ROTAVIRUS VACCINES

Abstract

The present invention provides mRNA sequences comprising at least one coding region, encoding for at least one epitope of a protein, or of a fragment, variant or derivative thereof, of a virus of the genus rotavirus. Particularly preferred is the protein respectively the protein cleavage product VP8* of rotavirus. The mRNA sequence may be used as a vaccine or generally as a pharmaceutical composition for prophylaxis or treatment of rotavirus infections.

Claims

1. mRNA sequence comprising at least one coding region, encoding at least one epitope of a protein, or of a fragment, variant or derivative thereof of a virus of the genus rotavirus.

2. The mRNA sequence according to claim 1, wherein the rotavirus is selected from the species A or B or C.

3. The mRNA sequence according to claim 1 or claim 2, wherein the rotavirus is selected from the species A.

4. The mRNA sequence according to any one of the preceding claims, wherein the rotavirus is selected from the G-serotypes or P-serotypes G1 or G2 or G3 or G4 or G9 or G12 or P[4] or P[6] or P[8].

5. The mRNA sequence according to any one of the preceding claims, wherein the mRNA sequence encodes for at least one epitope of a protein, or of a fragment, variant or derivative thereof of a virus of the genus rotavirus, which is at least 60% identical, more preferably at least 70% identical, more preferably at least 80% identical, more preferably at least 90% identical, more preferably at least 95% identical, or most preferably at least 99% identical to the wild type protein or to the respective fragment of the wild type protein.

6. The mRNA sequence according to any one of the preceding claims, wherein the protein of a virus of the genus rotavirus is selected from a structural protein of rotavirus, preferably VP1 or VP2 or VP3 or VP4 or VP6 or VP7, or from a non-structural protein of rotavirus, preferably NSP1 or NSP2 or NSP3 or NSP4 or NSP5 or NSP6, more preferably VP2 or VP4 or VP6 or VP7 or NSP2 or NSP4.

7. The mRNA sequence according to claim 6, wherein the at least one epitope is derived from any of the protein sequences according to SEQ ID Nos. 1-80.

8. The mRNA sequence according to claim 6 or claim 7, wherein the protein of a virus of the genus rotavirus is selected from proteins derived from cleavage of VP4, wherein preferably the cleavage product is VP5* or VP8*, most preferably VP8*.

9. The mRNA sequence according to claims 6 to 8, wherein the protein VP7 is derived from the serotypes G1 or G2 or G3 or G4 or G9 or G12 and/or wherein the protein VP4 or the protein cleavage product VP5* or VP8* is derived from the serotypes P[4] or P[6] or P[8].

10. The mRNA sequence according to claim 9, wherein VP8* is a shortened protein form.

11. The mRNA sequence according to claim 9 or claim 10, wherein VP8* is selected from any one of the protein sequences according to SEQ ID NOs: 41, 45, 47, or 49 or from one of the shortened forms according to SEQ ID NOs: 173-176, 269-272, 365-368, 461-464, 557-560, 653-656, 749-750.

12. The mRNA sequence according to any one of the preceding claims, wherein the mRNA sequence additionally comprises at least one sequence section encoding at least one helper peptide and optionally at least one sequence section encoding a peptide linker for enhancement of immunogenicity.

13. The mRNA sequence according to claim 12, wherein the sequence section encoding the helper peptide is located at the 5-end of the coding region, encoding the at least one epitope of a protein, or of a fragment, variant or derivative thereof of a virus of the genus rotavirus.

14. The mRNA sequence according to claim 12 or claim 13, wherein the helper peptide is derived from tetanus toxin, or a fragment, variant or derivative thereof.

15. The mRNA sequence of according to claim 14, wherein the helper peptide comprises or consists of the amino acid sequence according to SEQ ID NO: 3147 or a fragment, variant or derivative thereof.

16. The mRNA sequence according to any one of claims 12 to 15, wherein the at least one coding region encodes at least one of the amino acid sequences as shown in Table 3.

17. The mRNA sequence according to any one of the preceding claims, wherein the mRNA sequence additionally comprises at least one sequence section encoding at least one signal peptide.

18. The mRNA sequence of claim 17, wherein the signal peptide is derived from tissue plasminogen activator or albumin or CD5 or HLA-A2 or luciferase or immunoglobulin or IL-2 or chymotrypsinogen or a fragment, variant or derivative thereof.

