TW202421644A - Messenger RNA vaccines against poxviruses - Google Patents

Abstract

The present invention relates to an anti-poxvirus vaccine, which contains mRNA molecules encoding the following proteins and/or fusion proteins: (a) one or more monkeypox virus proteins selected from the following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein. The present invention also relates to a reagent kit for preparing an anti-poxvirus vaccine, which contains: (i) a DNA molecule encoding the above-mentioned 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 above-mentioned protein and fusion protein.

Description

Messenger RNA vaccines against poxviruses

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

Poxviruses (i.e., Poxviridae viruses) are a class of large, pathogenic double-stranded DNA viruses. Among them, the 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 number 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 the genetic information that encodes the pathogen protein, such as its ribonucleic acid sequence (RNA) or deoxyribonucleic acid sequence (DNA).

Since the genes and protein sequences of the viruses in the Poxviridae family are highly similar, the vaccine used 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 replicable 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 with it. JYNNEOS is a replication-deficient vaccinia virus, which means it cannot replicate in the human body. However, since it is still a whole virus, containing hundreds of proteins, the specific antigen information is unclear, and some proteins play an immunomodulatory function, the specific role is still unclear. Therefore, people are also worried about this vaccine. At present, there are relevant reports on animal experiments of subunit vaccines of poxviruses, such as protein and DNA vaccines. Proteins usually need to be expressed in vitro, and the purification process is relatively complicated. Moreover, since it is a product expressed in vitro, its 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 gene guns, which is complicated to operate and has the risk of integration into the human genome. Therefore, it is not the best vaccine choice.

The present invention relates to the following implementation methods:

1. An anti-poxvirus vaccine comprising mRNA molecules encoding the following proteins and/or fusion proteins: (a) one or more monkeypox virus proteins selected from the following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein.

2. The vaccine 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.

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

4. The vaccine according to the aforementioned 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. The vaccine according to the aforementioned embodiment, wherein the A35R protein is encoded by a DNA sequence comprising SEQ ID NO: 3, the M1R protein is encoded by a DNA sequence comprising SEQ ID NO: 7, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 15, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 19, the B6R protein is encoded by a DNA sequence comprising SEQ ID NO: 23, and the A29L protein is encoded by a DNA sequence comprising SEQ ID NO: 27.

6. The vaccine according to the above 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. The vaccine according to the aforementioned 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 above-mentioned embodiment, wherein the mRNA molecule is selected from: mRNA molecules encoding A35R protein, which comprises the RNA sequence shown in SEQ ID NO: 4, mRNA molecules encoding M1R protein, which comprises the RNA sequence shown in SEQ ID NO: 8, mRNA molecules encoding the fusion protein of A35R and M1R, which comprises the RNA sequence shown in SEQ ID NO: 16, mRNA molecules encoding the fusion protein of A35R and M1R, which comprises the RNA sequence shown in SEQ ID NO: 20, mRNA molecules encoding B6R protein, which comprises the RNA sequence shown in SEQ ID NO: 24, and mRNA molecules encoding A29L protein, which comprises the RNA sequence shown in SEQ ID NO: 28.

11. The vaccine according to the aforementioned embodiment, wherein, when the vaccine contains a mixture of two or more mRNA molecules, the two or more mRNA molecules are first 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 above embodiment, wherein the mRNA molecule is encapsulated as lipid nanoparticles (LNP).

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 following: 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 following: 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 above embodiment, wherein a signal peptide is attached to the 5' end of the fusion protein.

16. The use according to the above-mentioned 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 above-mentioned embodiment, wherein the A35R protein is encoded by a DNA sequence comprising SEQ ID NO: 3, the M1R protein is encoded by a DNA sequence comprising SEQ ID NO: 7, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 15, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 19, the B6R protein is encoded by a DNA sequence comprising SEQ ID NO: 23, and the A29L protein is encoded by a DNA sequence comprising SEQ ID NO: 27.

18. The use according to the above 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 above embodiments, 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 above-mentioned embodiment, wherein the mRNA molecule is selected from: mRNA molecules encoding A35R protein, which comprises the RNA sequence shown in SEQ ID NO: 4, mRNA molecules encoding M1R protein, which comprises the RNA sequence shown in SEQ ID NO: 8, mRNA molecules encoding the fusion protein of A35R and M1R, which comprises the RNA sequence shown in SEQ ID NO: 16, mRNA molecules encoding the fusion protein of A35R and M1R, which comprises the RNA sequence shown in SEQ ID NO: 20, mRNA molecules encoding B6R protein, which comprises the RNA sequence shown in SEQ ID NO: 24, and mRNA molecules encoding A29L protein, which comprises the RNA sequence shown in SEQ ID NO: 28.

23. The use according to the above-mentioned embodiment, wherein when a vaccine is prepared from a mixture of two or more mRNA molecules, the two or more mRNA molecules are first 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 above 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 virus), Rabbit 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 virus, 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.

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 following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein, and optionally (ii) a reagent for transcribing the DNA molecule of (i) into an mRNA molecule.

28. The reagent kit 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.

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

30. The reagent kit according to the aforementioned 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.

31. The reagent kit according to the aforementioned embodiment, wherein the A35R protein is encoded by a DNA sequence comprising SEQ ID NO: 3, the M1R protein is encoded by a DNA sequence comprising SEQ ID NO: 7, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 15, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 19, the B6R protein is encoded by a DNA sequence comprising SEQ ID NO: 23, and the A29L protein is encoded by a DNA sequence comprising SEQ ID NO: 27.

32. The reagent 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. The reagent kit according to the aforementioned 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, which comprises a promoter linked from 5' to 3', a 5'UTR coding DNA and a 3'UTR coding DNA, adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate, a 5' cap, and an RNA polymerase.

34. A reagent kit according to the aforementioned 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. The kit according to any preceding embodiment, wherein the uridine triphosphate is N1-methylpseudouridine triphosphate.

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

38. Use of the following substances in a kit for preparing an anti-pox virus vaccine: (i) a DNA molecule encoding: (a) one or more monkeypox virus proteins selected from the following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein, and optionally (ii) a reagent for transcribing the DNA molecule of (i) into an mRNA molecule.

39. 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.

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

41. The use according to the above-mentioned 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.

42. The use according to the above-mentioned embodiment, wherein the A35R protein is encoded by a DNA sequence comprising SEQ ID NO: 3, the M1R protein is encoded by a DNA sequence comprising SEQ ID NO: 7, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 15, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 19, the B6R protein is encoded by a DNA sequence comprising SEQ ID NO: 23, and the A29L protein is encoded by a DNA sequence comprising SEQ ID NO: 27.

43. The use according to the above 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 above-mentioned 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, which comprises a promoter linked from 5' to 3', a 5'UTR coding DNA and a 3'UTR coding DNA, adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate, a 5' cap, and an RNA polymerase.

45. The use according to the above embodiments, 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 above 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, Sheeppox virus, Goatpox virus, Lumpy skin disease virus, Deerpox virus, Swinepox virus, Myxoma virus, Rabbit fibroma virus, 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 The most common viruses in the world include 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. Fusion proteins formed by fusion of the following monkeypox virus proteins or parts of their 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 according to the preceding embodiment, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 14 or 18.

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

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

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

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

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

59. A DNA molecule encoding a B6R protein, comprising 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. An mRNA molecule encoding an A35R protein, comprising the RNA sequence shown in SEQ ID NO: 4.

62. An mRNA molecule encoding an M1R protein, comprising the RNA sequence shown in SEQ ID NO: 8.

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

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

65. An mRNA molecule encoding a B6R protein, comprising the RNA sequence shown in SEQ ID NO: 24.

66. An mRNA molecule encoding an A29L protein, comprising an RNA sequence as shown in SEQ ID NO: 28. [Technical Effects] 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 help 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, it is able to efficiently express proteins and produce effective immune responses.

