Enterovirus mRNA vaccine and application thereof

Abstract

The invention provides an enterovirus mRNA vaccine and application thereof. A nucleotide sequence of the mRNA vaccine comprises a Kozak sequence, a promoter sequence, a 5 '-terminal non-coding region, a secretory signal peptide coding sequence, a VP1 protein coding sequence, a 3'-terminal non-coding region and a 3 '-terminal PolyA tail. The vaccine can induce a body to generate high-level IgG binding antibodies, neutralizing antibodies and specific cellular immune responses, and the content of viruses in the body is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid vaccines, and in particular to an enterovirus mRNA vaccine and an application thereof. Background Art

[0002] Human enteroviruses (EVs) are non-enveloped positive-sense single-stranded RNA viruses of the Picornaviridae family. They are one of the most common viral pathogens and can cause a variety of diseases, including febrile illness, hand, foot and mouth disease, acute flaccid paralysis, aseptic meningitis, myocarditis and pneumonia. According to the molecular, biological and genetic characteristics of EVs, they are divided into four categories: enterovirus A (EV-A), B (EV-B), C (EV-C) and D (EV-D). At present, more than 100 human enteroviruses have been reported worldwide. Enterovirus 71 (EVA71) is a member of the genus Enterovirus of the Picornaviridae family and is the main pathogen causing hand, foot and mouth disease (HFMD) and herpangina.

[0003] The EVA71 virus particle is a non-enveloped, symmetrical, icosahedral body with a diameter of 20 to 30 nm. The viral genome is about 7,500 nucleotides long and can encode four viral structural proteins, including VP1, VP2, VP3 and VP4, and seven non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). The four structural proteins (VP1 to VP4) aggregate to form a protomer, and 60 protomers form a viral particle that surrounds the viral genome. The structural proteins VP1, VP2 and VP3 are exposed on the surface of the capsid, while VP4 is located inside the capsid.

[0004] Hand, foot and mouth disease caused by EV-A71 infection is usually self-limiting, manifested by fever, rash on the hands and feet, and blister ulcers in the mouth. There is currently no specific drug for hand, foot and mouth disease, and vaccination is an effective means to prevent EV-A71 infection and protect children's health. At present, the research on EVA71 vaccines mainly focuses on inactivated vaccines, live attenuated vaccines, virus-like particle vaccines, subunit vaccines and other research directions, but only the EVA71 whole virus inactivated vaccine has completed human clinical trials and has been approved to enter the market. However, due to the recombination and mutation of the virus, the effectiveness of inactivated vaccines in the face of these mutations is challenged, so it is necessary to explore other vaccine preparation technologies. Summary of the invention

[0005] The present invention provides an enterovirus mRNA vaccine and application thereof.

[0006] On the one hand, the present invention provides an enterovirus mRNA vaccine, the nucleic acid sequence of which comprises: a Kozak sequence, a promoter sequence, a 5' non-coding region, a secretion signal peptide coding sequence, a VP1 protein coding sequence, a 3' non-coding region and a 3' PolyA tail.

[0007] In one embodiment, the enterovirus is any one or more of enterovirus A, enterovirus B, enterovirus C or enterovirus D.

[0008] In one embodiment, the enterovirus is enterovirus type 71 (EVA71).

[0009] In one embodiment, the amino acid sequence of the VP1 protein is as shown in SEQ ID NO:1.

[0010] In one embodiment, the nucleic acid sequence of the VP1 protein is as shown in SEQ ID NO:2.

[0011] In one embodiment, the VP1 protein is an optimized VP1 protein; the optimized VP1 protein refers to a VP1 protein that has been codon optimized. In one embodiment, the amino acid sequence of the optimized VP1 protein after adding a secretory signal peptide sequence is shown in SEQ ID NO: 3. In one embodiment, the nucleic acid sequence of the optimized VP1 protein after adding a secretory signal peptide coding sequence is shown in SEQ ID NO: 4.

[0012] In one embodiment, the amino acid sequence of the secretion signal peptide is METDTLLLWVLLLWVPGSTGD.

[0013] In one embodiment, the nucleic acid sequence of the mRNA vaccine is as shown in SEQ ID NO:5.

[0014] In one embodiment, the nucleic acid sequence of the mRNA vaccine is capped.

[0015] In one embodiment, the mRNA vaccine comprises a delivery vector that encapsulates the RNA sequence.