19. The mRNA sequence according to claim 17 or claim 18, wherein the signal peptide comprises or consists of an amino acid sequence selected from any one of the amino acid sequences according to SEQ ID NOs: 3148-3159 or a fragment, variant or derivative thereof.

20. The mRNA sequence according to claim 19, wherein the mRNA sequence encodes at least one of the amino acid sequences as shown in Table 5.

21. The mRNA sequence according to claim 19, wherein the mRNA sequence additionally comprises at least one sequence section encoding the helper peptide derived from tetanus toxin, preferably according to SEQ ID NO. 3147 or a fragment, variant or derivative thereof.

22. The mRNA sequence according to claim 21, wherein the mRNA sequence encodes at least one of the amino acid sequences as shown in Table 7.

23. The mRNA sequence according to any one of the preceding claims, wherein the coding region, encoding at least one epitope of a protein, or a fragment, variant or derivative thereof, of a virus of the genus rotavirus is mutated to delete at least one predicted or potential glycosylation site.

24. The mRNA sequence according to claim 23, wherein at least one codon coding for an asparagine, arginine, serine, threonine, tyrosine, lysine, proline or tryptophan is mutated in such a way that a different amino acid is encoded to delete at least one predicted or potential glycosylation site.

25. The mRNA sequence according to claim 24, wherein the at least one codon codes for asparagine which is mutated to glutamine.

26. The mRNA sequence according to any one of the preceding claims, wherein the coding region, encoding at least one epitope of a protein, or a fragment, variant or derivative thereof, of a virus of the genus rotavirus is mutated to delete all predicted glycosylation sites.

27. The mRNA sequence according to any one of claims 23 to 26, wherein the coding region encodes at least one of the amino acid sequences as shown in Table 9.

28. The mRNA sequence according to any one of the preceding claims, wherein the mRNA sequence additionally comprises at least one sequence section encoding at least one transmembrane domain of a protein, or a fragment, variant or derivative thereof.

29. The mRNA sequence according to claim 28, wherein the transmembrane domain is selected from the transmembrane domain of hemagglutinin (HA) of influenza virus, or Env of HIV-1, or EIAV (equine infectious anemia), or MLV (murine leukemia), or mouse mammary tumor virus, or G protein of VSV (vesicular stomatitis virus), or rabies virus, or a fragment, variant or derivative thereof.

30. The mRNA sequence according to claim 23 or claim 24, wherein the at least one sequence section encoding a transmembrane domain of a protein, or a fragment, variant or derivative thereof, is located at the 3-end of the coding region, encoding at least one epitope of a protein, or of a fragment, variant or derivative thereof, of a virus of the genus rotavirus.

31. The mRNA sequence according to any one of claims 28 to 30, wherein the transmembrane domain comprises or consists of any of the amino acid sequences according to SEQ ID NOs: 3160-3171 or a fragment, variant or derivative thereof.

32. The mRNA sequence according to any of claims 28 to 31, wherein the coding region encodes at least one of the amino acid sequences as shown in Table 14.

33. The mRNA sequence according to any one of the preceding claims, wherein the mRNA sequence additionally comprises at least one sequence section encoding at least one peptide or protein enabling VLP (virus like particle) formation.

34. The mRNA sequence according to claim 33, wherein the peptide or protein enabling VLP formation is derived from non-human pathogenic viruses.

35. The mRNA sequence according to claim 33 or 34, wherein the peptide or protein enabling VLP formation is derived from Woodchuck hepatitis virus core antigen (WHcAg) or from Alfalfa mosaic virus CP (coat protein).

36. The mRNA sequence according to any one of claims 33 to 35, wherein the peptide or protein enabling VLP formation comprises or consists of any of the amino acid sequences according to SEQ ID NO: 3172 or SEQ ID NO: 3173.

37. The mRNA sequence according to claim 35 or claim 36, wherein the at least one sequence section encoding at least one peptide or protein enabling VLP formation derived from Woodchuck hepatitis virus core antigen (WHcAg) is located 5 of the coding region, encoding at least one epitope of a protein, or a fragment, variant or derivative thereof of a virus of the genus rotavirus.

