neonatal death, prior low birth weight or preterm delivery, prior history of at least 3 miscarriages, prior pregnancies numbering greater than 5, or previous infant with a known genetic disorder or major congenital anomaly, previous vaccination with any coronavirus vaccine, receipt of medications intended to prevent COVID-19, receipt of blood/plasma products or immunoglobulin from 60 days before administration of study intervention or planned receipt through delivery (with 1 exception, anti-D immunoglobulin (eg, RhoGAM), which can be given at any time), current alcohol abuse or illicit drug use, participants who receive treatment with immunosuppressive therapy (including cytotoxic agents or systemic corticosteroids, eg, for cancer or an autoimmune disease, or planned receipt through the postvaccination blood draw), participation in other studies involving study intervention within 28 days prior to study entry and/or during study participation, previous participation in other studies involving study intervention containing LNPs, current febrile illness, current symptoms of COVID-19 infection, Receipt of any seasonal or pandemic influenza vaccine in the previous 14 days, anticipated receipt of any seasonal or pandemic influenza vaccine in the 7 days after study intervention administration, receipt of a tetanus-, diphtheria-, and/or pertussis-containing vaccine in the previous 14 days, anticipated receipt of a tetanus-, diphtheria-, and/or pertussis-containing vaccine in the 7 days after study intervention administration, receipt of short-term (<14 days) systemic corticosteroids less than 28 days prior to dosing (inhaled/nebulized, intra-articular, intrabursal, or topical (skin or eyes) corticosteroids are permitted).

(1847) In some embodiments, a mother vaccinated as described herein may be taking or may begin taking, for example, an antipyretic or other pain medication to treat symptoms associated with the vaccination. Alternatively or additionally, in some embodiments, a mother may be taking or may begin taking a medication required for treatment of a preexisting stable condition and/or an inhaled, topical or localized injection of corticosteroids.

(1848) In some embodiments, a mother vaccinated as described herein may be given one or more antenatal corticosteroids, in particular if the pregnancy is at risk for preterm delivery. In some embodiments, the corticosteroid is a glucocorticoid. In some embodiments, the corticosteroid is betamethasone or progesterone, or a mixture thereof.

(1849) In some embodiments, vaccination as described in this Example reduces incidence of COVID-19 disease (and/or documented SARS-CoV-2 infection), or of severe COVID-19 disease, in mothers and/or infants born to them, for example relative to that observed in a comparable unvaccinated (e.g., having received placebo) population. In some embodiments, a mother is considered to have COVID-19 disease if least 1 of symptom of COVID-19 disease (fever; new or increased cough; new or increased shortness of breath; new or increased muscle pain; new loss of taste or smell; sore throat; diarrhea; vomiting; and/or in some embodiments fatigue, headache, nasal congestion or runny nose, nausea) is present and a SARS-CoV-2 NAAT-positive test is obtained during, or within 4 days before or after, the symptomatic period, either at the central laboratory or at a local testing facility (using an acceptable test). In some embodiments, a mother is considered to have severe COVID-19 disease if she has confirmed COVID-19 and one or more of the following is present: Clinical signs at rest indicative of severe systemic illness (RR ?30 breaths/min, HR ?125 beats/min, SpO2 ?93% on room air at sea level, or PaO2/FiO2 <300 mm Hg); Respiratory failure (defined as needing high-flow oxygen, noninvasive ventilation, mechanical ventilation, or ECMO); Evidence of shock (SBP <90 mm Hg, DBP <60 mm Hg, or requiring vasopressors); Significant acute renal, hepatic, or neurologic dysfunction*; Admission to an ICU; Death.

(1850) In some embodiments, an infant is considered to have COVID-19 disease if at least one symptom (fever, new or increased cough, new or increased shortness of breath, diarrhea, vomiting; and/or in some embodiments one or more of nasal congestion or runny nose, poor appetite or poor feeding, abdominal pain/colic) is present and a SARS-COV-2 NAAT-positive test result is obtained during, or within 4 days before or adter, the symptomatic period, either at the central laboratory or at a local testing facility (using an acceptable test). In some embodiments, an infant is considered to have severe COVID-19 disease if she has confirmed COVID-19 and one or more of the following is present: (i) Clinical signs at rest indicative of severe systemic illness: RR (breaths/min): >50 from birth to 1 week of age, 40 from 1 week to 1 month of age, 34 from 1 month to 6 months of age; HR (beats/min): >180; SpO2 ?92% on room air or >50% FiO2 to maintain ?92%, or PaO2/FiO2<300 mm Hg24; (ii) Respiratory failure (defined as needing high-flow oxygen including nasal CPaP/BiPaP, noninvasive ventilation, mechanical ventilation, or ECMO); (iii) Evidence of shock or cardiac failure: SBP (mm Hg) (<5th percentile for age): +<65 from birth to 1 week of age, <75 from 1 week to 1 month of age, <100 from 1 month to 6 months of age;

(1851) OR Requiring vasoactive drugs to maintain BP in the normal range;

(1852) (iv) Significant acute renal failure: serum creatinine >2 times ULN for age or 2-fold increase in baseline creatinine;

(1853) (v) Significant GI/hepatic failure: total bilirubin >4 mg/dL or ALT 2 times ULN for age;

(1854) (vi) Significant neurologic dysfunction: Glasgow Coma Scale score <11 or acute change in mental status with a decrease in Glasgow Coma Scale score ?3 points from abnormal baseline;

(1855) (vii) Admission to an ICU;

(1856) (viii) Death.

