An mRNA-mv2 vaccine of monkeypox virus and a preparation method thereof

Abstract

The application provides an mRNA-MV2 vaccine of monkeypox virus and a preparation method thereof, and relates to the technical field of vaccine preparation.The amino acid sequence of the mRNA-MV2 vaccine is shown as SEQ ID NO.1, the nucleic acid sequence is shown as SEQ ID NO.2, and the vaccine is obtained by cutting and reassembling two antigens of conservative A35R and conservative M1R in series through a cutting sequence P2A to form a single-chain dimer conformation.The application overcomes the defects of the prior art, and designs and prepares a vaccine which has a good preventive effect on MPXV infection, is safe and stable, has a short research and development cycle, and is low in cost.

Description

Technical Field

[0001] This invention relates to the field of vaccine preparation technology, specifically to a monkeypox virus mRNA-MV2 vaccine and its preparation method. Background Technology

[0002] Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus belonging to the genus Orthopoxvirus in the family Poxviridae. This virus has two distinct branches: branch I (with sub-branches Ia and Ib) and branch II (with sub-branches IIa and IIb). It was first discovered in Denmark in 1958 and caused a global monkeypox outbreak in 2022. Infection with monkeypox virus results in a painful rash that progresses through several stages: macules, papules, vesicles, pustules, and finally crusting. Vesicles and pustules are often spherical, firm in texture, and may be accompanied by significant itching and pain. After the crusts fall off, erythema or pigmentation may remain, and even scars can persist for several years. In addition, bacterial infections of the skin can lead to abscesses or severe skin damage, and a variety of complications may occur, such as pneumonia, corneal infection with vision loss, painful or difficult swallowing, dehydration or malnutrition due to vomiting and diarrhea, as well as blood infections (sepsis), brain infections (encephalitis), heart infections (myocarditis), rectal infections (proctitis), genital infections (balanitis), or urinary tract infections (urethritis).

[0003] In some cases, monkeypox can be fatal. Current research platforms for MPXV vaccines primarily include recombinant protein vaccines, inactivated and live attenuated vaccines, etc. Currently, three vaccines have been approved for use against monkeypox virus (ACAM2000, JYNNEOS, and Lc16m8), all of which are live attenuated vaccines. There are currently no mRNA vaccines available for MPXV.

[0004] MPXV exists in two forms: 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 involves immunizing with a mixture of multiple antigens. This method has been shown to have some protective efficacy in many studies, but it increases the complexity and cost of vaccine production to some extent. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a monkeypox virus mRNA-MV2 vaccine and its preparation method, designing and preparing a vaccine that has good preventive effect against MPXV infection, is safe and stable, has a short development cycle, and is low in cost.

[0006] To achieve the above objectives, the present invention provides the following technical solution: 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 includes the following steps: S1. Retrieve MPXV-A35R / M1R proteins from the database, 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. By reasonably excising and reassembling to form a single-chain dimer conformation, the two antigens A35R and M1R are tandemly connected using the cleavage sequence P2A to obtain the antigen amino acid sequence as shown in SEQ ID NO.1; S3. The antigen amino acids were codon optimized according to human codon preference to obtain the DNA sequence shown in SEQ ID NO.2. The DNA sequence was then constructed into plasmid PUC57 containing the T7 promoter, 5'UTR, 3'UTR and 100 PolyA, forming PUC57-T7-5UTR-E3-E2-6k-E1-3UTR-PolyA, thus completing the plasmid construction. S4. The plasmid successfully constructed above is amplified and transcribed to obtain pure mRNA; S5. The mRNA obtained above is prepared into lipid nanoparticles (LNPs) using a microfluidic method.

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

[0009] Preferably, the amplification method in step S4 is to amplify plasmids using Top 10 bacterial strains, extract the plasmids, linearize them using BsaI enzyme digestion, and recover the linearized fragments using DNA magnetic beads.

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

[0011] Preferably, the specific method for preparing lipid nanoparticles (LNPs) in step S5 includes the following steps: S5-1. Dissolve the mRNA in a 50mM citrate buffer solution with pH=4 to control the mRNA concentration at 108ng / μL, thus obtaining an mRNA-citric acid solution. S5-2. Prepare an anhydrous ethanol solution with 50% SM102, 1.5% DMG-PEG2000, 10% DSPC, and 38.5% cholesterol to obtain a lipid mixed solution. S5-3. The lipid mixture and the mRNA-citric acid solution were filtered separately through a 0.22μm microporous membrane. The mixture was then mixed using a microfluidic instrument at a ratio of 1:8 between the phosphorus content of the mRNA and the nitrogen content of SM102 to obtain lipid nanoparticles (LNP).

