Artificial nucleic acid molecules encoding a norovirus antigen and uses thereof

Abstract

The present invention is directed to an artificial nucleic acid and to polypeptides suitable for use in treatment or prophylaxis of an infection with Norovirus or a disorder related to such an infection. In particular, the present invention concerns a Norovirus vaccine. The present invention is directed to an artificial nucleic acid, polypeptides, compositions and vaccines comprising the artificial nucleic acid or the polypeptides. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial nucleic acid, polypeptides, compositions and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial nucleic acid, polypeptides, compositions and vaccines.

Claims

1. Artificial nucleic acid molecule comprising at least one coding region encoding a Norovirus VP1 polypeptide, said coding region having at least 90% identity to a sequence of SEQ ID NOs: 39713-39746, wherein the artificial nucleic acid molecule comprises at least one heterologous 5′ and/or 3′ untranslated region (UTR).

2. The artificial nucleic acid molecule of claim 1, wherein the artificial nucleic acid is monocistronic, bicistronic or multicistronic.

3. The artificial nucleic acid molecule of claim 1, wherein the artificial nucleic acid is an mRNA.

4. The artificial nucleic acid molecule of claim 3, wherein the artificial nucleic acid comprises a 5′-cap structure.

5. The artificial nucleic acid molecule of claim 1, wherein the G/C content of the coding region of the mRNA sequence is increased compared to the G/C content of the corresponding coding sequence of the wild type mRNA, or wherein the C content of the coding region of the mRNA sequence is increased compared to the C content of the corresponding coding sequence of the wild type mRNA, or wherein the codon usage in the coding region of the mRNA sequence is adapted to the human codon usage, or wherein the codon adaptation index (CAI) is increased or maximised in the coding region of the mRNA sequence, wherein the encoded amino acid sequence of the mRNA sequence is preferably not being modified compared to the encoded amino acid sequence of the wild type mRNA.

6. The artificial nucleic acid molecule of claim 1, wherein the artificial nucleic acid comprises at least one histone stem-loop.

7. The artificial nucleic acid molecule of claim 1, wherein the 3′-UTR comprises at least one heterologous 3′-UTR element.

8. The artificial nucleic acid molecule of claim 1, wherein the 3′-UTR comprises a poly(A) sequence and/or a poly(C) sequence.

9. The artificial nucleic acid molecule of claim 1, wherein the 5′-UTR comprises at least one heterologous 5′-UTR element.

10. The artificial nucleic acid molecule of claim 1, comprising the following elements: a) optionally a 5′-cap structure, b) the at least one coding region, c) a poly(A) tail comprising 10 to 200 adenosine nucleotides, d) optionally a poly(C) tail comprising 10 to 200 cytosine nucleotides, and e) optionally a histone stem-loop.

11. The artificial nucleic acid molecule of claim 10, comprising a 3′-UTR element comprising a nucleic acid sequence, which is derived from an α-globin gene.

12. The artificial nucleic acid molecule of claim 1 said coding region having at least 95% identity to a sequence of SEQ ID NOs: 39713-39746.

13. The artificial nucleic acid molecule of claim 10, comprising a 5′-UTR element, which comprises or consists of a nucleic acid sequence, which is derived from the 5′-UTR of a TOP gene.

14. The artificial nucleic acid molecule of claim 1, wherein the coding region comprises a chemically modified nucleotide.

15. A method of treating or preventing a Norovirus infection, wherein the method comprises administering to a subject in need thereof the artificial nucleic acid according to claim 1.

16. The artificial nucleic acid molecule of claim 1, said coding region having at least 90% identity to a sequence of SEQ ID NOs: 39743.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: shows that transfection of HeLa cells with mRNAs coding for Norovirus antigen VP1 leads to the expression of the encoded protein. For cell transfection, mRNA constructs (construct ID R2) and (construct ID R4) were used. Norovirus VP1 proteins were stained intracellularly with a specific anti-Norovirus GII.4 antibody and a FITC labelled secondary antibody and analyzed by FACS. A detailed description of the experiment is provided in the examples section, Example 2.

(2) FIG. 2: shows that transfection of HeLa cells with mRNAs coding for Norovirus antigen WI leads to the expression of the encoded protein. For cell transfection, mRNA constructs (construct ID R26), (construct ID R27), and (construct ID R28) were used. Norovirus VP1 proteins were stained with a specific anti-Norovirus GII.4 antibody and a FITC labelled secondary antibody and analyzed by FACS. A detailed description of the experiment is provided in the examples section, Example 2.

(3) FIG. 3: shows that transfection of HeLa cells with mRNAs coding for Norovirus antigen WI leads to protein expression. For cell transfection, mRNA constructs (construct ID R2) and (construct ID R4) were used. Western blot analysis was performed cell lysates of transfected cells. As a control, a commercial VLP preparation (Medigen; 59 kD) was used. Norovirus VP1 proteins were stained with a specific anti-Norovirus GII.4 antibody. M=marker lane: I=mRNA construct R2; 2=mRNA construct R4; 3=WFI control; 4=empty control; 5=commercial VLP control. A detailed description of the experiment is provided in the examples section, Example 3.

