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

1-78. (canceled)

79. A pharmaceutical composition comprising a purified RNA molecule comprising a region encoding a polypeptide comprising at least 100 amino acids of a VP8* cleavage product of a rotavirus VP4 protein, said RNA comprising a 5′ Cap structure and a poly(A) region of 60 to 250 adenosine nucleotides, wherein said RNA is formulated with a lipid nanoparticle (LNP).

80. The pharmaceutical composition of claim 79, wherein the 5′ Cap structure is a CAP1.

81. The pharmaceutical composition of claim 79, wherein the poly(A) region is located at the 3′ terminus of the RNA.

82. The pharmaceutical composition of claim 79, wherein the VP8* cleavage product is derived from the P[4], P[6], or P[8] serotype.

83. The pharmaceutical composition of claim 79, wherein the polypeptide comprises an amino acid substitution to remove at least one predicted glycosylation site.

84. The pharmaceutical composition of claim 79, wherein the RNA is a mRNA that comprises, from 5′ to 3′: (i) a 5′ Cap structure; (ii) a 5′ untranslated region (UTR); (iii) the region encoding the polypeptide; (iv) a 3′ UTR; and (v) the poly(A) region.

85. The pharmaceutical composition of claim 82, wherein the composition comprises at least a second RNA encoding a VP8* cleavage product of a rotavirus VP4 from a different rotavirus strain as the first RNA.

86. The pharmaceutical composition of claim 82, wherein the LNP comprises an ionizable amino lipid, phospholipid, cholesterol and a PEGylated lipid.

87. The pharmaceutical composition of claim 82, wherein the RNA further encodes a sequence encoding a VLP forming protein.

88. The pharmaceutical composition of claim 87, wherein the VLP forming protein is derived from an enveloped virus.

89. The pharmaceutical composition of claim 82, wherein the wherein the G/C content of the region encoding the polypeptide is increased compared to the G/C content of the coding region of a corresponding original polypeptide.

90. The pharmaceutical composition of claim 82, said RNA further comprising a sequence encoding a helper peptide, wherein the sequence encoding the helper peptide is located at the 5′ end of the polypeptide.

91. The pharmaceutical composition of claim 90, wherein the helper peptide is derived from tetanus toxin.

92. The pharmaceutical composition of claim 82, wherein the RNA molecule further comprises a sequence encoding a signal peptide derived from tissue plasminogen activator, albumin, CD5, HLA-A2, luciferase, immunoglobulin or IL-2.

93. The pharmaceutical composition of claim 82, wherein the RNA comprises 1-methyl-pseudouridine substitutions.

94. A kit comprising a pharmaceutical composition of claim 79 and instructions for administering said composition.

Description

SHORT DESCRIPTION OF THE FIGURES

[0402] 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: Woodchuck Hepatitis virus core antigen; L: linker; TM: transmembrane domain

[0403] FIG. 2 Humoral responses upon vaccination with the preferred constructs encoding the P2 VP8* protein. [0404] 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. [0405] 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. [0406] 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

[0407] 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

[0408] 1. Preparation of DNA and mRNA Constructs

[0409] 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.

[0410] 2. In Vitro Transcription

[0411] 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, Tübingen, Germany; WO2008/077592A1).

[0412] The following mRNA sequences were prepared:

TABLE-US-00021 TABLE 21 R SEQ ID AA in Helper Signal N Transmembrane VLP number NO. Serotype VP4 peptide peptide glycosylation 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

[0413] The mRNA sequences comprise in 5′- to 3′-direction: [0414] a) a 5′-CAP structure, consisting of m7GpppN; [0415] b) a 5′-UTR element comprising the corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NO: 3189; [0416] c) at least one sequence encoding a signal peptide [0417] d) optionally at least one sequence encoding at least one helper peptide (e.g. P2) [0418] e) optionally at least one sequence encoding at least one protein enabling VLP formation (e.g. WHcAg) [0419] f) at least one G/C optimized coding region encoding the protein of interest, preferably as shown in Table 2 Column B, [0420] g) a 3′-UTR element comprising the corresponding RNA sequence of a nucleic acid sequence according to SEQ ID NO: 3205; [0421] h) a poly(A) sequence, comprising 64 adenosines; [0422] i) a poly(C) sequence, comprising 30 cytosines; and [0423] j) a histone-stem-loop structure, comprising the RNA sequence according to SEQ ID NO: 3207.

[0424] 3. Preparation of the mRNA Vaccine

[0425] 3.1. Protamine Complexation:

[0426] The mRNA vaccine consisted of a mixture of 50% free m RNA and 50% m RNA 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.

[0427] 3.2. LNP Formulation

[0428] 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.

[0429] 3.3. CVCM Formulation:

[0430] The polyethylene glycol/peptide polymers (HO-PEG 5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)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).

[0431] 4. Analysis of VP8* Specific Antibodies by ELISA

[0432] ELISA plates were coated with 1 μg/ml (for P[4] P2-VP8* and P[6] P2-VP8*) or 10 μg/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.

[0433] 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

[0434] 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. 2A), P2 VP8* P[4] protein (FIG. 2B), or P2 VP8* P[8] protein (FIG. 2C) as a coating reagent (Wen et al., 2014. Vaccine 32, 4420-4427).

