Varicella-zoster virus mRNA vaccine as well as preparation method and application thereof

Abstract

The invention discloses an mRNA (messenger Ribonucleic Acid) vaccine of varicella-zoster virus as well as a preparation method and application of the mRNA vaccine. According to the mRNA vaccine provided by the invention, a gE antigen sequence is subjected to codon optimization; then selecting a proper promoter, 5 'UTR, 3' UTR and 3 '-terminal PolyA to combine into complete mRNA nucleic acid for coding the varicella-zoster virus, screening to obtain two groups of mRNA nucleic acid for coding the varicella-zoster virus with both expression level and stability, and adopting a novel cationic lipid imidazopyridyl lipid compound to prepare a vaccine liposome. The targeting property in drainage of lymph nodes is improved, and the humoral immunity and cellular immunity levels excited by the vaccine in animal bodies are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a varicella-zoster virus mRNA vaccine and a preparation method and application thereof. Background Art

[0002] Varicella-zoster virus (VZV) is a human alpha-herpes virus in the genus Varicella-herpesvirus, which causes chickenpox and herpes zoster. Chickenpox is a common disease in children that is highly contagious and occurs between the ages of 2 and 6. Herpes zoster is a disease caused by the reactivation of the varicella-zoster virus lurking in the body, and mainly occurs in adults and the elderly. Its main hazard is neuralgia. Because the virus invades the nerves, patients with herpes zoster often experience severe neuralgia, which may last for weeks or even months, seriously affecting the patient's normal sleep, diet, study and work. With the acceleration of population aging, the number of herpes zoster cases will increase year by year, and the economic burden on society will be further aggravated, which makes the prevention and treatment of herpes zoster particularly important. Vaccination is the most effective means to prevent and control herpes zoster and its complications. Currently, the attenuated live herpes zoster vaccine and the recombinant herpes zoster adjuvant subunit vaccine have been approved for marketing, and no mRNA vaccine has yet been marketed.

[0003] Studies have shown that the reduction of VZV-specific cell-mediated immunity (CMI) is highly correlated with the incidence of herpes zoster and the proportion of postherpetic neuralgia (PHN). On the contrary, high levels of VZV-specific antibodies cannot completely prevent herpes zoster. Therefore, how to design a vaccine to make the body produce specific antibodies and CMI has become the key to the development of herpes zoster vaccines. Summary of the invention

[0004] The purpose of the present invention is to provide an mRNA vaccine of varicella-zoster virus and a preparation method and application thereof, so as to significantly improve the humoral immunity and cellular immunity levels stimulated by the vaccine in animals.

[0005] The present invention adopts the following technical solution:

[0006] In a first aspect of the present invention, a varicella-zoster virus mRNA vaccine is provided, comprising mRNA expressing varicella-zoster virus gE protein.

[0007] Furthermore, the nucleotide sequence encoding the varicella-zoster virus gE protein is shown in SEQ ID NO.1-SEQ ID NO.2.

[0008] Furthermore, the nucleotide sequence encoding the varicella-zoster virus gE protein also includes:

[0009] A sequence in which one or more bases are substituted, deleted, added and / or replaced based on the above nucleotide sequence; or a sequence with a homology of more than 90% with the above nucleotide sequence.

[0010] Furthermore, the vaccine also includes: lipid nanoparticles encapsulating the mRNA.

[0011] Furthermore, the components of the lipid nanoparticles include: ionizable cationic lipids, distearoylphosphatidylcholine, polyethylene glycol lipids and cholesterol.

[0012] Furthermore, the ionizable cationic lipid is an imidazopyridyl lipid compound selected from the following compounds I-IV. The specific preparation method is shown in patent CN115232128B:

[0013]

[0014]

[0015] The compound I is abbreviated as cationic lipid molecule A4, the compound II is abbreviated as cationic lipid molecule A10, the compound III is abbreviated as cationic lipid molecule A20, and the compound IV is abbreviated as cationic lipid molecule A23.

[0016] Furthermore, the percentage of the ionizable cationic lipid is 40%-70%, preferably 45%-55%; the percentage of the neutral lipid is 5%-15%, preferably 8%-12%; the percentage of the cholesterol is 30%-50%, preferably 35%-45%.

[0017] Furthermore, the molar percentage of the polyethylene glycol lipid is 1%-5%, preferably 1%-3%.

[0018] Furthermore, the molar ratio of the cationic lipid, distearoylphosphatidylcholine, polyethylene glycol lipid and cholesterol is (40-70):(5-15):(30-50):(1-5).