38. The mRNA sequence according to claim 35 or claim 36, wherein the at least one sequence section encoding at least one peptide or protein enabling VLP formation derived from Alfalfa mosaic virus CP (coat protein) is located 3 of the coding region, encoding at least one epitope of a protein, or a fragment, variant or derivative thereof of a virus of the genus rotavirus.

39. The mRNA sequence according to claims 33 to 38, wherein the at least one sequence section encoding a peptide or protein enabling VLP formation is separated from the coding region, encoding at least one epitope of a protein, or a fragment, variant or derivative thereof, of a virus of the genus rotavirus by a at least one sequence section encoding a peptide linker.

40. The mRNA sequence according to claim 39, wherein the at least one sequence section encoding the peptide linker is coding for a flexible linker or a rigid inker.

41. The mRNA sequence according to claim 40, wherein the flexible linker comprises at least one stretch of glycine and/or serine residues, wherein preferably the flexible linker comprises more than one stretch of glycine and/or serine residues.

42. The mRNA sequence according to claim 40 or claim 41, wherein the peptide linker comprises at least one stretch consisting of four or five amino acids.

43. The mRNA sequence according to claims 39 to 42, wherein the peptide linker comprises or consists of any of the amino acid sequence according to SEQ ID NOs: 3174-3176.

44. The mRNA sequence according any of the claims 33 to 43, wherein the coding region encodes one of the amino acid sequences as shown in Table 16.

45. The mRNA sequence according to any of the preceding claims, wherein the mRNA sequence comprises at least two coding regions, each encoding at least one epitope of a protein, or of a fragment, variant or derivative thereof of a virus of the genus rotavirus.

46. The mRNA sequence according to claim 45, wherein the mRNA sequence comprises at least three or four coding regions.

47. The mRNA sequence according to claim 45 or claim 46, wherein each coding region encodes at least one epitope of a protein, or a fragment, variant or derivative thereof, of different serotypes of a virus of the genus rotavirus.

48. The mRNA sequence according to claim 47, wherein the at least one epitope of a protein, or a fragment, variant or derivative thereof, of different serotypes of a virus of the genus rotavirus is derived from the same protein, preferably from VP8*.

49. The mRNA sequence according to claim 45 or claim 46, wherein each coding region encodes at least one epitope of a protein, or a fragment, variant or derivative thereof, of the same serotype of a virus of the genus rotavirus, preferably from VP8*.

50. The mRNA sequence according to claim 49, wherein the sequence encodes one of the amino acid sequences as shown in Table 18.

51. The mRNA sequence according to any one of claims 45 to 49, wherein the at least two coding regions, each encoding at least one epitope of a protein, or of a fragment, variant or derivative thereof of a virus of the genus rotavirus, are separated by intermitting sequences.

52. The mRNA sequence according to claim 51, wherein the intermitting sequence is an internal ribosomal entry sites (IRES), wherein preferably the IRES is selected from the IRES of encephalomyocarditis virus and/or by the IRES of foot-and-mouth disease virus.

53. The mRNA sequence according to claim 51, wherein the intermitting sequence is a sequence section encoding a self-cleaving peptide.

54. The mRNA sequence according to claim 53, wherein the self-cleaving peptide is selected from F2A peptide derived from foot-and-mouth diseases virus, or self-cleaving peptides from equine rhinitis A virus, Thosea asigna virus or porcine teschovirus-1.

55. The mRNA sequence according to any one of the preceding claims, wherein the mRNA sequence comprises at least one sequence as shown in Tables 2, 4, 6, 8, 10-13, 15, 17, and 19.

56. The mRNA sequence according to any one of the preceding claims, wherein the G/C content of the coding region and/or coding sequence sections is increased compared with the G/C content of the coding region of the wild type mRNA, and wherein the coded amino acid sequence of said G/C-enriched mRNA is preferably not being modified compared with the coded amino acid sequence of the wild type mRNA.

57. The mRNA sequence according to any one of the preceding claims, wherein the mRNA sequence comprises additionally a 5-UTR element and/or a 3-UTR element and/or additionally at least one histone stem-loop sequence and/or additionally a 5-CAP structure and/or a poly(A) sequence and/or a poly(C) sequence.