(1857) In some embodiments, incidence of multisystem inflammatory syndrome is not significantly increased (e.g., relative to a comparable population whose mothers were not vaccinated, and/or who were not vaccinated according to a protocol as described herein) in infants whose mothers were vaccinated as described herein. In some embodiments, an infant is considered to have multisystem inflammatory syndrome if: the infant presents with fever (?38.0? C. for ?24 hours or report of subjective fever lasting ?24 hours); AND there is laboratory evidence of inflammation (based on local laboratory ranges) including, but not limited to, 1 or more of the following: an elevated CRP, ESR, fibrinogen, procalcitonin, D-dimer, ferritin, LDH, or IL-6, elevated neutrophils, reduced lymphocytes, and low albumin; AND there is evidence of clinically severe illness requiring hospitalization (definition as noted above for severe disease), with multisystem (2) organ involvement: Cardiac (eg, shock, elevated troponin, elevated BNP, abnormal echocardiogram, arrhythmia); Renal (eg, acute kidney injury or renal failure); Respiratory (eg, pneumonia, ARDS, pulmonary embolism); Hematologic (eg, elevated D-dimers, thrombophilia, or thrombocytopenia); GI/hepatic (eg, elevated bilirubin, elevated liver enzymes, or diarrhea); Dermatologic (eg, rash, mucocutaneous lesions); Neurological (eg, CVA, aseptic meningitis, encephalopathy); AND there is no alternative plausible diagnoses; AND the infant is determined to be positive for current or recent SARS-CoV-2 infection by RT-PCR, serology, or antigen test; OR the infant has had COVID-19 exposure within the 4 weeks prior to the onset of symptoms.

(1858) In some embodiments, vaccination of mothers as described herein does not materially increase incidence of preterm delivery of infany morbidity.

(1859) In some embodiments, incidence of COVID-19 disease (and/or documented SARS-CoV-2 infection) in infants whose mothers were vaccinated as described herein is reduced relative to that of infacts whose mothers were not so vaccinated. In some embodiments, incidence of COVID-19 disease (and/or documented SARS-CoV-2 infection) in infants whose mothers were vaccinated as described herein is comparable to that of infants who were directly vaccinated after their delivery.

(1860) In some embodiments, vaccination as described herein achieves one or more of the following Primary or Secondary Outcome measures:

(1861) Primary Outcome Measures: 1. Percentage of maternal participants reporting: Local reactions Pain at the injection site, redness, and swelling as self-reported on electronic diaries [Time Frame: For 7 Days after Dose 1 and Dose 2] 2. Percentage of maternal participants reporting systemic events Fever, fatigue, headache, chills, vomiting, diarrhea, new or worsened muscle pain, and new or worsened joint pain as self-reported on electronic diaries. [Time Frame: For 7 days after Dose 1 and Dose 2] 3. Percentage of maternal participants reporting adverse events As elicited by investigational site staff [Time Frame: From Dose 1 through 1 month after Dose 2] 4. Percentage of maternal participants reporting serious adverse events As elicited by investigational site staff [Time Frame: From Dose 1 through 6 months after delivery] 5. Demonstrate non inferiority of immune response in pregnant women compared to nonpregnant female participants from the C4591001 study without evidence of past SARS-CoV-2 infection. GMR, estimated by the ratio of the geometric mean of SARS CoV 2 neutralizing titers in pregnant women to those in nonpregnant female participants [Time Frame: 1 month after Dose 2] 6. Demonstrate non inferiority of immune response in pregnant women compared to nonpregnant female participants from the C4591001 study with and without evidence of prior SARS-CoV-2 infection GMR, estimated by the ratio of the geometric mean of SARS CoV 2 neutralizing titers in pregnant women to those in nonpregnant female participants [Time Frame: 1 month after Dose 2]

(1862) Secondary Outcome Measures: 7. Evaluate efficacy against confirmed COVID 19 in participants without evidence of infection prior to vaccination 1000 person years of follow-up [Time Frame: 7 days after Dose 2] 8. Evaluate efficacy against confirmed COVID 19 in participants without evidence of prior infection. 1000 person years of follow-up [Time Frame: 7 days after Dose 2]

Example 35: Neutralization of SARS-CoV-2 Lineage B.1.1.298 (Danish Strain; a.k.a., SARS-CoV-2/Hu/DK/CL-5/1 (Cluster 5)) and B.1.351 (South African Strain; a.k.a., 20H/501Y.V2 (501.V2)) Pseudovirus by BNT162b2 Vaccine-Elicited Human Sera

(1863) Sera of 12 younger adult participants in the previously reported German phase 1/2 trial drawn at 7 or 21 days after the booster immunization with 30 ?g BNT162b2, were tested for neutralization of SARS-CoV-2 Wuhan Hu-1 (reference), South African lineage B.1.351 (SA-strain), and Danish mink-related lineage B.1.1.298 (DNK-strain) spike protein pseudotyped VSV by a 50% neutralization assay (pVNT50). The SA-strain spike protein carries the following amino acid changes compared to the Wuhan reference: L18F, D80A, D215G, AL242-244, R246I, K417N, E484K, N501Y, D614G, A701V. The DNK-strain spike protein carries the following amino acid changes compared to the Wuhan reference: Y453F, D614G, I692V, M1229I.

(1864) BNT162b2-immune sera neutralized the DNK-strain pseudovirus almost as efficiently as the SARS-CoV-2 Wuhan Hu-1 pseudotyped reference. A decrease (5-fold) in neutralizing titers was measured against the SARS-CoV-2 lineage B.1.351 pseudovirus when comparing the titers to the Wuhan Hu-1 pseudotyped reference. Importantly, all tested BNT162b2-immune sera were still able to neutralize with no complete escape being noted (FIG. 127).

