MRNA-MV2 vaccine of monkey pox virus and preparation method of mRNA-MV2 vaccine

Abstract

The invention provides an mRNA-MV2 vaccine of monkey pox virus and a preparation method of the mRNA-MV2 vaccine, and relates to the technical field of vaccine preparation. The amino acid sequence of the mRNA-MV2 vaccine is as shown in SEQ ID NO.1, the nucleic acid sequence of the mRNA-MV2 vaccine is as shown in SEQ ID NO.2, and the vaccine is obtained by forming a single-chain dimer conformation through reasonable excision and reassembly and connecting two antigens of conservative A35R and conservative M1R in series by using a cutting sequence P2A. The invention overcomes the defects of the prior art, and designs and prepares a vaccine which has a good prevention effect on MPXV infection, and is safe, stable, short in research and development period and low in cost.

Description

Technical Field

[0001] The present invention relates to the technical field of vaccine preparation, and in particular to an mRNA-MV2 vaccine of monkeypox virus and a preparation method thereof. Background Art

[0002] Monkeypox virus (MPXV) is an enveloped, double-stranded DNA virus of the genus Orthopoxvirus in the family Poxviridae. It has two distinct clades: Clade I (with subclades Ia and Ib) and Clade II (with subclades IIa and IIb). First discovered in Denmark in 1958, MPXV caused a global monkeypox outbreak in 2022. Infection with MPXV causes a painful rash that progresses through macules, papules, herpes, and pustules, eventually forming crusts. The herpes and pustules are often spherical, firm, and may be accompanied by significant itching and pain. After the scabs fall off, they may leave erythema or hyperpigmentation, or even scarring, which can persist for years. In addition, bacterial infections may develop on the skin, leading to abscesses or severe skin lesions. Complications may include pneumonia, corneal infection with vision loss, painful or difficult swallowing, dehydration or malnutrition due to vomiting and diarrhea, and infections of the blood (sepsis), brain (encephalitis), heart (myocarditis), rectum (proctitis), reproductive organs (balanitis), or urinary tract (urethritis).

[0003] Monkeypox can be fatal in some cases. MPXV vaccine research focuses on recombinant protein vaccines, inactivated and live attenuated vaccines. Currently, there are three approved vaccines for monkeypox virus (ACAM2000, JYNNEOS, and Lc16m8), all of which are live attenuated vaccines. Currently, there are no mRNA vaccines available for MPXV.

[0004] MPXV has two forms of infectious virus particles: intracellular mature virus (IMV) and extracellular enveloped virus (EEV). Vaccines composed of a single antigen have poor protective efficacy. The current main strategy is cocktail therapy, which is to mix multiple antigens for immunization. It has been proven to have certain protective efficacy in many studies, but this method increases the complexity and cost of vaccine production to a certain extent. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides an mRNA-MV2 vaccine for monkeypox virus and a preparation method thereof, and designs and prepares a vaccine that has good preventive effect on MPXV infection and is safe and stable, with a short R&D cycle and low cost.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A monkeypox virus mRNA-MV2 vaccine, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleic acid sequence of which is shown in SEQ ID NO.2.

[0007] The preparation method of mRNA-MV2 vaccine comprises the following steps: S1. Search the database for MPXV-A35R / M1R protein to obtain conserved A35R and M1R for sequencing, wherein the amino acid sequence of A35R is shown in SEQ ID NO. 5, and the amino acid sequence of M1R is shown in SEQ ID NO. 6; S2. Through reasonable excision and reassembly to form a single-chain dimer conformation, the A35R and M1R antigens are connected in series using the cleavage sequence P2A to obtain the antigen amino acid sequence shown in SEQ ID NO.1; S3. Codon optimization was performed on the antigen amino acids according to human codon preference to obtain the DNA sequence shown in SEQ ID NO. 2. The DNA sequence was constructed into the plasmid PUC57 containing a T7 promoter, 5'UTR, 3'UTR, and 100 PolyA residues to form PUC57-T7-5UTR-E3-E2-6k-E1-3UTR-PolyA, thus completing the plasmid construction; S4. Amplify and transcribe the successfully constructed plasmid to obtain pure mRNA; S5. The mRNA obtained above is prepared into lipid nanoparticles LNP by a microfluidic method.

