WO2024103728A1 - Messenger ribonucleic acid vaccine against poxvirus - Google Patents

Abstract

The present invention relates to an anti-poxvirus vaccine, which comprises an mRNA molecule encoding the following proteins and/or fusion proteins: (a) more than one monkey poxvirus protein selected from the following: an A35R protein, an M1R protein, a B6R protein, and an A29L protein, and/or (b) a fusion protein formed by fusing two or more monkey poxvirus proteins or parts of proteins selected from the following: an A35R protein, an M1R protein, a B6R protein, and an A29L protein. The present invention also relates to a kit for preparing the anti-poxvirus vaccine, which comprises: (i) a DNA molecule encoding the described protein and/or fusion protein, and optionally (ii) a reagent for transcribing the DNA molecule of (i) into an mRNA molecule. The present invention also relates to a DNA molecule or mRNA molecule encoding the described protein and fusion protein.

Description

Messenger RNA vaccines against poxviruses [Technical field]

The present invention relates to an anti-pox virus vaccine and a kit for preparing the anti-pox virus vaccine. The present invention also relates to the use of the vaccine and the kit for preparing the vaccine.

【Background technique】

Poxviruses (i.e., Poxviridae viruses) are a class of large, pathogenic double-stranded DNA viruses. Among them, smallpox virus has caused great disasters to human society. Since 2022, the number of cases of monkeypox virus infection in humans has increased rapidly, with a cumulative total of more than 50,000 people. In order to curb the large-scale outbreak of the epidemic, corresponding vaccines are urgently needed.

An infectious disease vaccine is a substance that can induce an immune response in the body and protect the body from infection or serious infection. It can be the pathogen itself, such as a virus or bacteria, or a part of the pathogen, such as a protein, or it can be genetic information encoding the pathogen protein, such as its ribonucleic acid sequence (RNA) or deoxyribonucleic acid sequence (DNA).

Since the genes and protein sequences of various viruses in the Poxviridae family are highly similar, a vaccine for one poxvirus can also prevent another poxvirus. There are currently two poxvirus vaccines on the market, both of which are whole virus vaccines. They are ACAM2000 and JYNNEOS. ACAM2000 is a replication-competent vaccinia virus, which may cause significant side effects after vaccination, such as systemic infection, eczema, myocarditis and even death. Therefore, people are more concerned about the safety of this vaccine and are not very willing to get vaccinated. JYNNEOS is a replication-deficient vaccinia virus, which cannot replicate in the human body. However, since it is still a whole virus, it contains hundreds of proteins, the specific antigen information is unclear, and some proteins play an immunomodulatory function, and the specific role is still unclear. Therefore, people are also concerned about this vaccine. At present, there are reports on animal experiments on subunit vaccines of poxviruses, such as protein and DNA vaccines. Proteins usually need to be expressed in vitro cells, and the purification steps are relatively complicated. Moreover, since they are products of in vitro expression, their three-dimensional structure may be different from the protein structure during viral infection, so there is uncertainty about the immunogenicity. DNA vaccines are relatively simple to prepare, but their delivery requires the use of a gene gun, which is complex to operate and has the risk of integrating into the human genome. Therefore, it is not the best vaccine choice.

[Summary of the invention]

The present invention relates to the following embodiments:

1. An anti-pox virus vaccine comprising mRNA molecules encoding the following proteins and/or fusion proteins:

(a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

(b) A fusion protein formed by fusing two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein.

2. The vaccine according to the aforementioned embodiment, wherein the fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.

3. The vaccine according to the preceding embodiment, wherein a signal peptide is attached to the 5' end of the fusion protein.

4. A vaccine according to any preceding embodiment, wherein

The A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1,

The M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5,

The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14,

The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18,

The B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and

The A29L protein comprises the amino acid sequence shown in SEQ ID NO:25.

5. A vaccine according to any preceding embodiment, wherein

The A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.

The M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.

The fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15.

The fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19,

The B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and

The A29L protein is encoded by a DNA sequence shown in SEQ ID NO: 27.

6. The vaccine according to the preceding embodiment, wherein the mRNA molecule further comprises: a 5' cap, a 5' UTR, a 3' UTR and a poly A tail.

7. The vaccine according to the preceding embodiment, wherein the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

8. A vaccine according to any preceding embodiment, wherein

The 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29, and/or

The 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30.

9. The vaccine according to the preceding embodiment, wherein the uridine triphosphate in the mRNA molecule is N1-methylpseudouridine triphosphate.

10. The vaccine according to the preceding embodiment, wherein the mRNA molecule is selected from:

The mRNA molecule encoding the A35R protein comprises the RNA sequence shown in SEQ ID NO: 4,

The mRNA molecule encoding the M1R protein comprises the RNA sequence shown in SEQ ID NO: 8,

An mRNA molecule encoding a fusion protein of A35R and M1R, comprising the RNA sequence shown in SEQ ID NO: 16,

An mRNA molecule encoding a fusion protein of A35R and M1R, comprising the RNA sequence shown in SEQ ID NO: 20,

The mRNA molecule encoding the B6R protein comprises the RNA sequence shown in SEQ ID NO: 24, and

The mRNA molecule encoding A29L protein contains the RNA sequence shown in SEQ ID NO: 28.

11. The vaccine according to the preceding embodiment, wherein when the vaccine contains a mixture of two or more mRNA molecules,

First, the two or more mRNA molecules are encapsulated separately and then mixed with each other, or

The two or more mRNA molecules are first mixed with each other and then encapsulated as a whole.

12. The vaccine according to the preceding embodiment, wherein the mRNA molecules are encapsulated as lipid nanoparticles (LNPs).

13. Use of mRNA molecules encoding the following proteins and/or fusion proteins in the manufacture of anti-pox virus vaccines:

(a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

(b) A fusion protein formed by fusing two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein.

14. The use according to the above embodiment, wherein the fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.

15. The use according to the preceding embodiment, wherein a signal peptide is attached to the 5' end of the fusion protein.

16. The use according to the preceding embodiment, wherein

The A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1,

The M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5,

The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14,

The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18,

The B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and

The A29L protein comprises the amino acid sequence shown in SEQ ID NO:25.

17. The use according to the preceding embodiment, wherein

The A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.

The M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.

The fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15.

The fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19,

The B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and

The A29L protein is encoded by a DNA sequence shown in SEQ ID NO: 27.

18. The use according to the preceding embodiment, wherein the mRNA molecule further comprises: a 5' cap, a 5' UTR, a 3' UTR and a poly A tail.

