NUCLEIC ACID BASED COMBINATION VACCINES

Abstract

The present invention is inter alia directed to pharmaceutical compositions comprising at least one nucleic acid encoding at least one antigenic peptide or protein from a Coronavirus, preferably a pandemic Coronavirus, and at least one nucleic acid encoding at least one antigenic peptide or protein from a further virus, e.g. an Influenza virus or an RSV virus. Pharmaceutical compositions provided herein are suitable for use in treatment or prophylaxis of an infection with at least one Coronavirus and at least one further virus infection, and may therefore be comprised in a combination vaccine. The nucleic acid sequences of the pharmaceutical compositions and combination vaccines are preferably in association with a polymeric carrier, a polycationic protein or peptide, or a lipid nanoparticle (LNP). The invention is also directed to first and second and further medical uses of the pharmaceutical compositions and combination vaccines, and to methods of treating or preventing a Coronavirus infection and a further virus infection.

Claims

1. A pharmaceutical composition comprising: (A) at least one component A comprising a mRNA having a coding sequence encoding a SARS-CoV-2 spike protein (S) at least 90% identical to SEQ ID NO: 10 that is a pre-fusion stabilized spike protein (S_stab) comprising pre-fusion stabilizing K986P and V987P mutations; and (B) at least one component B comprising a mRNA having a coding sequence encoding a first influenza virus hemagglutinin (HA) protein or an immunogenic fragment thereof, wherein the mRNAs are complexed or associated with lipid nanoparticles (LNP) and wherein the LNP comprises: (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one one sterol; and (iv) at least one PEG-lipid, wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid, and wherein the mRNA from component A and component B each comprise a 5′ cap structure and a poly(A) sequence comprising 30 to 200 adenosine nucleotides.

2. The composition of claim 1, further comprising at least a second component B comprising a mRNA having a coding sequence encoding a second influenza virus HA protein, or an immunogenic fragment thereof, wherein the second influenza virus HA protein is from a different strain of influenza virus than the first influenza virus HA protein.

3. The composition of claim 2, further comprising at least a third component B comprising a mRNA having a coding sequence encoding a third influenza virus HA protein, or an immunogenic fragment thereof, wherein the third influenza virus HA protein is from a different strain of influenza virus than the first and the second influenza virus HA protein.

4. The composition of claim 1, further comprising at least a second component B comprising a mRNA having a coding sequence encoding a first influenza virus neuraminidase (NA) protein, or an immunogenic fragment thereof.

5. The composition of claim 2, wherein the composition comprises mRNAs encoding at least one influenza A HA and at least one influenza B HA.

6. The composition of claim 1, wherein the at least one neutral lipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

7. The composition of claim 6, wherein the at least one one sterol comprises cholesterol.

8. The composition of claim 7, wherein the LNP comprises elements (i) to (iv) in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-10% PEG-lipid.

9. The composition of claim 8, wherein the at least one cationic lipid of the LNP has the formula III: ##STR00013## or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L1 and L2 are each independently —O(C═O)— or —(O═O)O—; G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 cycloalkenylene; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl; R3 is H, OR5, CN, —C(═O)OR4, —OC(═O)R4 or —NR5C(═O)R4; R4 is C1-C12 alkyl; and R5 is H or C1-C6 alkyl.

10. The composition of claim 9, wherein the at least one cationic lipid has the formula III: ##STR00014## or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L1 and L2 are each independently —O(C═O)— or —(C═O)O—; G1 and G2 are each independently unsubstituted C1-C12 alkylene; G3 is C1-C24 alkylene; R1 and R2 are each independently C6-C24 alkyl; R3 is OR5; and R5 is H.

11. The composition of claim 10, wherein the at least one PEG-lipid comprises PEG-DMG or PEG-cDMA.

12. The composition of claim 8, wherein the at least one PEG-lipid has the formula IVa: ##STR00015## wherein n has a mean value ranging from 30 to 60.

13. The composition of claim 8, wherein the at least one cationic lipid comprises a cationic lipid according to formula III-3: ##STR00016##

14. The composition of claim 1, wherein the mRNA from component A and/or component B comprise a coding sequence that is a codon modified sequence, the codon modified sequence selected from a C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof.

15. The composition of claim 14, wherein the mRNA from component A and component B comprise a coding sequence that is modified to increase G/C content.

16. The composition of claim 15, wherein the mRNA from component A and/or component B comprise a coding sequence having a G/C content of at least about 50%.

17. The composition of claim 1, wherein the mRNA from component A and/or component B comprise a heterologous 5′ untranslated region (UTR) and/or a heterologous 3′ UTR.

18. The composition of claim 17, wherein the mRNAs from component A and component B comprise, from 5′ to 3′, the following elements: (I) a 5′ Cap having a m7G; (II) a coding sequence including a signal sequence; and (III) a poly(A) sequence comprising 30 to 200 adenosine nucleotides.

