A monkeypox virus antigen epitope mRNA composition and application thereof

Abstract

The application discloses a monkeypox virus antigen epitope mRNA composition and application thereof, and relates to the technical field of biological medicine. The monkeypox virus antigen epitope mRNA composition comprises mRNA coding an antigen epitope or an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity and the same immunogenicity. The monkeypox virus antigen epitope mRNA composition can produce a high level of immunoprotective effect against the monkeypox virus, can be used for preparing a medicine for preventing and treating the monkeypox virus, and has a wide application prospect in the clinic.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a monkeypox virus antigen epitope mRNA composition and its application. Background Technology

[0002] Monkeypox virus (MPXV) is a zoonotic virus that has experienced global outbreaks in recent years, particularly between 2022 and 2024, with the rapid spread of monkeypox posing a significant threat to global public health. Although vaccines against smallpox (such as JYNNEOS and ACAM2000) have been approved for monkeypox prevention, their protective efficacy is limited, and in-depth research on monkeypox virus-specific antigens is lacking. mRNA vaccine technology, due to its high efficiency and speed, has become a powerful tool for developing novel monkeypox vaccines. However, existing monkeypox mRNA vaccine research largely focuses on the design of previously identified antigens, failing to adequately consider the diversity of neutralizing antibodies and the protective role of cellular immune responses. Summary of the Invention

[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a monkeypox virus antigen epitope mRNA composition.

[0004] The present invention also provides a monkeypox virus antigen epitope protein composition.

[0005] The present invention also provides biological materials related to the above-described monkeypox virus antigenic epitope mRNA composition or the above-described monkeypox virus antigenic epitope protein composition.

[0006] The present invention also provides a vaccine.

[0007] The present invention also provides a method for preparing the above-mentioned vaccine.

[0008] The present invention also provides the use of the above-mentioned monkeypox virus antigenic epitope mRNA composition, the above-mentioned monkeypox virus antigenic epitope protein composition, the above-mentioned biological material or the above-mentioned vaccine in the preparation of medicaments for the prevention and / or treatment of monkeypox virus infection.

[0009] A monkeypox virus antigenic epitope mRNA composition according to a first aspect of the present invention comprises: an mRNA encoding an antigenic epitope or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with and having the same immunogenicity.

[0010] According to some embodiments of the present invention, the neutralization rate of the mRNA-immunized serum against monkeypox virus is not less than 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30%.

[0011] According to some embodiments of the present invention, the neutralization rate is not less than 80%.

[0012] According to some embodiments of the present invention, the serum is derived from a human or animal.

[0013] According to some embodiments of the present invention, the animal includes at least one of mice, rats, and monkeys.

[0014] According to some embodiments of the present invention, the method for obtaining the mRNA-immunized serum includes the following steps:

[0015] Humans or animals are immunized with the mRNA 1-3 times, blood is obtained, and serum is separated; the inoculation dose of the mRNA is 1μg-20μg each time.

[0016] According to some embodiments of the present invention, in the acquisition method, the mRNA is inoculated via intramuscular injection. The inoculated mRNA is liposome-encapsulated mRNA.

[0017] According to some embodiments of the present invention, in the acquisition method, the time interval between each immunization of a human or animal is 7-21 days. For example, it can be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days.

[0018] According to some embodiments of the present invention, in the acquisition method, the inoculation dose of the mRNA is 1-20 μg each time. For example, it can be 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, or 20 μg.

[0019] According to some embodiments of the present invention, in the method of obtaining blood, the timing of obtaining blood is 14-28 days after the last immunization. For example, it can be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days.

[0020] According to some embodiments of the present invention, the method for determining the neutralization rate is a plaque formation-based neutralization test. The method includes the following steps: co-incubating a serum sample with monkeypox virus, infecting monkeypox virus-susceptible cells with the incubation, culturing, examining plaque formation, and calculating the neutralization rate. The serum sample can be undiluted serum or an appropriately diluted serum sample. For example, the serum can be serially diluted 2-fold, 5-fold, 10-fold, or 50-fold.

[0021] According to some embodiments of the present invention, the dosage of monkeypox virus is 90-110 PFU. For example, it can be 90 PFU, 91 PFU, 92 PFU, 93 PFU, 94 PFU, 95 PFU, 96 PFU, 97 PFU, 98 PFU, 99 PFU, 100 PFU, 101 PFU, 102 PFU, 103 PFU, 104 PFU, 105 PFU, 106 PFU, 107 PFU, 108 PFU, 109 PFU, or 110 PFU.

[0022] According to some embodiments of the present invention, the monkeypox virus susceptible cells include, but are not limited to, Vero E6 cells.

[0023] According to some embodiments of the present invention, the content of monkeypox virus susceptible cells includes, but is not limited to, 1.0 × 10⁻⁶ cells. 4 -1.0×10 6 For example: it can be 1×10. 4 1, 2×10 4 1, 3×10 4 1, 4×10 4 5×10 4 6×10 4 7×10 4 8×10 4 9×10 4 1×10 5 1, 2×10 5 1, 3×10 5 1, 4×10 5 5×10 5 6×10 5 7×10 5 8×10 5 9×10 5 One or 1.0 × 106 indivual.

[0024] According to some embodiments of the present invention, the co-incubation temperature is 36°C-38°C.

[0025] According to some embodiments of the present invention, the co-incubation time is 1h-3h. For example, it can be 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, 2h, 2.1h, 2.2h, 2.3h, 2.4h, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, or 3h.

[0026] According to some embodiments of the present invention, the culture time is 3-4 days.

[0027] According to some embodiments of the present invention, the formula for calculating the neutralization rate is as follows:

[0028] Where a represents the number of plaques produced in the 50-fold diluted blank control serum treatment group, and b represents the number of plaques produced in the 50-fold diluted test serum treatment group.

[0029] According to some embodiments of the present invention, the antigenic epitopes include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen of the following: A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, I2, E8, C20.5, A14, E13, and monkeypox virus T-cell epitope peptides.

[0030] According to some embodiments of the present invention, the antigenic epitope further includes at least one of C15, M5, L5, A10, A15, A18, G4, G10, A27, C15, F2, C2, C19, and A36.

[0031] According to some embodiments of the present invention, the antigenic epitope comprises at least one of the following combinations:

[0032] (A1)A29, M1, B6, A35;

[0033] (A2)A29, M1, B6, A35, H3, A17, A30, H2;

[0034] (A3)A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, I2;

[0035] (A4)A29, M1, B6, A35, H3, A17, A30, H2, E8, C20.5, A14, E13;

[0036] (A5)A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, I2, E8, C20.5, A14, E13;

[0037] (A6) Monkeypox virus T-cell epitope peptide;

[0038] (A7)A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, I2, monkeypox virus T-cell epitope peptide.

[0039] According to some embodiments of the present invention, the amino acid sequence of A29 is shown in positions 1 to 110 of SEQ ID NO:1. The coding region sequence of the mRNA encoding A29 is shown in positions 133 to 462 of SEQ ID NO:1.

[0040] According to some embodiments of the present invention, the amino acid sequence of M1 is shown in positions 1 to 250 of SEQ ID NO:3. The coding region sequence of the mRNA encoding M1 is shown in positions 133 to 882 of SEQ ID NO:4.

[0041] According to some embodiments of the present invention, the amino acid sequence of B6 is shown in positions 1 to 317 of SEQ ID NO:5. The coding region sequence of the mRNA encoding B6 is shown in positions 133 to 1083 of SEQ ID NO:6.

[0042] According to some embodiments of the present invention, the amino acid sequence of A35 is shown as positions 1 to 181 of SEQ ID NO:7. The coding region of the mRNA sequence encoding A35 is shown as positions 133 to 675 of SEQ ID NO:8.

[0043] According to some embodiments of the present invention, the amino acid sequence of H3 is shown in positions 1 to 324 of SEQ ID NO:9. The coding region of the mRNA sequence encoding H3 is shown in positions 133 to 1104 of SEQ ID NO:10.

[0044] According to some embodiments of the present invention, the amino acid sequence of A17 is shown in positions 1 to 377 of SEQ ID NO:11. The coding region of the mRNA sequence encoding A17 is shown in positions 133 to 1263 of SEQ ID NO:12.

[0045] According to some embodiments of the present invention, the amino acid sequence of A30 is shown in positions 1 to 146 of SEQ ID NO:13. The coding region of the mRNA sequence encoding A30 is shown in positions 133 to 570 of SEQ ID NO:14.