[Poxvirus]

The genes and protein sequences of various viruses in the Poxviridae family are highly similar, and vaccines used for one poxvirus can often prevent another poxvirus. Therefore, the mRNA vaccine of the present invention can be used for immunization against various types of Poxviridae viruses listed in the table below. Genus Kind Orthopoxvirus 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 Capripoxvirus Sheeppox virus, Goatpox virus Cervidpoxvirus Lumpy skin disease virus, Deerpox virus Suipoxvirus Swinepox virus Leporipoxvirus Myxoma virus, Rabbit fibroma virus, Hare fibroma virus, Squirrel fibroma virus Molluscipoxvirus Molluscum contagiosum virus Yatapoxvirus Yabapox virus, Tanapox virus Avipoxvirus Fowlpox virus, Canarypox virus, Crowpox virus, Juncopox virus, Mynahpox virus, Pigeonpox virus, Psittacinepox virus, Quailpox virus, Sparrowpox virus, Starlingpox virus, Turkeypox virus Crocodylidpoxvirus Crocodilepox virus Parapoxvirus Orf virus, Pseudocowpox virus, Bovine papular stomatitis virus, Sealpox virus, Parapoxvirus of red deer in New Zealand Unclassified Carp edema virus, salmon gill poxvirus, squirrelpox virus [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 the Cap1 structure (m7GpppmN). Existing studies have found that the 5' end 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 nucleases, work in conjunction 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 eukaryotic initiation factor 4E (eIF4E) at the translation initiation stage 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 the technical effects of the present invention are not hindered, 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), i.e. 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 during in vitro transcription, including enzymatic capping, co-transcriptional capping, etc.

Enzymatic capping is a more traditional capping method. This method requires that after the IVT reaction involving T7 polymerase is completed, the uncapped mRNA is first purified, and then Cap0 is produced by vaccinia virus capping enzyme (which has RNA triphosphatase activity, guanosyltransferase activity and guanine methyltransferase activity). It is then converted to 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 to obtain mRNA containing Cap1 structure in one step, and only one purification is required throughout the process. This reaction method reduces the preparation steps, thereby effectively shortening the overall processing time, simplifying the purification steps, and reducing the amount of enzymes required. Therefore, chemical co-transcriptional capping is relatively simple in process, introduces less 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 processes. [Uridine triphosphate (UTP)]

As long as the technical effects of the present invention are not hindered, 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 by "Ψ"), whose molecular formula is _{C10H14N2Na3O15P3} , _and whose structural formula is as _follows : .

Incorporation of N1-methylpseudouridine triphosphate into the production of mRNA vaccines and drugs can improve the translation efficiency of mRNA and reduce the immunogenicity of mRNA in vivo. [5'UTR and 3'UTR]

As long as the technical effects of the present invention are not hindered, 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 the 5'UTR is as follows (SEQ ID NO: 29): gcttgttctt tttgcagaag ctcagaataa acgctcaact ttggccgcca cc The coding DNA sequence of the 3'UTR is as follows (SEQ ID NO: 30): tgaaaccagc ctcaagaaca cccgaatgga gtctctaagc tacataatac caacttacac tttacaaaat gttgtccccc aaaatgtagc cattcgtatc tgctcctaat aaaaagaaag tttcttcac 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, 99% or more identity with the above 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 following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein.

In a preferred embodiment, the fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of 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 by 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 shown in 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 the 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 the 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 the DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from Monkeypox virus. In a preferred embodiment, the present invention uses the 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 a codon-optimized DNA sequence and mRNA sequence of the above-mentioned protein and fusion protein derived from the Monkeypox virus Zaire79 strain. In a preferred embodiment, the present invention uses a codon-optimized DNA sequence and mRNA sequence of the above-mentioned protein and fusion protein derived from the Monkeypox virus Zaire79 strain 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]

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

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

• Coding DNA optimized for expression codon in humans (SEQ ID NO: 3) ATGATGACCC CTGAGAACGA CGAGGAACAG ACCAGCGTGT TCAGCGCCAC CGTGTACGGC GACAAGATCC AGGGCAAGAA CAAAAGAAAG AGAGTGATTG GACTGTGCAT CAGAATCAGC ATGGTTATCT CTCTGCTGAG CATGATCACC ATGAGCGCTT TTCTGATCGT GCGGCTGAAC CAGTGTATGA GCGCCAATGA GGCCGCCATC ACAGATAGCG CCGTCGCCGT GGCCGCTGCT AGCAGCACCC ACAGAAAAGT GGCCAGCTCC ACCACACAGT ACGACCACAA GGAAAGCTGC AACGGCCTCT ACTACCAAGG CAGCTGTTAC ATCCTGCACT CTGATTATAA GTCTTTCGAG GACGCCAAGG CCAATTGCGC CGCCGAGAGC TCTACACTGC CTAACAAGTC CGATGTGCTG ACCACCTGGC TGATCGACTA CGTGGAAGAT ACCTGGGGAT CTGACGGCAA CCCCATCACA AAGACCACAA GCGACTACCA GGACAGCGAC GTGTCCCAGG AGGTGCGGAA GTACTTCTGC ACATGA

• mRNA optimized for codon expression in humans (SEQ ID NO: 4) Cap1-GAG (3'OMe) gcΨΨgΨΨcΨΨ ΨΨΨgcagaag cΨcagaaΨaa acgcΨcaacΨ ΨΨggccgcca ccAΨGAΨGAC CCCΨGAGAAC GACGAGGAAC AGACCAGCGΨ GΨΨCAGCGCC ACCGΨGΨACG GCGACAAGAΨ CCAGGGCAAG AACAAAAGAA AGAGAGΨGAΨ ΨGGACΨGΨGC AΨCAGAAΨCA GCAΨGGΨΨAΨ CΨCΨCΨGCΨG AGCAΨGAΨCA CCAΨGAGCGC ΨΨΨΨCΨGAΨC GΨGCGGCΨGA ACCAGΨGΨAΨ GAGCGCCAAΨ GAGGCCGCCA ΨCACAGAΨAG CGCCGΨCGCC GΨGGCCGCΨG CΨAGCAGCAC CCACAGAAAA GΨGGCCAGCΨ CCACCACACA GΨACGACCAC AAGGAAAGCΨ GCAACGGCCΨ CΨACΨACCAA GGCAGCΨGΨΨ ACAΨCCΨGCA CΨCΨGAΨΨAΨ AAGΨCΨΨΨCG AGGACGCCAA GGCCAAΨΨGC GCCGCCGAGA GCΨCΨACACΨ GCCΨAACAAG ΨCCGAΨGΨGC ΨGACCACCΨG GCΨGAΨCGAC ΨACGΨGGAAG AΨACCΨGGGG AΨCΨGACGGC AACCCCAΨCA CAAAGACCAC AAGCGACΨAC CAGGACAGCG ACGΨGΨCCCA GGAGGΨGCGG AAGΨACΨΨCΨ GCACAΨgaΨg aaaccagccΨ caagaacacc cgaaΨggagΨ cΨcΨaagcΨa caΨaaΨacca acΨΨacacΨΨ ΨacaaaaΨgΨ ΨgΨcccccaa aaΨgΨagcca ΨΨcgΨaaΨcΨg cΨccΨaaΨaa aaagaaagΨΨ ΨcΨΨcacAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAg caΨaΨgacΨA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A [M1R]

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

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

• Coding DNA optimized for expression in humans (SEQ ID NO: 7) ATGGGCGCTG CAGCTTCTAT CCAGACCACC GTGAACACAC TGAGTGAAAG AATCAGCTCT AAGCTGGAAC AGGAGGCCAA TGCCAGCGCC CAGACAAAGT GCGACATTGA GATCGGAAAT TTCTACATCA GACAGAACCA CGGCTGTAAC ATCACCGTGA AAAACATGTG CAGCGCCGAC GCCGATGCTC AGCTGGACGC CGTGCTGAGC GCCGCTACCG AAACCTACAG CGGCCTGACC CCTGAGCAGA AAGCCTACGT GCCCGCCATG TTCACGGCCG CTCTGAACAT CCAGACCTCT GTTAACACCG TCGTGCGGGA CTTCGAGAAC TACGTGAAAC AAACCTGTAA CAGCAGCGCT GTGGTGGACA ACAAGCTCAA GATCCAGAAC GTGATCATCG ACGAGTGCTA CGGCGCCCCT GGCAGCCCCA CAAATCTGGA GTTCATCAAC ACCGGCTCCA GCAAGGGAAA TTGCGCCATC AAGGCCCTGA TGCAGCTGAC CACAAAGGCC ACAACACAGA TCGCCCCAAG ACAAGTGGCC GGCACCGGCG TCCAGTTCTA TATGATCGTG ATCGGCGTGA TTATCCTGGC CGCCCTGTTT ATGTACTACG CCAAGCGGAT GCTGTTTACC AGCACCAACG ATAAGATCAA GCTGATCCTG GCCAACAAGG AAAACGTGCA CTGGACCACA TACATGGACA CATTCTTCAG AACCTCCCCT ATGATCATCG CCACCACTGA TATGCAGAAC TGA