19. The composition of claim 18, wherein the mRNAs from component A and component B comprise, from 5′ to 3′, the following elements: (I) a 5′ Cap having a m7G; (II) a heterologous 5′ UTR; (III) a coding sequence including a signal sequence; (IV) a heterologous 3′ UTR; and (V) a poly(A) sequence comprising 30 to 200 adenosine nucleotides.

20. The composition of claim 18, wherein the mRNAs from component A and component B comprise at least one modified nucleotide selected from pseudouridine (ψ) and N1-methylpseudouridine (m1ψ).

21. The composition of claim 20, wherein the mRNAs from component A and component B are each complexed or associated with different LNP.

22. The composition of claim 1, wherein the composition is a lyophilized or spray-dried composition and has a water content of less than about 10%.

23. The composition of claim 1, wherein when administered to a subject the composition elicits antigen-specific immune responses to the encoded proteins of the mRNAs from component A and component B.

24. The composition of claim 23, wherein the antigen specific immune response to the encoded protein of the mRNA of component A is not reduced as compared to a subject administered a composition having only component A and without component B.

25. A pharmaceutical composition comprising: (A) at least one component A comprising a mRNA having a coding sequence encoding a SARS-CoV-2 spike protein (S) at least 90% identical to SEQ ID NO: 10 that is a pre-fusion stabilized spike protein (S_stab) comprising pre-fusion stabilizing K986P and V987P mutations; and (B) at least one component B comprising a mRNA having a coding sequence encoding a first influenza virus hemagglutinin (HA) protein, or an immunogenic fragment thereof, wherein the mRNAs are complexed or associated with lipid nanoparticles (LNP) and wherein the LNP comprises: (i) a cationic lipid of formula III: ##STR00017## or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: L1 and L2 are each independently —O(C═O)— or —(C═O)O—; G1 and G2 are each independently unsubstituted C1-C12 alkylene; G3 is C1-C24 alkylene; R1 and R2 are each independently C6-C24 alkyl; R3 is OR5; and R5 is H; (ii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) cholesterol; and (iv) a PEG-lipid, wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% DSPC, 25-55% cholesterol, and 0.5-10% PEG-lipid, wherein the mRNA from component A and component B each comprise a 5′ cap structure and a poly(A) sequence comprising 30 to 200 adenosine nucleotides, and wherein when administered to a subject the composition elicits antigen-specific immune responses to the encoded proteins of the mRNAs from component A and component B.

26. The composition of claim 25, wherein the mRNAs from component A and component B each have all uracil nucleotides replaced by m1ψ nucleotides.

27. The composition of claim 26, wherein the N/P ration for the cationic lipid relative to the mRNAs is about 1 to about 10.

28. The composition of claim 27, wherein the at least one PEG-lipid comprises DMG-PEG 2000.

29. The composition of claim 26, wherein component B comprises at least three different mRNAs each having a coding sequence encoding a different HA antigen, or an immunogenic fragment thereof, wherein each different HA protein is from a different strain of influenza.

30. A method of stimulating an immune response in a subject comprising administering a composition according to claim 25 to the subject, wherein the composition is administered by intramuscular administration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[1444] FIG. 1 Combination vaccines comprising component A-1 induce humoral immune responses against SARS-CoV-2 spike protein, shown by ELISA on day 21 after first vaccination (FIG. 1 A) and by VNT-Assay on day 42 after second vaccination (FIG. 1 B). Further details provided in Example 3 and Table 19.

[1445] FIG. 2 Combination vaccines comprising component B-3a induce humoral immune responses against RSV F protein, shown by ELISA on day 21 after first vaccination (FIG. 2 A) and on day 42 after second vaccination (FIG. 2 B). Further details provided in Example 3 and Table 19.

[1446] FIG. 3 Combination vaccines comprising component B-2 induce humoral immune responses against Influenza HA (A1 California/07/09), shown by ELISA on day 21 after first vaccination (FIG. 3 A) and on day 42 after second vaccination (FIG. 3 B). Further details provided in Example 3 and Table 19.

[1447] FIG. 4 Combination vaccines comprising component A-1 induce humoral immune responses against SARS-CoV-2 spike protein, shown by ELISA on day 21 after first vaccination (FIG. 4 A) and by VNT-Assay on day 42 after second vaccination (FIG. 4 B). Further details provided in Example 4 and Table 20.

[1448] FIG. 5 Combination vaccines comprising component B-3a induce humoral immune responses against RSV F protein, shown by ELISA on day 21 after first vaccination (FIG. 5 A) and on day 42 after second vaccination (FIG. 5 B). Further details provided in Example 4 and Table 20.

[1449] FIG. 6 Combination vaccines comprising component B-3b induce humoral immune responses against hMPV F protein, shown by FACS-based Immunoassay on day 42 after second vaccination for hMPV-A1 (FIG. 6 A) and for hMPV-B1 (FIG. 6 B). Further details provided in Example 4 and Table 20.

[1450] FIG. 7 Combination vaccines comprising component B-4a induce humoral immune responses against PIV3 F protein, shown by FACS-based Immunoassay on day 42 after second vaccination. Further details provided in Example 4 and Table 20.