19. The use according to the preceding embodiment, wherein the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

20. The use according to the preceding embodiment, wherein

The 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29, and/or

The 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30.

21. The use according to the preceding embodiment, wherein the uridine triphosphate in the mRNA molecule is N1-methylpseudouridine triphosphate.

22. The use according to the preceding embodiment, wherein the mRNA molecule is selected from:

The mRNA molecule encoding the A35R protein comprises the RNA sequence shown in SEQ ID NO: 4,

The mRNA molecule encoding the M1R protein comprises the RNA sequence shown in SEQ ID NO: 8,

An mRNA molecule encoding a fusion protein of A35R and M1R, comprising the RNA sequence shown in SEQ ID NO: 16,

An mRNA molecule encoding a fusion protein of A35R and M1R, comprising the RNA sequence shown in SEQ ID NO: 20,

The mRNA molecule encoding the B6R protein comprises the RNA sequence shown in SEQ ID NO: 24, and

The mRNA molecule encoding A29L protein contains the RNA sequence shown in SEQ ID NO: 28.

23. The use according to the preceding embodiment, wherein when a vaccine is manufactured from a mixture of two or more mRNA molecules,

First, the two or more mRNA molecules are encapsulated separately and then mixed with each other, or

The two or more mRNA molecules are first mixed with each other and then encapsulated as a whole.

24. The use according to the preceding embodiment, wherein the mRNA molecule is encapsulated as lipid nanoparticles (LNP).

25. The use according to the preceding embodiment, wherein the vaccine is used against poxviruses of one or more genera selected from the group consisting of Orthopoxvirus, Capripoxvirus, Cervidpoxvirus, Suipoxvirus, Leporipoxvirus, Molluscipoxvirus, Yatapoxvirus, Avipoxvirus, Crocodylidpoxvirus and Parapoxvirus.

26. The use according to the preceding embodiment, wherein the vaccine is used against one or more poxviruses selected from the group consisting of: Variola virus/Smallpox virus, Vaccinia virus, Cowpox virus, Camelpox virus, Ectromelia virus, Monkeypox virus, Uasin Gishu disease virus, Tatera poxvirus, Raccoonpox virus, Volepox virus, Skunkpox virus, Sheeppox virus, Goatpox virus, Lumpy skin disease virus, Deerpox virus, Swinepox virus, Myxoma virus, Rabbit fibroma virus fibroma virus), Hare fibroma virus, Squirrel fibroma virus, Molluscum contagiosum virus, Yabapox virus, Tanapox virus, Fowlpox virus, Canarypox virus, Crowpox virus, Juncopox virus, Mynahpox virus, Pigeonpox virus, Psittacinepox virus, Quailpox virus, Sparrowpox virus, Starlingpox virus, Turkeypox virus, Crocodilepox virus, Orf virus, Pseudocowpox virus, Bovine papular stomatitis virus virus, Sealpox virus, Parapoxvirus of red deer in New Zealand, Carp edema virus, Salmonid gill poxvirus and Squirrelpox virus.

27. A kit for preparing an anti-pox virus vaccine, comprising:

(i) a DNA molecule encoding:

(a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

(b) a fusion protein formed by fusing two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and

Optionally (ii) reagents for transcribing the DNA molecule of (i) into an mRNA molecule.

28. The kit according to the preceding embodiment, wherein the fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.

29. The kit according to the preceding embodiment, wherein a signal peptide is attached to the 5' end of the fusion protein.

30. A kit according to the preceding embodiment, wherein

The A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1,

The M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5,

The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14,

The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18,

The B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and

The A29L protein contains the amino acid sequence shown in SEQ ID NO: 25.

31. A kit according to the preceding embodiment, wherein

The A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.

The M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.

The fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15.

The fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19,

The B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and

The A29L protein is encoded by a DNA sequence shown in SEQ ID NO: 27.

32. The kit according to the preceding embodiment, wherein the reagent for transcribing the DNA molecule of (i) into an mRNA molecule is a reagent for transcribing the DNA molecule of (i) into an mRNA molecule in vitro.

33. A kit according to the preceding embodiment, wherein the reagent for in vitro transcription of the DNA molecule of (i) into an mRNA molecule comprises:

A nucleic acid vector for in vitro transcription, comprising a promoter linked from 5' to 3', a coding DNA of a 5'UTR and a coding DNA of a 3'UTR,

Adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate,

5' cap, and

RNA polymerase.

34. A kit according to the preceding embodiment, wherein

The 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29, and/or

The 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30.

35. The kit according to the preceding embodiment, wherein the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

36. A kit according to the preceding embodiment, wherein the uridine triphosphate is N1-methylpseudouridine triphosphate.

37. A kit according to the preceding embodiment, wherein the RNA polymerase is T7 RNA polymerase.

38. Use of the following substances in the manufacture of a kit for preparing an anti-pox virus vaccine:

(i) a DNA molecule encoding:

(a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

(b) a fusion protein formed by fusing two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and

Optionally (ii) reagents for transcribing the DNA molecule of (i) into an mRNA molecule.

39. The use according to the preceding embodiment, wherein the fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.

40. The use according to the preceding embodiment, wherein a signal peptide is attached to the 5' end of the fusion protein.

41. The use according to the preceding embodiment, wherein

The A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1,

The M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5,

The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14,

The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18,

The B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and

The A29L protein contains the amino acid sequence shown in SEQ ID NO: 25.

42. The use according to the preceding embodiment, wherein

The A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.

The M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.

The fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15,

The fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19,

The B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and

The A29L protein is encoded by a DNA sequence shown in SEQ ID NO: 27.

43. The use according to the preceding embodiment, wherein the reagent for transcribing the DNA molecule of (i) into an mRNA molecule is a reagent for transcribing the DNA molecule of (i) into an mRNA molecule in vitro.

44. The use according to the preceding embodiment, wherein the reagent for in vitro transcription of the DNA molecule of (i) into an mRNA molecule comprises:

A nucleic acid vector for in vitro transcription, comprising a promoter linked from 5' to 3', a coding DNA of a 5'UTR and a coding DNA of a 3'UTR,

Adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate,

5' cap, and

RNA polymerase.

45. The use according to the preceding embodiment, wherein

The 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29, and/or

The 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30.

46. The use according to the preceding embodiment, wherein the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

47. The use according to the preceding embodiment, wherein the uridine triphosphate is N1-methylpseudouridine triphosphate.