• mRNA optimized for codon expression in humans (SEQ ID NO: 8) Cap1-GAG (3'OMe) gcΨΨgΨΨcΨΨ ΨΨΨgcagaag cΨcagaaΨaa acgcΨcaacΨ ΨΨggccgcca ccAΨGGGCGC ΨGCAGCΨΨCΨ AΨCCAGACCA CCGΨGAACAC ACΨGAGΨGAA AGAAΨCAGCΨ CΨAAGCΨGGA ACAGGAGGCC AAΨGCCAGCG CCCAGACAAA GΨGCGACAΨΨ GAGAΨCGGAA AΨΨΨCΨACAΨ CAGACAGAAC CACGGCΨGΨA ACAΨCACCGΨ GAAAAACAΨG ΨGCAGCGCCG ACGCCGAΨGC ΨCAGCΨGGAC GCCGΨGCΨGA GCGCCGCΨAC CGAAACCΨAC AGCGGCCΨGA CCCCΨGAGCA GAAAGCCΨAC GΨGCCCGCCA ΨGΨΨCACGGC CGCΨCΨGAAC AΨCCAGACCΨ CΨGΨΨAACAC CGΨCGΨGCGG GACΨΨCGAGA ACΨACGΨGAA ACAAACCΨGΨ AACAGCAGCG CΨGΨGGΨGGA CAACAAGCΨC AAGAΨCCAGA ACGΨGAΨCAΨ CGACGAGΨGC ΨACGGCGCCC CΨGGCAGCCC CACAAAΨCΨG GAGΨΨCAΨCA ACACCGGCΨC CAGCAAGGGA AAΨΨGCGCCA ΨCAAGGCCCΨ GAΨGCAGCΨG ACCACAAAGG CCACAACACA GAΨCGCCCCA AGACAAGΨGG CCGGCACCGG CGΨCCAGΨΨC ΨAΨAΨGAΨCG ΨGAΨCGGCGΨ GAΨΨAΨCCΨG GCCGCCCΨGΨ ΨΨAΨGΨACΨA CGCCAAGCGG AΨGCΨGΨΨA CCAGCACCAA CGAΨAAGAΨC AAGCΨGAΨCC ΨGGCCAACAA GGAAAACGΨG CACΨGGACCA CAΨACAΨGGA CACAΨΨCΨC AGAACCΨCCC CΨAΨGAΨCAΨ CGCCACCACΨ GAΨAΨGCAGA ACΨgaΨgaaa ccagccΨcaa gaacacccga aΨggagΨcΨc ΨaagcΨacaΨ aaΨaccaacΨ ΨacacΨΨΨac aaaaΨgΨΨgΨ cccccaaaaΨ gΨagccaΨΨc gΨaΨcΨgcΨc cΨaaΨaaaaa gaaagΨΨΨcΨ ΨcacAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAgcaΨ aΨgacΨAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA [SP-A35R_IECD-M1R]

• Amino acid sequence of A35R_IECD (SEQ ID NO: 13): LNQCMSANEA AITDSAVAVA AASSTHRKVA SSTTQYDHKE SCNGLYYQGS CYILHSDYKS FEDAKANCAA ESSTLPNKSD VLTTWLIDYV EDTWGSDGNP ITKTTSDYQD SDVSQEVRKY FCT

• Amino acid sequence of the fusion protein (SEQ ID NO: 14): MDAMKRGLCC VLLLCGAVFV SPSLNQCMSA NEAAITDSAV AVAAASSTHR KVASSTTQYD HKESCNGLYY QGSCYILHSD YKSFEDAKAN CAAESSTLPN KSDVLTTWLI DYVEDTWGSD GNPITKTTSD YQDSDVSQEV RKYFCTGGGG SGGGGSGAAA SIQTTVNTLS ERISSKLEQE ANASAQTKCD IEIGNFYIRQ NHGCNITVKN MCSADADAQL DAVLSAATET YSGLTPEQKA YVPAMFTAAL NIQTSVNTVV RDFENYVKQT CNSSAVVDNK LKIQNVIIDE CYGAPGSPTN LEFINTGSSK GNCAIKALMQ LTTKATTQIA PRQVAGTGVQ FYMIVIGVII LAALFMYYAK RMLFTSTNDK IKLILANKEN VHWTTYMDTF FRTSPMIIAT TDMQN

• Coding DNA optimized for expression in humans (SEQ ID NO: 15) ATGGACGCCA TGAAAAGAGG CCTGTGCTGC GTGCTGCTGC TGTGTGGCGC CGTGTTCGTG TCCCCCAGCC TGAACCAGTG CATGAGCGCC AATGAGGCCG CTATCACCGA CAGCGCTGTT GCCGTGGCCG CCGCCTCCTC CACCCACAGA AAGGTGGCCA GCAGCACAAC CCAGTACGAC CACAAGGAGA GCTGTAACGG CCTGTACTAC CAGGGCAGCT GCTACATCCT GCATTCTGAT TATAAGAGCT TTGAGGACGC TAAAGCCAAT TGTGCCGCTG AAAGCAGTAC ACTGCCTAAC AAAAGCGATG TGCTGACAAC CTGGCTGATT GATTATGTGG AAGATACCTG GGGCAGCGAC GGCAATCCTA TCACCAAAAC CACCAGCGAC TACCAGGACT CCGACGTGTC TCAGGAGGTC CGGAAGTACT TCTGCACCGG CGGAGGCGGC TCTGGCGGAG GTGGCAGCGG CGCTGCTGCC AGCATCCAGA CCACAGTGAA CACACTGAGC GAGAGAATCA GCAGCAAGCT GGAACAGGAA GCCAACGCCT CTGCACAAAC CAAGTGCGAC ATCGAGATCG GCAACTTCTA CATCAGACAA AATCACGGCT GTAATATCAC AGTGAAGAAC ATGTGCAGCG CCGACGCTGA TGCCCAGCTG GACGCCGTGC TGTCTGCTGC CACCGAGACA TACAGCGGCC TCACCCCTGA GCAGAAGGCC TACGTGCCCG CCATGTTCAC CGCCGCTCTG AACATCCAAA CAAGCGTGAA CACCGTGGTG CGGGACTTTG AGAACTACGT GAAGCAGACC TGCAACAGCT CCGCCGTGGT CGACAACAAA CTGAAGATCC AGAACGTGAT TATCGACGAG TGCTACGGCG CCCCTGGCTC CCCAACAAAT CTGGAATTCA TCAACACCGG CAGCAGCAAG GGAAACTGCG CCATCAAGGC CCTGATGCAG CTGACCACCA AGGCCACAAC ACAGATCGCC CCTAGACAGG TGGCCGGAAC AGGAGTGCAG TTCTACATGA TAGTTATCGG CGTGATCATC CTGGCCGCTC TGTTCATGTA CTACGCCAAG CGGATGCTGT TTACCAGCAC CAACGACAAG ATCAAGCTGA TCCTGGCCAA CAAGGAAAAC GTGCACTGGA CCACCTACAT GGATACATTC TTCAGAACCA GCCCCATGAT CATCGCCACA ACCGATATGC AGAACTGA