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

[0013] This invention provides a monkeypox virus mRNA-MV2 vaccine and its preparation method, which has the following advantages compared with the prior art: In this invention, the transmembrane regions of the conserved sequences A35R and M1R are excised, and their structures are predicted using AlphaFold3. Based on these predictions, appropriate excision and reassembly are performed to create a dimer-like structure. This single-chain dimer design allows the immunogen to possess more antigenic epitopes. To ensure the stability of the 2A35Rs and 2M1Rs structures, a P2A peptide is used for amino acid sequence linking. This design is applied to an mRNA vaccine platform, where lipid nanoparticles (LNPs) are prepared using microfluidic methods to obtain mRNA-MV2. Balb / c mice are then immunized with this mRNA-MV2. ELISA assays are used to detect the level of binding antibodies produced after immunization, and neutralization assays are used to detect the neutralizing antibodies against monkeypox virus in the mouse serum at the immunization endpoint. Immunogenicity evaluation results show that mice, after two immunizations, produced high binding antibody titers, a certain level of neutralizing antibody titers, and a high level of T-cell immune response. In subsequent challenge protection experiments, immunized mice controlled viremia and significantly reduced lung viral load and lung damage. Vaccine virus (VACV) and monkeypox virus belong to the same genus. The mRNA-MV2 vaccine induced high neutralizing antibody titers against VACV in mice and protected them from a lethal dose (30LD50) of VACV, significantly reducing viral load and pathological damage in various organs. This indicates that the obtained vaccine mRNA-MV2 possesses good immunogenicity, eliciting favorable humoral and cellular immune responses, and exhibits some protective efficacy against both MPXV and VACV. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the mRNA-MV2 design strategy of the present invention; Figure 2This is a schematic diagram showing the particle size and encapsulation efficiency of lipid nanoparticles generated by LNP encapsulation in an embodiment of the present invention; Figure 3 This is a schematic diagram of RNA electrophoresis transcribed in vitro in an embodiment of the present invention; Figure 4 This is a schematic diagram illustrating the verification of mRNA-MV2 cell-level expression in an embodiment of the present invention; Figure 5 This is a schematic diagram illustrating the weight changes of BALB / c mice after vaccination in an embodiment of the present invention. Figure 6 This is a schematic diagram showing the antibody titers (a) against A35R and (b) against M1R in serum in an embodiment of the present invention. Figure 7 This is a schematic diagram showing the neutralizing antibody titers against MPXV (a) and VACV (b) in serum at the immune endpoint (28d) in this embodiment of the invention. Figure 8 This is a schematic diagram showing that MV2 vaccine immunization elicited strong T-cell immunity in mice, inducing high IFN-γ levels. Figure 9 This is a schematic diagram illustrating how MV2 vaccine immunization elicited strong T-cell immunity in mice, inducing high IL-2 levels. Figure 10 This is a schematic diagram of the weight changes of BaLB / c mice after immunization and MPXV challenge in an embodiment of the present invention. Figure 11 This is a schematic diagram illustrating the changes in viremia in BaLB / c mice after immunization and MPXV challenge in an embodiment of the present invention. Figure 12 This is a schematic diagram illustrating the changes in viral load in multiple organs of BaLB / c mice after immunization and MPXV challenge in an embodiment of the present invention. Figure 13 This is a schematic diagram of the pathological damage score of BaLB / c mice after immunization and MPXV challenge experiment in an embodiment of the present invention; Figure 14 This is a schematic diagram of the staining of pathological sections of various tissues damaged in BaLB / c mice after MPXV challenge experiment in an embodiment of the present invention. Figure 15 A schematic diagram of body weight changes in BaLB / c mice after immunization and VCV challenge in an embodiment of the present invention; Figure 16 This is a schematic diagram illustrating the changes in viral load in multiple organs of BaLB / c mice after immunization in a VCV challenge experiment, as described in this embodiment of the invention. Figure 17This is a schematic diagram of the pathological damage score of BaLB / c mice after immunization in the VCV challenge experiment in an embodiment of the present invention; Figure 18 This is a schematic diagram of the staining of pathological sections of BaLB / c mice after immunization and VCV challenge experiment in an embodiment of the present invention. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1:

[0016] Preparation of mRNA-MV2 vaccine: MPXV-A35R / M1R protein was retrieved from the NCBI database, yielding 7258 A35R amino acid sequences and 7011 M1R amino acid sequences from different strains. Sequence alignment was performed using snapgene software, and the most frequent amino acid at each amino acid site was selected. The amino acids were then reassembled in sequence to obtain the conserved amino acid sequences of A35R (as shown in SEQ ID NO.5) and M1R (as shown in SEQ ID NO.6). The transmembrane regions of the conserved sequences A35R and M1R were excised, and their structures were predicted using AlphaFold3. Based on the structural prediction, reasonable excision and reassembly were performed to give it a dimer-like structure. The two antigens were tandemly linked using the cleavage sequence P2A to maximize the stability of the two antigen structures, resulting in the antigen amino acid sequence 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: sequences 1-89 are removed; the second part: sequences 1-99 are removed. They are then assembled sequentially.

[0017] The M1R conserved sequence is divided into two parts. Part 1: excision of the sequence from 182 to 250; Part 2: excision of the sequences from 1 to 2 and from 182 to 250. The sequences are then assembled sequentially.

[0018] P2A sequence: (GSG)ATNFSLLKQAGDVEENPGP, where GSG promotes P2A autocleavage.

[0019] Codon optimization was performed according to human codon preferences, resulting in the DNA sequence shown in SEQ ID NO.2. This DNA sequence was then constructed onto plasmid PUC57, which contains a T7 promoter, a 5' UTR (as shown in SEQ ID NO.3), a 3' UTR (SEQ ID NO.4), and 100 PolyA molecules (PUC57-T7-5UTR-E3-E2-6k-E1-3UTR-PolyA). (For detailed design ideas, see...) Figure 1 ); Plasmid amplification was performed using the Top 10 bacterial strains. After plasmid extraction, linearization was achieved by BsaI restriction enzyme digestion, and the linearized fragments were recovered using DNA magnetic beads. Transcription was performed using a commercial transcription kit (vazyme#DD4203), and RNA was purified using RNA magnetic beads to obtain pure RNA. The plasmid was introduced into Top10 competent cells, followed by 800 μL of LB medium. After initial amplification, 400 μL of the amplified plasmid was added to 500 mL of LB medium, and ampicillin was added at a ratio of 1:1000. The mixture was shaken in a shaker (37°C, 220 rpm) for 16 h. Plasmid extraction was performed using a commercial plasmid large-scale extraction kit. The correctly sequenced plasmid was used for linearization: the mixture was homogenized and digested at 37°C for 16 h using the following method (plasmid: 50 μg, BsaI: 10 μL, 10×BsaI buffer: 50 μL, enzyme-free water to 500 μL). Linearized DNA was recovered using commercially available DNA magnetic beads (vazyme#N411-01): 0.5 times the volume (250 μL) of DNA magnetic beads were added to the linearization reaction solution and stirred until homogeneous. After incubating at room temperature for 5 minutes, the solution was placed in a magnetic rack. After 5 minutes, the liquid was aspirated. 600 μL of 80% ethanol was added, and the liquid was aspirated after 30 seconds. This process was repeated once. The liquid was discarded, and the solution was incubated at room temperature for 7 minutes to dry. After drying, the solution was removed from the magnetic rack, and 40 μL of enzyme-free water was added to dissolve the adsorbed magnetic beads. The solution was incubated at room temperature for 5 minutes, and the solution was placed in the magnetic rack again. After 5 minutes, the liquid was aspirated; this was 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 a final volume of 20 μL. 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 The reaction was carried out in a metal bath at 37°C for 2 hours, followed by the addition of 1 μL DNase I. Purification was performed using commercial RNA magnetic beads (vazyme#N412-01) to obtain pure RNA: 1.8 times the volume (36 μL) of RNA magnetic beads were added to the transcription reaction solution and mixed thoroughly by pipetting. After incubating at room temperature for 5 minutes, the solution was placed in a magnetic rack. After 5 minutes, the liquid was aspirated, and 200 μL of 80% ethanol was added. After 30 seconds, the liquid was aspirated again. This process was repeated once. The liquid was discarded, and the solution was incubated at room temperature for 7 minutes to dry. After removing the solution from the magnetic rack, 50 μL of enzyme-free water was added to dissolve the adsorbed magnetic beads. The solution was incubated at room temperature for 5 minutes, and the solution was placed in the magnetic rack again. After 5 minutes, the liquid was aspirated, yielding the purified transcription product.