48. The use according to the preceding embodiment, wherein the RNA polymerase is T7 RNA polymerase.

49. The use according to the preceding embodiment, wherein the vaccine is used against poxviruses of one or more genera selected from the group consisting of Orthopoxvirus, Capripoxvirus, Cervidpoxvirus, Suipoxvirus, Leporipoxvirus, Molluscipoxvirus, Yatapoxvirus, Avipoxvirus, Crocodylidpoxvirus and Parapoxvirus.

50. The use according to the preceding embodiment, wherein the vaccine is used against one or more poxviruses selected from the group consisting of smallpox virus, vaccinia virus, cowpox virus, camelpox virus, ectromelia virus, monkeypox virus, Uasin Gishu disease virus, Tatera poxvirus, Raccoonpox virus, Volepox virus, Skunkpox virus virus), Sheeppox virus, Goatpox virus, Lumpy skin disease virus, Deerpox virus, Swinepox virus, Myxoma virus, Rabbit fibroma virus, Hare fibroma virus, Squirrel fibroma virus, Molluscum contagiosum virus, Yabapox virus Yabapox virus, Tanapox virus, Fowlpox virus, Canarypox virus, Crowpox virus, Juncopox virus, Mynahpox virus, Pigeonpox virus, Psittacinepox virus, Quailpox virus, Sparrowpox virus, Starlingpox virus, Turkeypox virus poxvirus, Crocodilepox virus, Orf virus, Pseudocowpox virus, Bovine papular stomatitis virus, Sealpox virus, Parapoxvirus of red deer in New Zealand, Carp edema virus, Salmonid gill poxvirus and Squirrelpox virus.

51. A fusion protein formed by the fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein, M1R protein, B6R protein and A29L protein.

52. A fusion protein formed by the fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.

53. The fusion protein described in the above embodiment, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 14 or 18.

54. The fusion protein described in the aforementioned embodiment, wherein the fusion protein is encoded by a DNA sequence shown in SEQ ID NO: 15 or 19.

55. A DNA molecule encoding A35R protein, which comprises the DNA sequence shown in SEQ ID NO: 3.

56. A DNA molecule encoding M1R protein, which comprises the DNA sequence shown in SEQ ID NO: 7.

57. A DNA molecule encoding a fusion protein of A35R and M1R, which comprises the DNA sequence shown in SEQ ID NO: 15.

58. A DNA molecule encoding a fusion protein of A35R and M1R, which comprises the DNA sequence shown in SEQ ID NO: 19.

59. A DNA molecule encoding B6R protein, which comprises the DNA sequence shown in SEQ ID NO: 23.

60. A DNA molecule encoding A29L protein, comprising the DNA sequence shown in SEQ ID NO: 27.

61. The mRNA molecule encoding A35R protein contains the RNA sequence shown in SEQ ID NO: 4.

62. The mRNA molecule encoding M1R protein contains the RNA sequence shown in SEQ ID NO: 8.

63. The mRNA molecule encoding the fusion protein of A35R and M1R contains the RNA sequence shown in SEQ ID NO: 16.

64. The mRNA molecule encoding the fusion protein of A35R and M1R comprises the RNA sequence shown in SEQ ID NO: 20.

65. The mRNA molecule encoding B6R protein contains the RNA sequence shown in SEQ ID NO: 24.

66. The mRNA molecule encoding A29L protein contains the RNA sequence shown in SEQ ID NO: 28.

【Technical Effect】

Through the above implementation, the present invention achieves at least the following technical effects:

(1) The mRNA vaccine of the present invention does not require the aid of cell expression and has no risk of integration into the human genome. The preparation of mRNA is also simple and easy. Vaccines can be produced without contacting poxviruses that are capable of infection and reproduction, thus avoiding biosafety risks.

(2) The mRNA vaccine of the present invention has no obvious side effects and is highly safe.

(3) Thanks to the optimized sequence and combination with translation elements, proteins can be expressed efficiently and effective immune responses can be generated.

【Brief Description of the Figures】

[Figure 1] Schematic diagram of the mRNA molecular structure, from 5' to 3': 5' cap-5' UTR-coding sequence-3' UTR-poly A tail. The coding sequence is the RNA sequence of the following protein (or fusion protein) antigens: A35R, M1R, SP-A35R_IECD-M1R, SP-A35R_sECD-M1R, B6R and A29L, where "SP" represents signal peptide.

[Figure 2] Figures 2A to 2D show the levels of total anti-A35R antibodies, total anti-M1R antibodies, total anti-B6R antibodies, and total anti-A29L antibodies in the blood collected on the 29th day after each group of mRNA vaccines were administered to mice. Figure 2E shows the level of neutralizing antibodies to vaccinia virus in the serum collected on the 29th day after each group of mRNA vaccines were administered to mice (PFU/well). Figure 2F shows the continuous weight changes of mice administered with each group of mRNA vaccines after intranasal challenge with vaccinia virus. Figure 2G shows the viral load in the lungs of mice administered with each group of mRNA vaccines after intranasal challenge with vaccinia virus.

[Figure 3] Figures 3A and 3B show the levels of total anti-A35R antibodies and total anti-M1R antibodies in the blood collected at 1 month, 2 months, 3 months, 4 months and 5 months after each group of mRNA vaccines were administered to mice. Figure 3C shows the level (%) of neutralizing antibodies to vaccinia virus in the serum collected at 1 month, 2 months, 3 months, 4 months and 5 months after each group of mRNA vaccines were administered to mice. Figure 3D shows the continuous weight changes of mice administered with each group of mRNA vaccines after intranasal challenge with vaccinia virus at 5.5 months after vaccination.

[Figure 4] Figures 4A to 4E show the weight change (%) relative to the initial body weight of mice intravenously injected with mixed sera obtained from mice administered with DPBS, A-B Group 1, A-B Group 2, and A+B Group 1 vaccines and VACV-WR when challenged with vaccinia virus. Figure 4F compares the weight change (%) relative to the initial body weight of mice intravenously injected with mixed sera obtained from mice administered with different vaccines when challenged with vaccinia virus.

[Figure 5] Figures 5A and 5B show the levels of total anti-A35R antibodies and total anti-M1R antibodies in the blood collected on the 7th day after each group of mRNA vaccines were administered to mice. Figure 5C shows the level (%) of neutralizing antibodies to vaccinia virus in the serum collected on the 7th day after each group of mRNA vaccines were administered to mice. Figure 5D shows the continuous weight changes of mice administered with each group of mRNA vaccines after intranasal challenge with vaccinia virus on the 8th day after vaccination.