[0046] According to some embodiments of the present invention, the amino acid sequence of H2 is shown in positions 1 to 189 of SEQ ID NO:15. The coding region of the mRNA sequence encoding H2 is shown in positions 133 to 699 of SEQ ID NO:16.

[0047] According to some embodiments of the present invention, the amino acid sequence of A28 is shown in positions 1 to 509 of SEQ ID NO:17. The coding region of the mRNA sequence encoding A28 is shown in positions 133 to 1659 of SEQ ID NO:18.

[0048] According to some embodiments of the present invention, the amino acid sequence of A21 is shown as positions 1 to 115 of SEQ ID NO:19. The coding region of the mRNA sequence encoding A21 is shown as positions 133 to 477 of SEQ ID NO:20.

[0049] According to some embodiments of the present invention, the amino acid sequence of G2 is shown as positions 1 to 111 of SEQ ID NO:21. The coding region of the mRNA sequence encoding G2 is shown as positions 133 to 465 of SEQ ID NO:22.

[0050] According to some embodiments of the present invention, the amino acid sequence of I2 is shown in positions 1 to 73 of SEQ ID NO:23. The coding region of the mRNA sequence encoding I2 is shown in positions 133 to 351 of SEQ ID NO:24.

[0051] According to some embodiments of the present invention, the amino acid sequence of E8 is shown in positions 1 to 304 of SEQ ID NO:25. The coding region of the mRNA sequence encoding E8 is shown in positions 133 to 1044 of SEQ ID NO:26.

[0052] According to some embodiments of the present invention, the amino acid sequence of C20.5 is shown in positions 1 to 49 of SEQ ID NO:27. The coding region of the mRNA sequence encoding C20.5 is shown in positions 133 to 279 of SEQ ID NO:28.

[0053] According to some embodiments of the present invention, the amino acid sequence of A14 is shown in positions 1 to 70 of SEQ ID NO:29. The coding region of the mRNA sequence encoding A14 is shown in positions 133 to 342 of SEQ ID NO:30.

[0054] According to some embodiments of the present invention, the amino acid sequence of E13 is shown in positions 1 to 551 of SEQ ID NO:31. The coding region of the mRNA sequence encoding E13 is shown in positions 133 to 675 of SEQ ID NO:32.

[0055] According to some embodiments of the present invention, the amino acid sequence of the monkeypox virus T-cell epitope peptide is shown in positions 77 to 1823 of SEQ ID NO:69. The coding region of the mRNA sequence encoding the monkeypox virus T-cell epitope peptide is shown in positions 292 to 5532 of SEQ ID NO:70.

[0056] According to some embodiments of the present invention, the mRNA further includes at least one of a 5'UTR, a Kozak sequence, a sequence encoding a signal peptide, a sequence encoding a tag, a stop codon, a sequence encoding ubiquitin, a 3'UTR, and a PolyA sequence.

[0057] According to some embodiments of the present invention, from 5' to 3', the mRNA sequentially comprises a 5'UTR, a kozak sequence, a sequence encoding a signal peptide, a coding region, a stop codon, a 3'UTR, and a polyA.

[0058] According to some embodiments of the present invention, from 5' to 3', the mRNA sequentially comprises a 5' UTR, a sequence encoding ubiquitin, a coding region, a stop codon, a 3' UTR, and polyA. The amino acid sequence corresponding to the ubiquitin sequence is shown in positions 2 to 76 of SEQ ID NO:69; the nucleotide sequence encoding ubiquitin is shown in positions 67 to 291 of SEQ ID NO:70.

[0059] According to some embodiments of the present invention, the label includes at least one of the following: Flag label, His label, MBP label, HA label, myc label, GST label, and SUMO label.

[0060] According to some embodiments of the present invention, the signal peptide includes at least one of tPA signal peptide, IgG signal peptide, GnRH signal peptide, and ALB signal peptide.

[0061] According to some embodiments of the present invention, each mRNA includes a 5' end capping modification.

[0062] According to some embodiments of the present invention, the antigenic epitopes are a combination (A1), and the mass ratio of the mRNAs encoding antigenic epitopes A29, M1, B6, and A35 is 1:(0.5-3):(0.5-3):(0.5-3).

[0063] According to some embodiments of the present invention, the antigenic epitopes are a combination (A2), and the mass ratio of the mRNAs encoding antigenic epitopes A29, M1, B6, A35, H3, A17, A30, and H2 is 1:(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3).

[0064] According to some embodiments of the present invention, the antigenic epitopes are a combination (A3), and the mass ratio of mRNA encoding antigenic epitopes A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, and I2 is 1:(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3).

[0065] According to some embodiments of the present invention, the antigenic epitopes are a combination (A4), and the mass ratio of mRNA encoding antigenic epitopes A29, M1, B6, A35, H3, A17, A30, H2, E8, C20.5, A14, and E13 is 1:(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3).

[0066] According to some embodiments of the present invention, the antigenic epitopes are a combination (A5), and the mass ratio of mRNA encoding antigenic epitopes A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, I2, E8, C20.5, A14, and E13 is 1:(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3).

[0067] According to some embodiments of the present invention, the antigenic epitopes are a combination (A7) encoding the mRNAs of antigenic epitopes A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, I2, and monkeypox virus T-cell epitope peptides in a mass ratio of 1:(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-3):(0.5-8). Combination (A7) achieves synergistic activation of humoral and cellular immunity, providing highly effective protection against monkeypox virus infection.

[0068] A monkeypox virus antigenic epitope protein composition according to a second aspect of the present invention comprises a protein encoded by the monkeypox virus antigenic epitope mRNA composition described in the first aspect embodiment.

[0069] According to a third aspect of the present invention, the biological material relating to the monkeypox virus epitope mRNA composition of the first aspect embodiment or the monkeypox virus epitope protein composition of the second aspect embodiment is any one of (B1) to (B5):

[0070] (B1) A nucleic acid molecule encoding the monkeypox virus antigenic epitope mRNA composition described in the first aspect embodiment or the monkeypox virus antigenic epitope protein composition described in the second aspect embodiment;

[0071] (B2) An expression cassette containing the nucleic acid molecule described in (B1);

[0072] (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2);

[0073] (B4) A recombinant microorganism containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3);

[0074] (B5) A transgenic cell line containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3).

[0075] According to some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the monkeypox virus antigenic epitope mRNA composition or the monkeypox virus antigenic epitope protein composition in host cells.

[0076] According to some embodiments of the present invention, the recombinant vector is selected from plasmids, granules, bacteriophages or viral vectors.

[0077] According to some embodiments of the present invention, the recombinant microorganisms and the transgenic cells do not contain reproductive material.

[0078] According to some embodiments of the present invention, the recombinant microorganism includes at least one of bacteria and fungi. For example, it can be Escherichia coli.

[0079] According to some embodiments of the present invention, the transgenic cells include at least one of mammalian cells and insect cells. For example, they can be 293T cells.

[0080] A vaccine according to a fourth aspect of the present invention comprises a monkeypox virus epitope mRNA composition as described in the first aspect embodiment or a monkeypox virus epitope protein composition as described in the second aspect embodiment; and optionally, an adjuvant.

[0081] According to some embodiments of the present invention, the adjuvant includes at least one of lipid nanoparticle adjuvants, oil-in-water adjuvants, polymer and water adjuvants, water-in-oil adjuvants, and aluminum hydroxide adjuvants.

[0082] According to some embodiments of the present invention, the adjuvant includes protonable cationic lipids, structural lipids, auxiliary lipids, and polyethylene glycol-modified lipids.

[0083] According to some embodiments of the present invention, the protonable cationic lipid includes SM102.

[0084] According to some embodiments of the present invention, the structural lipids include cholesterol or its derivatives.

[0085] According to some embodiments of the present invention, the auxiliary lipids include at least one of distearate phosphatidylcholine (DSPC), DOPE, DOPG, and DOPS.

[0086] According to some embodiments of the present invention, the PEGylated lipid comprises DMG-PEG2000.

[0087] According to some embodiments of the present invention, the molar ratio of the protonable cationic lipid, auxiliary lipid, structural lipid and polyethylene glycol-modified lipid is 50:(2-14.5):(35-45):(0.5-3).

[0088] According to some embodiments of the present invention, the vaccine is a dosage form for intramuscular, intradermal, or subcutaneous administration.

[0089] The method for preparing a vaccine according to the fourth aspect of the fifth aspect embodiment of the present invention includes the following steps:

[0090] The vaccine is obtained by preparing the monkeypox virus antigenic epitope mRNA composition or the mixture of the monkeypox virus antigenic epitope protein composition and the adjuvant. Those skilled in the art may choose to mix each mRNA separately with the adjuvant to obtain a complex, and then mix the complexes together; or they may choose to mix the mRNA mixture directly with the adjuvant.