58. The mRNA sequence according to claim 57, wherein the mRNA sequence comprises, preferably in 5- to 3-direction: a 5-CAP structure, preferably m7GpppN; a coding region encoding at least one epitope of a protein, or a fragment, variant or derivative thereof, of a virus of the genus rotavirus; a 3-UTR element comprising or consisting of a nucleic acid sequence which is derived from a -globin gene, preferably comprising the RNA according to SEQ ID NO: 3199 or a homolog, a fragment or a variant thereof; a poly(A) sequence, preferably comprising 64 adenosines; a poly(C) sequence, preferably comprising 30 cytosines; and a histone-stem-loop, preferably comprising the RNA sequence according to SEQ ID NO: 3207.

59. The mRNA sequence according to claim 57, wherein the mRNA sequence comprises, preferably in 5- to 3-direction: a 5-CAP structure, preferably m7GpppN; a 5-UTR element which comprises or consists of a nucleic acid sequence which is derived from the 5-UTR of a TOP gene, preferably comprising or consisting of the RNA sequence according to SEQ ID NO: 3189 or 3191 or a homolog, a fragment or a variant thereof; a coding region encoding at least one epitope of a protein, or a fragment, variant or derivative thereof, of a virus of the genus rotavirus; a 3-UTR element comprising or consisting of a nucleic acid sequence which is derived from a gene providing a stable mRNA, preferably comprising or consisting of any of the RNA sequences according to SEQ ID NOs: 3193, 3195, 3197, 3199, 3201, or 3203 or a homolog, a fragment or a variant thereof; a poly(A) sequence preferably comprising 64 adenosines; a poly(C) sequence, preferably comprising 30 cytosines; and a histone-stem-loop, preferably comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 3207.

60. The mRNA sequence according to any one of the preceding claims, wherein the mRNA sequence is prepared for use as a vaccine.

61. Composition comprising one or more mRNA sequences as defined according to any one of the preceding claims and optionally a pharmaceutically acceptable carrier.

62. The composition according to claim 61, wherein the mRNA sequence according to claims 1 to 59 is at least partially associated with or complexed with a cationic or polycationic compound and/or a polymeric carrier, optionally in a weight ratio selected from a range of about 6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1 (w:w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w) to about 2:1 (w/w) of mRNA to cationic or polycationic compound and/or with a polymeric carrier; or optionally in a nitrogen/phosphate ratio of mRNA to cationic or polycationic compound and/or polymeric carrier in a range of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1, and most preferably in a range of about 0.5-1 or 0.7-1, and even most preferably in a range of about 0.3-0.9 or 0.5-0.9.

63. The composition according to claim 61 or claim 62, preferably comprising one or more mRNA sequences as defined according to any one of claims 1 to 59 and at least one VLP forming protein or peptide, or a fragment, variant or derivative thereof, and/or a nucleic acid encoding a VLP forming protein or peptide, or a fragment, variant or derivative thereof.

64. The composition according to claim 63, wherein the VLP forming protein or peptide is a viral matrix protein or peptide derived from an enveloped virus.

65. The composition according to claim 64, wherein the matrix protein is a gag protein derived from an enveloped virus selected from HIV-1 or EIAV or MLV.

66. The composition according to claim 64, wherein the matrix protein is the matrix protein of vesicular stomatitis virus (VSV), Rabies virus or the VP40 protein derived from an Ebola virus.

67. Pharmaceutical composition comprising one or more mRNA sequences as defined according to any one of claims 1 to 59 or a composition according to any one of claims 60 to 66 and optionally a pharmaceutically acceptable carrier.

68. The pharmaceutical composition according to claim 67, wherein at least one mRNA sequence is at least partially associated with or complexed with a cationic or polycationic compound and/or a polymeric carrier, preferably cationic proteins or peptides and most preferably protamine.

69. The pharmaceutical composition according to claim 68, wherein the ratio of complexed mRNA to free mRNA is selected from a range of about 5:1 (w/w) to about 1:10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1:8 (w/w), even more preferably from a range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio of complexed mRNA to free mRNA is selected from a ratio of 1:1 (w/w).

70. Vaccine comprising a composition as defined according to any one of claims 60 to 66 or a pharmaceutical composition as defined according to any one of claims 67 to 69.

71. The composition as defined according to any one of claims 60 to 66, the pharmaceutical composition as defined according to any one of claims 67 to 69 or the vaccine as defined according to claim 70 further comprising at least one adjuvant component.