【Detailed ways】

Poxvirus

The gene and protein sequences of each virus in the Poxviridae family are highly similar, and a vaccine for one poxvirus can often prevent another poxvirus. Therefore, the mRNA vaccine of the present invention can be used for each of the following listed Immunity against Poxviridae viruses.

【5' cap】

The 5' end of eukaryotic mRNA usually has a bridged 7-methylguanosine (m7G) cap structure (Cap0). The 2' hydroxyl group of the first nucleoside after m7G in the Cap0 structure is methylated to form a Cap1 structure (m7GpppmN). Existing studies have found that the 5' cap structure can regulate the splicing and maturation of mRNA and help RNA transcription products pass through the selective pores of the nuclear membrane and enter the cytoplasm. In addition, the 5' cap structure can also protect mRNA from degradation by nuclease exonucleases, work with translation initiation factor proteins, recruit ribosomes, and assist ribosomes in binding to mRNA, so that translation starts from AUG. Normally, the Cap structure can recognize each other with the eukaryotic initiation factor 4E (eIF4E) at the initiation stage of translation and start the subsequent translation process. At the same time, the Cap1 structure can greatly reduce the immunogenicity of mRNA in vivo.

As long as it does not hinder the realization of the technical effects of the present invention, the 5' cap that can be used in the present invention is not particularly limited. In a preferred embodiment, the 5' cap is Cap1-GAG (3'OMe), that is, m7 (3'OMeG) (5') ppp (5') (2'OMeA) pG, whose molecular formula is C ₃₃ H ₄₅ N ₁₅ O ₂₄ P ₄ and whose structural formula is as follows;

There are different "capping" methods for preparing mRNA by in vitro transcription, including enzymatic capping, co-transcriptional capping, etc.

Enzymatic capping is a more traditional capping method. After the IVT reaction involving T7 polymerase is completed, the uncapped mRNA is purified first, and then Cap0 is produced by vaccinia virus capping enzyme (which has RNA triphosphatase activity, guanylyltransferase activity and guanine methyltransferase activity), which is then converted into Cap1 by 2'-O-methyltransferase and S-adenosylmethionine, and purified again to obtain the final mRNA.

One-step co-transcriptional capping is to directly add a cap analog to the IVT reaction system involving T7 polymerase, so as to obtain mRNA containing Cap1 structure in one step, and only one purification is required in the whole process. This reaction method reduces the preparation steps, thereby effectively shortening the overall processing time, simplifying the purification steps, and reducing the number of enzymes required. Therefore, the chemical co-transcriptional capping is relatively simple in process, introduces few impurities, and can quickly increase the production capacity of mRNA vaccines and drugs. At present, one-step co-transcriptional capping is gradually becoming the mainstream technical route for mRNA preparation technology.

【Uridine triphosphate (UTP)】

As long as it does not hinder the realization of the technical effects of the present invention, the uridine triphosphate that can be used in the present invention is not particularly limited, and can be natural uridine triphosphate or any modified uridine triphosphate commonly used in the art. In _a preferred embodiment, UTP is _{N1- methylpseudouridine} triphosphate (N1-Me-pUTP, usually represented as "Ψ"), whose molecular formula is _{C10H14N2Na3O15P3} , _and whose structural formula is as _follows :

Incorporation of N1-methylpseudouridine triphosphate during the production of mRNA vaccines and drugs can increase the translation efficiency of mRNA and reduce the immunogenicity of mRNA in vivo.

【5'UTR and 3'UTR】

As long as it does not hinder the realization of the technical effects of the present invention, the 5'UTR and 3'UTR that can be used in the present invention are not particularly limited. In a preferred embodiment,

The coding DNA sequence of 5'UTR is as follows (SEQ ID NO: 29):

The coding DNA sequence of 3'UTR is as follows (SEQ ID NO: 30):

The present invention also relates to 5'UTR and 3'UTR having 80% or more identity, 85% or more identity, 90% or more identity, 91% or more identity, 92% or more identity, 93% or more identity, 94% or more identity, 95% or more identity, 96% or more identity, 97% or more identity, 98% or more identity, or 99% or more identity with the above-mentioned 5'UTR and 3'UTR, respectively.

【Antigen and fusion antigen】

The poxvirus immunogen/antigen used in the present invention is preferably:

(a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

(b) A fusion protein formed by fusing two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein.

In a preferred embodiment, the fusion protein is a fusion protein formed by the fusion of the following monkeypox virus proteins or parts of the proteins: A35R and M1R. In a further preferred embodiment, the integral extracellular domain (IECD) of A35R is fused with M1R to form a fusion protein (A35R_IECD-M1R). In another further preferred embodiment, the small extracellular domain (sECD) of A35R is fused with M1R to form a fusion protein (A35R_sECD-M1R). In a preferred embodiment, a signal peptide (SP) may be attached to the 5' end of the fusion protein. In a further preferred embodiment, SP has the amino acid sequence shown in SEQ ID NO: 9 or 10.

In one embodiment, the fusion protein is connected via a peptide linker. In a preferred embodiment, the peptide linker has a structure represented by (G ₄ S) _n , wherein G represents glycine (Gly), S represents serine (Ser), and n is an integer from 1 to 7. In a further preferred embodiment, the peptide linker has an amino acid sequence represented by SEQ ID NO: 11 or 12.

As long as the technical effects of the present invention are not hindered, the DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins that can be used in the present invention are not particularly limited, and can be DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins of any virus from the Poxviridae family. In a preferred embodiment, the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the genus Orthopoxvirus. In a preferred embodiment, the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from Monkeypox virus. In a preferred embodiment, the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the Zaire79 strain of Monkeypox virus. In a preferred embodiment, the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the Zaire79 strain of Monkeypox virus. In a preferred embodiment, the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the Zaire79 strain of Monkeypox virus that are codon-optimized. In a preferred embodiment, the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the Zaire79 strain of Monkeypox virus that are codon-optimized for expression in humans. In a further preferred embodiment, the amino acid sequence, DNA sequence and mRNA sequence of the above-mentioned protein and fusion protein are as follows:

【A35R】

The amino acid sequence of the protein of Zaire79 strain (GenBank: AAN78222.1, https://www.ncbi.nlm.nih.gov/protein/AAN78222.1);

The wild-type nucleotide sequence of the gene of Zaire79 strain (GenBank: AY160188.1, https://www.ncbi.nlm.nih.gov/nuccore/AY160188.1);

Codon-optimized coding DNA for expression in humans (SEQ ID NO: 3)

mRNA codon-optimized for expression in humans (SEQ ID NO: 4)