(4) FIG. 4: shows that immunization of mice with formulated Norovirus mRNA vaccine (Norovirus GC-optimized VP1_X124V; construct ID R4; protamine formulated) induced binding IgG1 and IgG2 antibodies, both in a homologous ELISA design (FIG. 4A; coating material VLP GII.4) and in a heterologous ELISA design (FIG. 4B; coating material VLP GII.4 2011). 1=group vaccinated with Norovirus mRNA vaccine; 2=buffer control group. A detailed description of the experiment is provided in the examples section, Example 4.1.

(5) FIG. 5: shows that immunization of mice with formulated Norovirus mRNA vaccine (Norovirus GC-optimized VP1_X124V; construct ID R4; protamine formulated) induced heterologous blocking antibodies. The results of a Histo-Blood Group Antigen (HBGA) assay in serum dilution 1:12.5 are shown. I=group vaccinated with Norovirus mRNA vaccine; 2=buffer control group. A detailed description of the experiment is provided in the examples section, Example 4.2.

(6) FIG. 6: shows that immunization of mice with formulated Norovirus mRNA vaccine (Norovirus GC-optimized VP1_X124V; construct ID R4; protamine formulated) induced antigen specific T-cell responses. The results of an ICS assay are shown (CD8+ T-cells). 1=group vaccinated with Norovirus mRNA vaccine; 2=buffer control group. A detailed description of the experiment is provided in the examples section, Example 4.3.

EXAMPLES

(7) The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

Example 1: Preparation of mRNA for In Vitro and In Vivo Experiments

(8) 1.1. Preparation of DNA and mRNA Constructs:

(9) For the present examples, DNA sequences encoding Norovirus antigenic proteins, derived from three or more different Norovirus strains were prepared and used for subsequent RNA in vitro transcription reactions. The prepared RNA constructs (coding sequences (cds) and mRNA sequences) are listed in Table 4 below.

(10) Most DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a codon modified sequence or GC-optimized sequence for stabilization, using three or more different in silico algorithms that e.g. increase the GC content of the respective coding sequence (indicated as “GC opt 1”, “GC opt 2”, “GC opt 3”, “GC opt 4”, “opt 5”, “opt 6”, “opt 7” in Table 4; further details relating to sequence modifications are provided in the specifications of the invention). Some DNA sequences were used as a wild type coding sequence, without altering the GC content and without altering the codon usage of the coding sequence (indicated as “wt” in Table 4).

(11) DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization, using an in silica algorithms that increase the GC content of the respective coding sequence (e.g., indicated as “opt1” in Table 4, see explanation in the paragraph above).

(12) Moreover, sequences were introduced into a pUC19 derived vector and modified to comprise stabilizing sequences derived from alpha-globin-3′-UTR, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3′-terminal end (poly-A-tail), indicated as “design 1” in Table 4. Other sequences were introduced into a pUC19 derived vector to comprise stabilizing sequences derived from 32L4 5′-UTR ribosomal FOP UTR and 3′-UTR derived from albumin 7, a stretch of 30 cytosines, a histone-stem-loop structure, and a stretch of 64 adenosines at the 3′-terminal end (poly-A-tail), indicated as “design 2” in Table 4. Further details are relating mRNA construct design are provided in the specifications of the invention)

(13) The obtained plasmid DNA constructs were transformed and propagated in bacteria (Escherichia coli) using common protocols known in the art.

(14) TABLE-US-00003 TABLE 4 VP1 coding sequences, protein sequences and mRNA constructs Construct RNA ID description Norovirus strain RNA design SEQ ID NO R1 mRNA VP1_(X124V) GII.4-031693-USA-2003 design 1, wt 39713 R2 mRNA VP1_(X124V) GII.4-031693-USA-2003 design 2, wt 39714 R3 mRNA VP1_(X124V) GII.4-031693-USA-2003 design 1, GC opt 1 39715 R4 mRNA VP1_(X124V) GII.4-031693-USA-2003; C1N1 design 2, GC opt 1 39716 R5 protein VP1_(X124V) GII.4-031693-USA-2003 Protein* 2358 R6 cds VP1_(X124V) GII.4-031693-USA-2003 wild type, wt 6768 R7 cds VP1_(X124V) GII.4-031693-USA-2003 GC opt 1 39717 R8 cds VP1_(X124V) GII.4-031693-USA-2003 GC opt 2 11178 R9 cds VP1_(X124V) GII.4-031693-USA-2003 opt 5 15588 R10 cds VP1_(X124V) GII.4-031693-USA-2003 opt 6 19998 R11 cds VP1_(X124V) GII.4-031693-USA-2003 opt 7 24408 R12 cds VP1_(X124V) GII.4-031693-USA-2003 GC opt 3 28818 R13 cds VP1_(X124V) GII.4-031693-USA-2003 GC opt 4 33228 R14 mRNA Capsidprotein GII.4 Farmington Hills-2002-USA design 1 39718 R15 mRNA Capsidprotein GII.4 Farmington Hills-2002-USA design 2 39719 R16 mRNA Capsidprotein GII.4 Farmington Hills-2002-USA design 1, GC opt 1 39720 R17 mRNA Capsidprotein GII.4 Farmington Hills-2002-USA design 2, GC opt 1 39721 R18 protein Capsidprotein GII.4 Farmington Hills-2002-USA Protein* 1487 R19 cds Capsidprotein GII.4 Farmington Hills-2002-USA wild type 5897 R20 cds Capsidprotein GII.4 Farmington Hills-2002-USA GC opt 2 10307 R21 cds Capsidprotein GII.4 Farmington Hills-2002-USA opt 5 10307 R22 cds Capsidprotein GII.4 Farmington Hills-2002-USA opt 6 19127 R23 cds Capsidprotein GII.4 Farmington Hills-2002-USA opt 7 23537 R24 cds Capsidprotein GII.4 Farmington Hills-2002-USA GC opt 3 27947 R25 cds Capsidprotein GII.4 Farmington Hills-2002-USA GC opt 4 32357 R26 mRNA VP1 GII.4-2006b 092895-USA-2008 design 2, GC opt 1 39729 R27 mRNA VP1 GII.4-GZ2010-L87-Guangzhou-2011 design 2, GC opt 1 39734 R28 mRNA VP1 GII.4-USA-1997 design 2, GC opt 1 39738 R29 mRNA VP1 GI.1-USA-1968-Capsidprotein design 2, GC opt 1 39725 *protein sequence is back translated into RNA according to the above paragraph “G/C content modification”