[0016] In one embodiment, the delivery vehicle is a nanoparticle.

[0017] In one embodiment, the delivery vehicle is a lipid nanoparticle, and the lipid nanoparticle comprises a cationic lipid, polyethylene glycol, a phospholipid, and cholesterol.

[0018] On the other hand, the present invention provides a polynucleotide encoding the above-mentioned mRNA vaccine.

[0019] In one embodiment, the polynucleotide comprises any one or more of the following:

[0020] (I) the nucleic acid sequence shown in SEQ ID NO: 2, 4 or 5;

[0021] (II) a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the nucleic acid sequence shown in SEQ ID NO: 2, 4, or 5;

[0022] (III) a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 3;

[0023] (IV) A nucleic acid sequence encoding an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids compared to the amino acid sequence shown in SEQ ID NO: 1 or 3.

[0024] In another aspect, the present invention provides an expression vector comprising the above-mentioned polynucleotide.

[0025] In another aspect, the present invention provides a host cell, comprising the above-mentioned vector.

[0026] On the other hand, the present invention provides a method for constructing an enterovirus mRNA vaccine, which comprises the steps of constructing a recombinant plasmid expressing the mRNA vaccine, and obtaining the mRNA vaccine by nanoparticle encapsulation.

[0027] In one embodiment, the preparation method comprises the steps of:

[0028] (1) dissolving mRNA in an aqueous solution as the aqueous phase;

[0029] (2) dissolving the nanoparticles in an organic solvent as an organic phase;

[0030] (3) Mixing the aqueous phase and the organic phase and packaging to obtain the mRNA vaccine.

[0031] In one embodiment, the aqueous solution is sodium acetate buffer; and the organic solvent is ethanol.

[0032] In one embodiment, the mRNA sequence is shown as SEQ ID NO:5.

[0033] In one embodiment, the mRNA sequence is obtained by capping the sequence shown in SEQ ID NO:5.

[0034] On the other hand, the present invention provides the use of the above-mentioned mRNA vaccine, or the above-mentioned polynucleotide, or the above-mentioned expression vector, or the above-mentioned host cell in the preparation of a vaccine for preventing or treating diseases caused by enterovirus, or hand, foot and mouth disease, or herpetic pharyngitis.

[0035] In one embodiment, the above-mentioned mRNA vaccine, or the above-mentioned polynucleotide, or the above-mentioned expression vector, or the above-mentioned host cell is used in the preparation of a vaccine for preventing or treating a disease caused by enterovirus.

[0036] In one embodiment, the above-mentioned mRNA vaccine, or the above-mentioned polynucleotide, or the above-mentioned expression vector, or the above-mentioned host cell is used in the preparation of a vaccine for preventing or treating hand, foot and mouth disease, or herpetic pharyngitis.

[0037] Beneficial Effects

[0038] The enterovirus mRNA vaccine described in the present invention can achieve the expression of the target protein, and induce the body to produce high levels of IgG binding antibodies, neutralizing antibodies and specific cellular immune responses, reduce the virus content in the body, alleviate and eliminate clinical symptoms, and has clinical application value and industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 .Comparison of VP1 protein expression before and after optimization.

[0040] Figure 2 .Western blot method was used to detect the protein expression of EVA71-VP1 mRNA.

[0041] Figure 3 .The IFA method was used to detect the protein expression of EVA71-VP1 mRNA.

[0042] Figure 4 .EVA71-VP1 mRNA nanoparticle characterization assay.

[0043] Figure 5 .EVA71-VP1 specific antibody determination.

[0044] Figure 6 .EVA71 virus neutralizing antibody assay.

[0045] Figure 7 .CD4 + Cytokine detection in T cells.

[0046] Figure 8 .CD8 + Cytokine detection in T cells.

[0047] Fig. 9.Weight changes of mice treated with different methods.

[0048] Fig.10 .Clinical symptom results of mice treated with different methods.

[0049] Fig.11 .Survival rate of mice treated with different methods.

[0050] Fig.12 .The results of virus content in mice treated with different methods.