【M1R】

The amino acid sequence of the protein of Zaire79 strain (GenBank: AAN78221.1, https://www.ncbi.nlm.nih.gov/protein/AAN78221.1);

The wild-type nucleotide sequence of the gene of Zaire79 strain (GenBank: AY160187.1, https://www.ncbi.nlm.nih.gov/nuccore/AY160187.1);

Codon-optimized coding DNA for expression in humans (SEQ ID NO: 7)

mRNA codon-optimized for expression in humans (SEQ ID NO: 8)

【SP-A35R_IECD-M1R】

The amino acid sequence of A35R_IECD (SEQ ID NO: 13):

Amino acid sequence of the fusion protein (SEQ ID NO: 14):

Codon-optimized coding DNA for expression in humans (SEQ ID NO: 15)

mRNA codon-optimized for expression in humans (SEQ ID NO: 16)

【SP-A35R_sECD-M1R】

Amino acid sequence of A35R_sECD (SEQ ID NO: 17):

Amino acid sequence of the fusion protein (SEQ ID NO: 18):

Codon-optimized coding DNA for expression in humans (SEQ ID NO: 19)

mRNA codon-optimized for expression in humans (SEQ ID NO: 20)

【B6R】

The amino acid sequence of the protein of Zaire79 strain (GenBank: AAN78223.1, https://www.ncbi.nlm.nih.gov/protein/AAN78223.1);

The wild-type nucleotide sequence of the gene of Zaire79 strain (GenBank: AY160189.1, https://www.ncbi.nlm.nih.gov/nuccore/AY160189.1);

Codon-optimized coding DNA for expression in humans (SEQ ID NO: 23)

mRNA codon-optimized for expression in humans (SEQ ID NO: 24)

【A29L】

The amino acid sequence of the protein of Zaire79 strain (GenBank: AAN78220.1, https://www.ncbi.nlm.nih.gov/protein/AAN78220.1);

The wild-type nucleotide sequence of the gene of Zaire79 strain (GenBank: AY160186.1, https://www.ncbi.nlm.nih.gov/nuccore/AY160186.1);

Codon-optimized coding DNA for expression in humans (SEQ ID NO: 27)

mRNA codon-optimized for expression in humans (SEQ ID NO: 28)

The present invention also relates to amino acid sequences, DNA sequences and mRNA sequences that are more than 80% identical, more than 85% identical, more than 90% identical, more than 91% identical, more than 92% identical, more than 93% identical, more than 94% identical, more than 95% identical, more than 96% identical, more than 97% identical, more than 98% identical, or more than 99% identical to the above-mentioned amino acid sequences, DNA sequences and mRNA sequences, respectively.

[mRNA encapsulation vector and delivery method using the vector]

Since naked mRNA cannot effectively enter the cells of the body for protein expression, and the stability of mRNA is poor and easy to degrade, the mRNA vaccine of the present invention is preferably encapsulated in a protective carrier. As long as it is sufficient to keep the mRNA vaccine of the present invention from degrading for a sufficiently long time and does not hinder the realization of the technical effects of the present invention, there is no particular limitation on the encapsulation carrier of mRNA that can be used in the present invention. In a preferred embodiment, nanoparticle-type carriers are used to encapsulate mRNA in the present invention. In a further preferred embodiment, nanoparticles containing lipids (also referred to as "lipid nanoparticles (lipid nanopartical, LNP)") are used in the present invention to encapsulate mRNA. In a further preferred embodiment, LNP may include but is not limited to liposomes and micelles. In a specific embodiment, the lipid nanoparticles may include cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphiphilic lipids, pegylated lipids and/or structural lipids.

In one embodiment, the LNP may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids. "Cationic lipid" generally refers to a lipid that carries any number of net positive charges at a certain pH (e.g., physiological pH). The cationic lipids may include, but are not limited to, SM102, 3-(didodecylamino)-N1,N1,4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(triadecylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctaporane (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, octyl-CLinDMA, octyl-CLinDMA (2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA and DMRIE.

In certain embodiments, the molar ratio of the cationic lipid in the lipid nanoparticle is about 40-70%, for example, about 40-65%, about 40-60%, about 45-55% or about 48-53%.

In a specific embodiment, the LNP may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) non-cationic lipids. The non-cationic lipids may include anionic lipids. Anionic lipids suitable for lipid nanoparticles of the present application may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylphosphatidylethanolamine, and other neutral lipids having anionic groups connected thereto.

In a more specific embodiment, the non-cationic lipid may include a neutral lipid, which may include, for example, a phospholipid, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (PO ...phosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (DPPG), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleo In some embodiments, the present invention relates to a lipid having a mixture of saturated and unsaturated fatty acid chains. The lipid having a mixture of saturated and unsaturated fatty acid chains can be used. For example, the neutral lipid described in the present application can be selected from DOPE, DSPC, DPPC, POPC or any related phosphatidylcholine.

In certain embodiments, the molar ratio of the phospholipid in the lipid nanoparticles is about 5-20%.

In some embodiments, the LNP may include lipid conjugates, for example, polyethylene glycol (PEG)-modified lipids and derived lipids. PEG-modified lipids may include, but are not limited to, polyethylene glycol chains covalently linked to lipids with alkyl chains of lengths of C6 to C20, up to a length of 5 kDa. The addition of these components can prevent lipid aggregation, increase circulation duration, facilitate lipid-nucleic acid composition delivery to target cells, or quickly release nucleic acids. For example, the polyethylene glycol (PEG)-modified lipid molecule can be a PEG-ceramide with a shorter acyl chain (e.g., C14 or C18). In some embodiments, the polyethylene glycol (PEG)-modified lipid molecule has a molar ratio of about 0.5 to 2% in lipid nanoparticles, for example, about 1 to 2%, about 1.2 to 1.8%, or about 1.4 to 1.6%. In some embodiments, the polyethylene glycol (PEG)-modified lipid molecule can be PEG2000-DMG.

In certain embodiments, the LNP may further comprise cholesterol. The molar ratio in the lipid nanoparticles is about 30-50%, for example, about 35-45%, or about 38-42%.

In some embodiments, the LNP may include cationic lipids, cholesterol, phospholipids and lipid molecules modified with polyethylene glycol. In some embodiments, the molar ratio of the cationic lipids, cholesterol, phospholipids and lipid molecules modified with polyethylene glycol may be 45-55:35-45:5-15:0.5-2.

There is no particular limitation on the delivery method using the above-mentioned encapsulation carrier, and any delivery method conventionally used in the art may be adopted, for example, the delivery method mentioned in US20160376224A1 or WO2015199952A1 may be adopted.