[0091] According to some embodiments of the present invention, the adjuvant includes protonable cationic lipids, structural lipids, auxiliary lipids, and polyethylene glycol-modified lipids; the preparation method includes the following steps:

[0092] A mixture of an organic solvent containing protonable cationic lipids, structural lipids, auxiliary lipids, and polyethylene glycol-modified lipids, and a buffer containing the monkeypox virus antigen epitope mRNA composition, was prepared to obtain liposome encapsulations.

[0093] According to some embodiments of the present invention, the average hydrated particle size of the liposome encapsulation is 50nm-200nm. For example, it can be 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, or 200nm.

[0094] According to some embodiments of the present invention, the preparation method further includes replacing the buffer solution, concentration, and / or sterilization.

[0095] According to some embodiments of the present invention, the organic solvent includes, but is not limited to, ethanol.

[0096] According to some embodiments of the present invention, the buffer solution includes, but is not limited to, citrate buffer solution.

[0097] According to some embodiments of the present invention, the volume ratio of the organic solvent to the buffer solution is 1:3.

[0098] According to some embodiments of the present invention, the adjuvant includes aluminum hydroxide adjuvant; the preparation method includes the following steps:

[0099] Prepare a mixture of aluminum hydroxide adjuvant with the monkeypox virus antigenic epitope mRNA composition and / or the monkeypox virus antigenic epitope protein composition.

[0100] The use of the monkeypox virus antigenic epitope mRNA composition described in the first aspect embodiment of the sixth aspect embodiment of the present invention, the monkeypox virus antigenic epitope protein composition described in the second aspect embodiment, the biological material described in the second aspect embodiment, or the vaccine described in the fourth aspect embodiment in the preparation of a medicament for the prevention and / or treatment of monkeypox virus infection.

[0101] The present invention has at least the following beneficial effects:

[0102] The monkeypox virus antigenic epitope mRNA composition and monkeypox virus antigenic epitope protein composition of the present invention can produce a high level of immune protection against monkeypox virus and can be used to prepare drugs for the prevention and treatment of monkeypox virus, and have broad application prospects in clinical practice.

[0103] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0104] Figure 1 The results show the expression of each protein; the left lane M finger is the protein marker (Thermo, CAT: 26619), and the Mock finger is the blank control group.

[0105] Figure 2 The results show the levels of neutralizing antibodies produced in mice after immunization with 35 antigenic epitopes.

[0106] Figure 3 The graph shows the particle size analysis results for Mix-4, Mix-8, and Mix-12.

[0107] Figure 4 The results of neutralizing antibody titers in the serum of mice immunized with Mix-4, Mix-8, and Mix-12 are presented.

[0108] Figure 5 Results of viral load detection in the lungs of suckling mice in Mix-4, Mix-8, and Mix-12 groups;

[0109] Figure 6 The results of IFN-γ detection in the spleen of suckling mice in Mix-4, Mix-8 and Mix-12 groups after passive immunization;

[0110] Figure 7 The results show the neutralizing antibody titers in the serum of mice immunized with Mix-12 and Mix-12-2, respectively; where ## indicates a highly significant difference (p<0.01);

[0111] Figure 8 The results show the neutralizing antibody titers in the serum of mice immunized with Mix-12 and Mix-16, respectively; where ## indicates a highly significant difference (p<0.01);

[0112] Figure 9 CD4 in mice immunized with MPX-EPs + With CD8 + T cell content detection results; A: CD4 + T cell content, B:CD8+ T cell count;

[0113] Figure 10 Results of IFN-γ detection in the spleen of mice immunized with MPX-EPs;

[0114] Figure 11 Results of IFN-γ and TNF-α detection in the spleen of mice immunized with MPX-EPs; A: Cell clustering diagram, B: TNF-α in diagram A. + IFN-γ + Cell count;

[0115] Figure 12 Results of viral load detection in the lungs of mice immunized with MPX-EPs after challenge;

[0116] Figure 13 The results of lung pathological staining in mice immunized with MPX-EPs after challenge.

[0117] Figure 14 The graph shows the particle size analysis results for MPX-m-Mix.

[0118] Figure 15 The results of detection of binding antibodies (IgG) and neutralizing antibodies (nAb) in MPX-m-Mix immunized mice; A: binding antibody, B: neutralizing antibody;

[0119] Figure 16 The results of IFN-γ and IL-4 detection in the spleen of mice immunized with MPX-m-Mix and MPX-p-Mix; A: IFN-γ, B: IL-4;

[0120] Figure 17 Results of viral load detection in the lungs of mice immunized with MPX-m-Mix and MPX-p-Mix after challenge;

[0121] Figure 18 The results show the pathological staining of the lungs of mice immunized with MPX-m-Mix and MPX-p-Mix after challenge. Detailed Implementation

[0122] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0123] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0124] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0125] Unless otherwise specified, in this invention, the term "identity" refers to the similarity between amino acid sequences or nucleotide sequences. "Identity" can be evaluated by computer software (e.g., the computer program BLAST using default parameters).

[0126] Unless otherwise specified, in this invention, "about" or "around" indicates that the allowable error is within ±5%.

[0127] Unless otherwise specified, the mRNA preparation steps in this invention are as follows:

[0128] (1) The target gene sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. The target gene sequence, from 5' to 3' end, consisted of the T7 promoter, 5' untranslated region (5'UTR), kozak sequence, tPA signal peptide sequence, coding region, stop codon, 3' untranslated region (3'UTR), polyadenylated acid (polyA), and BsaI restriction site. The coding region encoded the corresponding antigen; the addition of the BsaI restriction site facilitated plasmid linearization. Using genetic engineering, the target gene sequence was constructed into the cloning vector pUC57, plasmids were constructed, and transformed into competent E. coli cells for bacterial amplification and plasmid extraction.

[0129] The nucleotide sequence of the T7 promoter is TAATACGACTCACTATAGG;

[0130] The nucleotide sequence of the 5'UTR is GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG;

[0131] The nucleotide sequence of the kozak sequence is GCCACC;

[0132] The nucleotide sequence of the tPA signal peptide is ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGT TCGTGAGCGCCTCC;

[0133] The nucleotide sequence of the 3'UTR is GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCC AGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG;

[0134] The nucleotide sequence of polyA is AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA;

[0135] (2) The extracted plasmids were linearized by digestion with Bsa I restriction endonuclease (catalog number: GMP4303-PC-01, Nanjing Novizan Biotechnology Co., Ltd.) (reaction at 37℃ for 1 h, digestion system: 10×Bsa I Digestion Buffer 2 μL, Bsa I 1 μL, plasmid 5 μg, water to 20 μL). The linearized plasmids were recovered using a 5-minute DNA rapid purification kit (catalog number: EP101-01, Beijing TransGen Biotech Co., Ltd.).

[0136] (3) mRNA was synthesized in vitro using the T7 RNA Synthesis Kit (catalog number: 10673ES, Yisheng Biotechnology (Shanghai) Co., Ltd.), and a capping structure was added to the mRNA using 7MeG(3'Ome)-5'-ppp-5'-A(OMe)G (catalog number: Cap101, Cangzhou Weikexin Biochemical Technology Co., Ltd.). After the reaction, 1.5 volumes of lithium chloride solution were added to the system, and the mixture was incubated at -20℃ for 1 h to precipitate the mRNA. Subsequently, the mixture was centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the mRNA was redissolved in DEPC-treated water. The concentration of RNA was determined using a Nanodrop device and stored at -80℃.

[0137] (4) 293T cells were evenly seeded into 24-well culture plates. 0.8 μg of mRNA was introduced into HEK-293T cells per well using PEI transfection reagent (catalog number: FT401-01, Beijing TransGen Biotech Co., Ltd.). After culturing for 48 hours, cell samples were collected, and their expression capacity was examined using a Western blot experiment. 293T cells that did not transcribe mRNA served as a blank control group.

[0138] Partial protein expression results are as follows Figure 1 As shown.

[0139] Compared with the blank control group, the experimental group transfected with mRNA showed the target band on the Western blot map, and the size was consistent with the expected protein size, which confirmed that the mRNA could successfully express the protein in the cell.

[0140] Unless otherwise specified, the preparation steps of the mRNA vaccine (LNP-mRNA) in this invention are as follows:

[0141] (1) Preparation of alcohol phase: 35.4 mg lipid (SM102), 7.9 mg distearate phosphatidylcholine, 14.8 mg cholesterol and 3.76 mg DMG-PEG2000 (molar ratio = 50:10:38.5:1.5) were dissolved in 13.5 mL of anhydrous ethanol.