[0051] The sequence involved in the present invention is as follows

[0052] Amino acid sequence of VP1 protein (SEQ ID NO: 1):

[0053] GDRVADVIESSIGDSVSRALTHALPAPTGQNTQVSSHRLDTGKVPALQAAEI

[0054] GASSNASDESMIETRCVLNSHSTAETTLDSFFSRAGLVGEIDLPLKGTTNPNG

[0055] YANWDIDTTGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMF

[0056] VPPGAPKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYD

[0057] GYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGTSKSKYPLVIRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNSIKPTGASRTAITTL;

[0058] Nucleic acid sequence of VP1 protein (SEQ ID NO: 2):

[0059] GGAGACAGGGTGGCAGATGTAATAGAAAGCTCCATAGGAGATAGCGTGA

[0060] GCAGAGCCCTCACTCACGCTCTACCAGCACCCACAGGTCAGAACACACA

[0061] GGTGAGCAGCCATAGACTGGATACAGGCAAGGTTCCAGCACTCCAAGCT

[0062] GCTGAGATTGGAGCATCATCAAATGCTAGTGATGAGAGCATGATTGAAAC

[0063] ACGCTGTGTTCTTAATTCGCACAGCACAGCTGAGACCACTCTTGATAGTT

[0064] TCTTCAGCAGGGCGGGGTTAGTTGGAGAAATAGATCTCCCTCTTAAGGGC

[0065] ACAACTAACCCAAATGGTTATGCCAACTGGGACATAGATACAACAGGTTA

[0066] CGCGCAAATGCGCAGAAAGGTAGAGCTATTCACCTACATGCGCTTTGATG

[0067] CAGAGTTCACTTTTGTTGCGTGCACGCCCACTGGGGAAGTTGTTCCTCAA

[0068] TTGCTCCAATATATGTTTGTGCCGCCTGGAGCCCCTAAGCCAGACTCAAG

[0069] GGAATCTCTTGCATGGCAAACCGCCACTAACCCCTCAGTTTTTGTCAAGT

[0070] TGTCAGACCCTCCAGCGCAGGTTTCAGTGCCATTCATGTCACCTGCGAGT

[0071] GCCTACCAATGGTTTTATGACGGATATCCCACATTCGGAGAACACAAACA

[0072] GGAGAAAGATCTTGAATACGGAGCATGTCCTAATAACATGATGGGCACGT

[0073] TCTCAGTGCGGACTGTGGGGACCTCCAAGTCTAAATATCCTTTAGTAATTA

[0074] GGATCTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATG

[0075] CGTAACCAGAACTATCTATTCAAAGCCAACCCAAATTATGCTGGCAACTCTATTAAGCCAACTGGTGCTAGTCGCACAGCGATCACCACCCTT;

[0076] Amino acid sequence of the optimized VP1 protein (SEQ ID NO: 3): METDTLLLWVLLLWVPGSTGDGDRVADVIESSIGDSVSRALTHALPAPTGQNTQVSSHRLDTGKVPALQAAEIGASSNASDESMIETRCVLNSHSTAETTLDSFFSRAGLVGEIDLPLKGTTNPNGYANWDIDTTGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGAPKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGTSKSKYPLVIRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNSIKPTGASRTAITTL;

[0077] Nucleic acid sequence of the optimized VP1 protein (SEQ ID NO: 4): ATGGAAACTGATACACTGCTGCTGTGGGTGCTGCTGCTCTGGGTCCCCGGCAGCACCGGCGACGGCGATAGAGTGGCCGACGTGATCGAGTCTAGCATCGGCGACAGCGTGTCTCGGGCCCTGACCCACGCCCTGCCTGCCCCTACAGGCCAGAACACCCAGGTGTCTAGCCATAGACTGGACACAGGAAAAGTGCCTGCTCTGCAGGCCGCCGAAATTGGCGCCTCCAGCAACGCCAGCGACGAGAGCATGATCGAGACAAGATGCGTGCTGAACTCCCACAGCACCGCCGAAACCACCCTGGATAGCTTCTTCTCTAGAGCTGGCCTGGTCGGAGAGATCGACCTGCCTCTGAAAGGAACAACAAATCCCAACGGCTACGCTAATTGGGACATCGACACCACCGGCTACGCCCAGATGCGGAGAAAGGTGGAACTGTTTACCTACATGCGATTTGACGCCGAGTTCACCTTCGTGGCTTGTACACCTACCGGCGAGGTGGTGCCTCAGCTGCTGCAATACATGTTCGTGCCACCTGGCGCTCCTAAGCCTGATAGCAGGGAGAGCCTGGCCTGGCAGACCGCCACCAACCCCAGCGTGTTCGTTAAGCTGAGCGATCCCCCAGCTCAAGTGTCCGTGCCATTCATGAGCCCTGCCAGCGCCTACCAGTGGTTCTACGACGGCTATCCTACCTTCGGCGAGCACAAGCAGGAGAAGGACCTGGAATACGGCGCCTGCCCTAACAACATGATGGGCACATTTTCTGTGCGGACCGTGGGAACAAGCAAGTCCAAATACCCCCTGGTGATCAGAATCTACATGAGAATGAAGCACGTGCGGGCCTGGATCCCCAGACCTATGCGGAACCAGAACTATCTGTTCAAGGCCAACCCTAATTACGCCGGCAACAGCATCAAGCCCACCGGAGCCTCTAGAACCGCCATCACCACACTG;