[0008] Preferably, the sequence of the 5'UTR in step S3 is shown as SEQ ID NO. 3, and the sequence of the 3'UTR is shown as SEQ ID NO. 4.

[0009] Preferably, the amplification method in step S4 is to use Top10 bacterial strains to amplify the plasmid, extract the plasmid, use BsaI to perform enzyme digestion and linearization, and use DNA magnetic beads to recover the linearized fragments.

[0010] Preferably, the transcription in step S4 is performed using a commercial transcription kit vazyme#DD4203, and purified using RNA magnetic beads to obtain pure RNA.

[0011] Preferably, the specific method of preparing lipid nanoparticles LNP in step S5 includes the following steps: S5-1. Dissolve the mRNA in 50 mM citric acid buffer at pH 4 to control the mRNA concentration to 108 ng / μL to obtain an mRNA-citric acid solution. S5-2, prepare an anhydrous ethanol solution with a SM102 mass concentration of 50%, a DMG-PEG2000 mass concentration of 1.5%, a DSPC mass concentration of 10%, and a cholesterol mass concentration of 38.5% to obtain a lipid mixed solution; S5-3. The lipid mixed solution and the mRNA-citric acid solution were filtered through a 0.22 μm microporous filter membrane respectively, and mixed using a microfluidic instrument according to the ratio of the phosphorus content in the mRNA to the nitrogen content in SM102 at 1:8 to obtain lipid nanoparticles LNP.

[0012] Preferably, in step S5-3, the flow rate ratio of the mixing performed by the microfluidic instrument is 15 mL / min: 5 mL / min for the mRNA-citric acid solution: lipid mixed solution.

[0013] The present invention provides a monkeypox virus mRNA-MV2 vaccine and a preparation method thereof, which has the following advantages over the prior art: In this study, the conserved transmembrane regions of the A35R and M1R sequences were excised and their structures were predicted using AlphaFold3. Based on this structure prediction, rational excision and reassembly were performed to achieve a dimer-like structure. This single-chain dimer design strategy allows the immunogen to possess more antigenic epitopes. To ensure the structural stability of the 2A35Rs and 2M1Rs, the amino acid sequences were linked using a P2A peptide. This design was then applied to an mRNA vaccine platform, which was prepared into lipid nanoparticles (LNPs) using a microfluidic method to obtain mRNA-MV2. This mRNA was then immunized into Balb / c mice. Elisa assays were used to assess the level of binding antibodies produced by immunization, and neutralization assays were used to measure neutralizing antibodies against monkeypox virus in the serum of mice at the end of the immunization period. Immunogenicity evaluation results showed that after two immunizations, mice produced high binding antibody titers, moderate neutralizing antibody titers, and a high-level T cell immune response. In subsequent challenge and protection experiments, the immunized mice controlled viremia and significantly reduced lung load and lung damage. Vaccinia virus (VACV) and monkeypox virus belong to the same genus. mRNA-MV2 elicited high neutralizing antibody titers against VACV in mice, protected mice against a lethal dose (30 LD50) of VACV, and significantly reduced viral loads and pathological damage in various organs. The resulting vaccine, mRNA-MV2, exhibited strong immunogenicity, eliciting robust humoral and cellular immune responses, and demonstrated protective efficacy against both MPXV and VACV. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the mRNA-MV2 design strategy of the present invention; Figure 2Schematic diagram of the particle size and encapsulation efficiency of lipid nanoparticles produced by LNP encapsulation in an embodiment of the present invention; Figure 3 Schematic diagram of RNA electrophoresis of in vitro transcription in an embodiment of the present invention; Figure 4 Schematic diagram of verification of mRNA-MV2 cell level expression in an embodiment of the present invention; Figure 5 This is a schematic diagram of the weight changes of BALB / c mice after vaccination in an embodiment of the present invention; Figure 6 Schematic diagram of the titer of binding antibodies against A35R (a) and M1R (b) in serum according to an embodiment of the present invention; Figure 7 Schematic diagram of neutralizing antibody titers against MPXV (a) and VACV (b) in serum at the immunization endpoint (28 days) in an embodiment of the present invention; Figure 8 Schematic diagram showing that MV2 vaccine induces strong T cell immunity and high IFN-γ levels in mice after immunization; Figure 9 Schematic diagram showing that MV2 vaccine induces strong T cell immunity in mice and induces higher IL-2 levels; Figure 10 This is a schematic diagram of the weight changes of BaLB / c mice after immunization and MPXV challenge experiment in the embodiment of the present invention; Figure 11 This is a schematic diagram of the changes in viremia in the MPXV challenge experiment in BaLB / c mice after immunization in an embodiment of the present invention; Figure 12 This is a schematic diagram of the changes in viral loads in multiple organs of BaLB / c mice undergoing MPXV challenge experiments after immunization in an embodiment of the present invention; Figure 13 This is a schematic diagram of the pathological lesion scoring of the MPXV challenge experiment in BaLB / c mice after immunization in the embodiment of the present invention; Figure 14 This is a schematic diagram of the staining of pathological lesion sections of various tissues in the MPXV challenge experiment in BaLB / c mice after immunization in the embodiment of the present invention; Figure 15 Schematic diagram of weight changes in BaLB / c mice subjected to VACV challenge experiments after immunization in the embodiment of the present invention; Figure 16 This is a schematic diagram of the changes in viral loads in multiple organs of VACV challenge experiments in BaLB / c mice after immunization in an embodiment of the present invention; Figure 17This is a schematic diagram of the pathological lesion scoring of the VACV challenge experiment on immunized BaLB / c mice in an embodiment of the present invention; Figure 18 This is a schematic diagram of the staining of pathological damage sections of various tissues in the VACV challenge experiment in immunized BaLB / c mice in an example of the present invention. DETAILED DESCRIPTION