[0019] In a second aspect of the present invention, a method for preparing the mRNA vaccine is provided, the method comprising:

[0020] Connecting the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 to the plasmid to obtain a recombinant plasmid;

[0021] The recombinant plasmid is linearized using a restriction endonuclease to obtain linearized DNA;

[0022] Using the linearized DNA as a template for transcription to prepare mRNA;

[0023] obtaining a lipid mixed solution;

[0024] The mRNA and the lipid mixed solution are mixed and then diluted, ultrafiltered and concentrated to obtain the varicella-zoster virus mRNA vaccine.

[0025] Furthermore, the plasmid is pUC57-Kan.

[0026] Furthermore, the restriction endonucleases are BglI and MluI.

[0027] Furthermore, the lipid mixed solution is a solution obtained by mixing ionizable cationic lipids, neutral lipids, steroid compounds, polyethylene glycol lipids and organic solvents.

[0028] Furthermore, the volume ratio of the mRNA to the lipid mixed solution is (2-4):1, preferably 3:1.

[0029] In the third aspect of the present invention, provided is the use of the varicella-zoster virus mRNA vaccine in the preparation of a medicament for preventing or ameliorating varicella and / or herpes zoster and / or postherpetic neuralgia.

[0030] The varicella-zoster virus mRNA vaccine of the present invention can increase the varicella-zoster virus antibody titer and specific cellular immune response produced in vivo.

[0031] The present invention has the following advantages and beneficial effects:

[0032] 1. The mRNA vaccine of varicella-zoster virus of the present invention is prepared by codon-optimizing the gE antigen sequence, selecting a suitable promoter, 5'-UTR, 3'-UTR and 3'-end PolyA to form a complete mRNA nucleic acid encoding varicella-zoster virus, and screening to obtain two groups of mRNA nucleic acids encoding varicella-zoster virus that take into account both expression level and stability.

[0033] 2. The present invention improves the components of LNP and adopts an imidazopyridine lipid compound with better targeting to the draining lymph nodes. The mRNA vaccine prepared produces a strong and stable immune effect after immunizing mice, especially the level of neutralizing antibodies and cellular immunity, and does not affect the normal growth of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0035] Figure 1 is the capillary electrophoresis diagram in Example 1;

[0036] Figure 2 This is a schematic diagram of the animal experiment scheme flow for mRNA-LNP administration and blood collection in Example 3;

[0037] Figure 3 This is a schematic diagram of the Anti-gE antibody titer detection results in Example 3;

[0038] Figure 4 This is a schematic diagram of the Anti-gE neutralizing antibody titer detection results in Example 3;

[0039] Figure 5 This is a schematic diagram of the IFN-r ELISPOT detection results in Example 3. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0041] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0042] The present application will be described in detail below with reference to embodiments and experimental data.

[0043] Example 1: Varicella-zoster virus mRNA and preparation method thereof

[0044] 1. VZV-mRNA sequence design

[0045] The reference sequence for expressing the antigen protein VZV gE is from ORF 68 of the VZV whole genome (GenBank sequence number: AAF61669.1), including five mutations: Y569A, S593A, S595A, T596A, and T598A. The sequence was codon optimized, including adjusting the codon preference for expression in mice and humans, while taking into account the expression level (codon adaptation index (CAI)) and stability (minimum free-energy change (MFE)), and the nucleotide sequence was obtained as shown in SEQ ID No. 1-SEQ ID No. 2.

[0046] 2. Preparation of VZV-mRNA stock solution

[0047] The above sequence and reference sequence are synthesized into the pUC57-T7 plasmid vector, located between the T7 promoter binding sequence and the PolyA site. The synthesized plasmid is transferred into E. coli (E.Coli) for culture, and the positive clones with stable PolyA are selected. The cells are collected after amplification by shaking flask culture, and high-quality templates are obtained after alkaline lysis, neutralization, clarification, concentration and liquid replacement, purification, linearization and enzyme digestion, and template purification for in vitro transcription. The template is transcribed in vitro by optimized T7 RNA polymerase and transcription conditions to obtain respective mRNAs.

[0048] The synthesized mRNA was purified by oligo-dT affinity, buffer exchanged to sodium acetate by lateral flow filtration, and sterile filtered. The integrity of the mRNA was determined by microfluidic parallel capillary electrophoresis (Fragment Analyzer system 5200, Agilent) (see Figure 1 ), the solution concentration, pH value, residual DNA, protein and double-stranded RNA impurities were also measured. Finally, mRNAs that meet the requirements of preclinical research were obtained and named VZV-1, VZV-2 and VZV-WT (sequence without codon optimization).