72. Kit or kit of parts comprising one or more of the m RNA sequences as defined according to any one of claims 1 to 59 or the composition as defined according to any one of claims 60 to 66 or the pharmaceutical composition as defined according to any one of claims 67 to 69 or the vaccine as defined according to claim 70 and optionally technical instructions with information on the administration and dosage of the components.

73. The mRNA sequence as defined according to any one of claims 1 to 59 or the composition as defined according to any one of claims 60 to 66 or the pharmaceutical composition as defined according to any one of claims 67 to 69 or the vaccine as defined according to claim 70 or the kit or kit of parts as defined according to 72 for use as a medicament.

74. The mRNA sequence as defined according to any one of claims 1 to 59 or the composition as defined according to any one of claims 60 to 66 or the pharmaceutical composition as defined according to any one of claims 67 to 69 or the vaccine as defined according to claim 70 or the kit or kit of parts as defined according to claim 72 for use in the treatment or prophylaxis of rotavirus infections.

75. The mRNA sequence or the composition or the pharmaceutical composition or the vaccine or the kit or kit of parts for use according to claim 73 or claim 74, wherein the mRNA sequence, the composition, the pharmaceutical composition, the vaccine or the kit or kit of parts is administered by subcutaneous, intramuscular, intradermal, topical or transdermal application, more preferably by intradermal injection.

76. A method of treatment or prophylaxis of rotavirus infections comprising the steps: providing one or more of the mRNA sequences as defined according to any one of claims 1 to 59 or the composition as defined according to any one of claims 60 to 66 or the pharmaceutical composition as defined according to any one of claims 67 to 69 or the vaccine as defined according to claim 70 or the kit or kit of parts as defined according to claim 72; and applying or administering the mRNA sequence(s) or the composition or the pharmaceutical composition or the vaccine or the kit or kit of parts to a tissue or an organism.

77. The method according to claim 76, wherein the mRNA sequence(s) or the composition or the pharmaceutical composition or the vaccine or the kit or kit of parts is administered to the tissue or to the organism by subcutaneous, intramuscular, intradermal, topical or transdermal application, more preferably by intradermal injection.

78. The method according to claim 77, wherein the injection is carried out by using conventional needle injection or jet injection, preferably by using jet injection.

Description

SHORT DESCRIPTION OF THE FIGURES

[0393] FIG. 1 Schematic drawings of preferred VP8* constructs. P2: P2 helper peptide from Tetanus toxin; VP8*: Virus protein 8* (cleavage product of rotavirus VP4 protein); SP: signal peptide; WhcAg: Wodchuck Hepatitis virus core antigen; L: linker; TM: transmembrane domain

[0394] FIG. 2 Humoral responses upon vaccination with the preferred constructs encoding the P2 VP8* protein. [0395] A: IgG1 and IgG2a antibody titers assessed by ELISA using P2 VP8* P[6] protein as a coating reagent. The experiment was performed as described in Example 2. Statistically significant IgG1 and IgG2a responses were detectable for most groups vaccinated with the mRNA vaccine encoding P2 VP8*. The best antibody responses were detectable in secreted and VLP designs. Each dot represents an individual animal and horizontal lines represent median values. [0396] B: IgG1 and IgG2a antibody titers assessed by ELISA using P2 VP8* P[4] protein as a coating reagent. The experiment was performed as described in Example 2. This figure shows cross-reactive responses in mice vaccinated with P[6] designs with P[4] serotype protein. Comparison of the different groups shows that the trend seen for P[6] coating remains unaltered. Each dot represents an individual animal and horizontal lines represent median values. [0397] C: IgG1 and IgG2a antibody titers assessed by ELISA using P2 VP8* P[8] protein as a coating reagent. The experiment was performed as described in Example 2. This figure shows cross-reactive responses in mice vaccinated with P[6] designs with P[8] serotype protein. Comparison of the different groups shows that the trend seen for P[6] and P[4] coating remains unaltered. Each dot represents an individual animal and horizontal lines represent median values.

EXAMPLES

[0398] 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 the Rotavirus mRNA Vaccine

[0399] 1. Preparation of DNA and mRNA Constructs

[0400] For the present examples DNA sequences encoding the VP8* protein of different serotypes of rotavirus were prepared and used for subsequent in vitro transcription. Schematics of the constructs are shown in FIG. 1.