(1865) Materials and Methods:

(1866) A recombinant replication-deficient VSV vector that encodes green fluorescent protein (GFP) and luciferase (Luc) instead of the VSV-glycoprotein (VSV-G) was pseudotyped with Wuhan-Hu-1 isolate SARS-CoV-2 spike (S) (GenBank: QHD43416.1), a variant harbouring four mutations found in the S protein of the Danish mink-related lineage B.1.1.298 (Y453F, D614G, I692V, M1229I), or variants harbouring ten mutations (L18F, D80A, D215G, R246I, ?242/243/244, K417N, E484K, N501Y, D614G, A701V) found in the South African lineage B.1.351 S protein according to published pseudotyping protocols. In brief, HEK293T/17 monolayers transfected to express the respective SARS-CoV-2 S truncated of the C-terminal cytoplasmic 19 amino acids (SARS-CoV-2-S(CA19)) were inoculated with VSV?G-GFP/Luc vector. After incubation for 1 h at 37? C., the inoculum was removed, and cells were washed with PBS before medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast) was added to neutralise residual input virus. VSV-SARS-CoV-2 pseudovirus-containing medium was collected 20 h after inoculation, 0.2-?m-filtered and stored at ?80? C. For pseudovirus neutralisation assays, 40,000 Vero 76 cells were seeded per 96-well. Sera were serially diluted 1:2 in culture medium starting with a 1:10 dilution (dilution range of 1:10 to 1:2,560). VSV-SARS-CoV-2-S pseudoparticles were diluted in culture medium for a fluorescent focus unit (ffu) count in the assay of ?1,000 TU in the assay. Serum dilutions were mixed 1:1 with pseudovirus for 30 minutes at room temperature prior to addition to Vero 76 cell monolayers in 96-well plates and incubation at 37? C. for 24 hours. Supernatants were removed, and the cells were lysed with luciferase reagent (Promega). Luminescence was recorded, and neutralisation titers were calculated in GraphPad Prism version 9 by generating a 4-parameter logistical (4PL) fit of the percent neutralisation at each serial serum dilution. The 50% pseudovirus neutralisation titre (pVNT50) was reported as the interpolated reciprocal of the dilution yielding a 50% reduction in luminescence.

Example 36: Neutralization of N501Y Mutant SARS-CoV-2 by BNT162b2 Vaccine-Elicited Sera

(1867) Rapidly spreading variants of SARS-CoV-2 have arisen in the United Kingdom and South Africa (Volz E. et al. Report 42-Transmission of SARS-CoV-2 Lineage B.1.1.7 in England: Insights from linking epidemiological and genetic data. www.imperial.ac.uk/mrc-global-infectious-disease-analysis/covid-19/report-42-sars-cov-2-variant/; Tegally H. et al. Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Afric. medRxiv 2020. doi.org/10.1101/2020.12.21.20248640). These variants have multiple mutations in their S glycoproteins, which are key targets of virus neutralizing antibodies. These rapidly spreading variants share the spike N501Y substitution. This mutation is of particular concern because it is located in the viral receptor binding site for cell entry, increases binding to the receptor (angiotensin converting enzyme 2), and enables the virus to expand its host range to infect mice (Gu H. et al. Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy. Science 2020; 369:1603-7; Chan K. K. et al. An engineered decoy receptor for SARS-CoV-2 broadly binds protein S sequence variants. Cold Spring Harbor Laboratory 2020.doi: 10.1101/2020.10.18.344622).

(1868) We generated an isogenic Y501 SARS-CoV-2 on the genetic background of the N501 clinical strain USA-WA1/2020, which also provided the genetic background of the BNT162b2-encoded spike antigen. Sera of 20 participants in the previously reported trial (Walsh E. E. et al. Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. N Engl J Med 2020; Polack F. P. et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Eng. J Med 2020. DOI: 10.1056/NEJMoa2034577), drawn 2 or 4 weeks after immunization with two 30-?g doses of BNT162b2 spaced three weeks apart, were tested for neutralization of N501 and Y501 viruses by a 50% plaque reduction neutralization assay (PRNT.sub.50; FIG. 128). The ratio of the 50% neutralization GMT of the sera against the Y501 virus to that against the N501 virus was 1.46, indicating no reduction in neutralization activity against the virus bearing the Y501 spike.

(1869) Materials and Methods

(1870) Construction of Isogenic Viruses

(1871) We prepared an isogenic pair of SARS-CoV-2 containing the N501 or Y501 spike protein (FIG. 129). The N501Y mutation was generated by an A-to-T substitution at nucleotide 23,063 of the viral genome using an infectious cDNA clone of clinical strain WA1 (2019-nCoV/USA_WA1/2020) (Xie X. et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 2020; 27:841-8 e3). Following a previously reported mutagenesis protocol (Plante J. A. et al. Spike mutation D614G alters SARS-CoV-2 fitness. Nature 2020), we recovered N501 and Y501 viruses with titers of >10.sup.7 plaque-forming units (PFU) per ml. The two viruses developed similar plaque morphologies on Vero E6 cells (FIG. 130).

(1872) Serum Specimens and Neutralization Assay

(1873) The immunization and serum collection regimen is illustrated schematically in FIG. 131. For measuring neutralization titers, each serum was 2-fold serially diluted in culture medium with the first dilution of 1:40 (dilution range of 1:40 to 1:1280). The diluted serum was incubated with 100 PFU of N501 or Y501 virus at 37? C. for 1 h, after which the serum-virus mixtures were inoculated onto Vero E6 cell monolayer in 6-well plates. A conventional (non-fluorescent) plaque reduction neutralization assay was performed to quantify the serum-mediated virus suppression as previously reported (Muruato A. E. et al. A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation. Nat Commun 2020; 11:4059). A minimal serum dilution that suppressed >50% of viral plaques is defined as PRNT.sub.50. A table of the neutralization titers is provided (Table 28). The ratio for each serum of the PRNT.sub.50 against N501 and Y501 virus is plotted in FIG. 132.