[1451] FIG. 8 Combination vaccines comprising component B-2 induce humoral immune responses against Influenza HA (A1 California/07/09), shown by ELISA on day 42 after second vaccination. Further details provided in Example 4 and Table 20.

ABBREVIATIONS

[1452] FLUAV: Influenza A virus [1453] FLUBV: Influenza B virus [1454] RSV: Human respiratory syncytial virus [1455] HRSV-A, RSV-A: Human respiratory syncytial virus A [1456] HPIV, hPIV: Human respirovirus (Human parainfluenzavirus) [1457] HPIV3: Human respirovirus 3 [1458] HMPV, hMPV: Human metapneumovirus [1459] MERS-CoV: Middle East respiratory syndrome-related coronavirus [1460] SARS-CoV: Severe acute respiratory syndrome-related coronavirus [1461] HCoV: Human coronavirus [1462] Bat SARS-like CoV: Bat SARS-like coronavirus [1463] BatCoV: Rousettus bat coronavirus [1464] PDCoV: Porcine deltacoronavirus [1465] PEDV: Porcine epidemic diarrhea virus [1466] MHV: Murine hepatitis virus

EXAMPLES

[1467] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods, which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Preparation of DNA and RNA Constructs, Compositions, and Vaccines

[1468] The present example provides methods of obtaining the RNA of the invention as well as methods of generating a composition or a vaccine of the invention.

1.1. Preparation of DNA and RNA Constructs:

[1469] DNA sequences encoding different virus antigen designs were prepared and used for subsequent RNA in vitro transcription reactions. Said DNA sequences were prepared by modifying the wild type or reference encoding DNA sequences by introducing a G/C optimized or modified coding sequence (e.g., “cds opt1”) for stabilization and expression optimization. Sequences were introduced into a pUC derived DNA vector to comprise stabilizing 3′-UTR sequences and 5′-UTR sequences, additionally comprising a stretch of adenosines (e.g. A64 or A100), and optionally a histone-stem-loop (hSL) structure, and optionally a stretch of 30 cytosines (e.g. C30) (see Table 17). The obtained plasmid DNA constructs were transformed and propagated in bacteria using common protocols known in the art. Eventually, the plasmid DNA constructs were extracted, purified, and used for subsequent RNA in vitro transcription.

1.2. RNA In Vitro Transcription from Plasmid DNA Templates:

[1470] DNA plasmids prepared according to section 1.1 were enzymatically linearized using a restriction enzyme and used for DNA dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (e.g. m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG)) under suitable buffer conditions. The obtained RNA constructs were purified using RP-HPLC (PureMessenger®, CureVac AG, Tubingen, Germany; WO2008077592) and used for in vitro and in vivo experiments. The generated RNA sequences/constructs are provided in Table 17 with the encoded antigenic protein and the respective UTR elements indicated therein.

TABLE-US-00018 TABLE 17 Overview of mRNA constructs used in Examples SEQ ID SEQ ID SEQ ID RNA NO: NO: CDS NO: ID Name Protein (gc) RNA R1 FLUAV/H1N1/A/California/7/2009 hemagglutinin (HA) 14178 14230 14438 R2 FLUAV/H3N2/A/Hong Kong/4801/2014 hemagglutinin (HA) 14181 14233 14441 R3 FLUBV/H0N0/B/Brisbane/60/2008 hemagglutinin (HA) 14182 14234 14442 R4 FLUBV/H0N0/B/Phuket/3073/2013 hemagglutinin (HA) 14183 14235 11113 R5 FLUAV/H1N1/A/California/7/2009 neuraminidase (NA) 14210 14262 14470 R6 FLUAV/H3N2/A/Hong Kong/4801/2014 neuraminidase (NA) 14213 14265 14473 R7 FLUBV/H0N0/B/Brisbane/60/2008 neuraminidase (NA) 14214 14266 14474 R8 HRSV-A/A2 fusion glycoprotein (F-del_DSCav1_mut5) 14550 14560 14600 R9 HPIV3/Homo sapiens/PER/FLA4571/2008 fusion glycoprotein (F0) 14612 14614 14622 R10 HPIV3/BJ/001/08 hemagglutinin-neuraminidase (HN) 14613 14615 14623 R11 HMPV/TN/92-4 fusion glycoprotein (F0) 14626 14650 14746 R12 MERS-CoV/MERS-CoV-Jeddah-human-1 spike protein (1-1353)(V1060P_L1061P) 14795 14811 14875 R13 SARS-CoV/Tor2 spike protein (1-1255)(K968P_V969P) 14910 14955 15135 R14 SARS-CoV-2/Wuhan/WIV05/2019 spike protein (1-1273)(K986P_V987P); R9515 10 137 163 R15 SARS-CoV-2/Wuhan/WIV05/2019 spike protein (1-1273)(K986P_V987P); R9709 10 137 149 R16 SARS-CoV-2/Wuhan/WIV05/2019 spike protein (1- 22961 23091 23531 1273)(K986P_V987P_L18F_D80A_D215G_L242del_A243del_L244del_R246I_K4 17N_E484K_N501Y_D614G_A701V); R10384 R17 SARS-CoV-2/Wuhan/WIV05/2019 spike protein (1- 22959 23089 23529 1273)(K986P_V987P_H69del_V70del_Y144del_N501Y_A570D_D614G_P681H_T 716I_S982A_D1118H); R10357 R18 FLUAV/H1N1/A/California/7/2009 hemagglutinin (HA); R8849 14178 14230 26949 R19 FLUAV/H3N2/A/Hong Kong/4801/2014 hemagglutinin (HA); R8853 14181 26946 26950 R20 FLUBV/H0N0/B/Brisbane/60/2008 hemagglutinin (HA); R8852 14182 14234 26951 R21 FLUBV/H0N0/B/Phuket/3073/2013 hemagglutinin (HA); R8854 14183 14235 26952 R22 FLUAV/H1N1/A/California/7/2009 neuraminidase (NA); R8858 14210 14262 26953 R23 FLUAV/H3N2/A/Hong Kong/4801/2014 neuraminidase (NA); R8856 14213 14265 26954 R24 FLUBV/H0N0/B/Brisbane/60/2008 neuraminidase (NA); R8860 14214 14266 26955 R25 HRSV-A/A2 fusion glycoprotein (F-del_DSCav1_mut5); R8898 14550 14560 14580 R26 HPIV3/USA/629-D01959/2007 fusion glycoprotein (variant); R10331 26957 26959 26962 R27 HMPV/00-1 fusion glycoprotein (variant); R10326 26971 26977 26984 R28 HMPV/NL/1/99 fusion glycoprotein (variant); R10329 26968 26974 26981
1.4. Preparation of an LNP Formulated mRNA Composition:

[1471] LNPs were prepared using cationic lipids, structural lipids, a PEG-lipids, and cholesterol. Lipid solution (in ethanol) was mixed with RNA solution (aqueous buffer) using T-piece formulation as outlined below. Obtained LNPs were re-buffered in a carbohydrate buffer via dialysis, and optionally up-concentrated to a target concentration using ultracentrifugation tubes. LNP-formulated mRNA was stored at −80° C. prior to use in in vitro or in vivo experiments.

[1472] In short, lipid nanoparticles were prepared and tested according to the general procedures described in PCT Pub. Nos. WO2015199952, WO 2017004143 and WO2017075531, the full disclosures of which are incorporated herein by reference. Lipid nanoparticle (LNP)-formulated mRNA was prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid (lipids see Table A). LNPs were prepared as follows. Cationic lipid according to formula III-3 (ALC-0315), DSPC, cholesterol and PEG-lipid according to formula IVa (ALC-0159) were solubilized in ethanol at a molar ratio of approximately 47.5:10:40.8:1.7. Lipid nanoparticles (LNP) comprising compound 111-3 (ALC-0315) were prepared at a ratio of mRNA to total Lipid of 0.03-0.04 w/w. Briefly, the mRNA was diluted to 0.05 to 0.2 mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15 ml/min. The ethanol was then removed and the external buffer replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 μm pore sterile filter. Lipid nanoparticle particle diameter size was 60-90 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

TABLE-US-00019 TABLE A Lipid-based carrier composition of the examples Ratio Compounds (mol%) Structure Mass 1 Cholesterol 40.9 [00009] 386.4 2 1,2-distearoyl- sn-glycero-3- phosphocholine (DSPC) 10 [00010] 789.6 3 Cationic Lipid 47.4 [00011] 765.7 4 PEG Lipid 1.7 [00012] 2010.1 Average n = ~49
1.5. Preparation of Combination mRNA Vaccines:

[1473] Combination mRNA vaccines were formulated with LNPs either in a separate or co-formulated way. For separately mixed or formulated mRNA vaccines, each mRNA component was prepared and separately LNP formulated as described in Example 1.4, followed by mixing of the different LNP-formulated components. For co-formulated mRNA vaccine, the different mRNA components were firstly mixed together, followed by a co-formulation in LNPs as described in Example 1.4.

Example 2: Vaccination of Mice with Nucleic Acid Based Combination Vaccines

[1474] Preparation of LNP Formulated mRNA Vaccine:

[1475] mRNA constructs for a combination vaccine are prepared as described in Example 1 (RNA in vitro transcription). HPLC purified mRNA is formulated with LNPs according to Example 1.4 and Example 1.5 prior to use in in vivo vaccination experiments.

Immunization:

[1476] Female BALB/c mice (6-8 weeks old) are injected intramuscularly (i.m.) with mRNA combination vaccine (respective RNA identifiers see Table 17) with doses as indicated in Table 18. As a negative control, one group of mice is vaccinated with buffer. All animals are vaccinated on day 0 and 21. Blood samples are collected on day 21 (post prime) and 42 (post boost) for the determination of antibody titers. After vaccination experiments, the efficiency of the combination vaccines is determined.