(15) 1.2. RNA In Vitro Transcription:

(16) The DNA plasmids prepared according to paragraph 1.1 were enzymatically linearized using EcoRI and transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture and cap analog (m7GpppG) under suitable buffer conditions. The obtained mRNAs were purified using PureMessenger® (CureVac, Tübingen, Germany: WO 2008/077592 A1) and used for in vitro and in vivo experiments.

(17) 1.3. Preparation of Protamine Formulated RNA Vaccine:

(18) The obtained mRNA, e.g. HPLC purified RNA, was complexed with protamine by addition of protamine-trehalose solution to RNA solution at a RNA:protamine weight to weight ratio of 2:1. Then, complexed RNA was mixed with non-complexed RNA in a ratio of 50% free RNA and 50% complexed RNA to obtain formulated RNA. Formulated RNA was used for in viva vaccination experiments.

(19) 1.4. Preparation of LNP Formulated RNA Vaccine:

(20) RNA is encapsulated in lipid nanoparticle (LNP) using established protocols known in the art. Briefly, LNP-encapsulated RNA is prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. Cationic lipid, DSPC, cholesterol and PEG-lipid are solubilized in ethanol. RNA is diluted to a total concentration of about 0.05 mg/mL in 50 mM citrate buffer pH 4. Syringe pumps are used to mix the ethanolic lipid solution with RNA at a ratio of about 1:6 to 1:2 (vol/vol). Ethanol is then removed and the external buffer replaced with PBS by dialysis. Lipid nanoparticles are filtered through a 0.2 μm pore sterile filter. Lipid nanoparticle particle diameter size may be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).

Example 2: Expression of Norovirus VP1 Antigens in HeLa Cells and Analysis by FACS

(21) To determine in vitro protein expression of the inventive Norovirus mRNA constructs, Hela cells were transfected with mRNA constructs encoding Norovirus VP1 antigens and analyzed by intracellular FACS staining. For cell transfection, an mRNA comprising VP1_X124V (GII.4-031693-USA-2003) wild type coding sequence (SEQ ID NO: 39714; construct ID R2) an mRNA comprising VP1_X124V (GII.4-031693-USA-2003) GC-optimized coding sequence (SEQ ID NO: 39716; construct ID R4), an mRNA comprising VP1 (GII.4-2006b 092895-USA-2008) GC-optimized coding sequence (SEQ ID NO: 39729; construct ID R26), an mRNA comprising VP1 (GII.4-GZ2010-L87-Guangzhou-2011) GC-optimized coding sequence (SEQ ID NO: 39734; construct R27) and an mRNA comprising VP1 NOV(GII.4-USA-1997)-Capsidprotein GC-optimized coding sequence (SEQ ID NO: 39738; construct ID R28) were used. The detailed description of the performed experiment is provided below.

(22) HeLa cells were seeded in a 6-well plate at a density of 400,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24 h prior to transfection. Cells were transfected with 1 μg and 2 μg mRNA per construct using lipofectamine 2000 (Invitrogen) as transfection reagent. As a negative control, water for injection (WFI) was used.

(23) 24 hours post transfection, transfected HeLa cells were stained with a commercial mouse anti-Norovirus GII.4 antibody [2000-G5] (Abcam; 1:500) and an anti-mouse FITC labelled secondary antibody (F5262 from Sigma; 1:500) after Cytofix/Cytoperm (BD Biosciences) treatment according to manufacturer's protocol. Subsequently, cells were analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal was performed using the FlowJo software package (Tree Star, Inc.). The results of the FACS expression analysis are shown in FIG. 1 and FIG. 2.

(24) Results:

(25) FIG. 1 and FIG. 2 show that the Norovirus proteins were expressed in Ma cells transfected with the mRNA constructs R2, R4, R26, R27 and R28. Overall, around 80%-90% of transfected cells showed positive FITC signal, indicating that the inventive constructs tested here were able to efficiently drive protein expression without affecting cell viability. Of note, the data suggests that analogous mRNA constructs encoding other Norovirus VP1 or VP2 antigens (as defined in the specifications or listed in Table 1 and Table 3) may also drive protein expression in a similar manner.

Example 3: Analysis of Protein Expression Using Western Blot

(26) To determine in vitro protein expression upon HeLa cell transfection with the inventive mRNA constructs, HeLa cells were transiently transfected with an mRNA constructs comprising VP1_X124V coding sequences. Cell lysates were prepared and analyzed using western blot. The detailed description of the performed experiment is provided below.