[0021] The purified RNA was dissolved in 50 mM citrate buffer (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 a ratio of 50%, 1.5%, 10%, and 38.5% respectively to obtain a lipid mixture. The lipid mixture and the mRNA-citric acid solution were filtered separately through a 0.22 μm microporous membrane. The phosphorus content of the mRNA was mixed with the nitrogen content of SM102 at a ratio of 1:8 using a microfluidic instrument at a flow rate of 15 mL / min:5 mL / min for the mRNA-citric acid solution to the lipid mixture to obtain mRNA-LNP.

[0022] Detection:

[0023] The obtained mRNA-LNP was immediately diluted with 15 mL of the above citrate buffer and ultrafiltered using a 100 KD ultrafiltration tube at a centrifugation force of 3000 g. After ultrafiltration to 1 / 4 volume, 20 mM Tris-HCl buffer (pH=7.5) was added to 15 mL. This process was repeated twice, and the particle size and encapsulation efficiency were measured. It was found that the particle size was around 100 nm, and the encapsulation efficiency reached over 95% (e.g., Figure 2 (as shown) The results of in vitro transcribed RNA electrophoresis 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.

[0024] After preparing mRNA-MV2, transfection experiments were performed on 293T cells. 24 hours later, total cell protein was collected, and Western blotting (WB) with MPXV A35R and M1R antibodies was used to detect the expression of the target antigen, ensuring successful antigen expression in the mRNA-MV2 group. Specifically: First, 500,000 293T cells were added to a 6-well plate and cultured for 16 hours. RNA transfection was performed using a commercially available RNA transfection reagent. Specifically: 3.75 μL of transfection reagent was added to 125 μL of Opti-MEM, mixed, and incubated for 10 minutes. Simultaneously, 5 μg of RNA was added to 250 μL of Opti-MEM, mixed, and then 125 μL was added to the dilution of the transfection reagent obtained in the previous step. After mixing and incubation for 5 minutes, the mixture was evenly added to the 6-well plate. The control group received no treatment. After culturing the cells for another 24 hours, the culture medium in the wells was aspirated, and 200 μL / well of cell lysis buffer and 2 μL / well of protease inhibitor were added. After placing the membrane on ice for 30 min, aspirate the lysis buffer from 80 μL of each well, add 20 μL of 5× loading buffer, mix well, and incubate in molten metal at 95°C for 10 min. 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 min, 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 min. Discard the blocking buffer, add 10 mL of the corresponding primary antibody diluted with the blocking buffer, and incubate at 4°C for 16 h. Discard the primary antibody, add 15 mL of 1×TBST and wash the membrane for 10 min, repeating 3 times. Add 10 mL of the corresponding secondary antibody diluted with the 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 min, repeating 3 times. Develop the membrane using developing buffer and a developing apparatus, and save the developed images. To ensure all samples were normal, the experiment was repeated using an anti-β-Actin antibody. Unlike the previous method, this time only the primary antibody was added, and the membrane was incubated at room temperature for 1 hour, followed by washing with 15 ml of 1×TBST for 10 minutes, repeated three times. Then, developing solution was added for development. In our results, compared to the control group, both anti-A35R and anti-M1R antibodies showed specific bands at 20 kDa, and β-Actin was used in the Western blot experiment.

[0025] Specific results are as follows Figure 4 As shown, the internal reference in both the control and experimental groups was expressed normally. This indicates that both components of the vaccine MV2 (A35R and M1R) can be expressed in 293T cells.

[0026] The mRNA-MV2 vaccine was administered twice, on day 0 and day 14, with a high dose of 10 μg / animal, a low dose of 5 μg / animal, and PBS as a control. mRNA-LNP was prepared according to the above method. Female Balb / c immunized 4-6 week old animals with high dose (10ug / animal), low dose (5ug / animal), and PBS as control, by intramuscular injection.