[Example]

[Example 1: Preparation of mRNA vaccine]

The sequences of DNA encoding several protein (or fusion protein) antigens derived from monkeypox virus listed in Table 1 below were obtained.

[Table 1: Several protein (or fusion protein) antigens from monkeypox virus and their encoding DNA sequences]

The coding DNA sequence listed in Table 1 above was inserted between the 5' untranslated region (UTR) (SEQ ID NO: 29) and 3'UTR (SEQ ID NO: 30) downstream of the T7 promoter in the T7 RNA polymerase-based in vitro transcription (IVT) vector and used as an IVT template.

Prepare 20 μL of IVT reaction system according to Table 2 below.

[Table 2: 20μL IVT reaction system]

IVT was performed, and the transcription products were purified to obtain the mRNAs shown in the following Table 3 ( FIG. 1 ).

【Table 3: mRNA】

The mRNA or its combination listed in Table 3 above is dissolved in citrate buffer, and then mixed with a lipid mixture dissolved in ethanol (ionizable lipid ALC-0315: distearoylphosphatidylcholine: cholesterol: PEG lipid ALC-0159) using a microfluidic device NanoAssembler to obtain lipid nanoparticles (lipid nanopartical, LNP) (also called "LNP-mRNA") encapsulating the mRNA or its combination as shown in Table 4 below.

【Table 4: mRNA vaccines in each group】

[Example 2: Administration and efficacy of mRNA vaccine 1]

(1) Administration of mRNA vaccines

Each group of mRNA vaccines (LNP-mRNA) prepared in Example 1 was initially administered (Prime) to 7-week-old Balb/c female mice at a dose of 10 μg/dose/mouse by intramuscular injection on day 0, and boosted (Boost) was administered to the mice by intramuscular injection on day 14. The control group was administered with Dulbecco's phosphate buffered saline (DPBS, Thermo Fisher, 14190136).

(2) Determination of total antibody concentration

The blood collected from the mice on day 29 after vaccination as described above was measured for the concentrations of total anti-A35R antibodies, total anti-M1R antibodies, total anti-B6R antibodies and total anti-A29L antibodies by measuring the absorbance at 450 nm (OD450) ( FIGS. 2A to 2D ).

As shown in Figure 2A, compared with the blood of negative control mice administered with DPBS, the total anti-A35R antibody levels in the blood of mice administered with the vaccines of each experimental group were significantly increased, and the total anti-A35R antibody levels in the blood of mice administered with the vaccines of A-B Group 2, A+B Group 1, A+B Group 2, A+B+C+D Group 1 and A+B+C+D Group 2 increased more significantly, among which the total anti-A35R antibody levels in the blood of mice administered with the vaccines of A-B Group 2, A+B Group 1 and A+B Group 2 increased most significantly.

As shown in Figure 2B, compared with the blood of negative control mice administered with DPBS, the total anti-M1R antibody levels in the blood of mice administered with the vaccines of each experimental group were increased, and the increase in the total anti-M1R antibody levels in the blood of mice administered with the vaccines of A-B Group 1, A-B Group 2, A+B Group 2 and A+B+C+D Group 2 was more significant, among which the increase in the total anti-M1R antibody levels in the blood of mice administered with the vaccines of A-B Group 1, A-B Group 2 and A+B Group 2 was the most significant.

As shown in FIG. 2C , the total anti-B6R antibody level in the blood of mice administered with the vaccines of A+B+C+D Group 1 and A+B+C+D Group 2 was significantly increased compared with the blood of negative control mice administered with DPBS.

As shown in FIG. 2D , the total anti-A29L antibody level in the blood of mice administered with the vaccines of A+B+C+D Group 1 and A+B+C+D Group 2 was significantly increased compared with the blood of negative control mice administered with DPBS.

(3) Serum neutralization test

Blood was collected from the mice on day 29 after vaccination as described above, and serum was separated therefrom and diluted at 1: ²ⁿ (n=positive integer) to prepare a dilution series.

Take the live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) stock solution, This was diluted to 400 to 500 PFU (plaque forming units)/ml.

The serum dilution series and the virus dilution were mixed in equal volumes in a 96-well plate and incubated at 37°C for 1 hour. The PFU of each mixture was then determined. The serum dilution that reduced the PFU by 50% compared to the control group without serum addition was set as the neutralizing antibody titer of the serum.

The neutralizing antibody levels (PFU/well) against vaccinia virus in the sera diluted as above are shown in FIG. 2E .

As shown in Figure 2E, after each group of mRNA vaccines were administered to mice, the sera of mice administered with vaccines from Group A-B 1, Group A-B 2, and Group A+B 2 achieved complete neutralization of vaccinia virus, indicating that the level of neutralizing antibodies against vaccinia virus was the most abundant.

(4) Weight changes after viral attack

On the 36th day after the administration of each group of mRNA vaccines to mice, the mice vaccinated with each vaccine were challenged with live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) at a dose of 1×10 ⁶ PFU by intranasal administration. The mice challenged with vaccinia virus were weighed for several consecutive days thereafter, and the weight change (%) relative to the initial weight was calculated.

As shown in Figure 2F, in sharp contrast to the negative control mice administered with DPBS, the mice administered with each group of mRNA vaccines did not experience significant changes in body weight due to challenge with vaccinia virus.

(5) Changes in viral load in the lungs after viral attack

The mice described in (4) above were killed, and lung tissues were collected to measure the viral load (PFU/g) therein.

As shown in Figure 2G, in sharp contrast to the negative control mice administered with DPBS, the viral load in the lungs of mice administered with each group of mRNA vaccines was close to 0. This indicates that each group of mRNA vaccines exhibited significant anti-vaccinia virus immune efficacy.

[Example 3: Administration and efficacy of mRNA vaccine 2]

(1) Administration of mRNA vaccines

The vaccines of AB Group 1, AB Group 2 and A+B Group 1 prepared in Example 1 (at a dose of 10 μg/dose/mouse), live vaccinia virus Western Reserve (VACV-WR, Cat. # VR-1354, ATCC) as a positive control (at a dose of 1×10 ⁴ PFU) and DPBS (Thermo Fisher, 14190136) as a negative control were initially administered (Prime) to 7-week-old Balb/c female mice by intramuscular injection on day 0, and boosted (Boost) to the mice by intramuscular injection on day 14 (0.5 month after vaccination).