(27) HeLa cells are transfected with 2 μg mRNA comprising wild type VP1_X124V coding sequence (SEQ ID NO: 39714; construct ID R2) and 2 μg mRNA comprising GC-optimized VP1_X124V coding sequence (SEQ ID NO: 39718; construct ID R4). As a negative control water for injection (WFI) was used. After 24 hours post transfection lysis buffer was added to the culture to prepare cellular lysates. Cellular lysates as well as a commercial Norovirus virus like particle (VLP; obtained from Medigen) were reduced by heating the samples to 95° C. for 10 minute. Subsequently, samples were subjected to SDS-PAGE under denaturating/reducing conditions followed by western blot detection. For the detection of Norovirus proteins, a commercial mouse anti-Norovirus GII.4 antibody [2002-85] (1:250; Abcam) was used as primary antibody followed by secondary goat anti mouse antibody coupled to IRDye 800CW (1:10000; Licor Biosciences). The results of the experiment are shown in FIG. 3.

(28) Results:

(29) FIG. 3 shows that the Norovirus proteins were expressed in HeLa cells transfected with the inventive mRNA constructs (SEQ ID NOs: 39714 and 39716). Of note, the data suggests that analogous mRNA constructs encoding other Norovirus antigens (as defined in the specifications or listed in Table 1 and Table 3) may also drive protein expression in a similar manner.

Example 4: Immunization of Mice and Evaluation of Norovirus Specific Immune Responses

(30) Female BALB/c mice were immunized intradermally (i.d.) with protamine formulated mRNA vaccine (construct ID R4) with doses, application routes and vaccination schedules as indicated in Table 5. As a negative control, one group of mice was injected with buffer (ringer lactate, RiLa). All animals were vaccinated on day 0, 21 and 35. Blood samples were collected on day 49 for the determination of binding antibody titers (using a homologous and heterologous ELISA assay), blocking antibody titers (using a heterologous HGBA assay) and T-cell responses (intracellular cytokine assay). Detailed descriptions of the performed experiments are provided below.

(31) TABLE-US-00004 TABLE 5 Vaccination regimen (Example 4) Group No of mice Treatment Dose Route/Volume Vaccination schedule 1 6 Norovirus GC-optimized VP1_X124V 80 μg i.d. d 0, d 21, d 35 SEQ ID NO: 39716; R4 2 × 50 μl Protamine formulated 2 6 100% RiLa Control i.m. d 0, d 21, d 35 1 × 25 μl

(32) 4.1. Determination of Homologous and Heterologous Immune Responses by ELISA:

(33) ELISA was performed using synthetically produced norovirus Virus like particles (VLP) as coating material. For the analysis of homologous immune responses, plates were coated with VLP of the same strain of genotype GII.4 (GII.4 CIN1). For the analysis of heterologous immune responses, plates were coated with VLPs of another strain of genotype GII.4 (GII.4 2011). Coated plates were incubated using respective serum dilutions, and binding of specific antibodies to the Norovirus coating material was detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies were measured by ELISA on day 49 after three vaccinations (see Table 5). The results are shown in FIG. 4A (homologous responses) and FIG. 4B (heterologous responses).

(34) 4.2. Determination of Blocking Antibody Titers Using a HBGA Blocking Assay:

(35) Respective sera (day 49 after three vaccinations) were pre-incubated with synthetic norovirus VLPs (VLP (GII.4 2011)) and subsequently added to HBGA coated plates. VLP binding to Histo-Blood Group Antigen (HBGA) was detected by norovirus specific antibodies. In the presence of functional blocking antibodies in serum of immunized animals, VLP binding to HBGA was blocked which results in a reduction of the detected antibody signal. The respective blocking index was calculated as commonly known in the art. The results of the assay are shown in FIG. 5.

(36) 4.3. Determination of Specific CD8 T-Cell Responses Using ICS:

(37) Splenocytes from vaccinated mice were isolated according to a standard protocol known in the art. Briefly, isolated spleens were 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 were seeded into 96-well plates (2×10.sup.6 cells per well). The cells were stimulated with ten Norovirus CD8 peptide epitopes (1 μg/ml of each peptide) in the presence of 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) and anti-CD107α-PE-Cy7 antibody, after one hour at 37° C. After stimulation, cells were washed and stained and for staining of intracellular cytokines Cytofix/Cytoperm reagent (BD Biosciences) was used according to the manufacturer's instructions. The following antibodies were used for staining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), 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 was used to distinguish live/dead cells (Invitrogen). Cells were acquired using a Canto II flow cytometer (Beckton Dickinson). Flow cytometry data was analyzed using FlowJo software package (Tree Star, Inc.). Results for CD8+ T-cells are shown in FIG. 6.

(38) Results:

(39) FIG. 4 shows that the tested Norovirus mRNA vaccine induced Norovirus specific IgG1 and IgG2 antibody titers in immunized mice. Humoral immune response was demonstrated in a homologous ELISA setting (see FIG. 4A) as well as in a heterologous ELISA setting (see FIG. 4B). Of note, the observed heterologous humoral immune response (against another strain of genotype GII.4) is of particular importance for a broad protection against Norovirus infections, as GII.4 strains are fast-evolving which is challenging in successful Norovirus vaccine development.

