A multivalent monkeypox virus mRNA vaccine and its preparation method and application

Abstract

The present invention provides a multivalent monkeypox virus mRNA vaccine, characterized in that the vaccine is obtained by co-expressing multiple antigenic proteins of the monkeypox virus in series through a flexible linker, and the multiple antigenic proteins are selected from B6R, A35R, A29L and M1R. The effectiveness and safety of the mRNA vaccine of the present invention are verified by humoral immunity and cellular immunity, as well as the protection effect and safety evaluation of the attack. The results of the study show that the monkeypox virus mRNA vaccine prepared by the present invention only requires one mRNA to produce a good immune protection effect, and has good safety, has the characteristics of simple industrial production, easy quality control, etc., and is an ideal choice for monkeypox virus vaccines.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a multivalent monkeypox virus mRNA vaccine and a preparation method and application thereof. Background Art

[0002] Monkeypox is a zoonotic infectious disease caused by infection with the monkeypox virus (MPXV). MPXV is divided into two different branches based on genetic differences: branch I and branch II, and the current branch II is prevalent. In 1958, MPXV was first discovered and named in crab-eating macaques in Danish laboratories, and can cause monkeypox in humans, whose symptoms are similar to smallpox. MPXV is transmitted through contact with respiratory secretions, blood and other body fluids of infected animals, or through bites or scratches of infected animals, and the incubation period is generally 6-13 days. In humans, the typical clinical manifestations of monkeypox include high fever, headache and myalgia, swollen lymph nodes, and a rash that starts with fever on the face, tongue and mouth and then spreads to the whole body. The disease is usually milder than smallpox, but can lead to death, with a mortality rate of up to 10%. Epidemiology shows that MPXV was originally found around Central and West African countries, but since 2022, monkeypox has seen large-scale epidemics and outbreaks in non-endemic countries. As of July 25, 2023, there have been more than 88,600 confirmed cases and 152 deaths in 113 countries and regions. The World Health Organization (WHO) has declared the 2022 monkeypox outbreak a public health emergency of international concern. The outbreak of monkeypox has a significant impact on human life and health, social stability and economic development.

[0003] MPXV belongs to the genus Orthopoxvirus in the family Poxviridae. In addition to MPXV, there are three other viruses in this genus that can infect humans and cause disease, including variola virus (VARV), monkeypox virus (MPXV), cowpox virus (CPXV) and vaccinia virus (VACV). Studies have shown that orthopoxvirus immune responses can provide cross-protection. The MPXV genome is highly similar to VARV. Due to the similarity of antigens, the smallpox vaccine is effective against MPXV. It is reported that during the African monkeypox outbreak in the 1980s, the smallpox vaccine, as a live attenuated vaccine, had a protection rate of about 85% against monkeypox, but it can cause side effects. Monkeypox spreads rapidly around the world, and there is currently no specific MPXV vaccine that has been clinically approved. Therefore, the development of a safe and effective preventive vaccine is the trend of monkeypox virus control.

[0004] mRNA vaccines have immeasurable potential application value in many medical fields such as preventing infectious diseases and treating tumors. It is a vaccine that synthesizes mRNA encoding antigens through in vitro transcription technology and transports them to specific tissues and cells through a delivery system. mRNA vaccines are translated by the host cell's own translation system, inducing humoral immunity and cellular immunity at the same time, and can effectively protect the body through multiple mechanisms. Compared with other types of vaccines, mRNA vaccines are easier to design and produce. For example, Pfizer / BioNTech's mRNA vaccine BNT162b2 and Moderna's mRNA-1273 are both designed for the surface spike protein of the SARS-CoV-2 coronavirus (COVID-19), and the results show that the protection of both vaccines is greater than 94.0%. Screening for the best target antigen for MPXV is the key to mRNA sequence design, which will largely determine the effectiveness and protective effect of the vaccine. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an MPXV mRNA vaccine that only requires one mRNA to produce a good immune protection effect, has good safety, is simple to industrialize and produce, and is easy to control quality. It is an ideal choice for MPXV vaccines.

[0006] The technical solution of the present invention is to provide a 4 S 1 ) 3 The MPXV mRNA vaccine, which expresses the four antigenic proteins of MPXV (B6R, A35R, A29L and M1R) in series, was tested in mice through experiments such as humoral immunity and cellular immunity, as well as attack protection effect evaluation and safety evaluation. The results showed that the vaccine has good immunogenicity, can protect mice from poxvirus infection, and has good safety.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] On the one hand, the present invention provides a multivalent monkeypox virus mRNA vaccine, characterized in that the vaccine is obtained by co-expressing multiple antigenic proteins of the monkeypox virus in series via a flexible linker.

[0009] In some embodiments, the plurality of antigenic proteins are selected from B6R, A35R, A29L, and M1R.

[0010] In some embodiments, the coding sequence of B6R comprises the sequence shown in SEQ ID NO:1, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or a nucleotide sequence having one or more (preferably 1, 2 or 3) nucleotide mutations (preferably substitutions, insertions or deletions) compared to the sequence, or consisting of the same.

[0011] In some embodiments, the coding sequence of A35R comprises the sequence shown in SEQ ID NO:2, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or a nucleotide sequence having one or more (preferably 1, 2 or 3) nucleotide mutations (preferably substitutions, insertions or deletions) compared to the sequence, or consisting of the same.

[0012] In some embodiments, the coding sequence of A29L comprises the sequence shown in SEQ ID NO:3, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or a nucleotide sequence having one or more (preferably 1, 2 or 3) nucleotide mutations (preferably substitutions, insertions or deletions) compared to the sequence, or consisting of the same.

[0013] In some embodiments, the coding sequence of M1R comprises the sequence shown in SEQ ID NO:4, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or a nucleotide sequence having one or more (preferably 1, 2 or 3) nucleotide mutations (preferably substitutions, insertions or deletions) compared to the sequence, or consisting of the same.

[0014] In some embodiments, the amino acid sequence of B6R comprises the sequence shown in SEQ ID NO:6, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, or consisting of the same.

[0015] In some embodiments, the amino acid sequence of A35R comprises the sequence shown in SEQ ID NO:7, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, or consisting of the same.

[0016] In some embodiments, the amino acid sequence of A29L comprises the sequence shown in SEQ ID NO:8, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, or consisting of the same.

[0017] In some embodiments, the amino acid sequence of M1R comprises the sequence shown in SEQ ID NO:9, a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence, or an amino acid sequence having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequence, or consisting of the same.

[0018] In some embodiments, the flexible linker comprises glycine and serine. Preferably, the amino acid sequence of the flexible linker is GGGGSGGGGSGGGGS.