[0142] (2) Preparation of aqueous phase: Dissolve 500 μg mRNA in 7.5 mL of 50 mM citrate buffer (pH 4.0).

[0143] (3) The alcohol and aqueous phases were mixed at a volume ratio of 1:3 using a microfluidic device for mRNA packaging. The mixture was then diluted with RNase-free PBS buffer and concentrated using a 30 kDa ultrafiltration tube. An equal volume of 20 wt% sucrose PBS solution was added to adjust the mRNA concentration to 100 μg / mL, and the sucrose concentration to 10 wt%. Finally, the mixture was filtered through a 0.22 μm filter to obtain the prepared LNP-mRNA vaccine, which was then aliquoted and stored at -20°C.

[0144] Unless otherwise specified, the MPXV virus (Genebank: PP778666.1) used in the embodiments of the present invention was isolated from a patient in Guangzhou, China.

[0145] Example 1: Immunological effects of different monkeypox virus surface proteins as antigens

[0146] 1. By analyzing the surface proteins of mature viral particles (MVs) and extracellular viral particles (EVs) of monkeypox virus, 34 major antigenic epitopes (C15, M5, A21, A29, M1, L5, H3, E8, A17, A30, A28, H2, I2, I5, E13, A10, A14, A15, A15.5, A18, C20.5, G2, G4, G10, A27, C18, F2, C2, C19, A35, A36, A38, B2, B6) were selected, and mRNA vaccines were constructed for each.

[0147] Among them, the amino acid sequence of A29 protein is MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKI TNITTKFEQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYEHHHHHH (SEQ ID NO:1), and the sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGACGGAACCCUGUUUCCCGGCGACGAUGAUCUGGCCAUUCCUGCUACAGAGUUCUUCAGCACCAAGGCCGCUAAAAACCCCGAAACCAAGCGCGAAGCCAUCGUCAAGGCCUACGGCGAUGACAACGAGGAAACACUGAAGCAACGGCUGACCAAUCUGGAAAAGAAGAUCACAAACAUCACCACCAAGUUCGAGCAGAUCGAGAAGUGCUGUAAAAGAAACGACGAGGUGCUGUUUCGGCUGGAAAACCACGCCGAGACACUGAGAGCCGCCAUGAUCAGCCUGGCUAAGAAAAUCGACGUGCAGACCGGCAGACACCCAUACGAGCACCACCACCACCAUCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:2).

[0148]

[0149]

[0150] The amino acid sequence of the A35 protein is MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCM SANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLI DYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCTHHHHHH (SEQ ID NO:7).The sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGAUGACCCCUGAGAACGACGAGGAACAGACCAGCGUGUUCAGCGCCACCGUGUACGGCGAUAAGAUCCAGGGCAAGAACAAGCGGAAGAGAGUGAUCGGCCUGUGCAUCAGAAUCAGCAUGGUGAUUAGCCUGCUGAGCAUGAUCACCAUGAGCGCCUUCCUGAUCGUGCGGCUGAACCAGUGCAUGUCUGCCAACAAAGCCGCCAUCACCGACUCUGCUGUGGCCGUGGCCGCUGCUUCUUCUACACACAGAAAGGUGGUGUCCAGCACAACACAGUACGACCACAAGGAAUCCUGUAAUGGCCUGUACUACCAAGGCAGCUGCUACAUCCUGCACAGCGAUUAUAAGAGCUUCGAGGAUGCCAAGGCCAAUUGCGCCGCUGAAAGCAGCACACUGCCUAACAAGUCCGAUGUGCUGACCACAUGGCUGAUCGACUACGUCGAGGACACCUGGGGCAGCGACGGCAACCCCAUCACCAAGACCACCAGCGACUACCAGGACAGCGACGUGUCUCAGGAGGUGCGGAAAUACUUCUGCACCCACCACCAUCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:8).

[0151]

[0152]

[0153] The amino acid sequence of the A30 protein is MNSLSIFFIVVATAAVCLLFIQSYSIYENYGNIKEFNATHAAFEYSKSIGGTPALDRRVQDVND TISDVKQKWRCVVYPGNGFVSASIFGFQAEVGPNNTRSIRKFNTMRQCIDFTFSDVINIDIYNPCIAPNINNTECQFLKSVLHH HHHH (SEQ ID NO:13).The sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA AGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGAACAGCCUGAGCAUCUUUUUCAUCGUGGUUGCCACAGCCGCUGUGUGCCUGCUGUUCAUCCAGAGCUACAGCAUCUACGAGAAUUACGGCAAUAUUAAGGAGUUCAACGCCACCCACGCCGCCUUCGAGUACUCCAAGUCCAUUGGCGGUACACCUGCUCUGGACAGGAGAGUGCAGGACGUGAACGACACCAUCAGCGAUGUGAAGCAGAAAUGGCGGUGCGUGGUCUACCCCGGCAACGGCUUCGUGUCUGCUAGCAUCUUCGGCUUUCAGGCCGAGGUGGGCCCUAACAACACAAGAAGCAUCAGAAAGUUCAACACCAUGAGACAAUGUAUCGACUUCACCUUCAGCGACGUGAUCAACAUCGAUAUCUACAACCCUUGUAUCGCCCCCAACAUCAACAACACCGAGUGCCAGUUCCUGAAAAGCGUGCUGCACCACCAUCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:14).,

[0154] The amino acid sequence of the H2 protein is MDKTTLSVNACNLEYVREKAIVGVQAAKTSTLIFFVIILAISALLLWFQTSDNPVFNELTRYMR IKNTVNDWKSLTDSKTKLESDRGRLLAAGKDDIFEFKCVDFGAYFIAMRLDKKTYLPQAIRRGTGDAWMVKKAAKVDPSA QQFCQYLIKHKSNNVITCGNEMLNELGYSGYFMSPHWCSDLSNMEHHHHHH (SEQ ID NO:15).The sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGACAAGACAACCCUGUCUGUGAAUGCCUGUAAUCUGGAAUACGUCAGAGAGAAGGCCAUCGUGGGCGUGCAGGCUGCCAAGACCUCCACACUGAUCUUUUUCGUGAUCAUCCUGGCCAUUAGCGCCCUGCUGCUGUGGUUCCAGACAAGCGACAACCCCGUGUUCAACGAGCUGACAAGAUACAUGCGGAUCAAGAACACCGUGAACGACUGGAAGUCCCUGACCGACAGCAAGACCAAGCUGGAAUCUGAUAGAGGCAGACUGCUGGCAGCUGGCAAGGAUGACAUCUUUGAGUUCAAGUGCGUGGACUUCGGCGCUUAUUUCAUCGCCAUGAGACUGGACAAAAAGACCUACCUGCCUCAGGCCAUCAGGCGGGGCACCGGCGACGCCUGGAUGGUGAAGAAAGCCGCUAAAGUCGACCCCAGCGCCCAGCAAUUUUGCCAGUACCUGAUCAAGCACAAGAGCAACAACGUGAUCACAUGCGGCAACGAGAUGCUGAACGAACUGGGCUACAGCGGCUACUUCAUGAGCCCACACUGGUGCAGCGAUCUGAGCAACAUGGAACACCACCAUCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:16).

[0155]

[0156] The amino acid sequence of A21 protein is MITLFLILCYFILIFNIIVPAISEKMRRERAAYVNYKRLNKNFICVDDRLFSYNFTTSGIKAKVA VNKNVPIPCSKINEVNNKDVDTLYCDKDRDDIPGFARSCYRAYSDLFFTTHHHHHH (SEQ ID NO:19), and the sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGAUCACACUGUUUCUGAUCCUGUGCUACUUCAUCCUGAUUUUCAACAUCAUCGUGCCCGCUAUCAGCGAGAAGAUGAGGCGGGAAAGAGCUGCUUAUGUGAAUUACAAGCGGCUGAACAAGAAUUUCAUCUGCGUGGACGACAGACUGUUCAGCUACAACUUCACCACCAGCGGCAUCAAGGCCAAGGUGGCCGUGAACAAGAACGUGCCUAUCCCAUGUUCUAAGAUCAACGAGGUGAACAACAAAGAUGUGGACACCCUGUACUGCGACAAGGAUAGAGAUGACAUCCCUGGCUUCGCCAGAAGCUGCUACAGAGCCUACAGCGACCUGUUCUUCACCACACACCACCAUCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:20).