TABLE-US-00020 TABLE 18 Overview of the nucleic acid based combination vaccines and vaccination scheme Component A Component B (mRNA) (mRNA) Formulation Dose 1 Combination vaccine: R14 R1-R7 LNP 5 μg SARS-CoV-2-Influenza co-formulated 2 Combination vaccine: R14 R1-R7 LNP 5 μg SARS-CoV-2-Influenza-RSV R8 co-formulated 3 Combination vaccine: R14 R8 LNP 5 μg SARS-CoV-2-RSV co-formulated 4 Combination vaccine: R14 R12 LNP 5 μg SARS-CoV-2-MERS-SARS-CoV-1 R13 co-formulated 5 Combination vaccine: R14 R9-10, LNP 5 μg SARS-CoV-2-PIV-hMPV R11 co-formulated 6 Buffer

Determination of IgG1 and IgG2 Antibody Titers Using ELISA:

[1477] ELISA is performed using recombinant antigenic proteins for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies coated protein are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with Amplex as substrate. Endpoint titers of antibodies (IgG1, IgG2a) are measured by ELISA on day 21, and 42 post vaccinations.

Detection of Spike Protein-Specific Immune Responses (SARS-CoV-1, SARS-CoV-2, MERS-CoV):

[1478] Hela cells are transfected with 2 μg of mRNA encoding the spike protein using lipofectamine. The cells are harvested 20 h post transfection, and seeded at 1×10.sup.5 per well into a 96 well plate. The cells are incubated with serum samples of vaccinated mice (diluted 1:50) followed by a FITC-conjugated anti-mouse IgG antibody. Cells were acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo.

Intracellular Cytokine Staining:

[1479] Splenocytes from vaccinated mice are isolated according to a standard protocol known in the art. Briefly, isolated spleens are grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS, splenocytes are seeded into 96-well plates (2×10.sup.6 cells per well). Cells are stimulated with a mixture of protein specific peptide epitopes (5 μg/ml of each peptide) in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of a protein transport inhibitor. After stimulation, cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: Thy1.2-FITC (1:200), CD8-APC-H7 (1:100), TNF-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data is analyzed using FlowJo software package (Tree Star, Inc.)

Determination of Virus Neutralization Titers:

[1480] Serum is collected for determination of neutralization titers (VNTs) detected via CPE (cytopathic effect) or via a pseudotyped particle-based assay.

Determination of HA Specific Immune Responses

[1481] Detection of an HA-specific immune response (B-cell immune response) is carried out by detecting IgG2a antibodies directed against the particular influenza virus. Therefore, blood samples is taken from the vaccinated mice three weeks after vaccination and sera is prepared. MaxiSorb plates (Nalgene Nunc International) are coated with the particular recombinant HA protein. After blocking with 1×PBS containing 0.05% Tween-20 and 1% BSA the plates are incubated with diluted mice serum (as indicated). Subsequently a biotin-coupled secondary antibody (anti-mouse-IgG2a, Pharmingen) was added. After washing, the plate was incubated with horseradish peroxidase-streptavidin, followed by addition of the Amplex UltraRed Reagent (Invitrogen) and subsequent quantification of the fluorescent product.

Determination of Virus Neutralization RSV Titers:

[1482] RSV virus neutralization titers (VNTs) are measured on serum samples using a plaque reduction neutralization test (PRNT). Diluted serum samples are incubated with RSV/A2 for 1 hour at room temperature and inoculated in duplicates onto confluent HEp-2 monolayers in 24 well plates. After one hour incubation at 37° C. in a 5% CO.sub.2 incubator, the wells are overlayed with 0.75% Methylcellulose medium. After 4 days of incubation, the overlays are removed and the cells are fixed and stained. The corresponding reciprocal neutralizing antibody titers are determined at the 60% reduction end-point of the virus control.

Example 3: Vaccination of Mice with Nucleic Acid Based Combination Vaccines (mRNA Vaccines for SARS-CoV-2, RSV and/or Influenza)

[1483] The present example shows that combination mRNA vaccines induce strong and antigen-specific immune responses against all components of the combination vaccine (mRNA comprising at least one coding sequence encoding at least one antigenic peptide or protein selected from component A and/or from component B).

Preparation of LNP Formulated mRNA Vaccine Compositions/Combinations:

[1484] mRNA constructs for a combination vaccine were prepared as described in Example 1 (RNA in vitro transcription). HPLC purified mRNA was formulated with LNPs according to Example 1.4 and/or Example 1.5 prior to use in in vivo vaccination experiments.

Immunization:

[1485] Female BALB/c mice (6-8 weeks old) were injected intramuscularly (i.m.) with mRNA combination vaccine (respective RNA identifiers see Table 17) with doses as indicated in Table 19. As a negative control, one group of mice was vaccinated with buffer (0.9% NaCl). All animals received an injection volume of 25 μl and were vaccinated on day 0 and 21. Blood samples are collected on day 21 (post first vaccination) and 42 (post second vaccination) for the determination of antibody titers.