TABLE-US-00022 TABLE 22 Animal groups and treatment Strain/ Treatment/ Route/ Vaccin. gender Nr. Vaccine dose Construct Serotype Volume schedule 1 BALB/c 8 80 μg R3718 Secreted P[6] i.d. d0/21/42 Female P2-VP8* 2 × 50 μl 2 BALB/c 8 80 μg R3720 Secreted repeat P[6] i.d. d0/21/42 Female P2-VP8* 2 × 50 μl 3 BALB/c 8 80 μg R3722 Transmembrane P[6] i.d. d0/21/42 Female P2-VP8* 2 × 50 μl 4 BALB/c 8 80 μg R3724 VLP P2-VP8* P[6] i.d. d0/21/42 Female 2 × 50 μl 5 BALB/c 8 100% RiLa — — i.d. d0/21/42 Female 2 × 50 μl

[0435] Results

[0436] As shown in FIG. 2A, 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.

[0437] FIG. 2B 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.

[0438] FIG. 2C 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. 2A and FIG. 2B, 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:

[0439] 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 μl 2 BALB/c 6 Positive control i.m. d0/21/42 female 4 × 25 μl 3 BALB/c 12 80 μg R3718 i.d. d0/21/42 female 2 × 50 μl 4 BALB/c 12 80 μg R5594 i.d. d0/21/42 female 2 × 50 μl 5 BALB/c 12 80 μg R5595 i.d. d0/21/42 female 2 × 50 μl 6 BALB/c 12 80 μg R5596 i.d. d0/21/42 female 2 × 50 μl 7 BALB/c 12 80 μg R5597 i.d. d0/21/42 female 2 × 50 μl 8 BALB/c 12 80 μg R5471 i.d. d0/21/42 female 2 × 50 μl 9 BALB/c 12 80 μg R5473 i.d. d0/21/42 female 2 × 50 μl 10 BALB/c 12 80 μg R5475 i.d. d0/21/42 female 2 × 50 μl 11 BALB/c 12 80 μg R5479 i.d. d0/21/42 female 2 × 50 μl

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 μl 2 BALB/c 6 Positive control i.m. d0/21/42 Female 4 × 25 μl 3 BALB/c 12 80 μg R3718 i.d. d0/21/42 Female 2 × 50 μl 4 BALB/c 12 80 μg R5481 i.d. d0/21/42 Female 2 × 50 μl 5 BALB/c 12 80 μg R5483 i.d. d0/21/42 Female 2 × 50 μl 6 BALB/c 12 80 μg R5485 i.d. d0/21/42 Female 2 × 50 μl 7 BALB/c 12 80 μg R5487 i.d. d0/21/42 Female 2 × 50 μl 8 BALB/c 12 80 μg R5489 i.d. d0/21/42 Female 2 × 50 μl 9 BALB/c 12 80 μg R5490 i.d. d0/21/42 Female 2 × 50 μl 10 BALB/c 12 80 μg R5491 i.d. d0/21/42 Female 2 × 50 μl

Example 4

Immunogenicity of New VP8* Formulations:

[0440] 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 — i.d. d0/21/42 female control 2 × 50 μl 2 BALB/c 6 Positive — i.m. d0/21/42 female control 4 × 25 μl 3 BALB/c 12 Design 1 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 4 BALB/c 12 Design 2 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 5 BALB/c 12 Design 3 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 6 BALB/c 12 Design 4 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 7 BALB/c 12 Design 5 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 8 BALB/c 12 Design 1 LNP i.m. d0/21/42 female 5 μg formulation 1 × 25 μl 9 BALB/c 12 Design 2 LNP i.m. d0/21/42 female 5 μg formulation 1 × 25 μl 10 BALB/c 12 Design 3 LNP i.m. d0/21/42 female 5 μg formulation 1 × 25 μl 11 BALB/c 12 Design 4 LNP i.m. d0/21/42 female 5 μg formulation 1 × 25 μl 12 BALB/c 12 Design 5 LNP i.m. d0/21/42 female 5 μg formulation 1 × 25 μl

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 — i.d. d0/21/42 female control 2 × 50 μl 2 BALB/c 6 Positive — i.m. d0/21/42 female control 4 × 25 μl 3 BALB/c 12 Design 1 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 4 BALB/c 12 Design 2 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 5 BALB/c 12 Design 3 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 6 BALB/c 12 Design 4 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 7 BALB/c 12 Design 5 Protamine i.d. d0/21/42 female 80 μg formulation 2 × 50 μl 8 BALB/c 12 Design 1 CVCM i.m. d0/21/42 female 10 μg formulation 1 × 25 μl 9 BALB/c 12 Design 2 CVCM i.m. d0/21/42 female 10 μg formulation 1 × 25 μl 10 BALB/c 12 Design 3 CVCM i.m. d0/21/42 female 10 μg formulation 1 × 25 μl 11 BALB/c 12 Design 4 CVCM i.m. d0/21/42 female 10 μg formulation 1 × 25 μl 12 BALB/c 12 Design 5 CVCM i.m. d0/21/42 female 10 μg formulation 1 × 25 μl