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example 1:

[0016] Preparation of mRNA-MV2 vaccine: The MPXV-A35R / M1R protein was searched in the NCBI database, and 7258 A35R amino acid sequences and 7011 M1R amino acid sequences from different strains were obtained. Sequence alignment was performed using snapgene software. The most frequently occurring amino acid at each amino acid position was selected and the amino acids were reassembled in order to obtain the conserved amino acid sequences of A35R (shown in SEQ ID NO. 5) and M1R (shown in SEQ ID NO. 6). The conserved sequences A35R and M1R transmembrane regions were removed and their structures were predicted using AlphaFold3. Rational removal and reassembly were performed based on the structure prediction to give them a dimer-like structure. The two antigens were connected in series using the cleavage sequence P2A to maximize the stability of the two antigen structures. The resulting antigen amino acid sequence is shown in SEQ ID NO.1: The specific method is as follows: for the A35R conserved sequence, it is divided into two parts: the first part: the sequence 1-89 is removed; the second part: the sequence 1-99 is removed. Then the assembly is carried out in order.

[0017] The M1R conserved sequence was divided into two parts: the first part removed the sequence from 182 to 250, and the second part removed the sequences from 1 to 2 and 182 to 250. The assembly was performed in this order.

[0018] P2A sequence: (GSG)ATNFSLLKQAGDVEENPGP, where GSG promotes P2A self-cleavage.

[0019] Codon optimization was performed according to human codon preference to obtain the DNA sequence SEQ ID NO.2. The DNA sequence was constructed into the plasmid PUC57 containing T7 promoter, 5'UTR (such as SEQ ID NO.3), 3'UTR (SEQ ID NO.4) and 100 PolyA (PUC57-T7-5UTR-E3-E2-6k-E1-3UTR-PolyA) (detailed design ideas are shown in Figure 1 ); Plasmids were amplified using Top10 strains, extracted, and linearized using BsaI. The linearized fragments were recovered using DNA magnetic beads. Transcription was performed using a commercial transcription kit (vazyme #DD4203), and purified using RNA magnetic beads to obtain pure RNA: Transduce the plasmid into Top10 competent cells, then add 800 μl of LB medium. After initial amplification, take 400 μl and add it to 500 ml of LB medium. Add ampicillin at a 1:1000 ratio and shake on a shaker (37°C, 220 rpm) for 16 hours. Use a commercial plasmid extraction kit for plasmid extraction. Correctly sequenced plasmids were linearized using the following system: 50 μg of plasmid, 10 μl of BsaI, 50 μl of 10× BsaI buffer, and add enzyme-free water to 500 μl. To recover linearized DNA using commercial DNA magnetic beads (vazyme #N411-01): Add 0.5 times the volume (250 μl) of DNA magnetic beads to the linearization reaction solution and pipette evenly. Incubate at room temperature for 5 minutes, then place on a magnetic rack. After 5 minutes, aspirate the liquid, add 600 μl of 80% ethanol, and repeat this process after 30 seconds. Discard the liquid, incubate at room temperature for 7 minutes, air dry, remove from the magnetic rack, and add 40 μl of enzyme-free water to dissolve the adsorbed magnetic beads. Incubate at room temperature for 5 minutes, place on the magnetic rack again, and aspirate the liquid after 5 minutes. This is the purified linearized product.