[0027] Mice were weighed every other day for five days following MV2 vaccination. Specific results are as follows: Figure 5 As shown in the figure. The results showed that after immunization, the mice only experienced a slight decrease in body weight, which quickly returned to normal, indicating that the MV2 vaccine has a certain degree of safety and does not cause a significant decrease in the weight of the mice; The immunogenicity of the vaccine was assessed by detecting serum-specific IgG binding antibodies against A35R and M1R proteins. Recombinant A35R and M1R proteins (Antibodysystem, #EVV13101 / #EVV13301, 1 μg / mL) were adsorbed at 4°C for 16 hours and then immobilized on 96-well microplates (Thermo Fisher Scientific, #442404). Serum samples were serially diluted 1:100 in detection buffer (0.05% BSA / PBST) and then subjected to primary incubation (37°C, 60 min). The washing steps were repeated before using horseradish peroxidase-conjugated polyclonal goat anti-mouse IgG (Invitrogen, #A-10668), which was diluted 1:30,000 (100 μL / well) in sample diluent. A second incubation (37°C, 60 min) followed by washing was performed under ambient conditions (15 min) with 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific, 100 μL / well). The enzymatic reaction was quenched using acidic stop solution (SolarBio, #C1058), and dual-wavelength densitometric measurements were recorded at 450 nm (dominant wavelength) and 630 nm (reference wavelength). Baseline values ​​for negative control wells were defined using test buffer instead of serum samples, while the serum positivity threshold was determined to be 2.1 times higher than that for positive control wells. The geometric mean titer (GMT) of antigen-specific IgG was calculated based on the maximum reciprocal absorbance value reaching the threshold.

[0028] BALB / c mice were immunized with MV2 vaccine on days 0 and 14, and blood samples were collected on days 7, 14, 21, and 28 to detect the titers of binding antibodies against A35R and M1R in the serum. The results showed that the titers of binding antibodies in the serum of mice increased significantly after immunization. Specific results are as follows: Figure 6 As shown.

[0029] Neutralizing antibodies were detected in mouse serum at the immunization endpoint. Neutralizing antibodies were detected in Vero cells using the CPE method with MPXV and VACV viruses, respectively. To quantify the neutralizing activity of serum against highly replicative mpox virus (MPXV) and vaccine virus (VACV), live virus microneutralization assays were performed under biosafety level 2 or 3 isolation protocols. Mouse serum was heat-inactivated (56°C, 30 min) before being serially diluted logarithmically in DuPont Modified Eagle Medium (DMEM) in 96-well microplates. In each experimental replicate, 50 μL of titrated serum dilution was aliquoted into the designated wells. The working stock of virus in DMEM was normalized to 2 × 10⁻⁶. 3 The target concentration of plaque-forming units (PFU) / mL was determined. Equimolar amounts (50 μL) of viral inoculum and serially diluted serum samples were incubated in parallel (37°C, 1 h) to promote antibody-virus complex formation. After incubation, 100 μL of Vero cell suspension (1.5 × 10⁻⁶) was added. 4 Cells (in complete culture medium) were seeded into each virus-serum reaction well and cultured continuously under standard conditions (37°C, 5% CO2) for 5–7 days. Neutralization efficacy 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 achieving ≥50% CPE inhibition relative to virus control wells, expressed as 50% neutralization titer (NT50).

[0030] BALB / c mice were immunized with MV2 vaccine on days 0 and 14. The titers of neutralizing antibodies against MPXV and VACSV in serum were measured at the immunization endpoint (day 28). The results showed that the MV2 vaccine induced good neutralizing antibody titers against MPXV and VACSV. (See attached table for details.) Figure 7 As shown; The ElisPot method was used to detect IL-2 and INF-γ-positive cells in spleen immune cells after antigen stimulation to assess 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), and experimental controls were established as follows: baseline responsiveness was assessed using unstimulated cells (negative control for each experimental replicate), while phytohemagglutinin (PHA)-activated cells served as a positive control. Subsequent procedural stages included equilibration (37°C, 5% CO2), automated washing cycles, and seeding of cells into pre-coated 96-well ELISpot plates (Mabtech®; #3321-4APT-10 membrane plate, #3441-4APW-10 cap). Final spot quantification and high-resolution image acquisition were performed using the IRIS™ Automated ELISpot Reader System (Mabtech®), with data normalized to 10-10. 6 Spot-forming units (SFUs) of cells.

[0031] Specific results are as follows Figure 8 and Figure 9 As shown, MV2 vaccine immunization elicited strong T-cell immunity in mice, inducing high levels of IFN-γ (a) and IL-2 (b).

[0032] MPXV virus attack protection experiment: Blood samples were collected at different time points after challenge, and the viral load in the blood was detected using RT-qPCR to monitor the dynamic changes in viremia. Seven days after challenge, mice in each group were dissected, and the viral load in the lungs was detected using RT-qPCR to determine whether the vaccine effectively inhibited viral replication. At the same time, HE staining was used to detect pathological damage in various tissues and organs, and the degree of pathological damage was analyzed by relative quantitative statistical analysis using corresponding pathological scores. Specifically, the intranasal challenge method was used, instilling 1×10⁶ drops into the nasal cavity of mice. 6 PFU MPXV (West African clade IIb) was administered to MPXV challenge mice, with body temperature and weight monitored every other day, and blood and oropharyngeal swabs collected simultaneously. All animals survived to day 7 post-challenge and were subsequently euthanized. Tissue samples from the heart, liver, spleen, lungs, kidneys, brain, duodenum, and rectum were collected for viral load quantification and histopathological analysis.