• mRNA optimized for codon expression in humans (SEQ ID NO: 16) Cap1-GAG (3'OMe) gcΨΨgΨΨcΨΨ ΨΨΨgcagaag cΨcagaaΨaa acgcΨcaacΨ ΨΨggccgcca ccAΨGGACGC CAΨGAAAAGA GGCCΨGΨGCΨ GCGΨGCΨGCΨ GCΨGΨGΨGGC GCCGΨGΨΨCG ΨGΨCCCCCAG CCΨGAACCAG ΨGCAΨGAGCG CCAAΨGAGGC CGCΨAΨCACC GACAGCGCΨG ΨΨGCCGΨGGC CGCCGCCΨCC ΨCCACCCACA GAAAGGΨGGC CAGCAGCACA ACCCAGΨACG ACCACAAGGA GAGCΨGΨAAC GGCCΨGΨACΨ ACCAGGGCAG CΨGCΨACAΨC CΨGCAΨΨCΨG AΨΨAΨAAGAG CΨΨΨGAGGAC GCΨAAAGCCA AΨΨGΨGCCGC ΨGAAAGCAGΨ ACACΨGCCΨA ACAAAAGCGA ΨGΨGCΨGACA ACCΨGGCΨGA ΨΨGAΨΨAΨGΨ GGAAGAΨACC ΨGGGGCAGCG ACGGCAAΨCC ΨAΨCACCAAA ACCACCAGCG ACΨACCAGGA CΨCCGACGΨG ΨCΨCAGGAGG ΨCCGGAAGΨA CΨΨCΨGCACC GGCGGAGGCG GCΨCΨGGCGG AGGΨGGCAGC GGCGCΨGCΨG CCAGCAΨCCA GACCACAGΨG AACACACΨGA GCGAGAGAAΨ CAGCAGCAAG CΨGGAACAGG AAGCCAACGC CΨCΨGCACAA ACCAAGΨGCG ACAΨCGAGAΨ CGGCAACΨΨC ΨACAΨCAGAC AAAAΨCACGG CΨGΨAAΨAΨC ACAGΨGAAGA ACAΨGΨGCAG CGCCGACGCΨ GAΨGCCCAGC ΨGGACGCCGΨ GCΨGΨCΨGCΨ GCCACCGAGA CAΨACAGCGG CCΨCACCCCΨ GAGCAGAAGG CCΨACGΨGCC CGCCAΨGΨΨC ACCGCCGCΨC ΨGAACAΨCCA AACAAGCGΨG AACACCGΨGG ΨGCGGGACΨΨ ΨGAGAACΨAC GΨGAAGCAGA CCΨGCAACAG CΨCCGCCGΨG GΨCGACAACA AACΨGAAGAΨ CCAGAACGΨG AΨΨAΨCGACG AGΨGCΨACGG CGCCCCΨGGC ΨCCCCAACAA AΨCΨGGAAΨΨ CAΨCAACACC GGCAGCAGCA AGGGAAACΨG CGCCAΨCAAG GCCCΨGAΨGC AGCΨGACCAC CAAGGCCACA ACACAGAΨCG CCCCΨAGACA GGΨGGCCGGA ACAGGAGΨGC AGΨΨCΨACAΨ GAΨAGΨΨAΨC GGCGΨGAΨCA ΨCCΨGGCCGC ΨCΨGΨΨCAΨG ΨACΨACGCCA AGCGGAΨGCΨ GΨΨΨACCAGC ACCAACGACA AGAΨCAAGCΨ GAΨCCΨGGCC AACAAGGAAA ACGΨGCACΨG GACCACCΨAC AΨGGAΨACAΨ ΨCΨΨCAGAAC CAGCCCCAΨG AΨCAΨCGCCA CAACCGAΨAΨ GCAGAACΨga Ψgaaaccagc cΨcaagaaca cccgaaΨgga gΨcΨcΨaagc ΨacaΨaaΨac caacΨΨacac ΨΨΨacaaaΨ gΨΨgΨccccc aaaaΨgΨagc caΨΨcgΨaΨc ΨgcΨccΨaaΨ aaaaagaaag ΨΨΨcΨΨcacA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AgcaΨaΨgac ΨAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAA [SP-A35R_sECD-M1R]

• Amino acid sequence of A35R_sECD (SEQ ID NO: 17): ASSTTQYDHK ESCNGLYYQG SCYILHSDYK SFEDAKANCA AESSTLPNKS DVLTTWLIDY VEDTWGSDGN PITKTTSDYQ DSDVSQEVRK YFCT

• Amino acid sequence of the fusion protein (SEQ ID NO: 18): MDAMKRGLCC VLLLCGAVFV SPSGASSTTQ YDHKESCNGL YYQGSCYILH SDYKSFEDAK ANCAAESSTL PNKSDVLTTW LIDYVEDTWG SDGNPITKTT SDYQDSDVSQ EVRKYFCTGG GGSGGGGSGA AASIQTTVNT LSERISSKLE QEANASAQTK CDIEIGNFYI RQNHGCNITV KNMCSADADA QLDAVLSAAT ETYSGLTPEQ KAYVPAMFTA ALNIQTSVNT VVRDFENYVK QTCNSSAVVD NKLKIQNVII DECYGAPGSP TNLEFINTGS SKGNCAIKAL MQLTTKATTQ IAPRQVAGTG VQFYMIVIGV IILAALFMYY AKRMLFTSTN DKIKLILANK ENVHWTTYMD TFFRTSPMII ATTDMQN

• Coding DNA optimized for expression in humans (SEQ ID NO: 19) ATGGACGCCA TGAAAAGAGG CCTGTGCTGC GTGCTGCTGC TGTGTGGCGC CGTGTTCGTG TCCCCTAGCG GAGCCAGCAG CACCACACAG TACGACCATA AGGAAAGCTG CAACGGCCTC TACTACCAGG GCTCTTGTTA CATCCTGCAC AGCGACTATA AGTCCTTCGA AGATGCCAAA GCCAATTGCG CCGCCGAGAG CAGCACCCTG CCTAACAAGA GCGATGTGCT GACAACCTGG CTGATCGACT ACGTCGAGGA CACCTGGGGC AGCGACGGCA ACCCCATCAC CAAGACCACC AGCGACTATC AGGACAGCGA CGTGTCCCAG GAGGTGCGGA AGTACTTCTG CACCGGAGGA GGCGGCAGCG GAGGCGGCGG TAGCGGCGCC GCTGCATCTA TCCAGACCAC AGTGAACACC CTGTCTGAGA GAATTAGCAG CAAGCTGGAA CAGGAGGCCA ACGCCTCTGC TCAGACCAAG TGCGACATCG AGATCGGCAA TTTCTACATC CGGCAGAACC ACGGCTGTAA TATCACCGTT AAGAACATGT GCAGCGCTGA CGCTGATGCC CAACTTGATG CTGTGCTGAG CGCCGCTACA GAAACCTACA GCGGCCTGAC CCCTGAGCAG AAGGCCTACG TGCCCGCCAT GTTCACAGCC GCCCTGAACA TCCAGACATC TGTGAACACC GTGGTCAGAG ATTTTGAGAA CTACGTGAAA CAGACCTGTA ATAGCAGCGC CGTGGTGGAC AACAAGCTGA AGATCCAAAA TGTGATCATT GACGAGTGCT ACGGCGCTCC TGGAAGCCCC ACCAACCTGG AATTCATCAA CACCGGCTCC TCCAAGGGCA ACTGCGCCAT CAAGGCCCTG ATGCAACTGA CAACAAAGGC CACCACTCAG ATCGCCCCTA GACAGGTGGC CGGCACCGGC GTGCAGTTTT ACATGATCGT GATTGGCGTG ATCATCCTGG CCGCCCTGTT TATGTACTAC GCCAAGCGGA TGCTGTTCAC CTCTACAAAC GACAAGATCA AACTGATCCT GGCTAACAAA GAAAACGTGC ACTGGACCAC ATACATGGAT ACATTCTTCA GAACCTCCCC AATGATCATC GCCACCACTG ATATGCAGAA CTGA