[0078] DETAILED DESCRIPTION

[0079] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention falls within the protection scope of the present invention.

[0080] Example 1. Preparation of enterovirus 71 (EVA71) mRNA vaccine

[0081] 1. Synthesis of EVA71 virus VP1 protein gene and vector construction

[0082] In this embodiment, the target antigen of the enterovirus 71 (EVA71) mRNA vaccine is the full-length viral VP1 protein, whose gene coding sequence is from the laboratory isolated strain EVA71 JN315R. The specific amino acid sequence is shown in SEQ ID NO:1, and the nucleic acid sequence is shown in SEQ ID NO:2.

[0083] By adding a mouse Ig kappa secretory model signal peptide sequence (METDTLLLWVLLLWVPGSTGD) to the N-terminus of the VP1 gene sequence, the VP1 protein was codon optimized. The amino acid sequence of the optimized VP1 protein is shown in SEQ ID NO:3, and the nucleic acid sequence is shown in SEQ ID NO:4.

[0084] The VP1 protein sequences before and after optimization were connected to the pCAGGS eukaryotic expression vector by Nanjing GenScript Biotechnology Co., Ltd. (Nanjing GenScript), and the gene expression was analyzed by eukaryotic expression in mammalian cells (HEK293T). It can be seen that the protein abundance after sequence optimization is higher than that before optimization. The results are shown in Figure 1 .exist Figure 1 In the expression vector, the pCAGGS-EV71-VP1 group was the VP1 protein before optimization, the pCAGGS-EV71-VP1-Optimized group was the VP1 protein after optimization, and the pCAGGS group was the pCAGGS expression vector without the VP1 protein.

[0085] A Kozak sequence was added before the codon ATG of the nucleotide sequence encoding the optimized VP1 protein, non-coding sequences (UTRs) were added upstream and downstream of the sequence respectively, a T7 promoter was added to the N-terminus of the upstream UTR sequence, and a poly A sequence (110 A's) was added to the C-terminus of the downstream UTR sequence to obtain the EV71VP1 mRNA vaccine. The specific nucleic acid sequence is shown in SEQ ID NO:5.

[0086] The nucleotide sequence of the EV71 VP1 mRNA vaccine was synthesized by Nanjing GenScript Company and cloned into the pUC57 plasmid to obtain the recombinant plasmid pUC57-EVA71-VP1.

[0087] The pUC57-EVA71-VP1 plasmid was linearized by SapI restriction endonuclease, and then the linearized plasmid was concentrated and recovered for subsequent in vitro transcription reaction.

[0088] Configure the mRNA in vitro transcription reaction system according to the following table (the enzymes and reagents used were purchased from NEB):

[0089]

[0090] After mixing the above system, react at 37°C for 4 hours, then add 1 μl of RNase-free DNase I and continue to react for 1 hour.

[0091] The obtained reverse transcription product was purified using the RNA purification kit (AM1908) of Invitrogen Company to obtain purified RNA.

[0092] The purified mRNA was incubated at 65°C for 5 minutes, and then capped using the capping kit of Novoprotein (M082). The reaction system is as follows:

[0093]

[0094]

[0095] After the above system is mixed, the reaction is carried out at 37° C. for 30 minutes to obtain capped mRNA, which is then purified to obtain EVA71-VP1 mRNA.