[0157] The amino acid sequence of G2 protein is MASLLYLILFLLFVCISYYFTYYPTNKLQAAVMETDRENAIIRQRNEEIPTRTLDTAIFTDASTV SSAQIHLYYNSNIGKIIMSLNGKKHTFNLYDDNDIRTLLPILLLSKHHHHHH (SEQ ID NO:21), and the sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGCUUCUCUGCUCUACCUGAUCCUGUUCCUGCUGUUUGUGUGCAUCAGCUACUACUUCACCUACUAUCCUACAAACAAGCUGCAGGCCGCUGUGAUGGAAACCGAUAGAGAGAAUGCCAUUAUCAGACAGCGGAACGAGGAAAUCCCCACAAGAACCCUGGACACCGCCAUCUUCACAGAUGCCAGCACCGUGUCCAGCGCCCAGAUCCACCUGUACUACAACAGCAAUAUCGGCAAGAUCAUCAUGAGCCUGAACGGCAAAAAGCACACCUUCAACCUGUACGACGACAACGACAUCCGGACCCUGCUGCCUAUCCUGCUGCUGAGCAAGCACCACCACCAUCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:22).

[0158] The amino acid sequence of the I2 protein is MDKLYAAIFGVFMGSQEDDLTDFIEIVKSVLSDEKTVTSTNNTGCWGWYWLIIIFFIVLILLLLIYLYLKVVWHHHHHH (SEQ ID NO:23), and the sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGAUAAGCUGUACGCCGCUAUCUUCGGCGUGUUCAUGGGCAGCCAGGAAGAUGACCUGACCGACUUCAUCGAGAUCGUUAAGAGCGUGCUGAGCGACGAGAAGACAGUGACCAGCACCAACAACACAGGCUGCUGGGGCUGGUACUGGCUGAUUAUCAUCUUUUUCAUCGUCCUGAUCCUGCUGCUGCUGAUCUACCUGUAUCUGAAGGUGGUGUGGCACCACCACCAUCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:24).

[0159]

[0160] The amino acid sequence of the C20.5 protein is MVIGLVIFVSVAATIVGVLSNVLDMIMYVEENNEEDAKIKEEQELLLLYHHHHHH (SEQ ID NO:27), and the corresponding mRNA sequence is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC GCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUGUGAGCGCCUCCAUGGUCAUCGGCCUGGUGAUCUUCGUGUCCGUGG CCGCUACCAUCGUGGGCGUGCUGAGCAAUGUGCUGGACAUGAUCAUGUACGUGGAAGAGAACAACGAGGAAGAUGCCAAGAUCAAGGAAGAGCAGGAGCUGCUGCUGCUGUACCAUCACCA CCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:28).

[0161] The amino acid sequence of A14 protein is MIGILLLIGICVAVTVTILYTLYNKIKNPQNPNPSPNLNSPPPETRNTKFVNNLEKDHISSLYNLVKSSAHHHHHH (SEQ ID NO:29), and the sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGAUCGGCAUUCUGCUGCUGAUCGGCAUCUGCGUCGCCGUGACCGUGACAAUCCUGUACACCCUGUACAACAAGAUCAAGAACCCCCAGAACCCCAACCCUUCUCCUAAUCUGAACAGCCCUCCACCUGAGACAAGAAACACCAAGUUCGUGAACAACCUGGAAAAGGACCACAUCAGCAGCCUGUACAAUCUGGUGAAGUCCAGCGCCCACCACCACCAUCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:30).

[0162]

[0163]

[0164] The amino acid sequence of M5 protein is MENVPNVYFNPVFIEPTFKHSLLSVYKHRLIVLFEVFVVFILIYVFFRSELNMFFMPKRKIPDPIDRLRRANLACEDDKLMIYGLPWITTQTSALSINSKPIVYKDCAKLLRSINGSQPVSLNDVLRRHHHHHH (SEQ ID NO:35), and the sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGAAAACGUGCCAAACGUCUACUUCAACCCCGUGUUUAUCGAGCCAACAUUCAAGCACAGCCUGCUGAGCGUGUACAAGCACAGACUGAUUGUGCUGUUCGAGGUGUUCGUGGUGUUCAUCCUGAUCUAUGUGUUUUUCAGAAGCGAGCUGAAUAUGUUCUUCAUGCCUAAGCGGAAGAUCCCUGAUCCUAUCGACAGACUGCGCAGAGCCAAUCUGGCUUGUGAAGAUGACAAGCUGAUGAUCUACGGCCUGCCUUGGAUCACCACACAGACCAGCGCUCUGUCUAUCAACAGCAAGCCCAUCGUGUACAAAGACUGCGCCAAGCUGCUGCGGAGCAUCAACGGCUCUCAGCCUGUUUCUCUGAACGACGUGCUGAGAAGACACCACCAUCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:36).

[0165] The amino acid sequence of L5 protein is MTDEQIYAFCDTNKDDIRCKCIYPDKSIVRIGIDTRLPYYCWYEPCKRSDALLPASLKKNISRC NVSDCTISLGNVSITDSKLDVNNVCDSKRVATENIAVRYLNQEIRYPIIDIKWLPIGLLALAILILAFFHHHHHH (SEQ ID NO:37), and the sequence of its corresponding mRNA is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAG CCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGACCGACGAGCAAAUCUACGCCUUCUGCGACACCAACAAGGACGACAUCCGGUGCAAAUGCAUCUACCCUGAUAAGAGCAUCGUUAGAAUCGGCAUCGAUACAAGACUGCCCUACUACUGCUGGUACGAGCCUUGCAAGCGGAGCGACGCUCUGCUGCCUGCUUCUCUGAAGAAGAACAUCAGCAGAUGUAAUGUGUCCGAUUGCACCAUCAGCCUGGGCAAUGUGUCCAUCACCGAUAGCAAGCUGGACGUGAACAACGUGUGCGACAGCAAGAGAGUGGCCACCGAGAACAUCGCCGUGAGAUACCUGAACCAGGAGAUCAGAUAUCCUAUCAUCGACAUUAAGUGGCUGCCAAUUGGCCUGCUCGCCCUGGCUAUCCUGAUCCUGGCCUUUUUCCACCACCAUCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:38).

[0166] The amino acid sequence of the I5 protein is MVDAITVLTAICITVLMLLMVISGTAMIVKELNPNDIFTMQSLKFNRTVTIFKYIGLFIYIPGTIILY ATYVKSLLMKNHHHHHH (SEQ ID NO:39), and the corresponding mRNA sequence is GAAAUAAGAGAGAAAAGAAGAGU (SEQ ID NO:40).

[0167] The amino acid sequence of A10 protein is MSCYTAILKSVGGLALFQVANGAIDLCRHFFMYFCEQKLRPNSFWFVVVRAIASMIMYLVL GIALLYISEQDDKKNTNNDSNSNNDKRNVSSINSNSSHKHHHHHH (SEQ ID NO:41), and the sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGAGCUGCUACACAGCCAUCCUGAAGAGCGUGGGCGGCCUGGCCCUGUUCCAGGUCGCCAACGGCGCUAUCGACCUGUGUAGACACUUCUUCAUGUACUUCUGUGAACAGAAACUGCGGCCUAACUCUUUUUGGUUCGUGGUUGUGCGGGCCAUUGCCAGCAUGAUCAUGUAUCUGGUGCUGGGCAUCGCCCUGCUGUACAUCAGCGAGCAGGACGAUAAGAAGAACACCAACAACGACAGCAAUUCUAACAAUGAUAAGCGGAACGUGUCCAGCAUCAACAGCAACAGCAGCCACAAGCAUCACCACCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:42).

[0168] The amino acid sequence of A15 protein is MDMMLMIGNYFSGVLIAGIILLILSCIFAFIDFSKSTSPTRTWKVLSIMAFILGIIITVGMLIYSM WGKHCAPHRVSGVIHTNHSDISMNHHHHHH (SEQ ID NO:43), and the sequence of the corresponding mRNA expressing it is GAAAUAAGAGAG AAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGAUAUGAUGCUGAUGAUCGGCAAUUACUUCAGCGGAGUCCUGAUCGCUGGCAUCAUUCUGCUGAUCCUGAGCUGCAUCUUCGCCUUUAUCGACUUCUCCAAGAGCACAAGCCCCACAAGAACCUGGAAGGUGCUGAGCAUCAUGGCCUUCAUCCUGGGCAUCAUCAUCACCGUGGGCAUGCUGAUCUACAGCAUGUGGGGCAAGCACUGCGCCCCUCACCGGGUGUCUGGCGUGAUCCACACCAACCACAGCGACAUCAGCAUGAACCAUCAUCACCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:44).