[0401] 2. In Vitro Transcription

[0402] The respective DNA plasmids prepared according to paragraph 1 were transcribed in vitro using T7 RNA polymerase in the presence of a CAP analogue (m7GpppG). Subsequently the mRNA was purified using PureMessenger (CureVac, Tbingen, Germany; WO2008/077592A1).

[0403] The following mRNA sequences were prepared:

TABLE-US-00021 TABLE 21 SEQ N glycos- Trans- R ID Sero- AA in Helper Signal ylation membrane VLP number NO. type VP4 peptide peptide sites domain domain 1. R3718 3114 P[6] +P2 N -> Q 2. R3720 3135 P[6] +P2 N -> Q 3. R3722 3121 P[6] +P2 N -> Q 4. R3724 3128 P[6] +P2 N -> Q WhcAg 5. R5471 2968 P[8] 65-223 +P2 wt 6. R5473 2872 P[8] 40-223 +P2 wt 7. R5475 2488 P[8] 2-230 +P2 wt 8. R5479 2484 P[8] 1-230 wt 9. R5481 2496 P[8] 2-230 +HSA wt 10. R5483 2592 P[8] 10-223 +HSA wt 11. R5485 2880 P[8] 40-223 +HSA wt 12. R5487 2900 P[8] 40-223 +P2 +HSA wt 13. R5489 2904 P[8] 40-223 +P2 +IgE wt 14. R5491 2736 P[8] 10-240 +HSA N -> Q 15. R5493 2928 P[8] 40-223 +HSA N -> Q 16. R5594 2875 P[6] 40-223 +HSA wt 17. R5595 2895 P[6] 40-223 +P2 +TPA wt 18. R5596 2491 P[6] 2-230 +TPA wt 19. R5597 2827 P[6] 20-240 +TPA N -> Q

[0404] The mRNA sequences comprise in 5- to 3-direction: [0405] a) a 5-CAP structure, consisting of m7GpppN; [0406] b) a 5-UTR element comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 3189; [0407] c) at least one sequence encoding a signal peptide [0408] d) optionally at least one sequence encoding at least one helper peptide (e.g. P2) [0409] e) optionally at least one sequence encoding at least one protein enabling VLP formation (e.g. WHcAg) [0410] f) at least one G/C optimized coding region encoding the protein of interest, preferably as shown in Table 2 Column B, [0411] g) a 3-UTR element comprising the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO: 3205; [0412] h) a poly(A) sequence, comprising 64 adenosines; [0413] i) a poly(C) sequence, comprising 30 cytosines; and [0414] j) a histone-stem-loop structure, comprising the RNA sequence according to SEQ ID NO: 3207.

[0415] 3. Preparation of the mRNA Vaccine

[0416] 3.1. Protamine Complexation:

[0417] The mRNA vaccine consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, free mRNA was added, and the final concentration of the vaccine was adjusted with Ringer's lactate solution.

[0418] 3.2. LNP Formulation

[0419] Lipid nano particle (LNP)-formulated mRNA was generated using an ionizable amino lipid, phospholipid, cholesterol and a PEGylated lipid, similar in composition as described in Thess et al. Mol Ther J Am Soc Gene Ther. 2015; 23(9):1456-1464.

[0420] 3.3. CVCM Formulation:

[0421] The polyethylene glycol/peptide polymers (HO-PEG 5000-S(SCHHHHHHRRRRHHHHHHCS-)7-S-PEG 5000-OH according to formula IV (referred to as PB83) is used for complexation of the inventive mRNA sequences, optional in combination with a lipid or lipidoid as disclosed in PCT/EP2016/063228 (incorporated herewith by reference).

[0422] 4. Analysis of VP8* Specific Antibodies by ELISA

[0423] ELISA plates were coated with 1 g/ml (for P[4] P2-VP8* and P[6] P2-VP8*) or 10 g/ml (for P[8] P2-VP8*) protein. Coated plates were blocked (in 1% milk; 0.05% Tween in PBS) and incubated with serum in different dilutions. Binding of specific antibodies to the P2 VP8* protein was detected using HRP (horse radish peroxidase) coupled rat monoclonal anti-mouse IgG1 or IgG2a using Amplex Ultra Red as a substrate for detection.