• mRNA optimized for codon expression in humans (SEQ ID NO: 20) Cap1-GAG (3'OMe) gcΨΨgΨΨcΨΨ ΨΨΨgcagaag cΨcagaaΨaa acgcΨcaacΨ ΨΨggccgcca ccAΨGGACGC CAΨGAAAAGA GGCCΨGΨGCΨ GCGΨGCΨGCΨ GCΨGΨGΨGGC GCCGΨGΨΨCG ΨGΨCCCCΨAG CGGAGCCAGC AGCACCACAC AGΨACGACCA ΨAAGGAAAGC ΨGCAACGGCC ΨCΨACΨACCA GGGCΨCΨΨGΨ ΨACAΨCCΨGC ACAGCGACΨA ΨAAGΨCCΨΨC GAAGAΨGCCA AAGCCAAΨΨG CGCCGCCGAG AGCAGCACCC ΨGCCΨAACAA GAGCGAΨGΨG CΨGACAACCΨ GGCΨGAΨCGA CΨACGΨCGAG GACACCΨGGG GCAGCGACGG CAACCCCAΨC ACCAAGACCA CCAGCGACΨA ΨCAGGACAGC GACGΨGΨCCC AGGAGGΨGCG GAAGΨACΨΨC ΨGCACCGGAG GAGGCGGCAG CGGAGGCGGC GGΨAGCGGCG CCGCΨGCAΨC ΨAΨCCAGACC ACAGΨGAACA CCCΨGΨCΨGA GAGAAΨΨAGC AGCAAGCΨGG AACAGGAGGC CAACGCCΨCΨ GCΨCAGACCA AGΨGCGACAΨ CGAGAΨCGGC AAΨΨΨCΨACA ΨCCGGCAGAA CCACGGCΨGΨ AAΨAΨCACCG ΨΨAAGAACAΨ GΨGCAGCGCΨ GACGCΨGAΨG CCCAACΨΨGA ΨGCΨGΨGCΨG AGCGCCGCΨA CAGAAACCΨA CAGCGGCCΨG ACCCCΨGAGC AGAAGGCCΨA CGΨGCCCGCC AΨGΨΨCACAG CCGCCCΨGAA CAΨCCAGACA ΨCΨGΨGAACA CCGΨGGΨCAG AGAΨΨΨΨGAG AACΨACGΨGA AACAGACCΨG ΨAAΨAGCAGC GCCGΨGGΨGG ACAACAAGCΨ GAAGAΨCCAA AAΨGΨGAΨCA ΨΨGACGAGΨG CΨACGGCGCΨ CCΨGGAAGCC CCACCAACCΨ GGAAΨΨCAΨC AACACCGGCΨ CCΨCCAAGGG CAACΨGCGCC AΨCAAGGCCC ΨGAΨGCAACΨ GACAACAAAG GCCACCACΨC AGAΨCGCCCC ΨAGACAGGΨG GCCGGCACCG GCGΨGCAGΨΨ ΨΨACAΨGAΨC GΨGAΨΨGGCG ΨGAΨCAΨCCΨ GGCCGCCCΨG ΨΨΨAΨGΨACΨ ACGCCAAGCG GAΨGCΨGΨΨC ACCΨCΨACAA ACGACAAGAΨ CAAACΨGAΨC CΨGGCΨAACA AAGAAAACGΨ GCACΨGGACC ACAΨACAΨGG AΨACAΨΨCΨΨ CAGAACCΨCC CCAAΨGAΨCA ΨCGCCACCAC ΨGAΨAΨGCAG AACΨgaΨgaa accagccΨca agaacacccg aaΨggagΨcΨ cΨaagcΨaca ΨaaΨaccaac ΨΨacacΨΨa caaaaΨgΨΨg Ψcccccaaaa ΨgΨagccaΨΨ cgΨaΨcΨgcΨ ccΨaaΨaaaa agaaagΨΨΨc ΨΨcacAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAgca ΨaΨgacΨAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA [B6R]

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

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

• Coding DNA optimized for expression in humans (SEQ ID NO: 23) ATGAAAACCA TCAGCGTGGT CACACTGCTG TGCGTGCTGC CCGCCGTGGT GTACTCCACA TGTACCGTGC CAACCATGAA CAACGCCAAG CTGACAAGCA CGGAAACAAG CTTCAACGAC AAGCAGAAGG TGACCTTTAC CTGTGATAGC GGCTACCACA GCCTGGACCC TAACGCTGTC TGCGAGACCG ACAAATGGAA GTACGAGAAC CCCTGTAAAA AGATGTGCAC AGTGTCCGAC TACGTGTCCG AGCTGTACGA CAAGCCTCTG TATGAAGTGA ACAGCACAAT GACACTGTCT TGCAACGGCG AGACAAAGTA CTTCAGATGT GAAGAGAAGA ACGGCAACAC CTCTTGGAAT GATACAGTGA CCTGTCCTAA TGCCGAGTGC CAGCCTCTGC AGCTGGAACA CGGCTCTTGC CAACCTGTGA AGGAAAAGTA TTCTTTTGGC GAATACATGA CCATCAACTG CGACGTGGGC TACGAGGTGA TTGGAGTGTC CTACATCAGC TGCACCGCCA ATAGCTGGAA TGTGATCCCC AGCTGCCAGC AGAAATGCGA TATCCCTAGC CTGAGCAACG GCCTGATCAG CGGATCTACC TTCAGCATCG GCGGCGTTAT CCACCTGTCC TGCAAGAGCG GCTTCACCCT GACCGGCAGC CCTAGCAGCA CCTGCATCGA CGGCAAGTGG AACCCCATCC TGCCTACCTG CGTGCGGAGC AACGAGGAAT TCGACCCCGT GGACGATGGA CCTGATGACG AGACAGACCT GAGCAAGCTT TCTAAGGACG TGGTGCAGTA CGAACAGGAG ATCGAGAGCC TCGAGGCCAC CTACCATATC ATCATTATGG CTCTGACCAT CATGGGAGTG ATCTTCCTGA TCAGTATCAT CGTTCTGGTG TGCAGCTGTG ATAAGAACAA CGACCAGTAC AAGTTCCACA AGCTGCTGCC ATGA

• mRNA optimized for codon expression in humans (SEQ ID NO: 24) Cap1-GAG (3'OMe) gcΨΨgΨΨcΨΨ ΨΨΨgcagaag cΨcagaaΨaa acgcΨcaacΨ ΨΨggccgcca ccAΨGAAAAC CAΨCAGCGΨG GΨCACACΨGC ΨGΨGCGΨGCΨ GCCCGCCGΨG GΨGΨACΨCCA CAΨGΨACCGΨ GCCAACCAΨG AACAACGCCA AGCΨGACAAG CACGGAAACA AGCΨΨCAACG ACAAGCAGAA GGΨGACCΨΨΨ ACCΨGΨGAΨA GCGGCΨACCA CAGCCΨGGAC CCΨAACGCΨG ΨCΨGCGAGAC CGACAAAΨGG AAGΨACGAGA ACCCCΨGΨAA AAAGAΨGΨGC ACAGΨGΨCCG ACΨACGΨGΨC CGAGCΨGΨAC GACAAGCCΨC ΨGΨAΨGAAGΨ GAACAGCACA AΨGACACΨGΨ CΨΨGCAACGG CGAGACAAAG ΨACΨΨCAGAΨ GΨGAAGAGAA GAACGGCAAC ACCΨCΨΨGGA AΨGAΨACAGΨ GACCΨGΨCCΨ AAΨGCCGAGΨ GCCAGCCΨCΨ GCAGCΨGGAA CACGGCΨCΨΨ GCCAACCΨGΨ GAAGGAAAAG ΨAΨΨCΨΨΨΨG GCGAAΨACAΨ GACCAΨCAAC ΨGCGACGΨGG GCΨACGAGGΨ GAΨΨGGAGΨG ΨCCΨACAΨCA GCΨGCACCGC CAAΨAGCΨGG AAΨGΨGAΨCC CCAGCΨGCCA GCAGAAAΨGC GAΨAΨCCCΨA GCCΨGAGCAA CGGCCΨGAΨC AGCGGAΨCΨA CCΨΨCAGCAΨ CGGCGGCGΨΨ AΨCCACCΨGΨ CCΨGCAAGAG CGGCΨΨCACC CΨGACCGGCA GCCCΨAGCAG CACCΨGCAΨC GACGGCAAGΨ GGAACCCCAΨ CCΨGCCΨACC ΨGCGΨGCGGA GCAACGAGGA AΨΨCGACCCC GΨGGACGAΨG GACCΨGAΨGA CGAGACAGAC CΨGAGCAAGC ΨΨΨCΨAAGGA CGΨGGΨGCAG ΨACGAACAGG AGAΨCGAGAG CCΨCGAGGCC ACCΨACCAΨA ΨCAΨCAΨΨAΨ GGCΨCΨGACC AΨCAΨGGGAG ΨGAΨCΨΨCCΨ GAΨCAGΨAΨC AΨCGΨΨCΨGG ΨGΨGCAGCΨG ΨGAΨAAGAAC AACGACCAGΨ ACAAGΨΨCCA CAAGCΨGCΨG CCAΨgaΨgaa accagccΨca agaacacccg aaΨggagΨcΨ cΨaagcΨaca ΨaaΨaccaac ΨΨacacΨΨΨa caaaaΨgΨΨg Ψcccccaaaa ΨgΨagccaΨΨ cgΨaΨcΨgcΨ ccΨaaΨaaaa agaaagΨΨΨc ΨΨcacAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAgca ΨaΨgacΨAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA [A29L]