[0096] 2. Identification of EV71-VP1 mRNA expression

[0097] HEK293T cells were inoculated into 12-well plates and 96-well plates. When the cells covered about 80% of the bottom of the plate, EVA71-VP1 mRNA was transferred to the cells using Lipofectamine 2000 transfection reagent. 24 hours after transfection, the cells in the 12-well plates were collected, and the total intracellular protein was extracted. The protein expression was detected by Western blot using EVA71 VP1 antibody (prepared by Nanjing GenScript) as the primary antibody. The results are shown in Figure 2 :The target protein VP1 band was detected at 37kd in the EVA71-VP1 mRNA group; while no target protein band was detected in the blank control group mock without EVA71-VP1 mRNA ( Figure 2 At the same time, cells in 96-well plates were fixed with 4% paraformaldehyde, and protein expression was detected by IFA using EVA71 VP1 antibody as primary antibody and A488-labeled goat anti-rabbit IgG as secondary antibody. The results are shown in Figure 3 :Green fluorescence signal can be detected in the EVA71-VP1 mRNA group, while no green fluorescence signal can be detected in the blank control group mock without EVA71-VP1 mRNA. The above results show that the obtained EVA71-VP1 mRNA is successfully expressed in the cells.

[0098] 3. mRNA Nanoparticle Packaging

[0099] The mRNA was packaged in nanoparticles using microfluidic technology. The mRNA was dissolved in the aqueous phase (50mM sodium acetate buffer, PH 4.0), and the nanoparticle component was dissolved in the ethanol phase using the Shanghai Myanna commercial kit (GenNano-E0011). The total flow rate during packaging was 20ml / min, and the flow rate ratio was 1:3. The packaged mRNA vaccine was replaced with PBS using a dialysis bag to obtain the EVA71-VP1 mRNA vaccine. The encapsulation efficiency of the vaccine was determined to be 96% using the RiboGreen reagent, and then stored in a 4°C refrigerator for later use.

[0100] The obtained vaccine solution was tested for the diameter of vaccine nanoparticles using a dynamic light scattering intensity analyzer. The results are shown in Figure 4 : The prepared nanoparticle vaccine has a diameter of about 84 nanometers and good uniformity.

[0101] Example 2: Immunological evaluation of EV71-VP1 mRNA vaccine

[0102] 1. Evaluation of vaccine humoral immunity effect

[0103] Twenty-four 6-8 week old female Balb / c mice were randomly divided into three groups, each with 8 mice. The three groups were negative control group (Empty LNP), high-dose group (20μg) of EV71-VP1 mRNA vaccine and low-dose group (5μg) of EV71-VP1 mRNA vaccine. Both the high-dose group (20μg) of EV71-VP1 mRNA vaccine and the low-dose group (5μg) of EV71-VP1 mRNA vaccine were immunized by hind leg muscle injection. The first immunization was performed on the first day of the experiment, the booster immunization was performed on the 21st day after the first immunization, and the third immunization was performed on the 42nd day after the first immunization. The immunization dose was the same for each time. The negative control group (Empty LNP) was injected with PBS at the same time.

[0104] Determination of EVA71-VP1 specific antibodies in serum: EV71 VP1 protein (prepared by the laboratory through the 293F eukaryotic expression platform) was coated on a 96-well ELISA plate, with 500 ng coated per well. After sealing the plate with a sealing film, the plate was placed in a 4°C refrigerator overnight for coating. The next day, the ELISA plate was blocked with 5% skim milk, and then the diluted mouse serum was added to the plate, incubated at 37°C for 1 hour, and the HRP-labeled goat anti-mouse IgG secondary antibody was added after washing the plate, and incubated at 37°C for 1 hour. After washing the plate, TMB single-component colorimetric solution was added, and incubated at 37°C in the dark for 15 minutes, and then the color development was terminated with 2M sulfuric acid solution, and the OD450 value was measured by an enzyme reader.

[0105] The results are as follows Figure 5 As shown, the EV71 VP1 antibody content in the serum of mice in the negative control group (Empty LNP) was very low; the EV71 VP1 antibody content in the serum of mice in the low-dose group (5μg) of EV71-VP1 mRNA vaccine was higher than that in the negative control group, and the EV71 VP1 antibody content continued to increase with the increase in the number of immunizations; the EV71 VP1 antibody content in the serum of mice in the high-dose group (20μg) of EV71-VP1 mRNA vaccine was the highest, and the EV71 VP1 antibody content continued to increase with the increase in the number of immunizations. This shows that a higher level of EVA71-VP1 antibodies can be detected in the serum of mice immunized with EV71-VP1 mRNA vaccine, and it is dose-dependent, and the antibody level gradually increases with the increase in the number of immunizations. This shows that EVA71-VP1 mRNA vaccine can induce mice to produce high levels of IgG binding antibodies.