[0169] The amino acid sequence of the A15.5 protein is MISNYEPLLLLVITCCVLLFNFTISSKTKIDIIFAVQTIVFIWFIFHFVYSAIHHHHHH (SEQ ID NO:45), and the corresponding mRNA sequence is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC GCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGAUCAGCAACUACGAGCCUCUGCUGCUGCUCGUGA UCACCUGCUGCGUGCUGCUGUUCAACUUCACCAUUAGCAGCAAGACAAAGAUCGACAUCAUCUUCGCCGUGCAGACCAUCGUGUUCAUCUGGUUCAUCUUUCACUUCGUGUACAGCGCCAUCCA CCACCACCAUCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:46).

[0170]

[0171] The amino acid sequence of the G4 protein is MDAMKRGLCCVLLLCGAVFVSASMKNVLIIFGKPYCSICENVSEAVEELKSEYDILHVDILSFF LKDGDSSMLGDVKRGTLIGNFAAHLSNYIVSIFKYNPQTKQMAFVDINKSLDFTKTDKSLVNLEILKSEIEKATYGVWPPVTE HHHHHH (SEQ ID NO:49).The sequence of the corresponding mRNA expressing it is GAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGGACGCCAUGAAAAGAGGCCUGUGCUGCGUGCUGCUGCUGUGUGGCGCUGUGUUCGUGUCCGCCAGCAUGAAAAAUGUUCUGAUCAUCUUCGGCAAGCCUUACUGCAGCAUCUGCGAGAACGUGUCCGAGGCCGUCGAGGAACUGAAGUCUGAAUACGACAUCCUGCACGUGGACAUCCUCAGCUUCUUCCUGAAGGACGGCGACAGCAGCAUGCUGGGCGAUGUGAAGCGGGGCACCCUGAUCGGAAAUUUCGCCGCUCACCUGAGCAACUAUAUCGUGUCCAUCUUUAAGUACAACCCCCAGACCAAGCAGAUGGCCUUUGUGGAUAUUAACAAGAGCCUGGACUUCACAAAGACCGAUAAGAGCCUGGUGAACCUGGAAAUCCUGAAAAGCGAGAUCGAGAAGGCCACAUACGGCGUGUGGCCUCCAGUGACCGAGCACCACCACCACCAUCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:50).,

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178] The amino acid sequence of A36 protein is MKSLNRQTVSRFRKLSVPAAIMMLLSTIISGIGTFLHYREELMPSACANGWIQYDKHCYLDTNIKMSTDNAVYQCRKLRARLPRPDTRHLRVLFSIFYKDYWVSLKKTNDKWLDINNDKDIDISKLTNFKQLNSTTDSEACYIYKSGKLVKTVCKSTQSVLCVKRFYKHHHHHH(SEQ ID NO:63).The sequence of the corresponding mRNA expressing it is GAAAUAAGAGAG AAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCGCUAGCCUCGAGGCCACCAUGGACGCCAUGAAGAGGGGGCUGUGCUGCGUGCUGCUGCUGUGCGGAGCCGUGUUCGUGAGCGCCUCCAUGAAGAGCCUGAAUAGACAGACCGUGAGCAGAUUCAGAAAGCUUUCUGUGCCAGCCGCUAUCAUGAUGCUGCUGAGCACAAUCAUCUCCGGCAUCGGCACAUUCCUGCACUACCGGGAAGAGCUGAUGCCUAGCGCUUGUGCCAACGGCUGGAUUCAGUACGACAAGCACUGCUACCUGGACACCAACAUCAAGAUGAGCACAGACAACGCCGUGUACCAGUGCAGGAAGCUGAGAGCCAGACUGCCCAGACCUGAUACAAGACACCUGCGGGUGCUGUUUAGCAUCUUCUACAAGGACUACUGGGUGAGCCUGAAGAAGACAAAUGAUAAGUGGCUGGAUAUUAACAACGACAAAGAUAUCGACAUCAGCAAGCUGACCAACUUCAAGCAGCUGAACAGCACCACCGACAGCGAGGCCUGCUACAUCUACAAAAGCGGAAAGCUGGUGAAGACCGUUUGUAAAAGCACCCAGUCUGUGCUGUGUGUGAAACGGUUCUAUAAGCACCAUCACCACCACCACUGAGGUACCGAUAUCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO:64).

[0179]

[0180]

[0181] 2. One hundred and seventy-five SPF-grade female BALB / c mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd., 8 weeks old) were randomly divided into 35 groups of five mice each. Each group received an intramuscular injection of 10 μg mRNA vaccine. Three weeks after the initial immunization, a booster injection of the same dose of vaccine was administered. The control group received an intramuscular injection of 100 μL PBS. Three weeks after the booster immunization, blood was collected from the mice, serum was separated, and the levels of neutralizing antibodies produced after immunization of mice at each antigenic epitope were measured. The neutralizing activity of MPXV in the serum samples of immunized mice was assessed using a plaque-based neutralization assay. The assay was performed at a density of 1.0 × 10⁻⁶ cells per well. 5 Vero E6 cells were seeded at a density of [number] cells and cultured at 37°C for 2 hours. Serum samples were serially diluted 2-fold in 96-well plates with PBS, and then 100 μL of the diluted serum sample was incubated with 100 μL of PBS containing approximately 100 PFU MPXV at 37°C for 2 hours. 200 μL of the serum-virus mixture was added to Vero E6 cells. Plaque formation was examined 3-4 days post-infection, and the inhibition ratio was calculated.

[0182]

[0183] Where a represents the number of plaques produced in the 50-fold diluted blank control group mouse serum treatment group, and b represents the number of plaques produced in the 50-fold diluted test mouse serum treatment group.

[0184] The results are as follows Figure 2 As shown.

[0185] Different antigens produced different levels of neutralizing antibodies in mice immunized with. Among them, A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, and I2 were more conducive to producing higher levels of neutralizing antibodies.

[0186] Example 2: Comparison of the immune effects of Mix-4, Mix-8 and Mix-12 on mice

[0187] 1. Prepare mRNA vaccines and detect particle size distribution using dynamic light.

[0188] Mix-4: mRNA expressing A29, M1, B6, and A35 proteins are separately prepared into mRNA vaccines, mixed in equal weight (based on mRNA), filtered for sterilization, and packaged.

[0189] Mix-8: mRNA expressing A29, M1, B6, A35, H3, A17, A30, and H2 proteins are separately prepared into mRNA vaccines, mixed in equal weight (based on mRNA), filtered for sterilization, and packaged.

[0190] Mix-12: mRNA vaccines expressing A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, and I2 proteins are prepared separately, mixed in equal weight (based on mRNA), filtered for sterilization, and packaged.

[0191] The results are as follows Figure 3 As shown.

[0192] Mix-4, Mix-8 and Mix-12 have uniform particle size, with a particle size of around 100nm.

[0193] 2. Forty-eight SPF-grade female BALB / c mice (8 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into four groups of 12 mice each. The Mix-4, Mix-8, and Mix-12 groups were intramuscularly injected with 12 μg of the corresponding mRNA vaccine (calculated as mRNA). Three weeks after the initial immunization, they were boosted with the same dose of vaccine. The blank control group was intramuscularly injected with 100 μL of PBS. Two weeks after the booster immunization, blood was collected from the mice, serum was separated, and the level of neutralizing antibodies in the serum was measured. This serum was then used to immunize suckling mice to evaluate the passive immunization effect. The experimental procedure is as follows:

[0194] Twenty 3-week-old BALB / c suckling mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into four groups of five each. Serum samples (200 μL each) were injected intravenously into the fundus veins of the Mix-4, Mix-8, and Mix-12 groups. PBS served as the blank control group. Two hours later, serum samples were injected at a concentration of 1.0 × 10⁻⁶ μL. 6 The PFU / mouse content was determined by administering MPXV intranasal drops to mice in a challenge experiment, and then performing relevant tests on suckling mice 4 days later.

[0195] The neutralizing activity of neonatal rat serum samples against MPXV was assessed using a plaque-based neutralization assay. Vero E6 cells were loaded at 1.0 × 10⁻⁶ cells per cell line. 5 The serum samples were seeded at a density of 1 / 2 well in 96-well plates and incubated at 37°C for 2 hours. Serum samples were serially diluted 2-fold and co-incubated with a virus solution containing approximately 100 PFU MPXV at 37°C for 2 hours. The serum-virus mixture was then added to Vero E6 cells. Plaque formation was examined 3-4 days after infection, and neutralizing titers were calculated.