[0019] In some embodiments, the coding sequence of the mRNA vaccine includes the coding sequence of B6R+the coding sequence of the flexible linker+the coding sequence of A35R+the coding sequence of the flexible linker+the coding sequence of A29L+the coding sequence of the flexible linker+the coding sequence of M1R.

[0020] In some embodiments, the coding sequence of the mRNA vaccine also includes 5'UTR and 3'UTR sequences, and is cap-modified at the 5' end and poly(A) is added at the 3' end.

[0021] In some embodiments, the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO:11.

[0022] In some embodiments, the nucleotide sequence of the 3'UTR is as shown in SEQ ID NO:12.

[0023] In some embodiments, the coding sequence of the mRNA is shown as SEQ ID NO:5.

[0024] In some embodiments, the amino acid sequence of the mRNA is as shown in SEQ ID NO:10.

[0025] In another aspect, the present invention provides mRNA vaccine lipid complexes, wherein the lipids include ionizable cationic lipids, phospholipids, cholesterol and pegylated lipids.

[0026] In some embodiments, the ionizable cationic lipid is the cationic lipid SM-102.

[0027] In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0028] In some embodiments, the PEGylated lipid is DMG-PEG 2000.

[0029] In another aspect, the present invention provides a pharmaceutical composition comprising the vaccine as described above and a pharmaceutically acceptable carrier.

[0030] In another aspect, the present invention provides a method for preparing an mRNA vaccine lipid complex, comprising:

[0031] a. dissolving an ionizable cationic lipid, a phospholipid, cholesterol and a PEGylated lipid in anhydrous ethanol to obtain an organic phase;

[0032] b. dissolving the mRNA vaccine as described above in a citric acid buffer to obtain an aqueous phase;

[0033] c. Mix the organic phase solution and the aqueous phase solution to obtain the mRNA vaccine lipid complex.

[0034] On the other hand, the present invention provides the use of the mRNA vaccine as described above in the preparation of a medicament for preventing and / or treating monkeypox virus infection.

[0035] On the other hand, the present invention provides a method for preventing and / or treating monkeypox virus infection, characterized in that the method comprises administering the mRNA vaccine as described above to a subject.

[0036] In the present invention, amino acid residues are recorded using the following abbreviated symbols: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), valine (Val or V), and any amino acid residue (Xaa or X). Furthermore, in the present invention, the amino acid sequence is described in a manner such that the amino terminus (also referred to as the N terminus) is located on the left and the carboxyl terminus (also referred to as the C terminus) is located on the right.

[0037] In the present invention, conservative amino acid mutations are performed by replacing one amino acid in the amino acid sequence with another amino acid in the amino acid group having a side chain with similar chemical properties. Examples of amino acid groups having side chains with similar chemical properties include aliphatic amino acids, aromatic amino acids, and heterocyclic amino acids. Aliphatic amino acids include 1) neutral amino acids containing one amino group and one carboxyl group: glycine, alanine, valine, leucine, and isoleucine; 2) hydroxyl-containing amino acids: serine and threonine; 3) sulfur-containing amino acids: cysteine ​​and methionine; 4) amide-containing amino acids: asparagine and glutamine: 5) acidic amino acids containing one amino group and one carboxyl group: aspartic acid and glutamic acid; 6) basic amino acids containing two amino groups and one carboxyl group: lysine and arginine. Aromatic amino acids include phenylalanine and tyrosine. Heterocyclic amino acids include tryptophan, histidine, and proline.

[0038] Definitions (Please define the following terms in conjunction with this invention)

[0039] B6R: A target for neutralizing antibodies on the surface of MPXV.

[0040] A35R: A target for neutralizing antibodies on the surface of MPXV.

[0041] A29L: A target for neutralizing antibodies on the surface of MPXV.

[0042] M1R: A target for neutralizing antibodies on the surface of MPXV.

[0043] PRNT 50 : 50% serum plaque reduction neutralizing test (50% plaque reduction neutralizing test, PRNT 50 ), used to evaluate virus resistance.

[0044] LNP: Lipid nanoparticles, an mRNA delivery system that contains at least one lipid.

[0045] mRNA vaccine: mRNA is a naturally occurring molecule that carries the "blueprint" of human cells and can produce target proteins or immunogens to activate the body's immune response to fight various pathogens. mRNA vaccines use the genetic sequence of the virus rather than the virus itself, so mRNA vaccines do not contain viral components and do not pose a risk of infection.

[0046] Beneficial results

[0047] The research of the present invention shows that the mRNA vaccine of MPXV 4 antigenic proteins B6R, A35R, A29L and M1R co-expressed in series through a flexible connector can produce good humoral immunity and cellular immunity after immunizing mice, protect mice from poxvirus infection, and has good safety. In vitro detection by laser confocal and flow cytometry technology shows that after A29L protein is co-expressed in series, it can be expressed on the cell membrane surface together with the other three proteins B6R, A35R and M1R. The results of in vivo experiments show that compared with the group expressing MPXV 4 proteins alone or the four protein mixed group, the 1103mRNA immunization group has stronger humoral immunity and cellular immunity, can protect mice from vaccinia virus infection, and has good safety. Compared with the MPXVmRNA vaccines reported in the literature, such as single mRNA mixed vaccines expressing multiple antigen fragments separately, and single vaccines expressing multiple antigens separately after being connected in series, it has the advantages of simple production process, low production cost and good immune effect. Therefore, the MPXV mRNA vaccine provided by the present invention is more suitable for clinical application and has potential value in new drug development.

[0048] The present invention proposes for the first time an antigen design method in which the four antigenic proteins B6R, A35R, A29L and M1R of MPXV are co-expressed in series on the cell membrane via a flexible linker, and the nucleic acid sequences of the four antigens are prepared into an mRNA sequence, which is then encapsulated in liposomes and prepared into an MPXV mRNA vaccine. The vaccine produces good humoral immunity and cellular immunity after immunizing mice, can protect mice from infection by poxvirus vaccine strains, and has good safety. However, the existing MPXV mRNA vaccines are mostly composed of a mixture of multiple mRNA sequences expressing different antigenic proteins, which increases the difficulty of quality control and the production cost. There are literature reports that nucleic acid sequences expressing different antigenic proteins are connected in series into one mRNA sequence, but the antigenic proteins are expressed separately, and after multiple immunizations of mice, antibody production is still not detected for some antigenic proteins. The present invention innovatively designs the four antigenic proteins B6R, A35R, A29L and M1R of MPXV. Not only are the base sequences of the four antigenic proteins on one mRNA, but the four proteins are also expressed in fusion. The MPXV vaccine prepared from the mRNA sequence can produce a good immune effect in mice, protect mice from poxvirus infection, and has good safety, and has potential value for new drug development.