(40) FIG. 5 shows that the tested Norovirus mRNA vaccine induced Norovirus specific blocking antibody titers in immunized mice in a homologous and heterologous HGBA assay setup, showing that also functional antibodies were induced after immunization of mice with the inventive Norovirus mRNA vaccine. Of note, the induction of functional blocking antibodies also against another strain of genotype GII.4 demonstrates that the used mRNA Norovirus vaccine may also confer broad protection against different Norovirus strains of genotype GII.4.

(41) FIG. 6 shows that the tested Norovirus mRNA vaccine stimulated a robust CD8+ IFN-γ/TNF-α and CD8+CD107a/IFN-γ in spleen of immunized mice.

(42) Overall, the results of the immunization experiments in mice show that the inventive Norovirus mRNA vaccine induced a broad immune response engaging both the humoral-secretory and cellular immunity effector arms. Notably, heterologous immune responses were also observed (ELISA, HGBA). The data suggests that analogous mRNA constructs encoding other Norovirus antigens (as defined in the specifications or listed in Table 1 or Table 3) may also induce board immune responses in a similar manner.

Example 5: Immunization of Mice and Further Evaluation of Heterologous Immune Responses

(43) Female BALB/c mice are immunized intradermally (i.d.) and intramuscularily (i.m.) with protamine formulated or LNP formulated mRNA vaccines with doses, application routes and vaccination schedules as indicated in Table 6. As a negative control, one group of mice was injected with buffer (ringer lactate). All animals were vaccinated on day 0, 21 and 35. Blood samples are collected on day 49 for the determination of binding antibody titers (using a homologous and heterologous ELISA assay), blocking antibody titers (using a homologous and heterologous HGBA assay). Detailed descriptions of the performed experiments are provided below.

(44) TABLE-US-00005 TABLE 6 Vaccination regimen of mice (Example 5) Group No. of mice Treatment Dose Route/Volume Vaccination schedule 1 6 Norovirus GC-optimized VP1 80 μg i.d. d 0, d 21, d 35 GII.4-USA-1997 2 × 50 μl SEQ ID NO: 39738; R28 Protamine formulated 2 6 Norovirus GC-optimized VP1 80 μg i.d. d 0, d 21, d 35 GII.4-2006b 092895-USA-2008 2 × 50 μl SEQ ID NO: 39729; R26 Protamine formulated 3 6 Norovirus GC-optimized VP1 80 μg i.d. d 0, d 21, d 35 GII.4-GZ2010-L87-Guangzhou-2011 2 × 50 μl SEQ ID NO: 39734; R27 Protamine formulated 4 6 Norovirus GC-optimized VP1_X124V 80 μg i.d. d 0, d 21, d 35 SEQ ID NO: 39716; R4 2 × 50 μl Protamine formulated 5 6 Norovirus GC-optimized VP1 20 μg i.m. d 0, d 21, d 35 GII.4-USA-1997 2 × 25 μl SEQ ID NO: 39738; R28 LNP formulated 6 6 Norovirus GC-optimized VP1 20 μg i.m. d 0, d 21, d 35 GII.4-2006b 092895-USA-2008 2 × 25 μl SEQ ID NO: 39729; R26 LNP formulated 7 6 Norovirus GC-optimized VP1 20 μg i.m. d 0, d 21, d 35 GII.4-GZ2010-L87-Guangzhou-2011 2 × 25 μl SEQ ID NO: 39734; R27 LNP formulated 8 6 Norovirus GC-optimized VP1_X124V 20 μg i.m. d 0, d 21, d 35 SEQ ID NO: 39716; R4 2 × 25 μl LNP formulated 9 6 100% RiLa Control i.m. d 0, d 21, d 35 1 × 25 μl

(45) 5.1. Determination of Homologous and Heterologous Immune Responses by ELISA:

(46) ELISA is performed essentially as described in Example 4.1. Plates are coated with VLP 011.4 CIN1 and VLP GII.4 2011 to determine homologous and heterologous immune responses.

(47) 5.2. Determination of Blocking Antibody Titers Using a Heterologous HBGA Blocking Assay:

(48) The HGBA assay is performed essentially as described in Example 4.2. The respective blocking index are calculated as commonly known in the art to evaluate homologous and heterologous cross neutralizing capacities of the used mRNA vaccines.

(49) 5.3. Determination of Specific CD8 T-Cell Responses Using ICS:

(50) Multifunctional CD8 T-cell responses are analyzed as described in Example 4.3.

Example 6: Norovirus mRNA Vaccine Challenge Study in Gnotobiotic Pigs

(51) 6.1 Immunization of Gnotobiotic Pigs:

(52) Gnotobiotic pigs are derived by hysterectomy from near-term sows and maintained in germ-free isolator units. Pigs are fed commercial ultra-high-temperature-treated sterile food. All pigs are confirmed as seronegative for Norovirus and germ-free prior to immunization experiments. Gnotobiotic pigs are immunized with protamine formulated or LNP formulated mRNA vaccines (monovalent, bivalent, or tetravalent) with doses, application routes and vaccination schedules as indicated in Table 7. Analysis of immune responses is performed essentially as described in Example 4 (ELISA, HGBA, and ICS).