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

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

• Coding DNA optimized for expression codon in humans (SEQ ID NO: 27) ATGGACGGCA CCCTGTTTCC AGGAGATGAT GACCTGGCCA TTCCTGCTAC AGAATTCTTC TCTACCAAGG CCGCTAAAAA CCCCGAGACA AAGCGGGAAG CCATCGTGAA GGCCTACGGC GACGATAACG AGGAAACACT CAAGCAGAGA CTGACCAATC TGGAAAAGAA GATCACCAAC ATCACCACCA AGTTCGAGCA GATCGAGAAG TGCTGTAAAC ACAACGACGA GGTGCTGTTC CGGCTGGAAA ACCACGCCGA GACACTGAGA GCCGCCATGA TCAGCCTGGC CAAGAAAATC GACGTGCAGA CCGGCAGAAG ACCTTACGAG TGA

• Codon-optimized mRNA for expression in humans (SEQ ID NO: 28) Cap1-GAG (3'OMe) gcΨΨgΨΨcΨΨ ΨΨΨgcagaag cΨcagaaΨaa acgcΨcaacΨ ΨΨggccgcca ccAΨGGACGG CACCCΨGΨΨΨ CCAGGAGAΨG AΨGACCΨGGC CAΨΨCCΨGCΨ ACAGAAΨΨCΨ ΨCΨCΨACCAA GGCCGCΨAAA AACCCCGAGA CAAAGCGGGA AGCCAΨCGΨG AAGGCCΨACG GCGACGAΨAA CGAGGAAACA CΨCAAGCAGA GACΨGACCAA ΨCΨGGAAAAG AAGAΨCACCA ACAΨCACCAC CAAGΨΨCGAG CAGAΨCGAGA AGΨGCΨGΨAA ACACAACGAC GAGGΨGCΨGΨ ΨCCGGCΨGGA AAACCACGCC GAGACACΨGA GAGCCGCCAΨ GAΨCAGCCΨG GCCAAGAAAA ΨCGACGΨGCA GACCGGCAGA AGACCΨΨACG AGΨgaΨgaaa ccagccΨcaa gaacacccga aΨggagΨcΨc ΨaagcΨacaΨ aaΨaccaacΨ ΨacacΨΨΨac aaaaΨgΨΨgΨ cccccaaaaΨ gΨagccaΨΨc gΨaΨcΨgcΨc cΨaaΨaaaa gaaagΨΨΨcΨ ΨcacAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAgcaΨ aΨgacΨAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAA

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

Since naked mRNA cannot effectively enter the body's cells for protein expression, and mRNA has poor stability and is easily degraded, 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 mRNA encapsulation carrier 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 nanoparticles, 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 structured lipids.

In a specific 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 lipids" generally refer to lipids that carry any number of net positive charges at a certain pH (e.g., physiological pH). The cationic lipid may include but is not limited to SM102, 3-(didodecylamino)-N1,N1,4-triazolyl-1-piperazineethylamine (KL10), N1-[2-(docosylamino)ethyl]-N1,N4,N4-triazolyl-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 nanoparticles 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 contain 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-dodecylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylphosphoethanolamine, and other neutral lipids connected to anionic groups.

In a more specific embodiment, the non-cationic lipid may include a neutral lipid, which may include, for example, a phospholipid, such as distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dimalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleylphosphatidylcholine (POPC), palmitoyloleyl-phosphatidylethanolamine (P ...phosphatidylcholine (POPC), palmitoyloleyl-phosphatidylethanolamine (PPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylethanolamine (DPPG), palmitoylphosphatidylglycerol (DOPG), palmitoylphosphatidylglycerol (DPPG), palmitoylphosphatidylethanolamine (DOPE), palmitoyloleylphosphatidylcholine (POPC), palmitoyloleyl-phosphatidylethanolamine (PPE), palmi (POPE), dioleyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dimalcoholylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleyl-phosphatidylethanolamine (SOPE), or a mixture thereof. In addition, lipids 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 phospholipid acyl choline.

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

In certain embodiments, the LNP may include lipid conjugates, such as polyethylene glycol (PEG)-modified lipids and derivatized lipids. PEG-modified lipids may include, but are not limited to, polyethylene glycol chains with a length of up to 5 kDa covalently linked to lipids with alkyl chains of C6 to C20 in length. The addition of these components can prevent lipid aggregation, increase the duration of the cycle, facilitate the delivery of lipid-nucleic acid compositions 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 molar ratio of the lipid molecule modified with polyethylene glycol (PEG) in the lipid nanoparticles is about 0.5-2%, for example, about 1-2%, about 1.2-1.8% or about 1.4-1.6%. In some embodiments, the lipid molecule modified with polyethylene glycol (PEG) can be PEG2000-DMG.

In some embodiments, the LNP may also contain cholesterol. In some embodiments, the molar ratio of cholesterol 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 commonly used in the art may be adopted, for example, the delivery method mentioned in US20160376224A1 or WO2015199952A1 may be adopted. [Examples] [Example 1: Preparation of mRNA vaccine]

The sequences of DNA encoding several protein (or fusion protein) antigens 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] Protein (or fusion protein) antigen Sequences that encode DNA A35R SEQ ID NO: 3 M1R SEQ ID NO: 7 SP-A35R_IECD-M1R SEQ ID NO: 15 SP-A35R_sECD-M1R SEQ ID NO: 19 B6R SEQ ID NO: 23 A29L SEQ ID NO: 27

The coding DNA sequences listed in Table 1 above were inserted between the 5' untranslated region (UTR) (SEQ ID NO: 29) and 3'UTR (SEQ ID NO: 30) downstream of the T7 promoter in a 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: 20 μL of IVT reaction system] Components quantity 10× reaction buffer (Novozyme, DD4101R-02) 2 µL 100 mM ATP solution (Novagen, DD4106-PA-01) 2 µL 100 mM CTP solution (Novozyme, DD4107-PA-01) 2 µL 100 mM GTP solution (Novagen, DD4108-PA-01) 2 µL 100 mM N1-Me-pUTP solution (Novagen, DD4114-PA-01) 2 µL IVT Template 1 µg Cap1-GAG(3'OMe)（Novozyme, DD4119-PC-01） 2 µL T7 RNA polymerase (250 U/µL) (Novozyme, DD4101-02) 1 µL Inorganic pyrophosphatase (Novagen, D4103-PC-02) 0.04 U RNase-free ddH ₂ O (Biyuntian, R0022) Up to 20µL

IVT was performed and the transcription product was purified to obtain the mRNA shown in Table 3 below (Figure 1). [Table 3: mRNA] Protein (or fusion protein) antigen mRNA sequence A35R SEQ ID NO: 4 M1R SEQ ID NO: 8 SP-A35R_IECD-M1R SEQ ID NO: 16 SP-A35R_sECD-M1R SEQ ID NO: 20 B6R SEQ ID NO: 24 A29L SEQ ID NO: 28