[0106] 2. Determination of EVA71 virus neutralizing antibodies in serum

[0107] RD cells were inoculated into a 96-well plate one day in advance. The next day, the serum to be tested was diluted 2-fold and added to a new 96-well round-bottom plate, 50 μl per well. Then, the EVA71 virus was diluted to 100 TCID50 / 50μl. Add serum to virus in a 96-well round-bottom plate at a ratio of 1:1, mix well and incubate in a 37°C cell culture incubator for 2 hours. Then transfer the incubated serum-virus mixture to a 96-well RD cell culture plate prepared in advance, with 100μl of the mixture in each well. At the same time, set up a negative serum control, a virus-only infection control, and a cell-only control. Place the cells in a 37°C, 5% CO2 incubator for 5-6 days, observe the cytopathic effect (CPE) every 12 hours, and record the number of lesion wells.

[0108] The results are as follows Figure 6 As shown, the EVA71 virus neutralizing antibody titer in the serum of mice in the negative control group (Empty LNP) was very low; the EVA71 virus neutralizing antibody titer in the serum of mice in the low-dose group (5μg) of EV71-VP1 mRNA vaccine and the high-dose group (20μg) of EV71-VP1 mRNA vaccine was significantly higher than that in the negative control group, and the EVA71 virus neutralizing antibody titer continued to increase with the increase in the number of immunizations. This shows that a higher level of EVA71 virus neutralizing antibodies can be detected in the serum of mice immunized with EV71-VP1 mRNA vaccine, and the neutralizing antibody titer gradually increases with the increase in the number of immunizations. This shows that the EVA71-VP1 mRNA vaccine can induce mice to produce high levels of neutralizing antibodies.

[0109] 3. Vaccine Cellular Immunity Evaluation

[0110] Eighteen female mice aged 6-8 weeks were randomly divided into three groups, with 6 mice in each group, namely the negative control group (Empty LNP), the high-dose mRNA vaccine group (20μg) and the low-dose mRNA vaccine group (5μg). Both the high-dose mRNA vaccine group (20μg) and the low-dose mRNA vaccine group (5μg) were immunized by intramuscular injection of the hind legs. The first immunization was performed on the first day of the experiment, and then boosted every 3 weeks; the negative control group (Empty LNP) was injected with PBS at the same time. In the second week after the third immunization, the mice were euthanized, and the spleen was taken out. After the spleen was cut and ground, it was filtered through a 70μm filter to remove large cell clumps or tissues. All the obtained cells were transferred to a 50ml centrifuge tube, and the cells were collected by centrifugation at room temperature (1500 rpm). After discarding the supernatant, the cells were resuspended with 2ml red blood cell lysis buffer, lysed on ice for 5 minutes, 10ml 1640 culture medium was added, and the cells were collected by centrifugation, and then washed twice with 10ml 1640 culture medium. Finally, resuspend the cells in 2 ml of 1640 medium and count them using a cell counting plate, and adjust the cell density to 10 7 Pieces / ml.

[0111] Intracellular cytokine detection: The above cells (10 6 / 100μl) were inoculated into a 96-well round-bottom plate, and then 100μl of the pre-prepared VP1 peptide stimulation library (each peptide concentration was 3μg / ml) was added, mixed and incubated in a 37°C cell culture incubator for 6 hours, and then the cells were collected by centrifugation for intracellular cytokine staining, and then the cell fluorescence was detected using a BD FACS Canton flow cytometer.

[0112] The VP1-specific T cell immune response was analyzed by flow cytometry, and the results were as follows Figure 7-8 As shown, compared with the control group, the virus-specific CD4 + T and CD8 + T cells increased significantly. Further analysis showed that compared with the control group, the secretion of interferon gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin 2 (IL-2) in the spleen of mice immunized with EV71-VP1 mRNA vaccine increased significantly, while there was no significant difference in the secretion level of IL-4. This shows that immunization with EVA71-VP1 mRNA vaccine can significantly induce Th1 cell-biased immune response in mice.

[0113] 4. Evaluation of the protective effect of vaccine immune serum challenge.