(1874) TABLE-US-00047 TABLE 28 PRNT.sub.50 values of 20 BNT162b2 post-immunization sera against N501 and Y501 SARS-CoV-2. PRNT.sub.50 PRNT.sub.50 ratio Serum ID N501 Y501 (Y501/N501) 1 160 640 4 2 160 320 2 3 320 640 2 4 80 160 2 5 160 160 1 6 320 320 1 7 640 640 1 8 160 160 1 9 640 640 1 10 640 1280 2 11 160 640 4 12 320 320 1 13 640 1280 2 14 640 320 0.5 15 320 640 2 16 320 640 2 17 640 640 1 18 640 1280 2 19 640 640 1 20 640 640 1

Example 37: Neutralization of Spike 69/70 Deletion, E484K, and N501Y SARS-CoV-2 by BNT162b2 Vaccine-Elicited Sera

(1875) Rapidly spreading variants of SARS-CoV-2 have arisen in the United Kingdom (UK), South Africa (SA), and other regions (Volz E. et al. CMe. Report 42Transmission of SARS-CoV-2 Lineage B.1.1.7 in England: Insights from linking epidemiological and genetic data. wwwimperialacuk/mrc-global-infectious-disease-analysis/covid-19/report-42-sars-cov-2-variant/2021; Tegally H. et al. e. Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa medRxiv 2020. doi.org/10.1101/2020.12.21.20248640). These variants have multiple mutations in their spike glycoproteins, which are key targets of virus neutralizing antibodies. The emerged spike mutations have raised concerns of vaccine efficacy against these new strains. The goal of this study is to examine the effect of several key spike mutations from the UK and SA strains on BNT162b2 vaccine-elicited neutralization.

(1876) We engineered three SARS-CoV-2s containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion+N501Y+D614G from UK; and E484K+N501Y+D614G from SA. Neutralization geometric mean titers (GMTs) of twenty BTN162b2-vaccinated human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small mutational effects on neutralization by sera elicited by two BNT162b2 doses.

(1877) Using an infectious cDNA clone of SARS-CoV-2 (Xie X. et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 2020; 27:841-8 e3), we engineered three spike mutant viruses on the genetic background of clinical strain USA-WA1/2020 (FIG. 133). (i) Mutant N501Y virus contains the N501Y mutation that is shared by both the UK and SA variants. This mutation is located in the viral receptor binding domain (RBD) for cell entry, increases binding to the receptor (angiotensin converting enzyme 2), and enables the virus to expand its host range to infect mice (Xie X. et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 2020; 27:841-8 e3; Wrapp D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020; 367:1260-3). (ii) Mutant ?69/70+N501Y+D614G virus contains two additional changes from the UK variants: amino acid 69 and 70 deletion (A69/70) and D614G substitution. Amino acids 69 and 70 are located in the N-terminal domain of the spike S1 fragment; deletion of these residues may allosterically change the conformation of spike (Wrapp D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020; 367:1260-3). The D614G mutation is dominant in circulating strains around the world (Plante J A et al. Spike mutation D614G alters SARS-CoV-2 fitness. Nature 2020; Korber B. et al. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell 2020). (iii) Mutant E484K+N501Y+D614G virus contains the E484K substitution, which is also located in the viral RBD. The E484K substitution alone confers resistance to several monoclonal antibodies (Ku Z. et al. Molecular determinants and mechanism for antibody cocktail preventing SARS-CoV-2 escape. Nat Commun 2021; 12:469; Baum A. et al. Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies. Science 2020; 369:1014-8). Compared with the wild-type USA-WA1/2020 strain, the three mutant viruses showed similar plaque morphologies on Vero E6 cells (FIG. 134).

(1878) We tested a panel of human sera from twenty participants in the previously reported clinical trial (Walsh E E et al. Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. N Engl J Med 2020; Polack F P et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N Engl J Med 2020), drawn 2 or 4 weeks after immunization with two 30-?g doses of BNT162b2 spaced three weeks apart (FIG. 135). Each serum was tested for neutralization of wild-type USA-WA1/2020 strain and the three mutant viruses by a 50% plaque reduction neutralization assay (PRNT.sub.50; Tables 29 and 30).

(1879) TABLE-US-00048 TABLE 29 PRNT.sub.50s of twenty BNT162b2 post-immunization sera against wild-type (USA-WA1/2020) and mutant N501Y SARS-CoV-2s PRNT.sub.50 PRNT.sub.50 ratio Serum ID WT N501Y (N501Y/WT) 1 160 640 4 2 160 320 2 3 320 640 2 4 80 160 2 5 160 160 1 6 320 320 1 7 640 640 1 8 160 160 1 9 640 640 1 10 640 1280 2 11 160 640 4 12 320 320 1 13 640 1280 2 14 640 320 0.5 15 320 640 2 16 320 640 2 17 640 640 1 18 640 1280 2 19 640 640 1 20 640 640 1

(1880) TABLE-US-00049 TABLE 30 PRNT.sub.50s of twenty BNT162b2 post-immunization sera against wild-type (USA-WA1/2020), ?69/70 + N501Y + D614G, and E484K + N501Y + D614G SARS-CoV-2s PRNT.sub.50 PRNT.sub.50 ratio ?69/70 + E484K + ?69/70 + E484K + Serum N501Y + N501Y + N501Y + N501Y + ID WT D614G D614G D614G/WT D614G/WT 1 320 640 320 2 1 2 160 160 80 1 0.5 3 640 1280 640 2 1 4 160 160 80 1 0.5 5 320 320 320 1 1 6 640 640 640 1 1 7 640 1280 320 2 0.5 8 320 320 160 1 0.5 9 1280 1280 1280 1 1 10 640 1280 640 2 1 11 320 320 320 1 1 12 640 1280 320 2 0.5 13 1280 2560 1280 2 1 14 320 320 320 1 1 15 320 640 320 2 1 16 640 640 640 1 1 17 640 1280 640 2 1 18 320 640 320 2 1 19 640 640 320 1 0.5 20 640 1280 640 2 1

(1881) All sera showed equivalent neutralization titers between the wild-type and mutant viruses, with differences of ?4 fold (FIG. 136). Notably, ten out of the twenty sera had neutralization titers against mutant ?69/70+N501Y+D614G virus that were twice their titers against the wild-type virus (FIG. 136b), whereas six out of the twenty sera had neutralization titers against mutant E484K+N501Y+D614G virus that were half their titers against the wild-type virus (FIG. 136c). The ratios of the neutralization GMTs of the sera against the N501Y, ?69/70.sup.+N501Y+D614G, and E484K+N501Y+D614G viruses to their GMTs against the USA-WA1/2020 virus were 1.46, 1.41, and 0.81, respectively (FIG. 137).