The mRNA or its combination listed in Table 3 above was dissolved in a citrate buffer, and then mixed with a lipid mixture dissolved in ethanol (ionizable lipid ALC-0315: distearyl phospholipid acylcholine: cholesterol: PEG lipid ALC-0159) using a microfluidic device NanoAssembler to obtain lipid nanoparticles (LNP) (also referred to as "LNP-mRNA") encapsulating the mRNA or its combination as shown in Table 4 below. [Table 4: mRNA vaccines of each group] Test Group mRNA of the following antigens Packaging method Monovalent vaccines Group A A35R Encapsulated as LNP Group B M1R Encapsulated as LNP Group C B6R Encapsulated as LNP Group D A29L Encapsulated as LNP Fusion multivalent vaccine AB Group 1 SP-A35R_IECD-M1R Encapsulated as LNP AB Group 2 SP-A35R_sECD-M1R Encapsulated as LNP Mixed polyvalent vaccines A+B Group 1 A35R+M1R First encapsulate each into LNP and then mix A+B Group 2 A35R+M1R Mix first and then encapsulate into LNP A+B+C+D group 1 A35R+M1R+B6R+A29L First encapsulate each into LNP and then mix A+B+C+D group 2 A35R+M1R+B6R+A29L Mix first and then encapsulate into LNP [Example 2: Administration of mRNA vaccine and efficacy 1] (1) Administration of mRNA vaccine

Each group of mRNA vaccines (LNP-mRNA) prepared in Example 1 was administered to 7-week-old Balb/c female mice by intramuscular injection at a dose of 10 µg/dose/mouse on day 0, and by intramuscular injection on day 14. The control group was administered 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 was measured for the concentrations of total anti-A35R antibody, total anti-M1R antibody, total anti-B6R antibody, and total anti-A29L antibody 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 test group were significantly increased, and the increase in 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 was more significant. Among them, the increase in 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 was the most significant.

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 test 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 Figure 2D, the total anti-A29L antibody level in the blood of mice administered with the vaccines of Group A+B+C+D 1 and Group A+B+C+D 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 above, and serum was separated therefrom, which was 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 solution 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 measured. 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.

Figure 2E shows the neutralizing antibody levels (PFU/well) against vaccinia virus in the sera diluted as above.

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 neutralizing antibody level against vaccinia virus was the most abundant. (4) Weight changes after virus 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 the mRNA vaccines in each group did not experience significant weight changes due to the challenge with vaccinia virus. (5) Changes in viral load in the lungs after viral challenge

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

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 approached 0. This indicates that each group of mRNA vaccines exhibited significant anti-vaccinia virus immune efficacy. [Example 3: Administration and efficacy of mRNA vaccines 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 Scientific, 14190136) as a negative control were administered to 7-week-old Balb/c female mice by intramuscular injection for the first time on day 0 and by boosting injection on day 14 (0.5 months after vaccination). (2) Determination of Antibody Concentration

The concentrations of anti-A35R antibody and anti-M1R antibody in blood collected from the mice at 1 month, 2 months, 3 months, 4 months and 5 months after vaccination were measured 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 in 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 in 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 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 over time. (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 the vaccination as 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 solution 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 measured. 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.

Figure 3C shows the neutralizing antibody levels (%) against vaccinia virus in the sera diluted as above.

As shown in Figure 3C, 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 positive control mice administered with VACV-WR and 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 in the sera of positive control mice administered with VACV-WR and the sera of negative control mice administered with DPBS. The neutralizing antibody levels in the sera of mice administered with the vaccines of A-B Group 1, A-B Group 2, and A+B Group 1 were relatively constant over time. (4) Weight changes after virus attack

At 5.5 months after vaccination, 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 experience significant weight changes due to the attack of vaccinia virus. [Example 4: Antiviral activity of vaccine-induced antiserum] (1) Administration of mRNA vaccine

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 Scientific, 14190136) as a negative control were administered to 7-week-old Balb/c female mice by intramuscular injection for the first time on day 0 and by boosting (Boost) on day 14 (0.5 months after vaccination). (2) Preparation of mixed serum and its administration

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 them were mixed. 100µl of mixed serum was injected intravenously into a new batch of 7-8 week old Balb/c female mice. (3) Weight changes after virus 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 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 injected intravenously with pooled sera obtained from negative control mice administered DPBS at the time of vaccinia virus challenge.

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

Figure 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 at the time of 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 at the time of vaccinia virus challenge.

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 at the time of vaccinia virus challenge.

The weight changes (%) of mice intravenously injected with pooled sera obtained from mice administered with different vaccines as above relative to the initial weight at the time of challenge with vaccinia virus were compared. As shown in Figure 4F, mice intravenously injected with pooled sera obtained from mice administered with vaccines of Group A-B 2 and mice intravenously injected with pooled sera obtained from positive control mice administered with VACV-WR had significantly less weight changes due to challenge with vaccinia virus than mice intravenously injected with pooled sera obtained from negative control mice administered with DPBS. The weight changes of mice intravenously injected with mixed serum obtained from mice administered with the vaccine of group A-B 1, mice intravenously injected with mixed serum obtained from mice administered with the vaccine of group A+B 1, and mice intravenously injected with mixed serum obtained from negative control mice administered with DPBS were comparable to those of mice challenged with vaccinia virus. [Example 5: Administration and efficacy of mRNA vaccine 3] (1) Administration of mRNA vaccine

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 Scientific, 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 were measured by measuring the absorbance at 450 nm (OD450) ( FIG. 5A and FIG. 5B ).

As shown in Figure 5A, the concentrations of total anti-A35R antibodies in the blood of mice administered with vaccines of A-B Group 1, A-B Group 2, and A+B Group 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 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 vaccines 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

The blood was collected from the mouse on day 7 after vaccination as above, and serum was separated therefrom, which was 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 solution 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 measured. 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.

Figure 5C shows the neutralizing antibody levels (%) against vaccinia virus in the sera diluted as above.

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 virus challenge

On day 8 after the first vaccination, 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 DPBS, mice administered with vaccines from A-B Group 1, A-B 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.

without

[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 serial 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 from mice at 1 month, 2 months, 3 months, 4 months and 5 months after administration of each group of mRNA vaccines to mice. Figure 3C shows the levels (%) of neutralizing antibodies to vaccinia virus in the serum collected from mice at 1 month, 2 months, 3 months, 4 months and 5 months after administration of each group of mRNA vaccines 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 changes (%) relative to the initial 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 at the time of challenge with vaccinia virus. Figure 4F compares the weight changes (%) relative to the initial weight of mice intravenously injected with mixed sera obtained from mice administered with different vaccines at the time of challenge 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 administration of each group of mRNA vaccines to mice. Figure 5C shows the level (%) of neutralizing antibodies against 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.

TW202421644A_112127075_SEQL.xml

Claims (66)