[0196] Viral load in the lungs of suckling mice was detected using a dot-matrix assay, with a concentration of 1.0 × 10⁻⁶ cells per well. 5 Vero E6 cells were seeded at a density of [number] cells and cultured at 37°C for 2 hours. Lung tissue samples were homogenized and serially diluted 2-fold with PBS in 96-well plates. 100 μL of the diluted tissue homogenate was then incubated with 100 μL of PBS containing approximately 100 PFU MPXV at 37°C for 2 hours. 200 μL of the tissue-virus mixture was added to Vero E6 cells. Spot formation was examined 3-4 days post-infection, and viral load was calculated based on the dilution factor. The amount of IFN-γ produced by splenic lymphocytes in suckling mice was measured using an ELISpot (Abcam, Cat; No. #ab64029) to evaluate the vaccine's immunization efficacy, following the manufacturer's instructions.

[0197] The results are as follows Figure 4 , Figure 5 and Figure 6 As shown.

[0198] Neutralization NT of MPXV titers in serum of mice in Mix-4, Mix-8 and Mix-12 groups 50 The values ​​were 644±75, 937±133, and 1195±200, respectively. Among them, the neutralizing antibody titers in the serum of mice in the Mix-12 group were relatively higher.

[0199] Subsequent testing of viral load in the lungs of suckling mice revealed that the Mix-12 group had the lowest viral load, followed by the Mix-8 group, while the Mix-4 group had a higher viral load. The Mix-12 group also had the highest number of IFN-γ-secreting lymphocytes, followed by the Mix-8 group, while the Mix-4 group had a lower number of IFN-γ-secreting lymphocytes. This indicates that all three vaccines produced neutralizing antibodies, with the Mix-12 group showing the best efficacy.

[0200] Example 3: Comparison of the immunomodulatory effects of Mix-12 and Mix-12-2 on mice

[0201] 1. Preparation of mRNA vaccines.

[0202] Mix-12: Same as Example 2.

[0203] Mix-12-2: The mRNAs expressing A29, M1, B6, A35, H3, A17, A30, H2, E8, C20.5, A14, and E13 proteins are separately prepared into mRNA vaccines, mixed in equal weight (based on mRNA), filtered for sterilization, and packaged.

[0204] 2. Ten SPF-grade female BALB / c mice (8 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into two groups of five each. The Mix-12 and Mix-12-12 groups were injected intramuscularly with 12 μg of the corresponding mRNA vaccine (calculated as mRNA). Three weeks after the initial immunization, a booster immunization was administered with the same dose of vaccine. The blank control group was injected intramuscularly with 100 μL of PBS. Two weeks after the booster immunization, blood was collected from the mice, serum was separated, and neutralizing antibody levels were detected.

[0205] The results are as follows Figure 7 As shown.

[0206] Neutralization NT of MPXV titers in serum of mice in Mix-12 and Mix-12-2 groups 50 The values ​​were 1230±97 and 804±64, respectively. Among them, the neutralizing antibody titers in the serum of mice in the Mix-12 group were relatively higher.

[0207] Example 4: Comparison of the immunomodulatory effects of Mix-12 and Mix-16 on mice

[0208] 1. Preparation of mRNA vaccines.

[0209] Mix-12: Same as Example 2.

[0210] Mix-16: mRNA vaccines expressing A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, I2, E8, C20.5, A14, and E13 proteins are prepared separately, mixed in equal weight (based on mRNA), filtered for sterilization, and packaged.

[0211] 2. Ten SPF-grade female BALB / c mice (8 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into two groups of five each. The Mix-12 and Mix-12-12 groups were injected intramuscularly with 12 μg of the corresponding mRNA vaccine (calculated as mRNA). Three weeks after the initial immunization, a booster immunization was administered with the same dose of vaccine. The blank control group was injected intramuscularly with 100 μL of PBS. Two weeks after the booster immunization, blood was collected from the mice, serum was separated, and neutralizing antibody levels were detected.

[0212] The results are as follows Figure 8 As shown.

[0213] Neutralization NT of MPXV titers in serum of mice in Mix-12 and Mix-16 groups 50The values ​​were 1317±138 and 564±115, respectively. The neutralizing antibody titers in the serum of mice in the Mix-12 group were relatively higher. This may be due to a decrease in the content of antigens with high neutralizing activity. This also indicates that a higher number of antigens does not necessarily lead to higher neutralizing activity; rather, a higher level of immune response can be achieved through different combinations of antigens.

[0214] Example 5: The Immunogenic Effect of Cellular Immunotopes on Mice

[0215] 1. Using the NetMHCpan 4.1 server, HLA-I epitopes in monkeypox virus proteins were predicted, and T-cell epitope-rich antigens MPX-EPs were designed. A ubiquitin sequence was introduced at the N-terminus of MPX-EPs to promote proteasomal degradation of the antigen and enhance CD8 uptake. + Activation of T cells.

[0216] The amino acid sequence of the MPX-EPs antigen is MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNI QKESTLHLVLRLRGA CAAAUCUUCGUGAAAACCCUAACCGGCAAAACCAUUACACUCGAAGUGGAACCUUC AGAUACAAUCGAAAACGUGAAGGCCAAGAUCCAGGACAAGGAAGGCAUCCCACCUGAUCAGCAGAGACUGAUCUUC GCCGGAAAGCAGCUGGAGGACGGCAGAACCCUGUCUGACUACAACAUACAGAAGGAAAGCACGCUCCACCUAGUGC UGCGGCUGAGAGGAGCC

[0217] The mRNA expressing MPX-EPs was made into an mRNA vaccine, filtered and sterilized, and then packaged.

[0218] 2. Ten SPF-grade female HLA-A*02:01 / DR1 mice (4-5 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into two groups of five mice each. Mice in the MPX-EPs group received an intramuscular injection of 3 μg of MPX-EPs mRNA vaccine (based on mRNA). Three weeks after the initial immunization, they were given a booster injection of the same dose of vaccine. The blank control group received an intramuscular injection of 100 μL of PBS. Two weeks after the booster immunization, blood was collected from the mice, and serum and spleen cells were separated. The ability of lymphocytes to produce IFN-γ was detected using the ELISPOT assay, and the CD4+ levels in mouse spleen cells were analyzed using flow cytometry. + and CD8 + T cell count, TNF-α + Cells and IFN-γ + Cell count was measured.

[0219] The results are as follows Figure 9 , Figure 10 and Figure 11 As shown.

[0220] CD4 in mice immunized with MPX-EPs vaccine + and CD8 + The levels of T cells were significantly increased, as were the levels of IFN-γ cells and TNF-α cells in the spleen. + IFN-γ + The cell count increased significantly.

[0221] 3. Eighteen SPF-grade female HLA-A*02:01 / DR1 mice (4-5 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into 3 groups of 5 mice each. Mice in the MPX-EPs&Anti CD8a group were intramuscularly injected with 3 μg of MPX-EPs mRNA vaccine (based on mRNA). Three weeks after the initial immunization, a booster immunization was administered with the same dose of vaccine. Twenty days after the booster immunization, mice were injected with 200 mg / mouse of anti-CD8-α antibody (which clears CD8). + T cells were administered twice (24 hours apart); MPX-EPs group mice were intramuscularly injected with 3 μg MPX-EPs mRNA vaccine (calculated as mRNA). Three weeks after the initial immunization, they were given a booster dose of the same vaccine. Twenty days after the booster immunization, IgG2b isotype control antibody (which does not clear CD8) was injected at 200 mg / mouse. +T cells were administered twice (24 hours apart); the blank control group received an intramuscular injection of 100 μL PBS. Then, MPXV (1.0 × 10⁻⁶) was used. 7 Mice were infected with PFU (phenylephrine / phosphorus urea) via nasal instillation. Four days after infection, the viral load in the lungs of mice was detected by dot assay, and lung tissue pathology analysis was performed.

[0222] The results are as follows Figure 12 and Figure 13 As shown.

[0223] The viral load in the lungs of mice immunized with the MPX-EPs vaccine was significantly reduced, and it provided some protection.

[0224] In summary, MPX-EPs immunization induced a strong CD8 response. + T cell-mediated immune responses lead to CD8 + T cell proliferation and MPXV-targeting cytokine secretion have the potential to serve as vaccines against cellular immunity.

[0225] Example 6: Immunization effect of mixed cellular immunoepitaphs and Mix-12 on mice

[0226] 1. Vaccine preparation

[0227] MPX-m-Mix: Mix-12 was prepared in the same manner as in Example 2, and MPX-EPs were prepared in the same manner as in Example 5. Mix-12 and MPX-EPs were mixed at a mass ratio of 3:1 (based on mRNA), filtered for sterilization, and dispensed. Particle size distribution was detected by dynamic light. Results are as follows: Figure 14 As shown, the prepared mRNA vaccine particles are uniform in size, with a particle size of about 100 nm, similar to Mix-4, Mix-8, and Mix-12.