(53) TABLE-US-00006 TABLE 7 Vaccination regimen of pigs (Example B) Group No. of pigs Treatment Dose/Route Vaccination schedule 1 6 Monovalent vaccine: protamine formulated 240 μg d 0, d 21 GII.4-2006b 092895-USA-2008 i.d. SEQ ID NO: 39729; R26 2 × 200 μl 2 6 Bivalent vaccine: protamine formulated 240 μg d 0, d 21 GI.1-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.d. GII.4-GZ2010-L87-Guangzhou-2011 2 × 200 μl SEQ ID NO: 39734; R27 3 6 Tetravalent vaccine; protamine formulated 240 μg d 0, d 21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.d. GII.4-031693-USA-2003 2 × 200 μl SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729: R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 4 6 Monovalent vaccine: LNP formulated 60 μg d 0, d 21 GII.4-2006b 092895-USA-2008 i.m. SEQ ID NO: 39729; R26 2 × 100 μl 5 6 Bivalent vaccine: LNP formulated 60 μg d 0, d 21 GI.1-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.m. GII.4-GZ2010-L87-Guangzhou-2011 2 × 100 μl SEQ ID NO: 39734; R27 6 6 Tetravalent vaccine: LNP formulated 60 μg d 0, d 21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.m. GII.4-031693-USA-2003 2 × 100 μl SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 7 6 100% RiLa Control — d 0, d 21

(54) 6.2. Norovirus Challenge Experiment:

(55) At day 3D days post immunization, the vaccinated and buffer-injected control pigs are challenged orally with Norovirus GII.4 (isolated from human stool samples) to assess the protection against Norovirus-induced diarrhea and fecal virus shedding. After virus challenge, rectal swaps and feces samples are collected at day 1, 3, 5, 7 and 10. Norovirus loads in rectal swaps and feces samples are determined using quantitative PER. In addition, pigs are monitored for Norovirus-associated symptoms and fecal consistence scores are recorded to assess severity of the Norovirus infection.

Example 7: Immunization of Non-Human Primates and Evaluation of Immune Responses

(56) Non-human primates (NHPs) are immunized with protamine or LNP formulated mRNA vaccines with doses, application routes and vaccination schedules as indicated in Table 8. Analysis of immune responses is performed essentially as described in Example 4 (ELISA, HGBA, and ICS).

(57) TABLE-US-00007 TABLE 8 Vaccination regimen of NHPs (Example 7) Group Number of NHPs Treatment Dose/Route Vaccination schedule 1 6 Monovalent vaccine protamine formulated 240 μg d 0, d 21 GII.4-2006b 092895-USA-2008 i.d. SEQ ID NO: 39729; R26 2 × 200 μl 2 6 Bivalent vaccine: protamine formulated 240 μg d 0, d 21 GI.1-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.d. GII.4-GZ2010-L87-Guangzhou-2011 2 × 200 μl SEQ ID NO: 39734; R27 3 6 Tetravalent vaccine: protamine formulated 240 μg d 0, d 21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.d. GII.4-031693-USA-2003 2 × 200 μl SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 4 6 Monovalent vaccine: LNP formulated 60 μg d 0, d 21 GII.4-2006b 092895-USA-2008 i.m. SEQ ID NO: 39729: R26 2 × 100 μl 5 6 Bivalent vaccine: LNP formulated 60 μg d 0, d 21 GI.1-USA-1968-Capsidprotein (total) SEQ ID NO: 39725; R29 + i.m. GII.4-GZ2010-L87-Guangzhou-2011 2 × 100 μl SEQ ID NO: 39734; R27 6 6 Tetravalent vaccine: LNP formulated 60 μg d 0, d 21 GII.4-USA-1997 (total) SEQ ID NO: 39738; R28 + i.m. GII.4-031693-USA-2003 2 × 100 μl SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 7 6 100% RiLa Control i.m. d 0, d 21 1 × 100 μl

Example 8: Development of a Multivalent Norovirus mRNA Vaccine

(58) 8.1. Generation of Bivalent, Tetravalent and Multivalent Norovirus mRNA Vaccines

(59) For bivalent and tetravalent Norovirus mRNA vaccines, each mRNA construct is individually produced (as described in Example 1).

(60) Multivalent Norovirus vaccine compositions are produced according to procedures as disclosed in the PCT application PCT/EP2016/082487. In short, Norovirus DNA constructs (each of which comprising different norovirus coding sequences and a T7 promotor; e.g. synthetic DNA templates immobilized on a chip) are used as a matrix for simultaneous PER amplification. The obtained PER product mixture is purified and used as a template for simultaneous RNA in vitro transcription to generate a mixture of Norovirus mRNA constructs. The obtained Norovirus mRNA mixture is subjected to quantitative and qualitative measurements (e.g., RNA AGE, RT-qPCR, NGS, and Spectrometry). Following that, purification and formulation is performed (protamine formulation and LNP formulation). For the preparation of multivalent mRNA mixtures, Norovirus sequences as provided in Table 3 (see specifications) are used.

(61) The produced bivalent, tetravalent and multivalent Norovirus mRNA vaccines are used for in vitro and in vivo experiments.

(62) 8.2. Expression Analysis of Multivalent Norovirus mRNA Vaccines Using Quantitative Mass Spectrometry

(63) Hela cells are transfected with bivalent, tetravalent and multivalent mRNA mixtures (see Table 9) and protein expression is analyzed using quantitative mass spectrometry to show that every mRNA comprised in the respective mRNA mixture is efficiently translated into Norovirus protein/antigen.