(1882) Consistent with other recent reports of the neutralization of variant SARS-CoV-2 or corresponding pseudoviruses by convalescent or post-immunization sera (Wibmer C K et al. SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma. bioRxiv 2021:doi: doi.org/10.1101/2021.01.18.427166; Wang Z. et al. mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. bioRxiv 2021:doi: doi.org/10.1101/2021.01.15.426911), the neutralization GMT of the serum panel against the virus with three mutations from the SA variant (E484K+N501Y+D614G) was slightly lower than the neutralization GMTs against the N501Y virus or the virus with three mutations from the UK variant (?69/70+N501Y+D614G). However, the magnitude of the differences in neutralization GMTs against any of the viruses in this study was small, as compared to the 4-fold differences in hemagglutination-inhibition titers that have been used to signal potential need for a strain change in influenza vaccines (Smith D J et al. Mapping the antigenic and genetic evolution of influenza virus. Science 2004; 305:371-6).

(1883) Methods

(1884) Construction of isogenic viruses. Three recombinant SARS-CoV-2 mutants (N501Y, ?69/70-N501Y+D614G, E484K+N501Y+D614G in spike protein) were prepared on the genetic background of an infectious cDNA clone derived from clinical strain WA1 (2019-nCoV/USA_WA1/2020) (Xie X. et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 2020; 27:841-8 e3) by following the PCR-based mutagenesis protocol as reported previously (Plante J A et al. Spike mutation D614G alters SARS-CoV-2 fitness. Nature 2020). The full-length infectious cDNAs were in vitro ligated and used as templates to transcribe full-length viral RNA. Mutant viruses (P0) were recovered on day 2 from Vero E6 cells after electroporation of the in vitro RNA transcripts. P1 viruses were harvested as stocks by passaging the P0 virus once on Vero E6 cells. The titers of P1 viruses were determined by plaque assay on Vero E6 cells. The genome sequences of the P1 viruses were validated by Sanger sequencing. The detailed protocol was recently reported (Xie X. et al. Engineering SARS-CoV-2 using a reverse genetic system. Nature Protocols 2021:doi.org/10.1038/s41596-021-00491-8).

(1885) Serum specimens and neutralization assay. The immunization and serum collection regimen are illustrated schematically in FIG. 135. A conventional (non-fluorescent) plaque reduction neutralization assay was performed to quantify the serum-mediated virus suppression as previously reported (Muruato A E et al. A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation. Nat Commun 2020; 11:4059). Briefly, each serum was 2-fold serially diluted in culture medium with the first dilution of 1:40 (dilution range of 1:40 to 1:1280). The diluted sera were incubated with 100 plaque-forming units of wild-type or mutant viruses at 37? C. for 1 h, after which the serum-virus mixtures were inoculated onto Vero E6 cell monolayer in 6-well plates. After 1 h of infection at 37? C., 2 ml of 2% Seaplaque agar (Lonza) in Dulbecco's modified Eagle medium (DMEM) containing 2% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) was added to the cells. After 2 days of incubation, 2 ml of 2% Seaplaque agar (Lonza) in DMEM containing 2% FBS, 1% P/S and 0.01% neutral red (Sigma) were added on top of the first layer. After another 16 h of incubation at 37? C., plaque numbers were counted. The minimal serum dilution that inhibits 50% of plaque counts is defined as the 50% plaque reduction neutralization titer (PRNT.sub.50). Each serum was tested in duplicates. The PRNT.sub.50 assay was performed at the biosafety level-3 facility at the University of Texas Medical Branch.

Example 38: Neutralizing Activity of BNT162b2-Elicited Serum

(1886) New, highly transmissible SARS-CoV-2 variants that were first detected in the United Kingdom (B.1.1.7 lineage), South Africa (B.1.351 lineage), and Brazil (P.1 lineage) with mutations in the S gene are spreading globally. To analyze effects on neutralization elicited by BNT162b2, we engineered S mutations from each of the three new lineages into USA-WA1/2020, a relatively early isolate of the virus from January 2020 (FIG. 138). We subsequently produced five recombinant viruses. The first had all the mutations found in the S gene in the B.1.1.7 lineage (B.1.1.7-spike), the second had all the mutations found in the S gene in the P.1 lineage (P.1-spike), the third had all the mutations found in the S gene in the B.1.351 lineage (B.1.351-spike), the fourth had an N-terminal domain deletion found in the B.1.351 lineage and the globally dominant D614G substitution (B.1.351-?242-244.sup.+D614G), and the fifth had the three mutations from the B.1.351 lineage at the receptor-binding site (K417N, E484K, and N501Y) and a D614G substitution (B.1.351-RBD+D614G). The amino acid residues mutated in the B.1.351-RBD+D614G virus are also among those mutated in the P.1 lineage virus, though in the P.1 lineage virus, K417 is mutated to threonine rather than asparagine. All the mutant viruses yielded infectious viral titers exceeding 10 plaque-forming units per milliliter. The B.1.1.7-spike and B.1.351-spike virus formed plaques that were smaller than those of the other viruses (FIG. 139).