[0033] For details, please see [link / details]. Figure 10-14 As shown; To investigate the cross-protective efficacy of vaccines, a challenge protection experiment was conducted using a lethal dose (30LD50) of vaccinia virus Tian Tan strain (VACV-VTT): Body weight and temperature were measured on days 1, 3, 5, 7, 9, 11, and 12 post-infection. Mice in each group were dissected on day 12 post-infection (control group mice died on day 6, therefore dissecting them on day 6 post-infection). RT-qPCR was used to detect viral load in each organ, and HE staining was used to detect pathological damage in each tissue and organ. The degree of pathological damage was then analyzed using relative quantitative statistical analysis based on corresponding pathological scores. Specifically, an intranasal challenge method was used, in which mice were administered 30 LD50 of VAV via intranasal instillation. Body weight and temperature were measured on days 1, 3, 5, 7, 9, 11, and 12 post-challenge. All control mice died on day 6 post-infection, and their body weight and temperature were recorded. Specimens were collected from the deceased control mice, including the heart, liver, spleen, lungs, kidneys, brain, duodenum, and rectum. Prior to euthanasia, all vaccinated mice survived to day 12 post-challenge. Specific internal organs (heart, liver, spleen, lungs, kidneys, brain, duodenum, and rectum) were harvested from the vaccinated mice for subsequent viral load quantification and histopathological analysis.

[0034] Specific results are as follows Figures 15-18 .

[0035] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monkeypox virus mRNA-MV2 vaccine, characterized in that: The vaccine comprises lipid nanoparticles (LNPs) and mRNA, the nucleic acid sequence of which is shown in SEQ ID NO.

2.

2. A method for preparing an mRNA-MV2 vaccine as described in claim 1, characterized in that, The preparation method includes the following steps: S1. MPXV-A35R / M1R protein was retrieved from the database, and the conserved A35R and M1R were obtained and sequenced. 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. By reasonably excising and reassembling to form a single-chain dimer conformation, the two antigens A35R and M1R are tandemly connected using the cleavage sequence P2A to obtain the antigen amino acid sequence as shown in SEQ ID NO.1; S3. The antigen amino acids were codon optimized according to human codon preference to obtain the DNA sequence shown in SEQ ID NO.

2. The DNA sequence was then constructed into plasmid PUC57 containing the T7 promoter, 5'UTR, 3'UTR and 100 PolyA, forming PUC57-T7-5UTR-SP-A35R-A35R-P2A-M1R-M1R-3UTR-PolyA, thus completing the plasmid construction. S4. After amplification and transcription of the plasmid successfully constructed above, pure mRNA is obtained. S5. The mRNA obtained above is prepared into lipid nanoparticles (LNPs) using a microfluidic method.

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

4.

4. The preparation method according to claim 2, characterized in that: In step S4, the amplification method involves using Top 10 bacterial strains for plasmid amplification, extracting the plasmid, linearizing it using BsaI enzyme digestion, and recovering the linearized fragment using DNA magnetic beads.

5. The preparation method according to claim 2, characterized in that: In step S4, transcription is performed using the commercial transcription kit vazyme#DD4203, and the RNA is purified 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 (LNPs) in step S5 includes the following steps: S5-1. Dissolve the mRNA in a 50mM citrate buffer solution with pH=4 to control the mRNA concentration at 108 ng / μL, thus obtaining an mRNA-citric acid solution. S5-2. Prepare an anhydrous ethanol solution with 50% SM102, 1.5% DMG-PEG2000, 10% DSPC, and 38.5% cholesterol to obtain a lipid mixed solution. S5-3. The lipid mixture and the mRNA-citric acid solution were filtered separately through a 0.22μm microporous membrane. The mixture was then mixed using a microfluidic instrument at a ratio of 1:8 between the phosphorus content of the mRNA and the nitrogen content of SM102 to obtain lipid nanoparticles (LNP).

7. The preparation method according to claim 6, characterized in that: In step S5-3, the mRNA-citric acid solution and lipid mixture are mixed using a microfluidic instrument at a flow rate ratio of 15 mL / min to 5 mL / min.

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