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 following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein. The vaccine as described in claim 1, wherein the fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of proteins: A35R protein and M1R protein. The vaccine as described in claim 1 or claim 2, wherein a signal peptide is attached to the 5' end of the fusion protein. A vaccine as described in claim 1, 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. A vaccine as described in claim 4, wherein the A35R protein is encoded by a DNA sequence comprising SEQ ID NO: 3, the M1R protein is encoded by a DNA sequence comprising SEQ ID NO: 7, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 15, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 19, the B6R protein is encoded by a DNA sequence comprising SEQ ID NO: 23, and the A29L protein is encoded by a DNA sequence comprising SEQ ID NO: 27. The vaccine as described in claim 1, wherein the mRNA molecule further comprises: a 5' cap, a 5' UTR, a 3' UTR and a poly A tail. The vaccine of claim 6, wherein the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. A vaccine as described in claim 6, 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. A vaccine as described in claim 6, wherein the uridine triphosphate in the mRNA molecule is N1-methylpseudouridine triphosphate. A vaccine as described in any one of claims 6 to 9, wherein the mRNA molecule is selected from: mRNA molecules encoding A35R protein, which comprises the RNA sequence shown in SEQ ID NO: 4, mRNA molecules encoding M1R protein, which comprises the RNA sequence shown in SEQ ID NO: 8, mRNA molecules encoding the fusion protein of A35R and M1R, which comprises the RNA sequence shown in SEQ ID NO: 16, mRNA molecules encoding the fusion protein of A35R and M1R, which comprises the RNA sequence shown in SEQ ID NO: 20, mRNA molecules encoding B6R protein, which comprises the RNA sequence shown in SEQ ID NO: 24, and mRNA molecules encoding A29L protein, which comprises the RNA sequence shown in SEQ ID NO: 28. A vaccine as described in claim 1, wherein when the vaccine contains a mixture of two or more mRNA molecules, the two or more mRNA molecules are first 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. A vaccine as described in claim 1, wherein the mRNA molecule is encapsulated as lipid nanoparticles (LNP). Use of an mRNA molecule encoding the following proteins and/or fusion proteins in the manufacture of an anti-pox virus vaccine: (a) one or more monkeypox virus proteins selected from the following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein. The use as described in claim 13, 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. The use as described in claim 13 or claim 14, wherein a signal peptide is attached to the 5' end of the fusion protein. The use as described in claim 13, 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. The use as described in claim 16, wherein the A35R protein is encoded by a DNA sequence comprising SEQ ID NO: 3, the M1R protein is encoded by a DNA sequence comprising SEQ ID NO: 7, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 15, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 19, the B6R protein is encoded by a DNA sequence comprising SEQ ID NO: 23, and the A29L protein is encoded by a DNA sequence comprising SEQ ID NO: 27. The use as described in claim 13, wherein the mRNA molecule further comprises: a 5' cap, a 5' UTR, a 3' UTR and a poly A tail. The use as described in claim 18, wherein the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. The use as described in claim 18, 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. The use as described in claim 18, wherein the uridine triphosphate in the mRNA molecule is N1-methylpseudouridine triphosphate. The use as described in any one of claim 18 to claim 21, wherein the mRNA molecule is selected from: mRNA molecule encoding A35R protein, which comprises the RNA sequence shown in SEQ ID NO: 4, mRNA molecule encoding M1R protein, which comprises the RNA sequence shown in SEQ ID NO: 8, mRNA molecule encoding A35R and M1R fusion protein, which comprises the RNA sequence shown in SEQ ID NO: 16, mRNA molecule encoding A35R and M1R fusion protein, which comprises the RNA sequence shown in SEQ ID NO: 20, mRNA molecule encoding B6R protein, which comprises the RNA sequence shown in SEQ ID NO: 24, and mRNA molecule encoding A29L protein, which comprises the RNA sequence shown in SEQ ID NO: 28. The use as described in claim 13, wherein when a vaccine is prepared from a mixture of two or more mRNA molecules, the two or more mRNA molecules are first 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. The use as described in claim 13, wherein the mRNA molecule is encapsulated as lipid nanoparticles (LNP). The use as described in claim 13, wherein the vaccine is used against poxvirus of one or more genera selected from the group consisting of Orthopoxvirus , Capripoxvirus , Cervidpoxvirus , Suipoxvirus , Leporipoxvirus , Molluscipoxvirus , Yatapoxvirus , Avipoxvirus , Crocodylidpoxvirus and Parapoxvirus . The use as claimed in claim 13, 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 virus), Rabbit 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 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. 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 following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein, and optionally (ii) a reagent for transcribing the DNA molecule of (i) into an mRNA molecule. The reagent kit as described in claim 27, wherein the fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of proteins: A35R protein and M1R protein. The reagent kit of claim 27 or claim 28, wherein a signal peptide is attached to the 5' end of the fusion protein. The reagent kit as described in claim 27, 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. The reagent kit as described in claim 30, wherein the A35R protein is encoded by a DNA sequence comprising SEQ ID NO: 3, the M1R protein is encoded by a DNA sequence comprising SEQ ID NO: 7, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 15, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 19, the B6R protein is encoded by a DNA sequence comprising SEQ ID NO: 23, and the A29L protein is encoded by a DNA sequence comprising SEQ ID NO: 27. The reagent kit of claim 27, 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. A reagent kit as described in claim 32, 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, which comprises a promoter linked from 5' to 3', a 5'UTR coding DNA and a 3'UTR coding DNA, adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate, a 5' cap, and an RNA polymerase. A kit as claimed in claim 33, 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. The kit of claim 33, wherein the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. The kit of claim 33, wherein the uridine triphosphate is N1-methylpseudouridine triphosphate. The kit of claim 33, wherein the RNA polymerase is T7 RNA polymerase. Use of the following substances in a kit for preparing an anti-pox virus vaccine: (i) a DNA molecule encoding: (a) one or more monkeypox virus proteins selected from the following: 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 following: A35R protein, M1R protein, B6R protein and A29L protein, and optionally (ii) a reagent for transcribing the DNA molecule of (i) into an mRNA molecule. The use as described in claim 38, 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. The use as described in claim 38 or claim 39, wherein a signal peptide is attached to the 5' end of the fusion protein. The use as described in claim 38, 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. The use as described in claim 41, wherein the A35R protein is encoded by a DNA sequence comprising SEQ ID NO: 3, the M1R protein is encoded by a DNA sequence comprising SEQ ID NO: 7, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 15, the fusion protein of A35R and M1R is encoded by a DNA sequence comprising SEQ ID NO: 19, the B6R protein is encoded by a DNA sequence comprising SEQ ID NO: 23, and the A29L protein is encoded by a DNA sequence comprising SEQ ID NO: 27. The use as described in claim 38, 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. The use as described in claim 43, 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, which comprises a promoter linked from 5' to 3', a 5'UTR coding DNA and a 3'UTR coding DNA, adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate, a 5' cap, and an RNA polymerase. The use as described in claim 44, 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. The use as claimed in claim 44, wherein the 5' cap is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG. The use as described in claim 44, wherein the uridine triphosphate is N1-methylpseudouridine triphosphate. The use as described in claim 44, wherein the RNA polymerase is T7 RNA polymerase. The use as described in claim 38, wherein the vaccine is used against poxvirus of one or more genera selected from the group consisting of Orthopoxvirus , Capripoxvirus , Cervidpoxvirus , Suipoxvirus , Leporipoxvirus , Molluscipoxvirus , Yatapoxvirus , Avipoxvirus , Crocodylidpoxvirus and Parapoxvirus . The use of claim 38, 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 virus), Rabbit 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 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. A fusion protein formed by fusing the following monkeypox virus proteins or parts of the proteins: A35R protein, M1R protein, B6R protein and A29L protein. A fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein. The fusion protein as described in claim 52, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 14 or 18. The fusion protein as described in claim 53, wherein the fusion protein is encoded by a DNA sequence shown in SEQ ID NO: 15 or 19. A DNA molecule encoding A35R protein comprises the DNA sequence shown in SEQ ID NO:3. A DNA molecule encoding M1R protein comprises the DNA sequence shown in SEQ ID NO:7. A DNA molecule encoding a fusion protein of A35R and M1R comprises the DNA sequence shown in SEQ ID NO:15. A DNA molecule encoding a fusion protein of A35R and M1R comprises the DNA sequence shown in SEQ ID NO:19. A DNA molecule encoding B6R protein comprises the DNA sequence shown in SEQ ID NO: 23. A DNA molecule encoding A29L protein comprises the DNA sequence shown in SEQ ID NO: 27. An mRNA molecule encoding an A35R protein comprises the RNA sequence shown in SEQ ID NO:4. An mRNA molecule encoding an M1R protein comprises an RNA sequence shown in SEQ ID NO:8. An mRNA molecule encoding a fusion protein of A35R and M1R comprises the RNA sequence shown in SEQ ID NO: 16. An mRNA molecule encoding a fusion protein of A35R and M1R comprises the RNA sequence shown in SEQ ID NO: 20. An mRNA molecule encoding a B6R protein comprises the RNA sequence shown in SEQ ID NO: 24. An mRNA molecule encoding an A29L protein comprises the RNA sequence shown in SEQ ID NO: 28.