(1887) We performed 50% plaque reduction neutralization testing (PRNT.sub.50) using 20 serum samples that had been obtained from 15 participants in the pivotal trial (Polack F P et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med 2020; 383: 2603-15; Walsh E E et al. Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates. N Engl J Med 2020; 383: 2439-50) 2 or 4 weeks after the administration of the second dose of 30 ?g of BNT162b2 (which occurred 3 weeks after the first immunization) (FIG. 140). All the serum samples efficiently neutralized USA-WA1/2020 with almost all of them having titers higher than 1:40. Geometric mean neutralizing titers against USA-WA1/2020, B.1.1.7-spike, P.1-spike, B.1.351-spike, B.1.351-?242-244+D614G, and B.1.351-RBD+D614G viruses were 532, 663, 437, 194, 485, and 331, respectively (FIG. 141 and Table 31). Thus, as compared with neutralization of USA-WA1/2020, neutralization of B.1.1.7-spike and P.1-spike viruses was roughly equivalent, and neutralization of B.1.351-spike virus was still robust but ?2.7-fold lower. Our data are also consistent with lower neutralization titers against the virus with the full set of B.1.351-spike mutations than virus with either subset of mutations and suggest that mutations in the receptor-binding site (K417N, E484K, and N501Y) affect neutralization more than the 242-244 deletion in the N-terminal domain of the spike protein.

(1888) Because neutralization of the B.1.1.7-spike and P.1-spike viruses by BNT162b2-elicited sera is roughly equivalent to neutralization of USA-WA1/2020, the neutralization data provide strong support that BNT162b2 will continue to protect against the variants first detected in the UK or Brazil. Protection against B.1.351 lineage virus is also anticipated, given that, although neutralization titers against this variant are somewhat lower, they are still robust and much higher than the barely detectable titers observed after one dose of BNT162b2, when strong efficacy was already observed in the pivotal C4591001 efficacy trial (Polack F P et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med 2020; 383:2603-15; Walsh E E et al. Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates. N Engl J Med 2020; 383:2439-50; Sahin U et al. BNT162b2 induces SARS-CoV-2-neutralising antibodies and T cells in humans. Dec. 11, 2020 (www.-medrxiv.-org/-content/-10.-1101/-2020.-12.-09.-20245175v1). preprint.). In addition, T cell immunity may also be involved in protection (Liao M et al. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19. Nature Medicine 2020/), and BNT162b2 immunization elicits CD8+ T-cell responses that recognize multiple variants (Skelly D T et al. Vaccine-induced immunity provides more robust heterotypic immunity than natural infection to emerging SARS-CoV-2 variants of concern. Research Square 2021).

(1889) Materials and Methods

(1890) Construction of isogenic viruses. All recombinant SARS-CoV-2s with spike mutations (FIG. 138) were prepared on the genetic background of an infectious cDNA clone derived from clinical strain USA-WA1/2020 (Xie X et al. An Infectious cDNA Clone of SARS-CoV-2. Cell Host Microbe 2020; 27:841-8 e3). The mutations were introduced into the spike gene using a PCR-based mutagenesis protocol as reported previously (Plante J A et al. Spike mutation D614G alters SARS-CoV-2 fitness. Nature 2020. doi: 10.1038/s41586-020-2895-3; Xie X et al. Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and N501Y variants by BNT162b2 vaccine-elicited sera. Nat Med 2021. doi: 10.1038/s41591-021-01270-4). The full-length infectious cDNAs were ligated and used as templates to in vitro transcribe full-length viral RNAs. The original viral stocks (P0) were recovered from Vero E6 cells on day 2 post electroporation of the in vitro transcribed RNAs. The P0 viruses were propagated on Vero E6 cells for another round to produce P1 viruses for the neutralization assays. The titers of P1 viruses were measured by plaque assay on Vero E6 cells (FIG. 139). The complete spike sequences of the P1 viruses were confirmed by Sanger sequencing to have only the intended nucleotide changes from the USA-WA1/2020 sequence. A detailed protocol of the above experiments was recently reported (Xie X et al. Engineering SARS-CoV-2 using a reverse genetic system. Nature Protocols 2021:doi.org/10.1038/s41596-021-00491-8).

(1891) Serum specimens and neutralization assay. FIG. 140 illustrates the immunization and serum collection scheme. A conventional 50% plaque-reduction neutralization test (PRNT.sub.50) was performed to quantify the serum-mediated virus suppression as previously reported (Muruato A E et al. A high-throughput neutralizing antibody assay for COVID-19 diagnosis and vaccine evaluation. Nat Commun 2020; 11:4059). Briefly, individual sera were 2-fold serially diluted in culture medium with a starting dilution of 1:40 (dilution range of 1:40 to 1:1280). The diluted sera were incubated with 100 PFU of USA-WA1/2020 or mutant SARS-CoV-2. After 1 h incubation at 37? C., the serum-virus mixtures were inoculated onto a monolayer of Vero E6 cells pre-seeded on 6-well plates on the previous day. A minimal serum dilution that suppressed >50% of viral plaques is defined as PRNT.sub.50. The neutralization titers are presented in Table 31.