(2) Determination of antibody concentration

The concentrations of anti-A35R antibody and anti-M1R antibody in the blood collected from the mice at 1, 2, 3, 4 and 5 months after vaccination were determined by measuring the absorbance at 450 nm (OD450) ( FIGS. 3A and 3B ).

As shown in Figure 3A, the concentration of total anti-A35R antibodies in the blood of mice administered with the vaccine of A-B Group 1, A-B Group 2, and A+B Group 1 was significantly higher than that in the blood of positive control mice administered with VACV-WR and the blood of negative control mice administered with DPBS. In the blood of mice administered with the vaccine of A-B Group 1, A-B Group 2, and A+B Group 1, the concentration of anti-A35R antibodies decreased over time, but was still significantly higher than that in the blood of positive control mice administered with VACV-WR and the blood of negative control mice administered with DPBS.

As shown in Figure 3B, the concentration of total anti-M1R antibodies in the blood of mice administered with the vaccines of A-B Group 1 and A-B Group 2 was significantly higher than that of the blood of positive control mice administered with VACV-WR and the blood of negative control mice administered with DPBS. The concentration of total anti-M1R antibodies in the blood of mice administered with the vaccine of A+B Group 1 was between that of the blood of positive control mice administered with VACV-WR and the blood of negative control mice administered with DPBS. There was no obvious regularity in the changes over time in the concentration of total anti-M1R antibodies in the blood of mice administered with the vaccines of A-B Group 1, A-B Group 2, and A+B Group 1.

(3) Serum neutralization test

Serum was separated from blood collected from the mice at 1 month, 2 months, 3 months, 4 months and 5 months after vaccination as described above and diluted at 1: ²ⁿ (n=positive integer) to prepare a dilution series.

Take the live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) stock solution and dilute it to 400-500 PFU (plaque forming units)/ml.

The serum dilution series and the virus dilution were mixed in equal volumes in a 96-well plate and incubated at 37°C for 1 hour. The PFU of each mixture was then determined. The serum dilution that reduced the PFU by 50% compared to the control group without serum addition was set as the neutralizing antibody titer of the serum.

The neutralizing antibody levels (%) against vaccinia virus in the sera diluted as above are shown in FIG. 3C .

As shown in Figure 3C, the neutralizing antibody levels in the sera of mice administered with the vaccines of Group A-B 1 and Group A-B 2 were significantly higher than those of the positive control mice administered with VACV-WR and the negative control mice administered with DPBS. The neutralizing antibody levels in the sera of mice administered with the vaccine of Group A+B 1 were between those of the positive control mice administered with VACV-WR and the negative control mice administered with DPBS. The neutralizing antibody levels in the sera of mice administered with the vaccines of Group A-B 1, Group A-B 2, and Group A+B 1 were relatively constant over time.

(4) Weight changes after viral attack

At 5.5 months after vaccination, the mice vaccinated with each vaccine were challenged intranasally with live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) at a dose of 1×10 ⁶ PFU. The mice challenged with vaccinia virus were weighed on consecutive days thereafter, and the weight change (%) relative to the initial weight was calculated.

As shown in Figure 3D, in sharp contrast to the negative control mice administered with DPBS, the mice administered with vaccines of A-B Group 1, A-B Group 2, and A+B Group 1 and the positive control mice administered with VACV-WR did not undergo significant weight changes due to challenge with vaccinia virus.

[Example 4: Antiviral activity of vaccine-induced antiserum]

(1) Administration of mRNA vaccines

The vaccines of AB Group 1, AB Group 2 and A+B Group 1 prepared in Example 1 (at a dose of 10 μg/dose/mouse), live vaccinia virus Western Reserve (VACV-WR, Cat. # VR-1354, ATCC) as a positive control (at a dose of 1×10 ⁴ PFU) and DPBS (Thermo Fisher, 14190136) as a negative control were initially administered (Prime) to 7-week-old Balb/c female mice by intramuscular injection on day 0, and boosted (Boost) to the mice by intramuscular injection on day 14 (0.5 month after vaccination).

(2) Preparation and administration of mixed serum

Serum was separated from the blood collected from the mice at 1 month, 2 months, 3 months and 4 months after vaccination as above, and equal volumes of the blood were mixed. 100 μl of the mixed serum was intravenously injected into a new batch of 7-8 week old Balb/c female mice.

(3) Weight changes after viral attack

The mice injected with mixed serum were challenged with live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) at a dose of 1×10 ⁵ PFU via intranasal route on the next day. The mice challenged with vaccinia virus were weighed for 17 consecutive days thereafter, and the weight change (%) relative to the initial weight was calculated.

FIG. 4A shows the weight change (%) relative to the initial weight of mice intravenously injected with pooled sera obtained from negative control mice administered DPBS when challenged with vaccinia virus.

FIG. 4B shows the weight change (%) relative to the initial weight of mice injected intravenously with the pooled sera obtained from mice administered with the vaccine of Group A-B 1 upon challenge with vaccinia virus.

FIG. 4C shows the weight change (%) relative to the initial weight of mice injected intravenously with pooled sera obtained from mice administered with vaccines of Groups A-B 2 upon vaccinia virus challenge.

FIG. 4D shows the weight change (%) relative to the initial weight of mice intravenously injected with the pooled sera obtained from mice administered with the vaccine of Group A+B 1 when challenged with vaccinia virus.

FIG. 4E shows the weight change (%) relative to the initial weight of mice injected intravenously with pooled sera obtained from positive control mice administered VACV-WR when challenged with vaccinia virus.

The mice injected intravenously with pooled sera from mice given the above different vaccines were compared. Body weight change relative to initial body weight during vaccinia virus challenge (%). As shown in FIG4F , mice intravenously injected with pooled sera obtained from mice administered with AB group 2 vaccine and mice intravenously injected with pooled sera obtained from positive control mice administered with VACV-WR had significantly less weight change due to vaccinia virus challenge than mice intravenously injected with pooled sera obtained from negative control mice administered with DPBS. Mice intravenously injected with pooled sera obtained from mice administered with AB group 1 vaccine and mice intravenously injected with pooled sera obtained from mice administered with A+B group 1 vaccine had comparable weight changes due to vaccinia virus challenge as mice intravenously injected with pooled sera obtained from negative control mice administered with DPBS.

[Example 5: Administration and efficacy of mRNA vaccine 3]

(1) Administration of mRNA vaccines

The vaccines of AB Group 1, AB Group 2 and A+B Group 1 prepared in Example 1 (at a dose of 10 μg/dose/mouse), live vaccinia virus Western Reserve (VACV-WR, Cat. # VR-1354, ATCC) as a positive control (at a dose of 2×10 ⁴ PFU (VACV-WR-low) or 2×10 ⁵ PFU (VACV-WR-high)), and DPBS (Thermo Fisher, 14190136) as a negative control were administered to 7-week-old Balb/c female mice by intramuscular injection on day 0.