[0049] In addition, the present invention selects the full-length sequence of four antigenic proteins (B6R, A35R, A29L and M1R) of the monkeypox virus, and the nucleic acid sequences are connected in series to express the four proteins in fusion. However, some existing technologies, such as CN116036259A, select the antigenic protein nucleic acid sequences of the monkeypox virus antigens M1, H3, A29, A35, B6 and E8 to be prepared into mRNA respectively, and then mixed in equal proportions and encapsulated by lipid nanoparticles to prepare a monkeypox virus multi-antigen mRNA vaccine, which is different from the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The design pattern diagram of each mRNA sequence is shown. The top 4 are the full-length base sequences of B6R, A35R, A29L and M1R, and the fifth is the four protein base sequences of the above four fragments connected into one with a flexible linker. The upstream and downstream of the above five sequences are linked to the 5'UTR and 3'UTR sequences, and Cap modification is added at the 5' end and Poly (A) is added at the 3' end.

[0051] Figure 2 The particle size of each group of vaccine nanoparticles is shown.

[0052] Figure 3 The dispersion coefficient of each group of vaccine nanoparticles is shown.

[0053] Figure 4 The encapsulation efficiency of vaccine nanoparticles in each group is shown.

[0054] Figure 5 The results of observing the expression position of each antigen protein using a laser confocal microscope are shown.

[0055] Figure 6 The figure shows the expression of various antigen proteins on the cell membrane surface detected by flow cytometer.

[0056] Figure 7 The figure shows the expression of each protein detected by flow cytometer after cell permeabilization.

[0057] Figure 8 The graph shows the changes in the levels of IgG antibodies against MPXV B6R produced by different groups of mice.

[0058] Fig. 9 The graph shows the changes in the levels of IgG antibodies against MPXV A35R produced by different groups of mice.

[0059] Fig.10 The graph shows the changes in the levels of IgG antibodies against MPXV A29L produced by different groups of mice.

[0060] Fig.11 The graph shows the changes in the levels of IgG antibodies against MPXV M1R produced by different groups of mice.

[0061] Fig.12 The graph shows the level of neutralizing VACV antibodies produced by each group of mice 28 days after the first immunization.

[0062] Fig.13 The figure shows the changes in the number of splenic lymphocytes that secrete IFN-γ specifically against the B6R polypeptide pool produced by different groups of mice.

[0063] Fig.14 The figure shows the changes in the number of spleen lymphocytes that secrete IFN-γ specifically against the A35R polypeptide pool produced by different groups of mice.

[0064] Fig.15 The figure shows the changes in the number of spleen lymphocytes that secrete IFN-γ specifically against the A29L polypeptide pool produced by different groups of mice.

[0065] Fig.16 The figure shows the changes in the number of spleen lymphocytes that secrete IFN-γ specifically against the M1R polypeptide pool produced by different groups of mice.

[0066] Fig.17 The figure shows the changes in body weight of mice in each group after infection with VACV.

[0067] Fig.18 Shown are the survival status of mice in each group after infection with VACV.

[0068] Fig.19 The viral titers in the nasal turbinates of mice in each group on day 4 after VACV infection are shown.

[0069] Fig. 20 The viral titers in the lungs of mice in each group on day 4 after VACV infection are shown.

[0070] Fig.21 The figure shows the changes in routine blood indicators of mice after two immunizations with MPXV-1103.

[0071] Fig. 22 The graph shows the pathological changes of the main internal organs of mice after two immunizations with MPXV-1103. DETAILED DESCRIPTION

[0072] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0073] Example 1 Preparation of MPXV mRNA vaccine

[0074] 1. Whole gene synthesis of recombinant plasmid

[0075] The DNA sequences of different antigens ORF (open reading frame) sequences (SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5) were concatenated with the 5'UTR sequence (SEQ ID NO: 11), 3'UTR sequence (SEQ ID NO: 12) and poly (A) sequence (SEQ ID NO: 13) in the following manner, and then the whole gene synthesis was performed using pUC57 as a vector (Shanghai Bioengineering Co., Ltd.) to obtain a template plasmid (see the design pattern diagram). Figure 1 ). The coding sequence information of each antigen is as follows.

[0076] Table 1 Antigen coding sequence and amino acid sequence information

[0077] antigen Coding sequence Amino acid sequence B6R SEQ ID NO:1 SEQ ID NO:6 A35R SEQ ID NO:2 SEQ ID NO:7 A29L SEQ ID NO:3 SEQ ID NO:8 M1R SEQ ID NO:4 SEQ ID NO:9 1103 SEQ ID NO:5 SEQ ID NO:10

[0078] 2. Preparation and Purification of MPXV mRNAs

[0079] Take the enzyme-free tube, write a label, and add the reagents in sequence in the clean bench. The table below shows the amount of each component of the 20μl system. Other reaction volumes can be proportionally enlarged according to this method. After adding, use a pipette to blow and mix for 10 seconds, press the lid tightly, and centrifuge at low speed for 30 seconds to collect the liquid on the tube wall. The labeled enzyme-free tube is reacted at 37±3℃ in an intelligent constant temperature oscillator / PCR instrument for 2-5 hours. Use 2.5M lithium chloride to precipitate mRNA at -20℃ for 30min and then resuspend it in water. Centrifuge at 16,000×g for 20min at 43℃ to recover the purified mRNA. The concentration of mRNA is determined by a spectrophotometer. The sequences are named B6RmRNA, A35R mRNA, A29L mRNA, M1R mRNA and 1103mRNA.

[0080] Table 2 Reagents and volumes

[0081]

[0082]