[0114] Six cages of ICR mice were selected, and the number of mice in each litter was the same, 10. They were divided into three groups: negative control group (mock), vaccine protection group (20μg) and positive control group (Empty LNP), with 2 litters in each group. The treatment methods of each group are shown in the table below. The three immune serum samples were mixed and injected intraperitoneally into 1-day-old mice, 50μl / mouse, and a lethal dose of EV71 virus (10 7 TCID 50 The weight of mice was measured every day, and the clinical symptoms and survival rate of mice were recorded. At the same time, 4 suckling mice were randomly selected on the 2nd and 4th days after infection, and their muscle tissues were taken to detect the virus content in muscle.

[0115]

[0116] The weight changes of mice treated with different methods Fig. 9 As shown, the body weight changes of the negative control group (mock) and the vaccine protection group (20 μg) were similar, with a steady increase in body weight; the body weight of the mice in the positive control group (Empty LNP) was significantly lower than that of the negative control group (mock) and the vaccine protection group (20 μg) starting from the 2nd day.

[0117] Clinical symptoms of mice treated with different methods Fig.10As shown, the negative control group (mock) and the vaccine protection group (20 μg) had no obvious clinical symptoms; the clinical symptoms of the positive control group (Empty LNP) gradually increased with the extension of infection time.

[0118] The survival rate of mice treated with different methods is shown in Fig.11 As shown, all mice in the negative control group (mock) and vaccine protection group (20 μg) survived during the experiment; all mice in the positive control group (Empty LNP) died within 8 days.

[0119] Analysis of virus content in muscle tissue of mice treated with different methods Fig.12 As shown, a higher level of virus titer was detected in the positive control group (Empty LNP) mice; the serum of mice in the vaccine protection group (20 μg) could significantly reduce the virus titer in the mice; no virus was detected in the negative control group (mock) mice.

[0120] The above results show that EV71-VP1 mRNA vaccine can effectively reduce the virus content in infected mice, improve the survival rate of mice, and alleviate and eliminate clinical symptoms. Mice immunized with EVA71-VP1 mRNA vaccine can resist EVA71 infection, and EVA71-VP1 mRNA vaccine has a good protective effect.

[0121] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. An enterovirus mRNA vaccine, characterized in that: The nucleic acid sequence of the mRNA vaccine comprises: a Kozak sequence, a promoter sequence, a 5' non-coding region, a secretion signal peptide coding sequence, a VP1 protein coding sequence, a 3' non-coding region and a 3' PolyA tail; The amino acid sequence of the VP1 protein is shown in SEQ ID NO:

1.

2. The mRNA vaccine according to claim 1, characterized in that The nucleic acid sequence of the mRNA vaccine is shown in SEQ ID NO:

5.

3. The mRNA vaccine according to claim 2, characterized in that The nucleic acid sequence of the mRNA vaccine is capped.

4. The mRNA vaccine according to claim 1, characterized in that The enterovirus is enterovirus type 71 (EVA71).

5. The mRNA vaccine according to any one of claims 1 to 4, characterized in that The mRNA vaccine includes a delivery vector, which is capable of encapsulating the RNA sequence; Preferably, the delivery vehicle is a nanoparticle.

6. A polynucleotide, characterized in that The polynucleotide encodes the mRNA vaccine according to any one of claims 1 to 4; Preferably, the polynucleotide comprises any one or more of the following: (I) the nucleic acid sequence shown in SEQ ID NO: 2, 4 or 5; (II) a nucleic acid sequence having at least 90% homology to the nucleic acid sequence shown in SEQ ID NO: 2, 4 or 5; (III) a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 3; (IV) A nucleic acid sequence encoding an amino acid sequence obtained by modifying, replacing, deleting or adding one or more amino acids compared to the amino acid sequence shown in SEQ ID NO: 1 or 3.

7. An expression vector, characterized in that: The expression vector comprises the polynucleotide of claim 6.

8. A host cell, characterized in that The host cell comprises the vector of claim 7.

9. A method for constructing an enterovirus mRNA vaccine, the preparation method comprising the steps of: A recombinant plasmid expressing the mRNA vaccine according to any one of claims 1 to 4 is constructed, and the mRNA vaccine is obtained by nanoparticle encapsulation.

10. Use of the mRNA vaccine according to any one of claims 1 to 5, or the polynucleotide according to claim 6, or the expression vector according to claim 7, or the host cell according to claim 8 in the preparation of a vaccine for preventing or treating diseases caused by enterovirus, or hand, foot and mouth disease, or herpetic pharyngitis.

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