[0049] Example 2. Expression of VZV-1, VZV-2 and VZV-WT in HEK293

[0050] Transfected T-75cm 2 Taking 293T cells in culture flask as an example, the operation is as follows:

[0051] (1) Use low-passage, good and stable 293T cells and wait for the cell density in the culture flask to reach about 80% for transfection.

[0052] (2) 12 μg of VZV-1 and VZV-2 mRNA were gently mixed in 2.5 mL of empty culture medium; 36 μL of transfection reagent Lipofectamine 2000 was mixed in an equal volume of empty culture medium. After incubation at room temperature for 5 min, the two tubes of liquid were mixed evenly and incubated at room temperature for 20 min. VZV-WT was used as a control.

[0053] (3) Add the transfection reagent-mRNA mixture into the cell culture flask, and place the cell culture flask in a 37°C incubator for 4-6 hours.

[0054] (4) Discard the liquid, add 15 mL of DMEM medium containing 2% FBS into the cell culture flask, continue to culture in a 37° C. incubator for 24 hours, and then harvest the cells.

[0055] (5) After the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, the cells were permeabilized with 0.2% Saponin for 10 minutes, and the cells were incubated with mouse anti-VZV gE as the primary antibody (ab272686 from Abcam) and FITC-anti-Mouse IgG (bs-0296G-FITC from Bioss) as the secondary antibody, and the fluorescence intensity was detected by flow cytometry. The results are shown in Table 1.

[0056] Table 1 mRNA expression in HEK293

[0057]

[0058]

[0059] As shown in Table 1, the three mRNAs were expressed in more than 80% of HEK293T cells, and the average fluorescence intensities (MFI) of the three experiments were 5723.4±682.6, 9578.2±782.2, and 9360.2±911.6, respectively. It can be seen that the expression of gE protein after codon optimization was significantly higher than that of the wild type (VZV-WT), while there was no significant difference in the expression of VZV-1 and VZV-2.

[0060] Example 3 Preparation of mRNA vaccine

[0061] 1. A method for preparing lipid nanoparticles, comprising the following steps:

[0062] 1. Prepare a mixture of an imidazopyridyl lipid compound (ionizable cationic lipid A4, A10, A0, A23, see patent CN115232128B for the specific preparation method), an optional neutral lipid, a steroid compound, a polyethylene glycol lipid and an organic solvent; the organic solvent is a solvent miscible with water, such as ethanol, acetone, isopropanol, methanol, etc. As a specific embodiment of the present application, ethanol is specifically selected.

[0063] 2. Prepare an aqueous solution of the bioactive agent; the aqueous solution includes a buffer, preferably a physiologically acceptable buffer, such as a phosphate buffer, and optionally a PBS buffer. As a specific embodiment of the present application, a PBS buffer is specifically selected.

[0064] 3. Mix the mixed solution in steps 1 and 2 and the aqueous solution at a certain flow rate ratio (preferably 1:3) and volume ratio (preferably 1:3) through a microfluidic system, and dilute the solution mixed by the microfluidic chip with water (preferably 5 times) to obtain an intermediate solution; the preparation of the intermediate solution is carried out at room temperature, and the solution mixed by the microfluidic chip is immediately diluted with water to reduce the ethanol concentration and prevent ethanol from damaging the particles.

[0065] 4. The intermediate solution is ultrafiltered and concentrated to obtain a concentrated solution; the intermediate solution is ultrafiltered using an ultrafiltration tube to remove a large amount of ethanol in the solution, and at the same time, concentrated to obtain a concentrated solution with a higher concentration, which is convenient for the subsequent preparation of a cationic lipid nanoparticle solution of the required target concentration.

[0066] 5. The concentrated solution is filtered through a filter membrane to obtain the lipid nanoparticles. Specifically, the concentrated solution is filtered through a 0.22 μm water filter membrane to remove large particles that have aggregated in the system and obtain cationic lipid nanoparticles with better dispersion and uniform size.

[0067] The two optimized mRNA molecules obtained in Example 1 were combined with four novel cationic lipid molecules to obtain a total of 8 groups of mRNA vaccines. The products were characterized by analysis, including the determination of particle size and polydispersity, encapsulation efficiency, pH value, endotoxin and mRNA concentration (see Table 2).