[0083] 3. Preparation of MPXV mRNA vaccine formulation

[0084] Organic phase (lipid) preparation: Four lipid components were selected, namely, ionizable cationic lipid SM-102 (Shunna Medical, SN-M-CL23-100), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avitor, 816-94-4), cholesterol (Avitor, 57-88-5) and DMG-PEG 2000 (MCE, HY-112764). These lipids were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5 to obtain an organic phase. Preparation of aqueous phase (nucleic acid): Dissolve each mRNA sequence (B6RmRNA, A35R mRNA, A29L mRNA, M1R mRNA and 1103mRNA) in a weakly acidic citric acid buffer (weigh 9.6g of anhydrous citric acid and 2.7g of sodium hydroxide, add to 900mL of distilled water while stirring, and finally add distilled water to 1L; test the pH value and adjust the pH to 4.5 with citric acid or sodium hydroxide dilution solution) to obtain an aqueous phase. Preparation of microfluidic equipment: Use anhydrous ethanol and citric acid buffer to clean the organic phase pump and aqueous phase pump of the microfluidic instrument respectively. Microfluidic mixing: Inject the organic phase solution and the aqueous phase solution into the microfluidic injection pump respectively, and evenly mix the two phase solutions through the microfluidic chip at a flow rate ratio of 5:1 between the aqueous phase and the organic phase, and collect the mixed solution (i.e., LNPs solution). Dilution: The collected LNPs solution is diluted to 10 times the original volume with purified water. Ultrafiltration concentration: The diluted LNPs solution was further subjected to ultrafiltration concentration steps to remove residual ethanol and unencapsulated free mRNA. The concentrated LNPs solution was temporarily stored at 2-8°C for future use. The particle size and polydispersity index (PDI) of LNPs were detected by dynamic light scattering. The mRNA content and encapsulation efficiency of LNPs were detected by fluorescent dye method. After being prepared into vaccines, they were named B6R-LNPs, A35R-LNPs, A29L-LNPs, M1R-LNPs and MPXV-1103. Among them, B6R-LNPs, A35R-LNPs, A29L-LNPs and M1R-LNPs were mixed in a mass ratio of 1:1:1:1 to prepare a mixed vaccine group Mix 4-LNPs.

[0085] Dynamic light scattering was used to detect the particle size and polydispersity coefficient of LNPs. Figure 2 and 3 As shown, the average particle sizes of B6R-LNPs, A35R-LNPs, A29L-LNPs, M1R-LNPs and MPXV-1103 were 98.7nm, 94.9nm, 95.0nm, 105.8nm and 96.3nm, respectively, and the dispersion coefficients were 0.164, 0.050, 0.175, 0.106 and 0.207, respectively, and the particle sizes were relatively uniform.

[0086] The mRNA encapsulation efficiency of LNPs was detected by fluorescent dye method. The results are as follows Figure 4 As shown, the mRNA encapsulation efficiencies of B6R-LNPs, A35R-LNPs, A29L-LNPs, M1R-LNPs and MPXV-1103 were 96.48%, 96.26%, 95.83%, 97.29% and 97.62%, respectively, all above 95%.

[0087] The above results indicate that B6R-LNPs, A35R-LNPs, A29L-LNPs, M1R-LNPs, and MPXV-1103 vaccines have been successfully prepared.

[0088] Example 2 In vitro validation of MPXV mRNA vaccine

[0089] 1. Laser Confocal Microscope Observation

[0090] The day before transfection, 293T cells were seeded into a 24-well cell plate containing a cell slide (treated with poly-lysine) and transfected when the cells grew to 50-60% confluence. 1 μg of each mRNA sequence (B6R, A35R, A28L, M1R and 1103) was added to a 1.5 mL centrifuge tube containing Opti-MEM medium (Thermo Fisher Scientific, 11995065) to a final volume of 25 μl. At the same time, 2 μl of Lipofectamine TM2000 (Thermo Fisher Scientific, 11668019) was added to a 1.5 mL centrifuge tube containing 25 μl of Opti-MEM medium, gently mixed, and incubated at room temperature for 15 min. 293T cells were slowly washed 3 times with sterile PBS solution, 1 mL / time, and 450 μl / well of Opti-MEM medium was added after discarding the washing solution. The mRNA and liposome mixture incubated at room temperature was gently added dropwise to the cell culture well. 37°C cell culture incubator for 18-24 hours. Fix with 4% formalin solution overnight. The cell slides were incubated with the corresponding antibodies B6R, A35R, M1R (Nanjing Oukai Biotechnology Co., Ltd.) and A29L (Wuhan Pujian Biology) at a concentration of 0.5 μg / ml. After washing with PBS, the corresponding FITC-labeled goat anti-rabbit or goat anti-human IgG antibodies (1:1000; Proteintech) were incubated. Then use DAPI (1 μ g / ml) (Biosharp Life Sciences, BS097-10mg) to stain the slices for 10 min, and use Fluoro mount (Sigma, F4680) to seal the cell crawling slices on a microscope slide. Use a fluorescence microscope to image the samples. For protein expression positioning, the slices are additionally stained with Dil (a cell membrane red fluorescent probe) (Beyotime, C1991S) for 10 min. Use a laser scanning confocal microscope (Leica DMi8) to image the samples.

[0091] like Figure 5 As shown, after 1103 sequence transfection, compared with the single expression of B6R, A35R, A29L and M1R sequences, the cell membrane in the 1103 sequence group was stained with A29L antibody and yellow, while the A29L sequence group was not stained with A29L antibody. At the same time, the other three proteins B6R, A35R and M1R were all yellow in the 1103 sequence group and the single transfection group.

[0092] The above results show that the four proteins B6R, A35R, A29L and M1R in 1103 series can be expressed on the cell membrane surface, while the A29L sequence cannot express the A29L protein on the cell membrane surface.

[0093] 2. Flow Cytometry Analysis

[0094] The day before transfection, 293T cells were transferred to a 24-well cell plate and transfected when the cells grew to 80%-90% confluence. 1 μg of each mRNA sequence (B6R, A35R, A28L, M1R and 1103) was added to a 1.5 mL centrifuge tube containing Opti-MEM medium (ThermoFisher Scientific, 11995065) to a final volume of 25 μl. At the same time, 2 μl of Lipofectamine TM 2000 (Thermo Fisher Scientific, 11668019) was added to a 1.5 mL centrifuge tube containing 25 μl of Opti-MEM medium, gently mixed, and incubated at room temperature for 15 min. 293T cells were slowly washed 3 times with sterile PBS solution, 1 mL / time, and 450 μl / well of Opti-MEM medium was added after discarding the washing solution. The mRNA and liposome mixture incubated at room temperature was gently added dropwise to the cell culture well. Incubate at 37°C for 18-24 hours. Collect cells and perform subsequent experiments with and without transmembrane solution (to detect the protein displayed on the cell membrane surface).

[0095] The results are as follows Figure 6 and Figure 7 As shown, different mRNA sequences were transfected into 293T cells, and the expression of B6R, A35R, A29L and M1R proteins was detected by flow cytometry after membrane permeabilization and non-permeabilization. When the membrane was not permeated (living cells), the A29L sequence could not be detected to be expressed on the cell membrane surface, while the 1103 sequence could detect the expression of A29L, reaching 2.92%. At the same time, after the cells were permeated, the 1103 sequence and the 4 mRNA sequences expressing proteins alone could detect the expression of B6R, A35R, A29L and M1R proteins.

[0096] The above results further indicate that after fusion expression of the four proteins of MPXV (B6R, A35R, A29L and M1R), the A29L protein can be successfully expressed on the cell membrane surface.