[0424] Statistical analysis was done by Mann Whitney test. Asterisks represent the following p values: *p<0.05; **p<0.01; ***p<0.001.

Example 2: Induction of Humoral Responses Upon Vaccination with the mRNA Vaccine Encoding the P2 VP8* Protein

[0425] Balb/c mice were immunised with mRNA vaccines (as prepared in Example 1) encoding P2-VP8* from serotype P[6] or RiLa (Ringer lactate) as a negative control as indicated in Table 22 below. Intradermal (i.d.) vaccinations were performed on day 0, day 21 and day 42. Blood samples taken on day 56 were analysed for the presence of VP8* specific IgG1 and IgG2a antibodies by ELISA using P2 VP8* P[6] protein (FIG. 2 A), P2 VP8* P[4] protein (FIG. 2 B), or P2 VP8* P[8] protein (FIG. 2 C) as a coating reagent (Wen et al., 2014. Vaccine 32, 4420-4427).

TABLE-US-00022 TABLE 22 Animal groups and treatment Treat- ment/ Vaccin. Strain/ Vaccine Sero- Route/ sched- gender Nr. dose Construct type Volume ule 1 BALB/c 8 80 g Secreted P2- P[6] i. d. d0/21/42 Female R3718 VP8* 2 50 l 2 BALB/c 8 80 g Secreted repeat P[6] i. d. d0/21/42 Female R3720 P2-VP8* 2 50 l 3 BALB/c 8 80 g Transmembrane P[6] i. d. d0/21/42 Female R3722 P2-VP8* 2 50 l 4 BALB/c 8 80 g VLP P2-VP8* P[6] i. d. d0/21/42 Female R3724 2 50 l 5 BALB/c 8 100% i. d. d0/21/42 Female RiLa 2 50 l

[0426] Results

[0427] As shown in FIG. 2 A, statistically significant IgG1 and IgG2a responses were detectable for most groups vaccinated with the mRNA vaccine encoding P2 VP8* when P2 VP8* of serotype P[6] protein was used as a coating reagent. The best antibody responses were detectable in secreted and VLP designs.

[0428] FIG. 2 B shows cross-reactive responses in mice vaccinated with P[6] designs with the P[4] serotype P2 VP8* protein used as a coating reagent. Comparison of the different groups shows that the trend seen for P[6] coating in FIG. 2 remains unaltered. This result is very surprising as cross-protection between different serotypes of rotavirus would not have been expected.

[0429] FIG. 2 C shows cross-reactive responses in mice vaccinated with P[6] designs with the P[8] serotype P2 VP8* protein used as a coating reagent. Comparison of the different groups shows that the trend seen for P[6] and P[4] coating in FIG. 2 A and FIG. 2 B, respectively, remains unaltered. This result is very surprising as cross-protection between different serotypes of rotavirus would not have been expected.

Example 3: Immunogenicity of New VP8* Designs

[0430] Balb/c mice are immunised intradermally with the new mRNA vaccine designs encoding P2-VP8* (table 21). RiLa (Ringer lactate) and adjuvanted P2-VP8* protein are employed as a negative and positive control, respectively, as indicated in Table 23 and 24. Vaccinations are performed on day 0, day 21 and day 42. Blood samples taken on day 56 are analysed for the presence of VP8* specific IgG1 and IgG2a antibodies by ELISA using P2 VP8* P[8] or P[6] protein, respectively, as a coating reagent as described above.

TABLE-US-00023 TABLE 23 Animal groups and treatment part A Strain/ Route/ Gr. gender Number Treatment Volume Vaccin. schedule 1 BALB/c 6 Negative control i. d. d0/21/42 female 2 50 l 2 BALB/c 6 Positive control i. m. d0/21/42 female 4 25 l 3 BALB/c 12 80 g R3718 i. d. d0/21/42 female 2 50 l 4 BALB/c 12 80 g R5594 i. d. d0/21/42 female 2 50 l 5 BALB/c 12 80 g R5595 i. d. d0/21/42 female 2 50 l 6 BALB/c 12 80 g R5596 i. d. d0/21/42 female 2 50 l 7 BALB/c 12 80 g R5597 i. d. d0/21/42 female 2 50 l 8 BALB/c 12 80 g R5471 i. d. d0/21/42 female 2 50 l 9 BALB/c 12 80 g R5473 i. d. d0/21/42 female 2 50 l 10 BALB/c 12 80 g R5475 i. d. d0/21/42 female 2 50 l 11 BALB/c 12 80 g R5479 i. d. d0/21/42 female 2 50 l