[0068] Table 2. Key indicators of 8 groups of vaccines

[0069]

[0070] As shown in Table 2, these 8 groups of vaccines are suitable for in vivo and in vitro scientific research in the preclinical stage. Next, these 8 groups of vaccines will be tested for immune effects.

[0071] Example 4 Vaccine immune effect detection

[0072] 1. Experimental methods

[0073] A total of 45 female C57BL / 6 mice aged 6-8 weeks were selected and randomly divided into 9 groups (negative control group and 8 vaccine groups prepared in Example 3), 5 mice in each group. Muscle immunization was performed on days 0, 14, and 28, with an immunization dose of 5 μg / mouse and an immunization volume of 50 μL. 14 days after the third immunization, blood was collected from the eyeballs and the mice were euthanized to obtain the spleen.

[0074] (1) Collection of mouse serum samples: Blood was collected by orbital sampling, with about 300 μL of blood collected from each mouse each time. The whole blood was placed at 37°C for 1 hour and then at 4°C for 1 hour. The samples were centrifuged at 4000 rpm for 15 minutes, twice in total, and the serum samples were divided into aliquots. One tube was placed in a 56°C water bath for 30 minutes for complement inactivation treatment, which was used for subsequent neutralizing antibody detection. The serum samples were stored at -80°C.

[0075] (2) Serum antibody IgG detection

[0076] The VZV gE-specific IgG level in the serum samples of immunized mice was detected by indirect ELISA method. The specific experimental operation is as follows:

[0077] ① Coating: Use 0.5 μg / well gE protein as coating antigen, 100 μL / well, and coat overnight in a 4°C refrigerator.

[0078] ② Washing: Prepare PBST solution containing 0.5‰ Tween-20 for washing. Discard the coating solution, add 200μL PBST to each well, shake and wash the plate for 3 minutes, then discard the washing solution, and repeat 3 times.

[0079] ③ Blocking: prepare 3% BSA in PBS, 200 μL / well, and incubate at 37°C for 1 h.

[0080] ④Wash the plate: Same as step 2.

[0081] ⑤ Primary antibody: Prepare PBS with 1% BSA for diluting serum samples. Dilute the sample starting from 1:100 and perform a series of dilutions in a 5-fold gradient, for a total of 8 dilutions. Add 100 μL of diluted serum sample to each well and incubate at 37°C for 2 hours.

[0082] ⑥Wash the plate: Same as step 2.

[0083] ⑦ Secondary antibody: Prepare PBS with 1% BSA for antibody dilution. Use HRP-labeled goat anti-mouse IgG as secondary antibody, dilute 1:10000. 100 μL / well, incubate in 37°C incubator for 1 hour.

[0084] ⑧Wash the plate: Same as step 2.

[0085] ⑨ Color development: Add 100 μL of single-component TMB color development solution to each well and react for 10-30 minutes in the dark.

[0086] ⑩Termination: Add 50 μL 2M H2SO4 to each well to terminate color development.

[0087] Reading value: Read the absorbance value at OD450nm on the microplate reader and calculate the serum binding antibody titer.

[0088] (3) Serum neutralizing antibody detection

[0089] Fluorescent antibody to membrane antigen (FAMA) was used to detect the level of specific neutralizing antibodies in serum samples of immunized mice. The specific experimental procedures are as follows:

[0090] ①Use VZV-infected MRC-5 cells as coating antigen to prepare antigen slides.

[0091] ② The mouse serum was complement inactivated and then serially diluted in a 2-fold gradient from 1:10 to 1:2560, for a total of 8 dilutions.

[0092] ③ Add the diluted serum to the wells of the antigen slide in sequence, and then add negative and positive serum as controls, 30 μL / well. Incubate at 37°C for 60 min.

[0093] ④After incubation, quickly turn the slide over, shake off the liquid in the well, soak the slide in PBS and wash it three times, 5 minutes each time, and dry it.

[0094] ⑤ Use FITC-labeled goat anti-mouse IgG antibody (BIo SS bs-0296G-FITC) containing Evans blue staining solution as the secondary antibody, 30 μL / well. Incubate at 37°C for 30 min. Repeat step 4.

[0095] ⑥ Add 1 drop of buffered glycerol to each well of the slide, cover with a coverslip, and press lightly to cover tightly to avoid bubbles.

[0096] ⑦ Observe the prepared fluorescent slide under a fluorescent microscope. When the infected cells are combined with the serum containing VZV antibodies, a bright green fluorescent ring can be seen on the surface of the infected cells, which is judged as positive, otherwise it is judged as negative. The maximum dilution factor that produces a positive result is used as the titer of the neutralizing antibody.