[0097] Example 3 Analysis of the immunogenicity of MPXV mRNA vaccine

[0098] 1. Animal immunization

[0099] Fifty healthy female SPF Balb / C mice (Beijing Huafukang Biotechnology Co., Ltd.) aged 5 to 6 weeks were randomly divided into 10 groups, 5 mice / group, including MXPV-1103 low-dose group (1 μg / mouse), MXPV-1103 medium-dose group (5 μg / mouse), MXPV-1103 high-dose group (20 μg / mouse), Mix 4-LNP group (5 μg / mouse, 1.25 μg / mouse), B6R-LNP group (5 μg / mouse), A35R-LNP group (5 μg / mouse), A29L-LNP group (5 μg / mouse) and M1R-LNP group (5 μg / mouse), as well as LNP control group and PBS control group. After one week of adaptive breeding in the SPF animal room of the University of Science and Technology of China, the mice were injected with vaccines of each dose group once through the outer thigh muscle on day 0 and day 14, respectively. Blood was collected from the orbital venous plexus once on day 0 before immunization and 14 and 28 days after the first immunization. The IgG antibody titers of B6R, A35R, A29L and M1R were detected by ELISA. The neutralizing antibody titer was determined by Vaccinia virus strain (VACV).

[0100] 2. MPXV antigen-specific binding antibody detection

[0101] The ELISA method was used to detect the titer of MPXV antigen-specific antibody IgG in mouse serum.

[0102] The specific steps are as follows:

[0103] MPXV B6R, A35R, A29L and M1R proteins purified by nickel column in our laboratory (amino acid sequences are shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17) were diluted with coating solution (sodium bicarbonate buffer solution, PH = 9.6) to final concentrations of 1 μg / ml, 1 μg / ml, 2 μg / ml and 1 μg / ml, respectively. Each dilution was added to a 96-well ELISA plate (Corning, 9018), 60 μl / well, and coated overnight at 4°C. The liquid in the well was discarded, washed 3 times with PBST, and then a blocking solution containing 5% skim milk powder was added, 150 μl / well, and blocked at 37°C for 2h. The serum was diluted with the blocking solution, and the mouse serum was diluted 3 times from 1:100, 100 μL / well, and a blank control group was set up at the same time, and incubated at 37°C for 1h. After washing with PBST for 4 times, add HRP-labeled goat anti-mouse IgG antibody (1:20 000), add 100 μL / well of the ELISA plate, and incubate at 37°C for 1 hour. After washing with PBST again for 4 times, add 100 μL / well of TMB colorimetric solution in the dark and develop at room temperature for 30 minutes. Add 50 μL / well of H 2 SO4 (0.5M) to stop color development. Immediately after stopping color development, read the OD value of each well at 450nm on the microplate reader. 450 Value (P) / negative serum OD 450 The numerical value (N) was the highest serum dilution when P / N>2 as the antibody titer of ELISA.

[0104] The results are as follows Figure 8-11 As shown, different dose groups of MPXV 1103 vaccine can produce high levels of specific IgG antibodies against B6R, A35R, A29L and M1R, and all are dose-dependent. Among them, the production of A29L and M1R specific IgG antibodies can be detected in the medium and high dose groups of MPXV 1103 vaccine 14 days after the first immunization, while no antibodies were detected in the other groups. The above results show that MPXV 1103 vaccine can induce the production of high levels of IgG antibodies against each antigen (B6R, A35R, A29L and M1R), and has a good humoral immune effect.

[0105] 3. Neutralizing antibody detection

[0106] The 50% Plaque Reduction Neutralization Test (PRNT) 50 ) Detect the neutralizing antibody titer of serum samples on the 28th day after immunization. The specific steps are as follows:

[0107] The experiment was conducted when the cell confluence in the 24-well cell plate prepared with Vero E6 cells (ATCC, CRL-1586) the day before reached 80-90%. After the serum was inactivated at 56°C for 30 minutes, it was diluted 3-fold starting from 1:40 with serum-free DMEM medium (Gibco, C11995500BT), and 6 dilution gradients were made. An equal volume of diluted Vaccinia virus (VACV) containing 700 PFU / mL was added, mixed, and incubated at 37°C for 1 hour. The mixed solution after incubation was added to the 24-well cell plate, 200 μl / well, and incubated at 37°C for 1 hour. After the end, the incomplete culture medium (DMEM culture medium containing 2% fetal bovine serum and 1% double antibody (penicillin-streptomycin solution, Biosharp, catalog number BL505A)) containing 0.9% sodium carboxymethyl cellulose (Sodium salt of CaboxyMethyl Cellulose, CMC, Sigma, M0512) was replaced. At the same time, the serum of the non-immunized mice and VACV were used as negative and positive controls, respectively. The cells were incubated at 37°C and 5% CO 2The cells were cultured in a cell culture incubator for 2 days. After fixation with a tissue fixative containing 4% paraformaldehyde (Servicebio, G1101-500mL), the neutralizing antibody titer was determined using crystal violet (Beyotime Biotechnology, C0121-500mL) staining. The serum neutralization percentage was calculated, the neutralization curve was drawn, and the PRNT was calculated. 50 .

[0108] The results are as follows Fig.12 As shown in Figure 2, high levels of neutralizing antibodies were detected in mice immunized with low, medium, and high doses of MPXV 1103 vaccine in a dose-dependent manner. 50 1137, 2300 and 6241, respectively. PRNT of Mix 4-LNPs group 50 The PRNT of the other groups was 212, and that of the M1R-LNPs group was 309. 50 The above results show that MPXV 1103 vaccine can induce high levels of antibodies that neutralize VACV after immunization of mice.