TABLE-US-00024 TABLE 24 Animal groups and treatment part B Strain/ Route/ Gr. gender Number Treatment Volume Vaccin. schedule 1 BALB/c 6 Negative control i. d. d0/21/42 Female 2 50 l 2 BALB/c 6 Positive control i. m. d0/21/42 Female 4 25 l 3 BALB/c 12 80 g R3718 i. d. d0/21/42 Female 2 50 l 4 BALB/c 12 80 g R5481 i. d. d0/21/42 Female 2 50 l 5 BALB/c 12 80 g R5483 i. d. d0/21/42 Female 2 50 l 6 BALB/c 12 80 g R5485 i. d. d0/21/42 Female 2 50 l 7 BALB/c 12 80 g R5487 i. d. d0/21/42 Female 2 50 l 8 BALB/c 12 80 g R5489 i. d. d0/21/42 Female 2 50 l 9 BALB/c 12 80 g R5490 i. d. d0/21/42 Female 2 50 l 10 BALB/c 12 80 g R5491 i. d. d0/21/42 Female 2 50 l

Example 4: Immunogenicity of New VP8* Formulations

[0431] Five mRNA designs are tested as mRNA formulations with LNPs (lipid nanoparticle) and CVCMs formulations. For this, the respective mRNA sequences are formulated as described above and are tested upon intramuscular injection. RiLa (Ringer lactate) and adjuvanted P2-VP8* protein are employed as a negative and positive control, respectively, as indicated in Table 25 and 26 vaccinations are performed on day 0, day 21 and day 42. Blood samples taken on day 56 are analysed for the presence of VP8* specific IgG1 and IgG2a antibodies by ELISA using P2 VP8* P[8] or P[6] protein, respectively, as a coating reagent (FIG. 2)

TABLE-US-00025 TABLE 25 Animal groups and treatment LNP formulations Strain/ Vaccine Route/ Vaccin. Gr. gender Number Treatment formulation Volume schedule 1 BALB/c 6 Negative control i. d. d0/21/42 female 2 50 l 2 BALB/c 6 Positive control i. m. d0/21/42 female 4 25 l 3 BALB/c 12 Design 1 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 4 BALB/c 12 Design 2 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 5 BALB/c 12 Design 3 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 6 BALB/c 12 Design 4 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 7 BALB/c 12 Design 5 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 8 BALB/c 12 Design 1 LNP formulation i. m. d0/21/42 female 5 g 1 25 l 9 BALB/c 12 Design 2 LNP formulation i. m. d0/21/42 female 5 g 1 25 l 10 BALB/c 12 Design 3 LNP formulation i. m. d0/21/42 female 5 g 1 25 l 11 BALB/c 12 Design 4 LNP formulation i. m. d0/21/42 female 5 g 1 25 l 12 BALB/c 12 Design 5 LNP formulation i. m. d0/21/42 female 5 g 1 25 l

TABLE-US-00026 TABLE 26 Animal groups and treatment CVCM formulations Strain/ Vaccine Route/ Vaccin. Gr. gender Number Treatment formulation Volume schedule 1 BALB/c 6 Negative control i. d. d0/21/42 female 2 50 l 2 BALB/c 6 Positive control i. m. d0/21/42 female 4 25 l 3 BALB/c 12 Design 1 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 4 BALB/c 12 Design 2 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 5 BALB/c 12 Design 3 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 6 BALB/c 12 Design 4 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 7 BALB/c 12 Design 5 Protamine i. d. d0/21/42 female 80 g formulation 2 50 l 8 BALB/c 12 Design 1 CVCM formulation i. m. d0/21/42 female 10 g 1 25 l 9 BALB/c 12 Design 2 CVCM formulation i. m. d0/21/42 female 10 g 1 25 l 10 BALB/c 12 Design 3 CVCM formulation i. m. d0/21/42 female 10 g 1 25 l 11 BALB/c 12 Design 4 CVCM formulation i. m. d0/21/42 female 10 g 1 25 l 12 BALB/c 12 Design 5 CVCM formulation i. m. d0/21/42 female 10 g 1 25 l