[0097] (4) Isolation of mouse spleen cells

[0098] 14 days after the third immunization, the mice to be tested were killed using a carbon dioxide euthanasia system, disinfected by immersion in 75% alcohol solution, and then transferred to a clean bench for subsequent operations.

[0099] Tissue separation: Separate the spleen, place the spleen in a small dish, drop 500μL PBS, cover it with a 200-mesh screen, and use a 1mL syringe needle handle to grind the spleen through the screen. Grind and separate with a 200-mesh gauze to obtain single cells, resuspend the suspension in 4mL PBS, filter and transfer to a sterile centrifuge tube, and collect the cells by centrifugation at 350g for 10min at 4°C. Resuspend the cell pellet with 1mL ACK red blood cell lysis buffer, lyse at room temperature for 7 minutes, and then add 10mL PBS to stop the lysis. Centrifuge at 1800rpm and 4°C for 5min, resuspend the pellet with 1mL PBS, transfer the resuspended liquid to a 1.5mL EP tube for use, and pass through a 200-mesh screen again to remove the flocs visible to the naked eye. Count the cells of the spleen single cell suspension prepared above. Adjust the cell concentration to 4×10 6 cells / mL.

[0100] (5) IFN-γ ELISpot

[0101] ① Prepare RPMI1640 complete medium containing 10 μg / mL gE polypeptide peptide pool, and the final concentration of each peptide in the peptide pool is 0.2 μg / mL.

[0102] ② Remove the ELISPOT-96 well plate from the kit (Dakoway Biotechnology Co., Ltd.), add 200 μL 1640 culture medium to each well for activation, let it stand at room temperature for 10 minutes, and then remove the liquid.

[0103] ③ Add 1640 complete medium containing peptide pool to the activated ELISPOT plate, 150 μL / well. For the positive control group, add 140 μL 1640 complete medium and 10 μL positive stimulator in the kit (dissolved and prepared with 500 μL PBS according to the instructions) to the plate. For the negative control group, only 150 μL 1640 complete medium was added to the plate.

[0104] ④ Take out 50 μL (i.e. 2X10 5 cells) were added to the ELISPOT well plate, and efforts were made to ensure that the added cells were evenly dispersed.

[0105] ⑤ Place the ELISPOT plate in a cell culture incubator and incubate for 18-24 hours.

[0106] ⑥After incubation, drain the liquid in the well plate, add 200 μL of ice-cold deionized water, and let it stand at 4°C for 10 minutes to fully lyse the remaining cells.

[0107] ⑦ Remove the liquid from the plate, add 200 μL 1X washing buffer (the 50X washing buffer in the kit is diluted 50 times with deionized water), wash 3 times, and leave for 1 minute after adding 1x washing buffer each time.

[0108] ⑧ Add 100 μL of diluted primary antibody solution (biotin-labeled antibody provided by the kit, diluted 100 times with Dilutionbuffer R) and incubate at 37°C for 1 hour.

[0109] ⑨ Remove the liquid from the plate, add 200μL 1Xwashing buffer and wash 5 times, each time for 1 min.

[0110] ⑩ Add 100 μL of diluted enzyme-labeled avidin (diluted 100 times with Dilution buffer R in the kit) and incubate at 37°C for 1 hour.

[0111] Remove the liquid from the plate, add 200 μL 1washing buffer and wash 5 times, leaving the plate in 1washing buffer for 1 min each time.

[0112] Add 100 μL of prepared ACE colorimetric solution (mix 20XAEC solution I, 20XAEC solution II, 200XAEC solution III, and AEC dilution in the kit in a ratio of 10:10:1:180) and incubate at 37°C for 15-30 minutes.

[0113] Observe with the naked eye until the color develops to an appropriate level (observe every 5 minutes), remove the liquid in the wells, clean the well plate and base with deionized water, and place in a dark place to dry naturally.

[0114] The spots in the wells were counted using an automated ELISPOT plate reader (Mabtech IRIS FluoroSpot / ELISpot reader).

[0115] All experimental results were statistically analyzed and plotted using Graphpad Prim 8.0, and multiple groups of data were compared using one-way ANOVA. P < 0.05 was considered to be significantly different (ns: p>0.05; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0116] 2. Experimental Results

[0117] Anti-gE antibody titer test results are as follows Figure 3 As shown, all eight vaccine groups produced anti-gE antibodies after immunization of mice, among which the antibody titer of vaccine VZV-1-4 was the highest, significantly higher than the combination of the other three cationic lipid molecules; the antibody titer of vaccine VZV-2-4 was also significantly higher than the combination of the other three cationic lipid molecules, indicating that the vaccines prepared by the combination of sequences (as shown in SEQ ID NO.1 and SEQ ID NO.2) and cationic lipid A4 all produced humoral immune effects that were superior to A10, A20, and A23.