[0109] 4. Cellular Immune Response Detection

[0110] On the 10th day after the second immunization, 5 mice / group were killed and spleen lymphocytes were isolated. The mouse IFN-γ ELISpot kit (Dayou, 2210007) was used to detect the cytokine secretion of spleen lymphocytes and determine the cellular immune response. The specific steps were as follows: the plate was washed 4 times with sterile PBS, 200 μL / well; the washing solution was discarded, and 200 μL / well of RPMI 1640 culture medium containing 10% FBS (Thermo Fisher Scientific, 11875119) was added, and incubated at room temperature for at least 30 minutes; the liquid was discarded, and a cell density of 2.5×10 6 200 μL / well of spleen lymphocyte suspension with a concentration of 1 cell / mL was added, and A35R, B6R, M1R and A29L peptide libraries (Wuhan Dangang Biotechnology Co., Ltd., 15 peptides / strip, 10 amino acid repeats, purity>85%) were added respectively, and the final concentration of each peptide was 2.5 μg / mL. At the same time, negative control wells without stimulants and PMA (Phorbol12-myristate 13-acetate) as a positive control well for stimulants were set up, and the cells were placed at 37°C and 5% CO 2The cells were cultured in a cell culture incubator for 18-24 hours; the cell solution was discarded, and the plate was washed 5 times with PBS, 200 μL / well; the detection antibody was diluted with PBS containing 0.5% FBS to a final concentration of 1 μg / mL, 100 μL / well, and incubated at room temperature for 2 hours; the liquid in the well was discarded, and the plate was washed 5 times with PBS, 200 μL / well; Streptavidin-HRP (the reagent included in the mouse IFN-γ ELISpot kit) was diluted 1:1000 with PBS containing 0.5% FBS (fetal bovine serum), 100 μL / well, and incubated at room temperature for 1 hour; the liquid in the well was discarded, and the plate was washed 5 times with PBS, 200 μL / well; AEC color development solution (included in the IFN-γ ELISpot kit) color development solution, 100 μL / well, incubated until obvious spots appeared; the plate was rinsed with tap water to terminate the color development, and naturally air-dried at room temperature; the plate was read using an ELISpot plate reader (AID ELISPOT READER) to collect and read the spots in each well.

[0111] The results are as follows Figure 13-16 As shown, there were no obvious spots or very few spots in the spleen lymphocytes isolated from the LNP and PBS control groups after stimulation with the A35R, B6R, M1R and A29L peptide libraries, respectively, while different numbers of spots were produced in the MPXV 1103 vaccine immunization group after stimulation with different antigenic peptides. Among them, after stimulation with the B6R, A29L and M1R peptide libraries, the number of spots in the MPXV 1103 vaccine immunization group was significantly higher or higher than that in the other groups. Among the different dose groups of the MPXV 1103 vaccine, the medium dose group (5 μg / mouse) was higher than the low dose group (1 μg / mouse) and the high dose group (20 μg / mouse). Among them, in the B6R peptide library stimulation group, the average number of spots in the high, medium and low dose groups reached 44.4 SFC / 1×10 6 cells, 86.4SFC / 1×10 6 cells and 39.5SFC / 1×10 6 cells; in the A35R peptide library stimulation group, the average number of spots in the high, medium and low dose groups reached 207.2 SFC / 1×10 6 cells, 296.4SFC / 1×10 6 cells and 292.5 SFC / 1×10 6 cells; in the A29L peptide library stimulation group, the average number of spots in the high, medium and low dose groups reached 124 SFC / 1×10 6 cells, 136.8SFC / 1×10 6 cells and 71SFC / 1×10 6 cells; in the M1R peptide library stimulation group, the average number of spots in the high, medium and low dose groups reached 21.6 SFC / 1×10 6 cells, 56.5SFC / 1×10 6cells and 41.5SFC / 1×10 6 The above results show that MPXV 1103 vaccine can induce four antigen-specific cellular immune responses in mice, and the cellular immune effect produced by medium dose (5 μg / mouse) immunization is stronger.

[0112] Example 4 Evaluation of the protective effect of MPXV mRNA vaccine challenge

[0113] 1. Immunization and challenge of mice

[0114] Eighty healthy female SPF Balb / C mice (Beijing Huafukang Biotechnology Co., Ltd.) aged 5-6 weeks were randomly divided into 10 groups, 8 mice / group, including 1103mRNA low-dose group (1μg / mouse), 1103mRNA medium-dose group (5μg / mouse), 1103mRNA high-dose group (20μg / mouse), Mix 4LNPs group (5μg / mouse), B6R-LNPs group (5μg / mouse), A35R-LNPs group (5μg / mouse), A29L-LNPs group (5μg / mouse) and M1R-LNPs group (5μg / mouse), as well as LNP and PBS control groups. After one week of adaptive breeding in the SPF animal room of the University of Science and Technology of China, the mice were injected with vaccines of each dose group once through the outer thigh muscle on day 0 and day 14, respectively. Six weeks after the last booster immunization, 5×10 6 PFU of VACV, 100 μl / mouse, and the following indicators were tested after infection:

[0115] Clinical symptoms: body weight and survival rate;

[0116] Tissue virus content: On the 4th day after infection, 3 mice / group were randomly selected, and the virus titer of nasal turbinates and lung tissues was determined by plaque method.

[0117] 2. Clinical symptoms after infection

[0118] After each group of mice was infected with VACV, the body weight of each group of mice was measured every day and the survival and death of the mice were observed until the 28th day after infection.

[0119] The results of weight change are as follows Fig.17 As shown, the weight of mice in the LNP group and PBS group decreased significantly after VACV infection, and by the fifth day after infection, the weight had dropped to about 80%, while the weight of mice in each vaccine immunization group decreased slightly after VACV infection, and then quickly recovered to the weight before infection, and no significant decrease was observed. The above results show that each vaccine can reduce the effect of VACV infection on the weight of mice.

[0120] The survival rate changes were as follows Fig.18 As shown, mice in the LNP group and PBS group died on the 5th day after infection with VACV, and the mortality rate reached 100% on the 9th day, while mice in the vaccine immunization groups did not die after infection with VACV. The above results show that the MPXV 1103 vaccine and the other vaccines have the effect of protecting mice from VACV infection and death.

[0121] 3. Tissue Virus Content

[0122] To detect the protective effect of different vaccines against VACV infection, the virus content in tissues was determined by plaque method. The specific steps are as follows:

[0123] On the fourth day after infection, 3 mice were randomly selected per group. After isoflurane anesthesia, nasal turbinates and lung tissues were taken and placed in 2 mL EP tubes. Steel beads and 1 mL DMEM medium were added. After grinding, the supernatant was collected after centrifugation at 13 000g for 10 min at 4 °C. The virus content in tissues was determined by plaque method.

[0124] Vero E6 cells with good growth status were plated onto 12-well cell culture plates after trypsin digestion and grown to 90% confluence the next day. The samples to be tested were diluted 10 times with DMEM culture medium to obtain 4 concentrations of virus solution (1:1, 1:10, 1:100 and 1:1000). The cell culture medium was discarded, and 500 μL of the diluted virus solution of the sample to be tested was added to each well, and adsorbed at 37°C for 1 hour. After discarding the virus solution, the incomplete culture medium (containing 2% fetal bovine serum and 1% double antibody (penicillin-streptomycin solution, Biosharp, catalog number BL505A) containing 0.9% sodium carboxymethyl cellulose (Sodium salt of CaboxyMethyl Cellulose, CMC) was replaced. At the same time, the cell wells without virus solution were used as negative controls. Incubate at 37°C, 5% CO 2 Culture in a cell culture incubator for 2 days. Discard the semi-solid culture medium, add 500 μl / well of tissue fixative containing 4% paraformaldehyde, and fix at room temperature for 30 minutes. Wash the cells 3 times with PBST solution. Discard the washing solution, add 500 μl / well of crystal violet staining solution, and stain at room temperature for 20 minutes. Recover the crystal violet staining solution, rinse the plate with tap water, and observe and count.