[0228] MPX-p-Mix: The gene sequences of A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, and I2 proteins were constructed into the eukaryotic expression vector pcDNA3.1. Plasmids were extracted using a plasmid extraction kit and transfected into 293F cells. After culturing for 96 hours, the cell supernatant was harvested, and the target proteins were purified using a nickel column. The immunoepitaxypeptide (amino acid sequence as shown in SEQ ID NO:69) was synthesized by Beijing Qingke Biotechnology Co., Ltd. The purified A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, and I2 proteins were mixed by mass. The mixture and the immunoepitaxypeptide were then mixed at a mass ratio of 4:1 and adsorbed onto aluminum hydroxide adjuvant.

[0229] 2. Fifteen SPF-grade female HLA-A*02:01 / DR1 mice (4-5 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into three groups of five mice each. The MPX-m-Mix group was injected intramuscularly with 3 μg of MPX-m-Mix mRNA vaccine; the MPX-p-Mix group was subcutaneously immunized with 15 μg of protein per mouse; and the blank control group was injected intramuscularly with 100 μL of PBS. Three weeks after the initial immunization, mice were re-immunized with the same dose of vaccine as the initial immunization. Two weeks after the booster immunization, blood was collected from the mice, serum was separated, and the levels of binding antibodies and neutralizing antibodies in the mouse serum were detected. Spleen cells were isolated, and the ability of lymphocytes to produce IFN-γ and IL-4 was detected using the ELISPOT assay (Abcam, Cat; No. ab64029 and ab281966).

[0230] The results are as follows Figure 15 , Figure 16 As shown.

[0231] Following immunization with both protein and mRNA vaccines, the levels of binding and neutralizing antibodies in mouse serum increased, and the content of IFN-γ cells in the spleen of mice significantly increased, with the MPX-m-Mix group showing a greater increase than the MPX-p-Mix group. After immunization with both protein and mRNA vaccines, the content of IL-4 cells in the spleen of mice significantly increased, with the MPX-m-Mix group showing a weaker increase than the MPX-p-Mix group.

[0232] 3. Fifteen SPF-grade female HLA-A*02:01 / DR1 mice (4-5 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were randomly divided into three groups of five mice each. The MPX-m-Mix group received an intramuscular injection of 3 μg MPX-m-Mix mRNA vaccine; the MPX-p-Mix group received a subcutaneous injection of 15 μg protein / mouse. Three weeks after the initial immunization, a booster immunization was administered with the same dose of vaccine. The blank control group received an intramuscular injection of 100 μL PBS. Two weeks after the booster immunization, MPXV (1.0 × 10⁻⁶) was administered. 7 Mice were infected with PFU (phosphorus urea nitrogen) via nasal drip. Four days later, the viral load in the mice's lungs was detected by dot assay, and lung tissue pathology was performed.

[0233] The results are as follows Figure 17 , Figure 18 As shown.

[0234] The viral load in the lungs of mice in both the MPX-m-Mix and MPX-p-Mix groups was significantly reduced, with the MPX-m-Mix group showing superior protection compared to the MPX-p-Mix group. Analysis of lung histopathological changes indicated that the vaccine provided partial protection, with the MPX-m-Mix group showing better protection than the MPX-p-Mix group.

[0235] Overall, the MPX-m-Mix vaccination strategy effectively prevented MPXV infection in HLA transgenic mice. MPX-m-Mix achieved complete protection and was superior to the recombinant protein vaccine MPX-p-Mix.

[0236] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A monkeypox virus antigen epitope mRNA composition, characterized in that, include: mRNA encoding the amino acid sequences of antigenic epitopes A29, M1, B6, A35, H3, A17, A30, H2, A28, A21, G2, and I2; The amino acid sequence of A29 is shown in positions 1 to 110 of SEQ ID NO: 1; the amino acid sequence of M1 is shown in positions 1 to 250 of SEQ ID NO: 3; the amino acid sequence of B6 is shown in positions 1 to 317 of SEQ ID NO: 5; the amino acid sequence of A35 is shown in positions 1 to 181 of SEQ ID NO: 7; the amino acid sequence of H3 is shown in positions 1 to 324 of SEQ ID NO: 9; the amino acid sequence of A17 is shown in positions 1 to 377 of SEQ ID NO: 11; the amino acid sequence of A30 is shown in positions 1 to 146 of SEQ ID NO: 13; the amino acid sequence of H2 is shown in positions 1 to 189 of SEQ ID NO: 15; the amino acid sequence of A28 is shown in positions 1 to 509 of SEQ ID NO: 17; the amino acid sequence of A21 is shown in positions 1 to 115 of SEQ ID NO: 19; and the amino acid sequence of G2 is shown in positions 1 to 250 of SEQ ID NO:

19. As shown in positions 1 to 111 of SEQ ID NO: 23, the amino acid sequence of I2 is shown in positions 1 to 73 of SEQ ID NO:

23.

2. The monkeypox virus antigen epitope mRNA composition according to claim 1, characterized in that, The coding region sequence of the mRNA encoding A29 is shown as positions 133 to 462 of SEQ ID NO: 1; and / or the coding region sequence of the mRNA encoding M1 is shown as positions 133 to 882 of SEQ ID NO: 4; and / or the coding region sequence of the mRNA encoding B6 is shown as positions 133 to 1083 of SEQ ID NO: 6; and / or the coding region of the mRNA sequence encoding A35 is shown as positions 133 to 675 of SEQ ID NO: 8; and / or the coding region of the mRNA sequence encoding H3 is shown as positions 133 to 1104 of SEQ ID NO: 10; and / or the coding region of the mRNA sequence encoding A17 is shown as positions 133 to 1263 of SEQ ID NO: 12; and / or the coding region of the mRNA sequence encoding A30 is shown as positions 133 to 1263 of SEQ ID NO:

12. The coding region of the mRNA sequence encoding H2 is shown in positions 133 to 570 of SEQ ID NO: 14; and / or the coding region of the mRNA sequence encoding A28 is shown in positions 133 to 1659 of SEQ ID NO: 18; and / or the coding region of the mRNA sequence encoding A21 is shown in positions 133 to 477 of SEQ ID NO: 20; and / or the coding region of the mRNA sequence encoding G2 is shown in positions 133 to 465 of SEQ ID NO: 22; and / or the coding region of the mRNA sequence encoding I2 is shown in positions 133 to 351 of SEQ ID NO:

24.

3. The monkeypox virus antigen epitope mRNA composition according to claim 1, characterized in that, The mRNA also includes a 5' UTR, a Kozak sequence, a sequence encoding a signal peptide, a sequence encoding a tag, a stop codon, a sequence encoding ubiquitin, a 3' UTR, and a PolyA sequence.

4. A monkeypox virus antigenic epitope protein composition, characterized in that, The protein encoded by the monkeypox virus antigenic epitope mRNA composition as described in any one of claims 1 to 3.

5. A biomaterial relating to the monkeypox virus antigenic epitope mRNA composition of any one of claims 1 to 3 or the monkeypox virus antigenic epitope protein composition of claim 4, characterized in that, The biomaterial is any one of (B1) to (B5): (B1) A nucleic acid molecule encoding the monkeypox virus antigenic epitope mRNA composition of any one of claims 1 to 3 or the monkeypox virus antigenic epitope protein composition of claim 4; (B2) An expression cassette containing the nucleic acid molecule described in (B1); (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2); (B4) A recombinant microorganism containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3); (B5) A transgenic cell line containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2) or the recombinant vector described in (B3).

6. A vaccine, characterized in that, The composition includes the monkeypox virus antigenic epitope mRNA composition according to any one of claims 1 to 3 or the monkeypox virus antigenic epitope protein composition according to claim 4; and an adjuvant.

7. The method for preparing the vaccine according to claim 6, characterized in that, Includes the following steps: The vaccine is obtained by preparing the monkeypox virus antigenic epitope mRNA composition or the mixture of the monkeypox virus antigenic epitope protein composition and the adjuvant.

8. The use of the monkeypox virus antigenic epitope mRNA composition according to any one of claims 1 to 3, the monkeypox virus antigenic epitope protein composition according to claim 4, the biomaterial according to claim 5, or the vaccine according to claim 6 in the preparation of a medicament for preventing monkeypox virus infection.

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