[0020] Transcription was performed using a commercial transcription kit (vazyme#DD4203): Mix according to the following system Add RNase-free ddH2O to 20ul 10 × Co-Reaction Buffer 2ul N1-Me-Pseudo UTP (100 mM) 1.5ul ATP Solution (100 mM) 1.5ul CTP Solution (100 mM) 1.5ul GTP Solution (100 mM) 1.5ul CAG Trimer 1ul T7 RNA Polymerase Mix 2ul React in a 37°C metal bath for 2 hours, then add 1ul DNaseI Purify RNA using commercially available RNA magnetic beads (vazyme #N412-01): Add 1.8 times the volume (36 μl) of RNA magnetic beads to the transcription reaction mixture and pipette to mix thoroughly. Incubate at room temperature for 5 minutes, then place on a magnetic rack. After 5 minutes, aspirate the liquid, add 200 μl of 80% ethanol, and repeat after 30 seconds. Discard the liquid, incubate at room temperature for 7 minutes, air dry, remove from the magnetic rack, add 50 μl of enzyme-free water to dissolve the adsorbed beads, incubate at room temperature for 5 minutes, place on the magnetic rack again, and aspirate the liquid after 5 minutes. This is the purified transcript.

[0021] The purified RNA was dissolved in a 50 mM citric acid buffer at pH 4 to a final concentration of 108 ng / μL to obtain an mRNA-citric acid solution. SM102, DMG-PEG2000, DSPC, and cholesterol were mixed and dissolved in anhydrous ethanol at ratios of 50%, 1.5%, 10%, and 38.5% to obtain a lipid mixture solution. The lipid mixture solution and the mRNA-citric acid solution were filtered through a 0.22 μm microporous membrane and mixed using a microfluidic instrument at a ratio of 1:8, based on the phosphorus content of the mRNA and the nitrogen content of SM102. The flow rate ratio was 15 mL / min:5 mL / min for the mRNA-citric acid solution:lipid mixture solution to obtain mRNA-LNP. Detection:

[0022] The obtained mRNA-LNP was immediately diluted with 15 ml of the above citric acid buffer and ultrafiltered using a 100KD ultrafiltration tube with a centrifugal force of 3000 g. After ultrafiltration to 1 / 4 volume, 20 mM Tris-HCl buffer (PH = 7.5) was added to 15 mL. After repeating twice, the particle size and encapsulation efficiency were measured. It was found that the particle size was about 100 nm and the encapsulation efficiency was above 95% (such as Figure 2 shown); The electrophoresis results of in vitro transcribed RNA are as follows Figure 3 As shown; lane 2 is a DNA marker with a size of 5000, lanes 1, 3, and 6 are blank; lanes 4 and 5 are purified MV2-RNA.