(1892) TABLE-US-00050 TABLE 31 PRNT.sub.50's of twenty BNT162b2 post-immunization sera against USA-WA1/2020 and mutant SARS-CoV-2. *PRNT.sub.50 B.1.351- ?242- B.1.351- USA-WA1/2020 B.1.1.7- P.1-spike B.1.315-spike 244 + RBD + Serum ID Exp1 Exp2 Exp3 GMT spike Exp1 Exp2 GMT Exp1 Exp2 GMT D614G D614G 1 320 320 320 320 640 320 320 320 160 160 160 320 320 2 160 160 160 160 160 80 80 80 40 40 40 160 80 3 640 640 640 640 640 640 640 640 320 320 320 640 640 4 160 320 320 254 320 320 320 320 80 160 113 160 80 5 320 320 320 320 640 320 320 320 160 160 160 320 320 6 320 640 640 508 320 640 320 453 160 160 160 320 160 7 1280 640 640 806 1280 640 1280 905 320 320 320 1280 1280 8 320 320 320 320 320 160 160 160 80 80 80 160 160 9 1280 1280 1280 1280 1280 1280 640 905 640 640 640 1280 1280 10 640 640 1280 806 1280 640 320 453 640 320 453 1280 640 11 320 320 320 320 640 320 320 320 80 160 113 320 160 12 640 640 640 640 640 640 320 453 160 160 160 320 320 13 1280 1280 1280 1280 1280 640 640 640 160 320 226 1280 640 14 320 320 640 403 320 320 160 226 160 80 113 320 160 15 640 640 640 640 640 1280 640 905 320 320 320 640 320 16 320 320 640 403 1280 640 320 453 160 320 226 640 320 17 1280 1280 1280 1280 1280 1280 640 905 320 320 320 1280 640 18 640 640 640 640 640 640 320 453 160 320 226 320 320 19 640 640 640 640 640 1280 640 905 320 320 320 640 640 20 640 640 640 640 1280 640 320 453 160 160 160 640 320 .sup.GMT 502 520 577 532 663 520 368 437 184 204 194 485 331 .sup.#95% CI 371- 401- 443- 409- 497- 372- 275- 325- 133- 151- 144- 345- 228- 680 674 751 693 884 726 491 589 255 276 261 681 480 *The data for USA-WA1/2020 are from three experiments; the data for B.1.1.7-spike, B.1.351-?242-244 + D614G, and B.1.351-RBD-D614G viruses are from one experiment each; and the data for P.1-spike and B.1.351-spike viruses are from two experiments. For each independent experiment, individual PRNT50 value is the geometric mean of duplicate plaque assay results; no differences were observed between the duplicate assays. .sup.Geometric mean neutralizing titers. .sup.#95% confidence interval (95% CI) for the GMT.

Example 39: Durability of BNT162b2-Induced CD4+ and CD8+ T-Cell Responses

(1893) In a subset of 24 subjects across dose levels 10 to 30 ?g, samples collected at Day 85 and Day 184 (nine and 23 weeks post-boost, respectively) were analyzed in order to determine the durability of T-cell responses induced by BNT162b2. On Day 184 and after an initial contraction, both CD4.sup.+ and CD8.sup.+ T-cell responses were detectable in the majority of individuals, across the three dose levels tested. Kinetics of CD4.sup.+ and CD8.sup.+ responses observed in four older adult subjects vaccinated with 10 ?g BNT162b2 were comparable to younger adult subjects, with S protein-specific CD4.sup.+ T cells still detectable in all four subjects 23 weeks after boost vaccination. BNT162b2 induced CD4.sup.+ and CD8.sup.+ responses were either higher than or in the range of recall antigen memory responses (FIG. 142).

Example 40: MHC-I Binding Epitopes Recognized by CD8 T-Cells Induced by BNT162b2

(1894) Using MHC-class I multimer technology, several epitopes spread across the whole length of the S Protein and presented by a combination of common HLA-A and HLA-B alleles were identified to be recognized by CD8+ T-cells induced by BNT162-b2 (measured 7 days after the boost vaccination). Some peptide/HLA combinations were found in more than one subject.

(1895) TABLE-US-00051 TABLE 32 MHC-I binding epitopes recognized by CD8 T-cells induced by BNT162b2 Position in Identified in HLA Epitope SEQ ID NO S Protein No. Subjects B35:01 LPFNDGVYF 47 84-92 1 A03:01 GVYFASTEK 52 89-97 1 A02:01 YLQPRTFLL 40 269-277 3 B35:01 QPTESIVRF 45 321-329 1 A26:01 CVADYSVLY 53 361-369 1 B15:01 CVADYSVLY 53 361-369 1 A03:01 KCYGVSPTK 54 378-386 2 A24:02 NYNYLYRLF 43 448-456 3 B15:01 FQPTNGVGY 55 497-505 1 B35:01 IPFAMQMAY 46 896-904 1 A02:01 RLQSLQTYV 41 1000-1008 2 A68:01 GTHWFVTQR 56 1099-1108 1 C04:01 VYDPLQPEL 57 1137-1145 1 A24:02 QYIKWPWYI 42 1208-1216 3 A24:02 KWPWYIWLGF 44 1211-1220 1

Example 41: Histological Findings Following Administration of BNT162b2

(1896) Classical chromogenic immunohistochemistry (IHC) and chromogenic dual IHC-ISH (in situ hybridization) experiments were performed to investigate biodistribution of BNT162b2 in mouse tissues 6 h and 6d post-injection.

(1897) Protocol

(1898) After harvesting, tissue is fixed in 4% RotiHistofix overnight at 4? C. and embedded in paraffin wax after dehydration in Leica Tissue Processor. Chromogenic IHC is performed. Spike protein is detected with anti-Spike2 mouse monoclonal antibody (Genetex). Dual IHC-ISH assay is conducted according to a self-established protocol based on Document MK 51-149 from Advanced Cell Diagnostics using the company kits and reagents. BNT162b2 probe (modV9) for ISH is custom designed by Advanced Cell Diagnostics based on the sequence provided by TRON. IHC protocol for the immune cell markers CD11c (Cell Signaling), CD19 (Cell Signaling), CD169 (Thermo Fisher) and F4/80 (Cell Signaling) were in place at TRON and they are adapted to dual IHC-ISH assay for the project. Images are acquired using Vectra Polaris Multispectral Slide Scanner microscope (Akoya Bioscience) and analysed with PhenoChart software (Akoya Bioscience).