[0125] The results are as follows Fig.19 As shown, in the nasal concha, the virus contents in the LNP and PBS control groups reached 960 PFU / g and 813.33 PFU / g, respectively, on the 4th day after infection, and the virus contents in the B6R-5μg group and A29L-5μg group reached 546.67 PFU / g and 120 PFU / g, respectively, and no live virus particles were detected in the other groups.

[0126] The results are as follows Fig. 20 As shown, in the lungs, the virus contents in the LNP and PBS control groups reached 15722 PFU / g and 25222 PFU / g, respectively, on the 4th day after infection, and no live virus particles were detected in the other groups.

[0127] The above results indicate that 1103mRNA LNP vaccine can protect mice from VACV infection.

[0128] Example 5 Safety evaluation of MPXV mRNA vaccine

[0129] 1. Mouse Immunization

[0130] Fifteen healthy female SPF Balb / C mice (Beijing Huafukang Biotechnology Co., Ltd.) aged 5-6 weeks were randomly divided into three groups, 5 mice / group, and divided into low-dose group, medium-dose group (5μg / mouse) and high-dose group (20μg / mouse) of MPXV 1103, as well as a PBS control group. After one week of adaptive feeding in the SPF animal room of the University of Science and Technology of China, the vaccines of each dose group were injected once through the outer thigh muscle of the mice on day 0 and day 14, respectively. On the 6th day after booster immunization, blood was collected from each group of mice using anticoagulant tubes, and their blood routine indicators were detected by blood routine instruments. At the same time, 3 mice / group were randomly selected and killed by isoflurane anesthesia. The main internal organs such as the heart, liver, lungs and kidneys of the mice were taken, and the pathological changes of the tissues were observed after H&E staining (sent to Anhui Xinle Biotechnology Co., Ltd. for staining).

[0131] The results are as follows Fig.21 As shown, compared with the PBS control, no obvious abnormalities were found in the blood routine indicators of the medium-dose and high-dose groups of MPXV 1103.

[0132] The results are as follows Fig. 22 As shown, compared with the PBS control, no obvious pathological changes were observed in the major internal organs of the medium-dose and high-dose groups of MPXV 1103.

[0133] The above results indicate that MPXV 1103 shows good safety in mice.

[0134] sequence

[0135] SEQ ID NO:1 B6R base sequence

[0136] ATGAAAACGATTTCCGTTGTTACGTTGTTATGCGTACTACCTGCTGTTGTTTATTCAACATGTACTGTACCCACTATGAATAACGCTAAATTAACGTCTACCGAAACATCGTTTAATGATAAACAGAAAGTTACGTTTACATGTGATTCAGGATATCATTCTTTGGATCCAAATGCTGTCTGCGAAACAGATAAATGGAAATACGAAAATCCATGCAAGAAAATGTGCACAGTTTCTGATTATGTCTCTGAACTATATGATAAGCCATTATACGAAGTGAATTCCACCATGACACTAAGTTGCAACGGTGAAACAAAATATTTTCGTTGTGAAGAAAAAAATGGAAATACTTCTTGGAATGATACTGTCACGTGTCCTAATGCGGAATGTCAACCTCTTCAATTAGAACACGGATCGTGTCAACCAGTTAAAGAAAAATACTCATTTGGGGAATATATGACTATCAACTGTGATGTTGGATATGAGGTTATTGGTGTTTCGTATATAAGTTGTACGGCTAATTCTTGGAATGTTATTCCATCATGTCAACAAAAATGTGATATACCGTCTCTATCTAATGGATTAATTTCCGGATCTACATTTTCTATCGGTGGCGTTATACATCTTAGTTGTAAAAGTGGTTTTACACTAACGGGGTCTCCATCATCCACATGTATCGACGGTAAATGGAATCCCATACTCCCAACATGTGTACGATCTAACGAAGAATTTGATCCAGTGGATGATGGTCCCGACGATGAGACAGATCTGAGCAAACTCTCGAAAGACGTTGTACAATATGAACAAGAAATAGAATCGTTAGAAGCAACTTATCATATAATCATAATGGCGTTGACAATTATGGGTGTCATATTTCTAATCTCTATTATAGTATTAGTTTGTTCCTGTGACAAAAATAATGACCAATATAAGTTCCATAAATTGCTACCGTGA

[0137] SEQ ID NO:2 A35R base sequence

[0138] ATGATGACACCAGAAAACGACGAAGAGCAGACATCTGTGTTCTCCGCTACTGTTTACGGAGACAAAATTCAGGGAAAGAATAAACGCAAACGCGTGATTGGTCTATGTATTAGAATATCTATGGTTATTTCACTACTATCTATGATTACCATGTCCGCGTTTCTCATAGTGCGCCTAAATCAATGCATGTCTGCTAACAAGGCTGCTATTACTGACTCCGCTGTTGCCGTTGCTGCGGCATCATCTACTCATAGAAAGGTTGTGTCTAGCACTACACAATATGATCACAAAGAAAGCTGTAATGGTTTATATTACCAGGGTTCTTGTTATATATTACATTCAGACTATAAGTCATTCGAGGATGCTAAAGCAAACTGCGCTGCGGAATCATCAACACTACCCAATAAATCCGATGTCTTGACTACCTGGCTCATTGATTATGTTGAGGATACATGGGGATCTGATGGTAATCCAATTACAAAAACTACATCCGATTATCAAGATTCTGATGTATCACAAGAAGTTAGAAAGTATTTTTGTACATGA

[0139] SEQ ID NO:3 A29L base sequence

[0140] ATGGACGGAACTCTTTTCCCCGGAGATGACGATCTTGCAATTCCAGCAACTGAATTTTTCTCTACAAAGGCTGCTAAAAATCCAGAGACTAAACGCGAAGCAATTGTTAAAGCCTATGGAGACGATAATGAGGAAACTCTCAAACAACGGCTAACTAATTTGGAAAAAAAGATTACTAATATAACAACAAAGTTTGAACAAATAGAAAAGTGTTGTAAACGCAACGATGAAGTTCTATTTAGGTTGGAAAATCACGCTGAAACTCTAAGAGCGGCTATGATATCTCTGGCTAAAAAGATTGATGTACAGACTGGACGGCATCCATATGAGTGA