TABLE-US-00021 TABLE 19 Overview of the nucleic acid based combination vaccines and vaccination scheme Component Component Component A-1 B-2 B-3a Total Group Vaccine composition: (mRNA) (mRNA) (mRNA) Formulation Dose 1 SARS-CoV-2 mRNA vaccine R15 — — separately 0.5 μg formulated 2 RSV mRNA vaccine — R25 separately 0.5 μg formulated 3 Combination vaccine: R15 — R25 separately   1 μg SARS-CoV-2 mRNA vaccine + formulated (0.5 μg RSV mRNA vaccine each) 4 Heptavalent Influenza mRNA — R18-R24 — co-formulated 3.5 μg vaccine (0.5 μg each) 5 Combination vaccine: R15 R18-R24 — A-1 and B-2   4 μg SARS-CoV-2 mRNA vaccine + separately (0.5 μg heptavalent Influenza mRNA formulated each) vaccine 6 Combination vaccine: R15 R18-R24 R25 separately 4.5 μg SARS-CoV-2 mRNA vaccine + formulated (0.5 μg RSV mRNA vaccine + each) heptavalent Influenza mRNA vaccine 7 Control 0.9% NaCl — — — — —

Determination of Total IgG Antibody Titers for SARS-CoV-2 and RSV Using ELISA:

[1486] S.sub.RBD or RSV-F protein specific total IgG binding antibodies were quantified using an Amplex ELISA. Samples were pre-diluted 1:5 and consecutive 1:10 dilutions were tested (8 dilutions). SARS-CoV-2 (2019-nCoV) Spike RBD-His was used for coating (1 μg/ml) or 2.5 μg/mL rec. RSV-F GP A2 (SinoBiological, Cat: 11049-V08B). Plates were thoroughly washed after each incubation step. The coating was done at 37° C. for 4-5 hours. This was followed by an overnight blocking at 4° C. Serum samples were pre-diluted 1:5 in blocking buffer on a separate uncoated plate. The titrated sera samples were added into the pre-coated plate and incubated for 2-4h at RT. Detection antibodies were added into each well (1:300) and incubated for 1-1.5h at RT. Incubation with HRP-Streptavidin (1:1000) in sterile blocking buffer (1% BSA without NaN.sub.3) was carried out for 30 minutes. Amplex® UltraRed reagent (dissolved in DMSO; 1:200, 30% hydrogen peroxide H.sub.2O.sub.2 1:2000 in substrate buffer) was added into each well. The fluorescence was measured after a minimum of 45 minutes and a maximum of 24 hours. Endpoint titers of antibodies were measured by ELISA on day 21 post prime vaccination for SARS-CoV-2 and on day 21 and 42 for RSV.

Determination of Virus Neutralization Titers for SARS-CoV-2:

[1487] Serum was collected for determination of SARS-CoV-2 neutralization titers (VNTs) detected via CPE (cytopathic effect). Serial dilutions of heat-inactivated sera (56° C. for 30 min) tested in duplicates with a starting dilution of 1:10 followed by 1:2 serial dilutions were incubated with 100 TCID.sub.50 of wild type SARS-CoV-2 (strain 2019-nCov/Italy-INMI1) for 1 hour at 37° C. Every plate contained a dedicated row (8 wells) for cell control which contains only cells and medium, and a dedicated row of virus control which contain only cells and virus. Infectious virus was quantified upon incubation of 100 μl of virus-serum mixture with a confluent layer of Vero E6 cells (ATCC, Cat. 1586) followed by incubation for 3 days at 37° C. and microscopical scoring for CPE formation. A back titration was performed for each run in order to verify the correct range of TCID50 of the working virus solution. VN titres were calculated according to the method described by Reed & Muench. If no neutralization was observed (MNt<10), an arbitrary value of 5 was reported. Analyses were carried out at VisMederi srl (Siena, Italy). Virus neutralization titers were measured with sera collected on day 42.

Determination of Influenza HA Specific Immune Responses (Via Hemagglutinin Inhibition Assay)

[1488] Functional anti-HA antibody titers were analyzed by hemagglutination inhibition (HI) assay. Sera were incubated with receptor destroying enzyme (RDEII, Denka Seiken) at 37° C. overnight, inactivated (56° C., 60 min). Dilutions of pre-treated sera were incubated for 45 min with 4 HAU of inactivated influenza A H1N1 California/07/09 (NIBSC Material 09/174) and 50μl 0.5% erythrocyte solution was added. The plates were incubated for 45-60 minutes and HI titers are determined by the highest dilution of serum that leads to the complete inhibition of agglutination.

[1489] Results:

[1490] As shown in FIG. 1 A the vaccination with mRNA encoding full length S stabilized protein (S_stab) or combination vaccine comprising mRNA encoding full length S stabilized protein (S_stab) induced high titers of Spike specific binding antibodies after a single vaccination (day 21) (FIG. 1 A: total IgG). FIG. 1 B shows the induction of functional antibodies after the second vaccination (day 42) (FIG. 1 B: Virus neutralizing antibodies VNTs). The combination of component A-1 with component B-2 (Heptavalent Influenza mRNA vaccine (group 5)) and/or B-3a (RSV mRNA vaccine (group 3), (group 6)) does not influence the induction of humoral antibody responses in a negative way. The data shows that the observed immune responses of the respective combination vaccines comprising mRNA encoding full length S stabilized protein are comparable to those observed for the monovalent vaccine group (group 1).