(1899) Results

(1900) As can be seen in FIG. 143, a specific vaccine mRNA signal (red) is detected in the lymph nodes (LN) 6 h post injection using modV9 probe in dual IHC-ISH assay. Vaccine is mostly localized to subcapsular sinus (LN in 9 and 5 positions) and B cell follicles (LN in 12 and 1 positions). Dendritic cells are visualized by CD11c staining (turquoise, upper images) and only some of them uptake the vaccine. Majority of CD169+ macrophages (subcapsular sinus macrophages, turquoise, middle images) are positive for the vaccine. B cells (CD19+, turquoise, lower images) are the second major population showing vaccine signal.

(1901) A specific vaccine mRNA signal is still detectable in the LN 6d post injection using modV9 probe in dual IHC-ISH assay, albeit in very less amount (data not shown). Some CD11c+ DCs and subcapsular sinus macrophages are positive for the vaccine. Most of the vaccine signal detected is in the B cells (CD19+).

(1902) As can be seen in FIG. 144, a specific vaccine mRNA signal (red) is detected in the spleen 6 h post injection using modV9 probe in dual IHC-ISH assay. Majority of the vaccine signal is detected in the white pulp. Dendritic cells are visualized by CD11c staining (turquoise, upper images) and only some of them uptake the vaccine. A small portion of F4/80+ macrophages (turquoise, middle images) uptake the vaccine. B cells (CD19+, turquoise, lower images) are the major population showing the vaccine signal.

(1903) A specific vaccine mRNA signal is still detectable in the spleen 6d post injection using modV9 probe in dual IHC-ISH assay, albeit in very less amount (data not shown). The vaccine signal detected is solely in the B cells (CD19+). No DCs and macrophages show vaccine signal 6d post-injection.

(1904) After 6 h, using mouse anti-S2 mouse monoclonal antibody, we detected a signal in the muscle, especially in some muscle fibers and in the connective tissue perimysium. In the LNs, we detected cells expressing Spike protein in the T cell zone (data not shown).

(1905) After 6d, using mouse anti-S2 mouse monoclonal antibody, no Spike expression is detectable in the muscle. On the contrary, LNs are full of cells expressing the vaccine (data not shown).

(1906) No nonspecific staining is detected with the S2 mouse monoclonal antibody in the chromogenic IHC experiments.

(1907) Summary

(1908) A very strong vaccine signal is visible in the draining LNs and spleen 6 h post-injection. In the LN, vaccine is mostly detected in B cell follicles and subcapsular sinus, with some signal also in the T cell zone. By dual IHC-ISH, we showed that indeed the B cells (CD19+) and subcapsular sinus macrophages (CD169+) are the major cells that uptake the vaccine. Dendritic cells (CD11c+) in the T cell zone and intermediary sinus also uptake the vaccine. After 6d, some vaccine mRNA is still visible in the draining LNs. The signal observed in the T-cell zone after 6d is in the dendritic cells (CD11c+). Some B cells and LN macrophages also still have some vaccine at that stage.

(1909) Analysis of the spleen harvested 6 h post-injection showed that vaccine already reaches spleen within 6 h, most probably via blood circulation. The signal is located white pulp, where B cells and T cells form the major population and antigen presentation to T cells occur in the white pulp. With dual IHC-ISH assay, we showed that majority of B cells uptake the vaccine. Many DCs (CD11c+) surrounding the B cells are also positive. After 6d, signal is restricted to B cells. IHC protocol is established to detect spike protein expression using anti-Spike S2 mouse monoclonal antibody on the cell pellets treated with/without the vaccine. A specific signal is detected only in the cells treated with BNT162b2. No nonspecific staining was visible in the na?ve tissues tested. In the muscle, Spike expression is detected 6 h post injection in the muscle fibers and in the connective tissue perimysium. After 6d, no staining is detectable in the muscle. On the contrary, the massive Spike expression is visible 6d post injection in LN, in particular in the T-cell zone.

Example 42: Stability Studies

(1910) Stability assessments of BNT162b2 formulations at various concentrations (e.g., 0.5 mg/mL, 1 mg/mL, and 2 mg/mL) have been performed, and have included assessments of compositions, stored at various temperatures (e.g., ?70? C. [e.g., ?70?10? C.], ?20? C. [e.g., ?20?5? C.], +5? C. [e.g., 5?3? C.], or +25? C. [e.g., 25?2? C.]) and/or for various periods of time (e.g., 0.5 months, 1 month, 2 months, 3 months, 4 months, and in certain cases one or more intervening time points (e.g., 1.5 months, 2.5 months, etc).

(1911) In exemplary studies, mice were injected (single leg) at day 0 with 20 uL of a relevant formulation. Blood was collected and serum generated at days 14, 21, and 28 after the administration; spleen were isolated at day 28.

(1912) ELISAs were performed to detect presence of antibodies in serum that bind to S1 protein, or specifically to the RBD domain. FIG. 145 presents exemplary S1 ELISA results obtained with 28-day serum from mice injected with indicated formulations that had been stored under indicated temperature conditions for indicated periods of time. As can be seen with reference to FIG. 145, all stored samples performed well, and reasonably comparably, after one (1) month of storage. After two (2) or three (3) months of storage, some decreased activity was observed for samples stored at +25? C., but samples stored at ?70? C., ?20? C., +5? C. maintained significant performance.

(1913) At some time points, one or more parameters such as appearance, RNA content, RNA integrity, RNA encapsulation, lipid content (overall and/or of individual components and/or ratios thereof), particle size, particle polydispersity index, in vitro expressability, etc) were assessed; additional or alternative parameters may be or have been assessed.

(1914) Exemplary observations include that storage at +25? C. is not recommended for periods of time longer than about 2 weeks, and preferably not more than about 1 week as, among other things, RNA integrity was observed to decrease significantly. It was also observed that, in at least some cases, significant ability to induce antibodies was maintained even when in vitro expressability was materially reduced. Change in polydispersity index, particularly after about 3 months of storage, or after about 4 months of storage, were observed to be greater for formulations stored at +5? C. or above than for those stored at lower temperatures.