[0023] After preparing mRNA-MV2, transfection experiments were performed on 293T cells. After 24 hours, total cell protein was collected and Western blotting was performed using MPXV A35R and M1R antibodies, respectively, to detect target antigen expression. This ensured that the cells in the mRNA-MV2 group successfully expressed the antigen. Specifically, 50w 293T cells were added to a 6-well plate and then cultured for 16 hours. RNA transfection was performed using a commercial RNA transfection reagent. Specifically, 3.75ul of transfection reagent was aspirated, 125ul of Opti-MEM was added, mixed, and allowed to stand for 10 minutes. Simultaneously, 5ug of RNA was aspirated, 250ul of Opti-MEM was added, 125ul of the transfection reagent dilution obtained in the previous step was mixed, and 125ul of the transfection reagent was added to the dilution. The mixture was mixed and allowed to stand for 5 minutes. Finally, the cells were evenly distributed across the 6-well plate. The control group was left untreated. After culturing the cells for another 24 hours, the culture medium in the wells was aspirated, and 200ul / well of cell lysis buffer and 2ul / well of protease inhibitors were added. After incubating on ice for 30 minutes, aspirate 80 μl of lysate from each well and add 20 μl of 5× loading biffer. Mix thoroughly and incubate in 95°C electrophoresis for 10 minutes. After cooling, add the control and experimental groups to the wells of a 12-well 12% precast gel. Run the electrophoresis apparatus at 90V for 30 minutes, then at 180V for 1 hour. Transfer the membrane using a semi-dry transfer method, then add 20 ml of blocking buffer and incubate at room temperature for 15 minutes. Discard the blocking buffer, add 10 ml of the corresponding primary antibody diluted in blocking buffer, and incubate at 4°C for 16 hours. Discard the primary antibody, add 15 ml of 1× TBST, and wash the membrane for 10 minutes. Repeat three times. Add 10 ml of the corresponding secondary antibody diluted in blocking buffer and incubate at room temperature for 1 hour. Discard the secondary antibody, add 15 ml of 1× TBST, and wash the membrane for 10 minutes. Repeat three times. Develop the membrane using developer and a developer, and save the image. To ensure that all samples were normal, we repeated the experiment using an anti-β-actin antibody. Unlike the previous experiment, this time, only the primary antibody was added, followed by a 1-hour incubation at room temperature. The membrane was then washed with 15 ml of 1× TBST for 10 minutes, and this was repeated three times. The membrane was then developed using a developer. Our results showed that, compared to the control, specific bands at 20 kD were present when using either anti-A35R or anti-M1R antibodies, and Western blotting using β-actin was also performed.

[0024] Specific results such as Figure 4 As shown, the internal control of the control group and the experimental group were expressed normally. It shows that both components of the vaccine MV2 (A35R and M1R) can be expressed in 293T.

[0025] The mRNA-MV2 vaccine was administered twice, at a high dose of 10 μg / animal and a low dose of 5 μg / animal, and PBS was used as a control, at 0 and 14 days respectively. mRNA-LNP was prepared as described above, and female Balb / c aged 4 to 6 weeks were immunized with a high dose of 10 μg / animal, a low dose of 5 μg / animal, and PBS as a control, by intramuscular injection.

[0026] Within five days after immunization with vaccine MV2, the body weight of mice was measured every other day. The specific results are as follows: Figure 5 The results showed that after immunization, the mice's weight only decreased slightly and then quickly returned to normal, indicating that the vaccine MV2 has a certain degree of safety and does not cause a significant decrease in the mice's weight; The immunogenicity of the vaccine was assessed by testing the serum for specific IgG binding antibodies to A35R and M1R proteins: Recombinant A35R and M1R proteins (Antibody system, #EVV13101 / #EVV13301, 1 μg / mL) were adsorbed for 16 hours at 4°C and then immobilized in a 96-well microplate (Thermo Fisher Scientific, #442404). Serum samples were serially diluted 1:100 in detection buffer (0.05% BSA / PBST) and then incubated for 60 minutes at 37°C. The wash step was repeated before horseradish peroxidase-conjugated polyclonal goat anti-mouse IgG (Invitrogen, #A-10668) was applied at a 1:30,000 dilution in sample diluent (100 μL / well). Color development was performed with a secondary incubation (37°C, 60 minutes) and subsequent washes using 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific, 100 μL / well) under ambient conditions (15 minutes). The enzymatic reaction was quenched with an acidic stop solution (SolarBio, #C1058), and dual-wavelength optical density measurements were recorded at 450 nm (primary wavelength) and 630 nm (reference wavelength). Negative control wells were incubated with assay buffer instead of serum specimens to define the baseline value, while the seropositivity threshold was determined to be 2.1-fold higher than that of the positive control wells. The geometric mean titer (GMT) of antigen-specific IgG was calculated as the reciprocal absorbance value at the highest threshold.