[0141] SEQ ID NO:4 M1R base sequence

[0142] ATGGGTGCCGCAGCAAGCATACAGACGACTGTGAATACACTCAGTGAACGTATCTCGTCTAAATTAGAACAAGAAGCGAACGCTAGTGCTCAAACAAAATGTGATATAGAAATCGGAAATTTTTATATCCGACAAAACCATGGATGTAACATCACTGTTAAAAATATGTGCTCTGCGGACGCGGATGCTCAGTTGGATGCTGTGTTATCAGCCGCTACAGAAACATATAGTGGATTAACACCGGAACAAAAAGCATACGTACCAGCTATGTTTACTGCTGCGTTAAACATTCAGACGAGTGTAAACACTGTTGTTAGAGATTTTGAAAATTATGTGAAACAGACTTGTAATTCTAGCGCTGTTGTCGATAACAAATTAAAGATACAAAACGTAATTATAGATGAATGTTACGGAGCCCCAGGATCTCCAACAAATTTGGAATTTATTAATACAGGATCTAGCAAAGGAAATTGTGCCATTAAGGCGTTGATGCAATTGACTACTAAGGCCACTACTCAAATAGCACCTAGACAAGTTGCTGGTACAGGTGTTCAGTTTTATATGATTGTTATCGGTGTTATAATATTGGCAGCGTTGTTTATGTACTATGCCAAGCGTATGCTGTTCACATCCACCAATGATAAAATCAAACTTATTTTAGCCAATAAGGAAAACGTCCATTGGACTACTTACATGGACACATTCTTTAGAACTTCTCCGATGATTATTGCTACCACGGATATACAAAACTGA

[0143] SEQ ID NO:5 1103 (B6R - linker - A35R - linker - A29L - linker - M1R) base sequence

[0144]

[0145] Amino acid sequence of SEQ ID NO:6 B6R

[0146] MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIMALTIMGVIFLISIIVLVCSCDKNNDQYKFHKLLP

[0147] Amino acid sequence of SEQ ID NO:7 A35R

[0148] MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT

[0149] Amino acid sequence of SEQ ID NO:8 A29L

[0150] MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE

[0151] Amino acid sequence of SEQ ID NO:9 M1R

[0152] MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN

[0153] SEQ ID NO:10 Amino acid sequence of 1103 (B6R-Junction-A35R-Junction-A29L-Junction-M1R) (Junction amino acid sequences are underlined)

[0154] MKTISVVTLLCVLPAVVYSTCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYHIIIMALTIMGVIFLISIIVLVCSCDKNNDQYKFHKLLP GGGGSGGGGSGGGGS MMTPENDEEQTSVFSATVYGDKIQGKNKRKRVIGLCIRISMVISLLSMITMSAFLIVRLNQCMSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT GGGGSGGGGSGGGGS MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE GGGGSGGGGSGGGGS MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNK LKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTGVQFYMIVIGVIILAALFMYYAKRMLFTSTNDKIKLILANKENVHWTTYMDTFFRTSPMIIATTDIQN

[0155] SEQ ID NO:11 5'UTR sequence

[0156] GCACTGAGCCTAAGCAGCCGGTGATGGCGGCAGCGGCTGTGGTGGCTGCGGCGGGTCCGGGCCC

[0157] SEQ ID NO:12 3'UTR sequence

[0158] GCTGGAGCCTCGGTGGCCATGCTTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTG GGCGGCA

[0159] SEQ ID NO:13PolyA

[0160] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0161] SEQ ID NO:14B6R plate protein amino acid sequence (SR——XbaⅠ restriction site; GS——linker polypeptide; HHHHHH——His tag protein)

[0162] TCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDK PLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSCQPVKEKYSFGEYMTINCDVGYEVIG VSYISCTANSWNVIPSCQQKCDIPSLSNGLISGTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNE EFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYH SRGSHHHHHH

[0163] SEQ ID NO:15 A35R plate protein amino acid sequence (SR——XbaⅠ restriction site; GS——linker polypeptide; HHHHHH——His tag protein)

[0164] VRLNQCMSANKAAITDSAVAVAAASSTHRKVVSSTTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKAN CAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT SRGSHHHHHH

[0165] SEQ ID NO:16 Amino acid sequence of M1R plate protein (SR——XbaⅠ restriction site; GS——linker polypeptide; HHHHHH——His tag protein)

[0166] MGAAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVL SAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNL EFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAGTG SRGSHHHHHH

[0167] SEQ ID NO:17 A29L plate protein amino acid sequence (MGSS and SSG - linker polypeptide; HHHHHH - His tag protein; LVPRGS - thrombin recognition and cleavage site; MASMTGGQQMG - T7 tag peptide; RGSEFELRRQAC - multiple cloning restriction enzyme cleavage site)

[0168] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFELRRQAC MDGTLFPGDDDLAIPATEFFSTKAAK NPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKCCKRNDEVLFRLENHAETLRAAMISLAKKIDVQ TGRHPYE

[0169] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A multivalent monkeypox virus mRNA vaccine, characterized in that: The vaccine is obtained by co-expressing multiple antigenic proteins of monkeypox virus in series through a flexible linker, and the multiple antigenic proteins are selected from B6R, A35R, A29L and M1R, the amino acid sequence of B6R is shown in SEQ ID NO:6, the amino acid sequence of A35R is shown in SEQ ID NO:7, the amino acid sequence of A29L is shown in SEQ ID NO:8, and the amino acid sequence of M1R is shown in SEQ ID NO:

9.

2. The vaccine according to claim 1, characterized in that The coding sequence of B6R is shown in SEQ ID NO:

1.

3. The vaccine according to claim 1, characterized in that The coding sequence of A35R is as shown in SEQ ID NO:

2.

4. The vaccine according to claim 1, characterized in that The coding sequence of A29L is shown in SEQ ID NO:

3.

5. The vaccine according to claim 1, characterized in that The coding sequence of M1R is shown in SEQ ID NO:

4.

6. The vaccine according to any one of claims 1 to 5, characterized in that The flexible linker comprises glycine and serine.

7. The vaccine according to claim 6, characterized in that The amino acid sequence of the flexible linker is GGGGSGGGGSGGGGS.

8. The vaccine according to any one of claims 1 to 5, characterized in that The coding sequence of the mRNA vaccine also includes 5'UTR and 3'UTR sequences, and a Cap modification is added to the 5' end and a Poly (A) is added to the 3' end.

9. The vaccine according to claim 8, characterized in that The nucleotide sequence of the 5'UTR is shown in SEQ ID NO:11, and the nucleotide sequence of the 3'UTR is shown in SEQ ID NO:

12.

10. A pharmaceutical composition comprising the vaccine according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. Use of the vaccine according to any one of claims 1 to 9 in the preparation of a medicament for preventing monkeypox virus infection.

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