[1491] The induced antibody responses against RSV F protein (component B-3a) are shown in FIG. 2 A (day 21, after prime vaccination) and B (day 42, after second vaccination). All the combination vaccines comprising mRNA encoding RSV F protein induced high titers of IgG antibodies (group 3 and group 6). The data shows that the observed immune responses of the respective combination vaccines comprising mRNA encoding RSV F protein protein are comparable to those observed for the monovalent vaccine (group 2).

[1492] As shown in FIGS. 3 A and B the induction of functional antibodies against Influenza virus measured by Hemagglutinin Inhibition Assay was induced by vaccine combinations comprising the heptavalent Influenza vaccine component B-2. HI titers were determined on day 21 after one vaccination (FIG. 3 A) and on day 42 after two vaccinations (FIG. 3 B). The data shows that the observed immune responses of the combination vaccines comprising mRNA encoding influenza antigens (group 5 and group 6) are comparable to those observed for the heptavalent influenza vaccine (group 4).

[1493] As demonstrated in the present example, the tested combination vaccines are suitable to deliver the different mRNA components to the host cells to translate the mRNA components into antigenic proteins or antigenic peptides, which induces antigen-specific immune responses against all the encoded proteins.

[1494] Summarizing the above, the data demonstrates that the combination of antigens of SARS CoV-2, Influenza virus and RSV is possible and that the combination vaccines induce strong immune responses against each of the encoded antigens. In addition to that, all components of the tested combination vaccines induce efficient immune responses which indicates that single components were not immunodominant, or that the components of the combination vaccines do not show immune-interference.

Example 4: Vaccination of Mice with Nucleic Acid Based Combination Vaccines (mRNA Vaccines for SARS-CoV-2, RSV, Influenza, hMPV, and/or PIV3)

[1495] The present example shows that combination mRNA vaccines induce strong and antigen-specific immune responses against all components of the combination vaccine (mRNA comprising at least one coding sequence encoding at least one antigenic peptide or protein selected from component A and/or from component B). The combination vaccine induced high antibody responses against all encoded proteins, independent if co-formulated or separately mixed after LNP formulation. The present example also shows the combination of five different mRNA vaccine components (component A-1: SARS-CoV-2, component B-3a: RSV, component B-2: Influenza, heptavalent; component B-3b: hMPV (bivalent), and component B-4a: PIV). This combination vaccine includes 12 different antigens (12-valent).

Preparation of LNP Formulated mRNA Vaccine:

[1496] mRNA constructs for a combination vaccine were prepared as described in Example 1 (RNA in vitro transcription). HPLC purified mRNA was formulated with LNPs according to Example 1.4 and/or Example 1.5 prior to use in in vivo vaccination experiments.

[1497] The co-formulated 12-valent combination vaccine (see group 7, Table 20,) was prepared by mixing by molarity, so equal numbers of RNA molecules were mixed (equimolar). The separately formulated 12-valent combination vaccine (see group 6, Table 20), the total amount of RNA was used and no adjustment according to molarity was performed (equal mass).

Immunization:

[1498] Female BALB/c mice (6-8 weeks old) were injected intramuscularly (i.m.) with mRNA combination vaccine (respective RNA identifiers see Table 17) with doses as indicated in Table 20. As a negative control, one group of mice was vaccinated with buffer (0.9% NaCl). All animals received 25 μl and were vaccinated on day 0 and 21. Blood samples are collected on day 7 and day 21 (post first vaccination) and 42 (post second vaccination) for the determination of antibody titers.

TABLE-US-00022 TABLE 20 Overview of the nucleic acid based combination vaccines and vaccination scheme Component Component Component Component Component Vaccine A B-2 B-3a B-3b B-4a LNP Total composition: (mRNA) (mRNA) (mRNA) (mRNA) (mRNA) formulation Dose 1 SARS-CoV-2 mRNA R15 — — — — separately 0.5 μg vaccine formulated 2 Bivalent hMPV mRNA — — — R27 + R28 — co-formulated   1 μg vaccine 3 Combination vaccine: R15 — — R27 + R28 — separately 1.5 μg SARS-CoV-2 mRNA formulated (0.5 μg vaccine + Bivalent each) hMPV mRNA vaccine 4 PIV3 mRNA vaccine — — — — R26 0.5 μg 5 Combination vaccine: R15 — — — R26 separately   1 μg SARS-CoV-2 mRNA formulated (0.5 μg vaccine PIV3 mRNA each) vaccine 6 Combination vaccine: R15 R18-R24 R25 R27 + R28 R26 separately   6 μg 12-valent cocktail formulated; (0.5 μg mRNA vaccine equal mass. each) 7 Combination vaccine: R15 R18-R24 R25 R27 + R28 R26 co-formulated;   6 μg 12-valent cocktail equimolar mRNA vaccineL 8 Control 0.9% NaCl — — — — — — —

[1499] SARS-CoV-2, Influenza and RSV-specific immune responses (VNT, total IgG and HI titer) were analyzed as described in Example 3.