[0027] BALB / c mice were immunized with MV2 vaccine on day 0 and day 14, and blood was collected on day 7, day 14, day 21, and day 28 to detect the binding antibody titers against A35R and M1R in the serum. The results showed that the binding antibody titers in the serum of mice were significantly increased after immunization. The specific results are as follows: Figure 6 shown.

[0028] The sera of the endpoint mice were tested for neutralizing antibodies using the CPE method in vero cells using MPXV and VACV viruses for the neutralization test: To quantify the neutralizing activity of sera against the replication-competent mpox virus (MPXV) and the vaccine virus (VACV), live virus micro-neutralization tests were performed under biosafety level 2 or 3 containment protocols. Mouse sera were heat-inactivated (56°C, 30 min) before logarithmic two-fold serial dilutions in Dulbecco's modified Eagle's medium (DMEM) using 96-well microplates. In each experimental replicate, 50 μL of titrated serum dilutions were dispensed into designated wells. Virus working stocks in DMEM were normalized to a target concentration of 2x10 3 Plaque-forming units (PFU) per milliliter. Equal molar amounts (50 μL) of virus inoculum and serially diluted serum specimens were incubated in parallel (37°C, 1 h) to promote the formation of antibody-virus complexes. After incubation, 100 μL of Vero cell suspension (1.5x10 4 cells / well, complete medium) was inoculated into each virus-serum reaction well and incubated continuously under standard conditions (37°C, 5% CO2) for 5-7 days. Neutralization was determined by quantitative microscopic analysis of the progression of virus-induced cytopathic effects (CPE). Serum neutralization titers were calculated as the reciprocal endpoint dilution that achieved ≥50% inhibition of CPE relative to virus control wells, expressed as 50% neutralization titers (NT50).

[0029] MV2 vaccine immunized BALB / c mice at 0d and 14d, and the neutralizing antibody titers against MPXV and VACV in the sera of the endpoint (28d) were detected, the results showed that MV2 vaccine induced good neutralizing antibody titers against MPXV and VACV, see Figure 7 for specific results. The IL-2 and INF-γ positive cells of the spleen immune cells after antigen stimulation were detected by the ElisPot method to evaluate the level of cellular immunity: Enzyme-linked immunospot (ELISpot) assays were performed according to the manufacturer's standard protocol (Mabtech®). Mouse spleen tissue was aseptically dissected, and peripheral blood mononuclear cells (PBMCs) were isolated using a commercial lymphocyte isolation kit (SolarBio®, #P8860). Antigen-specific stimulation was achieved by incubation with recombinant A35R or M1R protein (2 μg / 50 μL per well). Experimental controls were established as follows: unstimulated cells (negative control in each experimental replicate) were used to assess baseline reactivity, while cells activated with phytohemagglutinin (PHA) served as a positive process control. Subsequent procedural stages included equilibration (37°C, 5% CO2), an automated wash cycle, and seeding of cells into pre-coated 96-well ELISpot plates (Mabtech®; #3321-4APT-10 membrane plates, #3441-4APW-10 plate covers). Final spot quantification and high-resolution image acquisition were performed using the IRIS™ automated ELISpot reader system (Mabtech®), and data were normalized to every 10 6 Spot-forming units (SFU) of cells.

[0030] Specific results such as Figure 8 and Figure 9 As shown, MV2 vaccine induced strong T cell immunity in mice after immunization, inducing high levels of IFN-γ (a) and IL-2 (b).

[0031] MPXV challenge protection experiment: Blood samples were collected at different time points after challenge and viral load was measured using RT-qPCR to monitor the dynamic changes in viremia. Seven days after challenge, mice in each group were dissected and lung viral load was measured using RT-qPCR to determine whether the vaccine effectively inhibited viral replication. Pathological damage in various tissues and organs was also detected using HE staining, and the corresponding pathological scores were used for relative quantitative statistical analysis of the extent of pathological damage. Specifically, the intranasal challenge method was used to give mice 1×10 6 In MPXV-challenged mice, body temperature and body weight were monitored every other day, and blood and oropharyngeal swabs were collected. All animals survived until day 7 post-challenge, after which they were euthanized. Heart, liver, spleen, lung, kidney, brain, duodenum, and rectum tissues were collected for viral load quantification and histopathological analysis.