(64) 8.3. Immunization of Mice and Evaluation of Norovirus Specific Immune Responses

(65) Female BALB/c mice are with protamine or LNP formulated monovalent, bivalent, tetravalent or multivalent mRNA vaccines with doses, application routes and vaccination schedules as indicated in Table 9. As a negative control, one group of mice is injected with buffer (ringer lactate, Rile). All animals are vaccinated on day 0, 21 and 35. Blood samples are collected on day 49 for the Ill determination of binding antibody titers (using an ELISA assay), blocking antibody titers (using a HGBA assay) and cellular immune responses (ICS) performed essentially as described in Example 4.

(66) TABLE-US-00008 TABLE 9 Vaccination regimen of mice (Example 8) Group Number of mice Treatment Dose/Route Vaccination schedule 1 6 Monovalent vaccine: Protamine formulated 40 μg d 0, d 21, d 35 GII.4-D31693-USA-2003 i.d. SEQ ID NO: 39716; R4 2 6 Bivalent vaccine: Protamine formulated 80 μg d 0, d 21, d 35 R4 or R26 or R27 or R28 + (40 μg each) R4 or R26 or R27 or R28 i.d. 3 6 Tetravalent vaccine; Protamine formulated 80 μg d 0, d 21, d 35 GII.4-USA-1997 (20 μg each) SEQ ID NO: 39738; R28 + i.d. GII.4-031693-USA-2003 SEQ ID NO: 397I6; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 + GII.4-GZ2010-L87-Guangzhou-2011 SEQ ID NO: 39734; R27 4 6 Bivalent vaccine; Protamine formulated 80 μg d 0, d 21, d 35 GI.1-USA-1968-Capsidprotein (40 μg each) SEQ ID NO: 39725; R29 + i.d. R4 or R26 or R27 or R28 5 6 Tetravalent vaccine: Protamine formulated 80 μg d 0, d 21, d 35 GI.1-USA-1968-Capsidprotein (20 μg each) SEQ ID NO: 39725; R29 + i.d. GII.4-USA-1997 SEQ ID NO: 39738; R28 + GII.4-031693-USA-2003 SEQ ID NO: 39716; R4 + GII.4-2006b 092895-USA-2008 SEQ ID NO: 39729; R26 6 6 Multivalent: Protamine formulated. 80 μg d 0, d 21, d 35 20 constructs encoding Norovirus antigens of (total) several genogroups, genotypes and strains i.d. (selected from Table 3). 7 6 Multivalent: Protamine formulated. 80 μg d 0, d 21, d 35 50 constructs encoding Norovirus antigens of (total) several genogroups, genotypes and strains i.d. (selected from Table 3). 8 6 Multivalent: LNP formulated. 80 μg d 0, d 21, d 35 20 constructs encoding Norovirus antigens of (total) several genogroups. genotypes and strains i.m. (selected from Table 3). 9 6 Multivalent: LNP formulated. 80 μg d 0, d 21, d 35 50 constructs encoding Norovirus antigens of (total) several genogroups, genotypes and strains i.m. (selected from Table 3). 10 6 100% RiLa Control — d 0, d 21, d 35

Example 9: Expression of Norovirus Proteins in HeLa Cells and Analysis by FACS

(67) To determine in vitro protein expression of the constructs, HeLa cells are transiently transfected with mRNA encoding Norovirus antigens and stained using suitable customized anti Norovirus-protein antibodies (raised in mouse) and a FITC-coupled secondary antibody (F5262 from Sigma).

(68) Hela cells are seeded in a 6-well plate at a density of 400000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24 h prior to transfection. Hela cells are transfected with 1 and 2 μg unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs are used in the experiment, including a negative control encoding an irrelevant protein. 24 hours post transfection. Hela cells are stained with suitable anti Norovirus-protein antibodies (raised in mouse; 1:500) and anti-mouse FITC labelled secondary antibody (1:500) and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal is performed using the FlowJo software package (Tree Star, Inc.).

Example 10: Expression and Secretion of Norovirus Proteins Using Western Blot

(69) For the analysis of Norovirus protein secretion, Hela cells are transfected with 1 μg and 2 μg unformulated mRNA (R1-R29, see Table 4) including a negative control encoding an irrelevant protein using Lipofectamine as the transfection agent. Supernatants, harvested 24 hours post transfection, are filtered through a 0.2 μm filter. Clarified supernatants are applied on top of 1 ml 20% sucrose cushion (in PBS) and centrifuged at 14000 rcf (relative centrifugal force) for 2 hours at 4° C. Horn virus protein content is analyzed by Western Blot suitable customized anti Norovirus-protein antibodies (raised in mouse; 1:500 diluted) as primary antibody in combination with secondary anti mouse antibody coupled to IRDye 800CW (Licor Biosciences). The presence of αβ-tubulin is also analyzed as control for cellular contamination (αβ-tubulin: Cell Signalling Technology; 1:1000 diluted) in combination with secondary anti rabbit antibody coupled to IRDye 680RD (Licor Biosciences).