FACS-Based ELISA for Determination of PIV-3 or hMPV-A1 and hMPV-B1 Specific Immune Responses

[1500] HeLa cells were transfected with 17 μg mRNA per T75 flask of R27 (R10326 (hMPV-A1 stabilized fusion protein with furin cleavage—F(19-539)PFs_GCv3.1)), R28 (R10329 (hMPV-B1 stabilized fusion protein with furin cleavage—F(19-539)_PFs_GCv3.1)), R26 (R10331 (PIV-3 fusion protein stabilized—F(19-539)DS2-Cav_GCv3.1)) using Lipofectamine 2000. Transfected cells were harvested 18-24h post lipofection and seeded in 96 V-bottom plate at 2×10.sup.5 cells/well The mouse serum samples collected in on day 42 were used as primary antibody followed by anti-mouse FITC-conjugated secondary antibody.

Results

[1501] As shown in FIG. 4 A the vaccination with mRNA encoding full length S stabilized protein (S_stab) alone or the combination vaccines comprising mRNA encoding full length S stabilized protein (S_stab) induced high titers of Spike specific binding antibodies after a single vaccination (day 21) (FIG. 4 A: total IgG, ELISA). FIG. 4 B shows the induction of functional antibodies after the second vaccination (d42) (FIG. 4 B: Virus neutralizing antibodies VNTs). The combination with component B-3b (hMPV, bivalent mRNA vaccine (A+D)) or B-4a (PIV-3 mRNA vaccine (A+E)) does not influence the induction of humoral antibody responses in a negative way. The data shows that the observed immune responses of the respective combination vaccines comprising mRNA encoding full length S stabilized protein are comparable to those observed for the monovalent vaccine group (group 1).

[1502] Even the 12-valent vaccine combinations (group 6 and group 7) induced high SARS-CoV-2 specific immune responses (binding antibodies and VNTs). The response of the LNP co-formulated vaccine combination (group 7) was slightly higher compared to separately LNP-formulated and mixed mRNA combinations (group 6).

[1503] RSV-F specific immune responses measured via ELISA are shown in FIGS. 5 A (day 21) and B (day 42). Both 12-valent combination vaccine (group 6 and group 7) induced high titers of total IgG. The titers on day 42 (FIG. 5 B) for both groups might be even higher (indicated by the arrow, due to assay conditions). Antibody titers against combination vaccines comprising component B-3b (comprising mRNA encoding hMPV antigens) raised also slight IgG titers (group 2 and 4). This might be due to cross-immunity between F proteins of related virus strains. RSV and hMPV both are belonging to Pneumoviridae virus family.

[1504] As shown in FIGS. 6 A and B all the vaccine combinations comprising component B3-b (comprising mRNA encoding bivalent hMPV antigen (group 2, 3, 6, and 7) induced strong humoral immune responses against F protein of hMPV A1 and B1 strains (FIG. 6 A: hMPV A1, 6 B: hMPV B1, day 42). FIG. 7 demonstrates the immune responses elicited against PIV3 F protein on day 42 after first vaccination. All vaccine combinations comprising component B-4a (comprising mRNA encoding PIV3 F protein (group 4, 5, 6, and 7) induced humoral immune responses against PIV3 F protein.

[1505] As shown in FIG. 8 the induction of functional antibodies against Influenza virus measured by Hemagglutinin Inhibition Assay was induced by vaccine combinations comprises the heptavalent Influenza vaccine component B-2 (group 6 and group 7). HI titers were determined on day 42 after two vaccinations.

[1506] As demonstrated in the present example, the tested combination vaccines are suitable to deliver the different mRNA components to the host cells to translate the mRNA components into antigenic proteins or antigenic peptides, which induces antigen-specific immune responses against all the encoded proteins.

[1507] Summarizing the above, the data demonstrates that the combination of antigens of SARS CoV-2, Influenza virus, hMPV, PIV, and RSV is possible and that the combination vaccines induce strong immune responses against each of the encoded antigens. In addition to that, all components of the tested combination vaccines induce efficient immune responses which indicates that single components were not immunodominant, or that the components of the combination vaccines do not show immune-interference.

Example 5: Clinical Development of a Nucleic Acid Based Combination Vaccine

[1508] To demonstrate safety and efficiency of the nucleic acid based combination vaccine, a clinical trial (phase 1) is initiated. In the clinical trial, a cohort of human volunteers is intramuscularly injected for at least two times (e.g. day 0 and day 28 with a dose of 2 μg, 6 μg, or 18 μg nucleic acid based combination vaccine (formulated in LNPs according to the invention). In order to assess the safety profile of the vaccine, subjects are monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis). The efficacy of the immunization is analyzed by determination of virus neutralizing titers (VNT) in sera from vaccinated subjects. Blood samples are collected on day 0 as baseline and after completed vaccination. Sera are analyzed for virus neutralizing antibodies.