[0032] Specific results can be found in Figure 10-14 As shown; To investigate the cross-protective efficacy of the vaccine, a challenge protection experiment was conducted using a lethal dose (30LD50) of vaccinia virus Tiantan strain (VACV-VTT): Body weight and temperature were measured at 1, 3, 5, 7, 9, 11, 12 days post-challenge. At 12 days post-challenge, all mice were euthanized (control mice were euthanized at 6 days post-challenge as they died at this time point), and organs were collected for viral load determination by RT-qPCR and histopathological analysis by HE staining. Specifically, the intranasal challenge method was used, and the mice were given 30 LD50 of VACV by nasal instillation. Body weight and temperature were measured at 1, 3, 5, 7, 9, 11, 12 days post-challenge. All control mice died at 6 days post-virus infection, and the terminal body weight and temperature were recorded. Specimens were collected from the dead control mice, including heart, liver, spleen, lung, kidney, brain, duodenum and rectum. All vaccinated groups survived to 12 days post-challenge before euthanasia. The determined internal organs (heart, liver, spleen, lung, kidney, brain, duodenum and rectum) were removed from the vaccinated mice for subsequent viral load quantification and histopathological analysis.

[0033] The specific results are as follows Figures 15-18 .

[0034] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A monkeypox virus mRNA-MV2 vaccine, characterized in that: The amino acid sequence of the vaccine is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.

2.

2. A method for preparing the mRNA-MV2 vaccine according to claim 1, characterized in that: The preparation method comprises the following steps: S1. Search the database for MPXV-A35R / M1R protein to obtain conserved A35R and M1R for sequencing, wherein the amino acid sequence of A35R is shown in SEQ ID NO. 5, and the amino acid sequence of M1R is shown in SEQ ID NO. 6; S2. Through reasonable excision and reassembly to form a single-chain dimer conformation, the A35R and M1R antigens are connected in series using the cleavage sequence P2A to obtain the antigen amino acid sequence shown in SEQ ID NO.1; S3. Codon optimization was performed on the antigen amino acids according to human codon preference to obtain the DNA sequence shown in SEQ ID NO.

2. The DNA sequence was constructed into the plasmid PUC57 containing a T7 promoter, 5'UTR, 3'UTR, and 100 PolyA residues to form PUC57-T7-5UTR-E3-E2-6k-E1-3UTR-PolyA, thus completing the plasmid construction; S4. Amplify and transcribe the successfully constructed plasmid to obtain pure mRNA; S5. The mRNA obtained above is prepared into lipid nanoparticles LNP by a microfluidic method.

3. The preparation method according to claim 2, wherein: The sequence of the 5'UTR in step S3 is shown as SEQ ID NO. 3, and the sequence of the 3'UTR is shown as SEQ ID NO.

4.

4. The preparation method according to claim 2, wherein: The amplification method in step S4 is to use Top10 bacterial strains to amplify the plasmid, extract the plasmid, use BsaI to linearize the enzyme, and use DNA magnetic beads to recover the linearized fragments.

5. The preparation method according to claim 2, wherein: The transcription method in step S4 is to use a commercial transcription kit vazyme#DD4203 for transcription, and to purify using RNA magnetic beads to obtain pure RNA.

6. The preparation method according to claim 2, characterized in that The specific method for preparing lipid nanoparticles LNP in step S5 includes the following steps: S5-1. Dissolve mRNA in 50 mM citric acid buffer at pH 4 to control the mRNA concentration to 108 ng / μL to obtain an mRNA-citric acid solution. S5-2, prepare an anhydrous ethanol solution with a SM102 mass concentration of 50%, a DMG-PEG2000 mass concentration of 1.5%, a DSPC mass concentration of 10%, and a cholesterol mass concentration of 38.5% to obtain a lipid mixed solution; S5-3. The lipid mixed solution and the mRNA-citric acid solution were filtered through a 0.22 μm microporous filter membrane respectively, and mixed using a microfluidic instrument according to the ratio of the phosphorus content in the mRNA to the nitrogen content in SM102 at 1:8 to obtain lipid nanoparticles LNP.

7. The preparation method according to claim 6, characterized in that: In step S5-3, the flow rate ratio of the mixing performed by the microfluidic instrument is 15 mL / min: 5 mL / min for the mRNA-citric acid solution: the lipid mixed solution.

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