(2) Determination of antibody concentration

The concentrations of anti-A35R antibody and anti-M1R antibody in the blood collected from the mice on day 7 after vaccination as described above were determined by measuring absorbance at 450 nm (OD450) ( FIGS. 5A and 5B ).

As shown in Figure 5A, the concentrations of total anti-A35R antibodies in the blood of mice administered with vaccines from Group A-B 1, Group A-B 2, and Group A+B 1 were significantly higher than those in the blood of positive control mice administered with VACV-WR-low and VACV-WR-high and the blood of negative control mice administered with DPBS.

As shown in Figure 5B, the concentration of total anti-M1R antibodies in the blood of mice administered with the vaccines of A-B Group 1 and A-B Group 2 was significantly higher than that in the blood of positive control mice administered with VACV-WR-low and VACV-WR-high and the blood of negative control mice administered with DPBS. The concentration of total anti-M1R antibodies in the blood of mice administered with the vaccine of A+B Group 1 was comparable to that in the blood of positive control mice administered with VACV-WR-low and VACV-WR-high and the blood of negative control mice administered with DPBS.

(3) Serum neutralization test

Blood was collected from the mice on day 7 after vaccination as described above, and serum was separated therefrom and diluted at 1: ²ⁿ (n=positive integer) to prepare a dilution series.

Take the live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) stock solution and dilute it to 400-500 PFU (plaque forming units)/ml.

The serum dilution series and the virus dilution were mixed in equal volumes in a 96-well plate and incubated at 37°C for 1 hour. The PFU of each mixture was then determined. The serum dilution that reduced the PFU by 50% compared to the control group without serum addition was set as the neutralizing antibody titer of the serum.

The neutralizing antibody levels (%) against vaccinia virus in the sera diluted as above are shown in FIG5C .

As shown in Figure 5C, the neutralizing antibody levels in the sera of mice administered with the vaccines of A-B Group 1 and A-B Group 2 were significantly higher than those in the sera of negative control mice administered with DPBS. The neutralizing antibody levels in the sera of mice administered with the vaccine of A+B Group 1 were between those of the sera of positive control mice administered with VACV-WR-low and VACV-WR-high and those of negative control mice administered with DPBS.

(4) Weight changes after viral attack

On day 8 after the first vaccination, the mice vaccinated with each vaccine were challenged intranasally with live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) at a dose of 1×10 ⁶ PFU. The mice challenged with vaccinia virus were weighed for 18 consecutive days thereafter, and the weight change (%) relative to the initial weight was calculated.

As shown in Figure 5D, in sharp contrast to the negative control mice administered with DPBS, the mice administered with vaccines from AB Group 1, AB Group 2, and A+B Group 1 and the positive control mice administered with VACV-WR-low and VACV-WR-high did not undergo significant weight changes in response to vaccinia virus challenge.

Claims (10)

1. An anti-pox virus vaccine comprising mRNA molecules encoding the following proteins and/or fusion proteins:

   (a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

   (b) A fusion protein formed by the fusion of two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, preferably a fusion protein formed by the fusion of the following monkeypox virus proteins or parts of proteins: A35R protein and M1R protein.
2. Use of mRNA molecules encoding the following proteins and/or fusion proteins in the manufacture of anti-poxvirus vaccines:

   (a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

   (b) A fusion protein formed by the fusion of two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, preferably a fusion protein formed by the fusion of the following monkeypox virus proteins or parts of proteins: A35R protein and M1R protein.
3. A kit for preparing an anti-pox virus vaccine, comprising:

   (i) a DNA molecule encoding:

   (a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

   (b) a fusion protein formed by fusion of two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, preferably a fusion protein formed by fusion of the following monkeypox virus proteins or parts of proteins: A35R protein and M1R protein, and

   Optionally (ii) reagents for transcribing the DNA molecule of (i) into an mRNA molecule.
4. Use of the following substances in the manufacture of a kit for preparing an anti-pox virus vaccine:

   (i) a DNA molecule encoding:

   (a) one or more monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or

   (b) a fusion protein formed by fusion of two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, preferably a fusion protein formed by fusion of the following monkeypox virus proteins or parts of proteins: A35R protein and M1R protein, and

   Optionally (ii) reagents for transcribing the DNA molecule of (i) into an mRNA molecule.
5. A vaccine, a kit or a use according to any preceding claim, wherein

   The A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1, and preferably the A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.

   The M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5, and preferably the M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.

   The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14, and preferably the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15.

   The fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18, and preferably the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19.

   The B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and preferably the B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and

   The A29L protein comprises the amino acid sequence shown in SEQ ID NO: 25. Preferably, the A29L protein Encoded by the DNA sequence shown in SEQ ID NO:27.
6. The vaccine, kit or use according to the preceding claims, wherein the mRNA molecule further comprises: a 5' cap, a 5' UTR, a 3' UTR and a poly A tail,

   Preferably, the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG;

   Preferably, the 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29;

   Preferably, the 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30; and/or

   Preferably, the uridine triphosphate in the mRNA molecule is N1-methylpseudouridine triphosphate.
7. The vaccine, kit or use according to the preceding claims, wherein the mRNA molecule is selected from:

   The mRNA molecule encoding the A35R protein comprises the RNA sequence shown in SEQ ID NO: 4,

   The mRNA molecule encoding the M1R protein comprises the RNA sequence shown in SEQ ID NO: 8,

   An mRNA molecule encoding a fusion protein of A35R and M1R, comprising the RNA sequence shown in SEQ ID NO: 16,

   An mRNA molecule encoding a fusion protein of A35R and M1R, comprising the RNA sequence shown in SEQ ID NO: 20,

   The mRNA molecule encoding the B6R protein comprises the RNA sequence shown in SEQ ID NO: 24, and

   The mRNA molecule encoding A29L protein contains the RNA sequence shown in SEQ ID NO: 28.
8. A fusion protein comprising the amino acid sequence shown in SEQ ID NO: 14 or 18, preferably encoded by a DNA sequence shown in SEQ ID NO: 15 or 19.
9. A DNA molecule comprising a DNA sequence selected from the following: SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23 and SEQ ID NO: 27.
10. An mRNA molecule comprising an RNA sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24 and SEQ ID NO:28.