[0118] Anti-gE neutralizing antibody titer test results are as follows Figure 4 As shown, all 8 groups of vaccines produced anti-gE neutralizing antibodies after immunizing mice. Among them, ZVZ-1-4, the combination of the DNA sequence as shown in SEQ ID NO.1 and the cationic lipid A4, was significantly higher than the combination of the sequence with the other three cationic lipid molecules A10, A20, and A23, and was also significantly better than the combination of SEQ ID NO.2 with the four cationic lipid molecules A4, A10, A20, and A23.

[0119] VZV-specific T cell immune response is the key to preventing the occurrence of HZ, among which IFN-γ is an important indicator for evaluating the level of vaccine cellular immune response. Figure 5 The results showed the level of specific cellular immune response after gE polypeptide peptide pool stimulated spleen cells after immunization with VZV vaccine in each group. The number of spots (i.e., the number of cells that specifically secreted IFN-γ) in the VZV-1-4 group was significantly higher than that in VZV-1-10, VZV-1-20, and VZV-1-23, while there was no significant difference among the three groups of VZV-1-10, VZV-1-20, and VZV-1-23; there was no significant difference among the four groups of VZV-2-4, VZV-2-10, VZV-2-20, and VZV-2-23, and they were all significantly lower than VZV-1-10.

[0120] The above results indicate that ZVZ-1-4 (a combination of mRNA with a sequence such as SEQ ID NO.1 and cationic lipid A20) produced unexpected optimal humoral and cellular immune effects after immunizing mice.

[0121] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0122] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0123] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A varicella-zoster virus mRNA vaccine, characterized in that: Includes mRNA expressing varicella-zoster virus gE protein.

2. A varicella-zoster virus mRNA vaccine according to claim 1, characterized in that: The nucleotide sequence encoding the varicella-zoster virus gE protein is shown in SEQ ID NO.1-SEQ ID NO.

2.

3. A varicella-zoster virus mRNA vaccine according to claim 2, characterized in that: The nucleotide sequence encoding the varicella-zoster virus gE protein also includes: A sequence in which one or more bases are substituted, deleted, added and / or replaced based on the above nucleotide sequence; or a sequence with a homology of more than 90% with the above nucleotide sequence.

4. The mRNA vaccine according to claim 1, characterized in that The vaccine also includes lipid nanoparticles encapsulating the mRNA.

5. The mRNA vaccine according to claim 4, characterized in that The components of the lipid nanoparticles include cationic lipids, distearoylphosphatidylcholine, polyethylene glycol lipids and cholesterol.

6. The mRNA vaccine according to claim 5, characterized in that The cationic lipid is an imidazopyridyl lipid compound selected from the following compounds I-IV:

7. The mRNA vaccine according to claim 5, characterized in that The molar ratio of the cationic lipid, distearoylphosphatidylcholine, polyethylene glycol lipid and cholesterol is (40-70):(5-15):(30-50):(1-5).

8. A method for preparing the mRNA vaccine according to any one of claims 1 to 7, characterized in that: The method comprises: Connecting the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2 to the plasmid to obtain a recombinant plasmid; The recombinant plasmid is linearized using a restriction endonuclease to obtain linearized DNA; Using the linearized DNA as a template for transcription to prepare mRNA; obtaining a lipid mixed solution; The mRNA and the lipid mixed solution are mixed by a microfluidic system to obtain a complex, which is then diluted, ultrafiltered and concentrated to obtain the varicella-zoster virus mRNA vaccine.

9. The preparation method according to claim 8, characterized in that: The plasmid includes a pUC57-Kan plasmid vector, the restriction endonucleases are BglI and MluI, the lipid mixed solution is a solution obtained by mixing ionizable cationic lipids, neutral lipids, steroid compounds, polyethylene glycol lipids and organic solvents; the volume ratio of the mRNA to the lipid mixed solution is (2-4):

1.

10. Use of the varicella-zoster virus mRNA vaccine according to any one of claims 1 to 7 in the preparation of a medicament for preventing or ameliorating varicella and / or herpes zoster and / or postherpetic neuralgia.

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