(70) For the analysis of Norovirus proteins in cell lysates, Hale cells are transfected with 1 μg and 2 μg unformulated mRNAs (R1-R29, see Table 4) including a negative control encoding an irrelevant protein using Lipofectamine as the transfection agent 24 hours post transfection, Held cells are detached by trypsin-free/EDTA buffer, harvested, and cell lysates are prepared. Cell lysates are subjected to SOS-PAGE under non-denaturating/non-reducting followed by western blot detection. Western Blot analysis is performed using a suitable customized anti Norovirus-protein antibodies antibody (raised in mouse; 1:500 diluted) as primary antibody in combination with secondary anti mouse antibody coupled to IRDye 800CW (Licor Biosciences).

Example 11: Preparation of Norovirus Vaccine Compositions

(71) For in vivo vaccination experiments, different compositions of Norovirus mRNA vaccine are prepared using Norovirus mRNA constructs (see Table 4). One composition comprises protamine-complexed mRNA, one composition comprises mRNA that is formulated with an aluminum phosphate adjuvant.

(72) 11.1. Preparation of Protamine Complexed mRNA (“Vaccine Composition 1”; RNActive®):

(73) Norovirus mRNA constructs are complexed with protamine prior to use in in vivo vaccination experiments. The mRNA complexation consists of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA is complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes are stably generated, free mRNA is added, and the final concentration of the vaccine is adjusted with Ringer's lactate solution.

(74) 11.2. Preparation of mRNA with Alum Phosphate (“Vaccine Composition 2”):

(75) mRNA constructs are mixed with the desired amount of aluminum phosphate adjuvant in Ringer's lactate solution (“naked mRNA”).

Example 12: Vaccination of Mice and Evaluation of Norovirus Specific Immune Response

(76) 12.1. Immunization

(77) Female BALB/c mice are injected intradermally (i.d.) and intramuscularly (i.m.) with respective mRNA vaccine compositions (prepared according to Example 11) with doses, application routes and vaccination schedules as indicated in Table 10. As a negative control, one group of mice is vaccinated with buffer (ringer lactate). All animals are vaccinated on day 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 for the determination of binding and neutralizing antibody titers (see below).

(78) TABLE-US-00009 TABLE 10 Vaccination regimen (Example 12) Number Route/ Vaccination Group of mice Vaccine composition Volume Schedule (day) 1 10 80 μg Norovirus i.d. 0/21/35 RNActive ® 2 × 50 μl Composition 1 2 10 40 μg Norovirus i.d. 0/21/35 RNActive ® 2 × 50 μl Composition 1 3 10 20 μg Norovirus i.d. 0/21/35 RNActive ® 2 × 50 μl Composition 1 4 10 40 μg Norovirus i.m. 0/21/35 naked RNA 2 × 25 μl Composition 2 5 10 40 μg Norovirus i.m. 0/21/35 naked RNA 2 × 25 μl Composition 2 6 10 40 μg Norovirus i.m. 0/21/35 naked RNA 2 × 25 μl Composition 2 7 10 100% RiLa Control i.d. 0/21/35 2 × 50 μl

(79) 12.2. Determination of Anti-Norovirus Protein Antibodies by ELISA:

(80) ELISA is performed using inactivated Norovirus infected cell lysate for coating. Coated plates are incubated using respective serum dilutions, and binding of specific antibodies to the Norovirus antigens are detected using biotinylated isotype specific anti-mouse antibodies followed by streptavidin-HRP (horse radish peroxidase) with ABTS as substrate. Endpoint titers of antibodies directed against the Norovirus antigens are measured by ELISA on day 63 after three vaccinations.

(81) 12.3. Intracellular Cytokine Staining

(82) 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). The cells are stimulated with a mixture of four Norovirus 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: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), 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.)

(83) 12.4. Norovirus Plaque Reduction Neutralization Test (PRNT50)

(84) Sera are analyzed by a plaque reduction neutralization test (PRNT50), performed as commonly known in the art. Briefly, obtained serum samples of vaccinated mice are incubated with Norovirus. That mixture is used to infect cultured cells, and the reduction in the number of plaques is determined.

Example 13: Clinical Development of a Norovirus mRNA Vaccine Composition

(85) To demonstrate safety and efficiency of the NoraviromRNA vaccine composition, a randomized, double blind, placebo-controlled clinical trial (phase I) is initiated.

(86) For clinical development, GMP-grade RNA is produced using an established GMP process, implementing various quality controls on DNA level and RNA level as described in detail in WO 2016/180430A1.

(87) In the clinical trial, a cohort of human volunteers is intradermally or intramuscularly injected for at least two times with a monovalent, or a bivalent, or a tetravalent or a multivalent mRNA based Norovirus vaccine as specified herein.

(88) In order to assess the safety profile of the Norovirus vaccine compositions according to the invention, subjects are monitored after administration (vital signs, vaccination site tolerability assessments, hematologic analysis).

(89) The efficacy of the immunization is analysed by determination of virus neutralizing titers (VNT) or HBGA blocking titers 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 or HBGA blocking antibodies.

(90) Furthermore, a subset of subjects is challenged with live GI.1 Norwalk virus or placebo by oral administration. Subjects are followed post-challenge for symptoms of Norovirus associated illness, infection and immune responses. There are multiple clinical assessments and collection of blood, emesis, saliva, and stool specimens.

(91) TABLE-US-00010 Lengthy table referenced here US11141474-20211012-T00001 Please refer to the end of the specification for access instructions.

(92) TABLE-US-LTS-00001 LENGTHY TABLES The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).