Mrna, methods of making and using the same, vaccines

Abstract

The application belongs to the technical field of biotechnology, discloses mRNA and a preparation method, use and vaccine thereof, a nucleotide sequence of a HA antigen coding gene of the mRNA is shown as SEQ ID NO:1, the mRNA is obtained by the following steps: adding a T7 promoter, a 5' end untranslated region and a Kozak sequence at the front end of a coding sequence of an HA protein of a swine influenza virus H1N2 after codon optimization, adding a 3' end untranslated region at the rear end, forming a DNA template sequence, and then obtaining the mRNA, and the vaccine prepared by using the mRNA has the advantages of good immunogenicity and strong protection.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to mRNA, its preparation methods, uses, and vaccines. Background Technology

[0002] Swine influenza (SI), also known as swine flu, is an acute, febrile, and highly contagious respiratory infectious disease in pigs caused by swine influenza virus (SIV), a virus belonging to the genus *Influenza* of the family Orthomyxoviridae. Clinically, it is characterized by sudden onset of high fever, cough, and difficulty breathing. SIV infection affects the production performance and disease resistance of infected pigs and is highly susceptible to secondary infections by other pathogens. Furthermore, pig respiratory epithelial cells contain receptors for both human and avian influenza viruses, making pigs a common susceptible host for both. Currently, the three main subtypes of SIV circulating worldwide are H1N1, H1N2, and H3N2.

[0003] Currently available influenza vaccines mainly include influenza split vaccines, live attenuated influenza vaccines, and protein subunit vaccines. Whether it's a traditional influenza split vaccine produced from chicken embryos or one produced using cell matrix or recombinant protein methods, the production time is relatively long. The development of influenza-related mRNA vaccines can bypass a series of complex biological activity processes such as cell fermentation, requiring only in vitro synthesis of mRNA, greatly improving development speed and simplifying the process. Whether it's a specific vaccine targeting a particular influenza strain or a universal vaccine, mRNA technology can rapidly advance candidate influenza vaccines to the clinical trial stage.

[0004] Chinese patent application 202010847434.0 discloses an HA-mRNA vaccine for preventing influenza A virus infection. The scheme provides that the HA-mRNA sequence consists of a 5'UTR, the nucleotide sequence shown in SEQ ID NO: 2, a 3'UTR, and a polyA tail. It can be seen that the preparation method of mRNA vaccine has been mentioned in the prior art. However, it is extremely difficult to prepare a high-titer vaccine against different types of influenza viruses.

[0005] The problem this application aims to solve is: how to provide an mRNA vaccine with good immunogenicity against the H1 subtype of swine influenza virus. Summary of the Invention

[0006] The purpose of this application is to provide a highly stable and highly efficient H1 subtype-based mRNA vaccine.

[0007] To achieve the above objectives, this application discloses an mRNA whose nucleotide sequence encoding the HA antigen gene is shown in SEQ ID NO: 1.

[0008] Preferably, the nucleotide sequence shown in SEQ ID NO: 1 is designed based on the gene sequence of swine influenza virus H1N2.

[0009] Preferably, the swine influenza virus H1N2 is the A / swine / Guangdong / A7 / 2016(H1N2) strain.

[0010] Preferably, it includes a Cap1 cap, a T7 promoter, a 5'UTR, the nucleotide sequence shown in SEQ ID NO: 1, a 3'UTR, and a polyA tail connected in sequence.

[0011] In addition, this application also discloses a method for preparing the mRNA as described above, comprising the following steps:

[0012] Step 1: Design a plasmid based on the gene sequence of the H1N2 strain, and then linearize the plasmid to obtain a linearized plasmid;

[0013] Step 2: Transcribe, purify, cap, and tail the linearized plasmid to obtain mRNA;

[0014] The transcription product of the linearized plasmid in step 2 includes the T7 promoter, 5'UTR, nucleotide sequence shown in SEQ ID NO: 1, and 3'UTR connected in sequence.

[0015] Preferably, during the transcription process in step 2, all uracil is replaced with pseudouracil.

[0016] In addition, this application also discloses the use of mRNA for preparing swine influenza vaccines as described above.

[0017] Preferably, the vaccine is a multivalent swine influenza vaccine.

[0018] In addition, this application also discloses a vaccine containing the mRNA described above.

[0019] The beneficial effects of this application are as follows: This application selects and optimizes the HA antigen coding sequence of swine influenza virus to construct an mRNA vaccine. After codon optimization of the HA protein coding sequence of swine influenza virus, a T7 promoter, a 5' untranslated region (5'UTR), and a Kozak sequence are added to the front end of the sequence, and a 3' untranslated region (3'UTR) is added to the back end to form the mRNA vaccine template DNA sequence. This sequence is then ligated into a plasmid vector to form the recombinant plasmid IVT-β2-mRNA. By linearizing, transcribing, capping, and tailing the recombinant plasmid, mRNA is obtained and packaged using LNP technology to produce a swine influenza mRNA vaccine. Immunoimmunization tests of the swine influenza mRNA vaccine have shown that it has significantly better immunogenicity than commercial vaccines and inactivated vaccines. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the Western Blot results;

[0021] Figure 2 A graph showing the HI titer test in mouse serum;

[0022] Figure 3 This is a schematic diagram of the gate system;

[0023] Figure 4 This is a schematic diagram of spleen T cell subsets in each group after the first immunization.

[0024] Figure 5 This is a schematic diagram of spleen T cell subsets in each group after secondary immunization.

[0025] Figure 6 This is a schematic diagram showing the levels of total IgG, IgG1, and IgG2a in serum.

[0026] Figure 7 This diagram illustrates the serum levels of IFN-γ, TNF-α, IL-2, and IL-6 cytokines.

[0027] Figure 8 This is a schematic diagram of virus titers in mouse lungs.

[0028] Figure 9 A schematic diagram of a pathological section of lung tissue from a mouse after viral challenge.

[0029] Figure 10 This diagram illustrates the weight and mortality rate of mice after challenge with the virus. Detailed Implementation

[0030] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0031] The raw material information during the experiment is as follows:

[0032] Relevant reagents: Vaccinia virus capping enzyme, homologous recombination kit, and Green Taq Mix were all purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0033] Citric acid, lithium chloride, sodium citrate, anhydrous ethanol, and DEPC were all purchased from McLean Company.

[0034] The A / swine / Guangdong / A7 / 2016(H1N2) swine influenza virus was preserved and provided by the Key Laboratory of Infectious Diseases, College of Veterinary Medicine, South China Agricultural University.

[0035] Gel recovery kit and plasmid extraction kit were purchased from Omega.

[0036] Ampicillin and PBS powder were purchased from Guangzhou Dingguo Biotechnology Co., Ltd.

[0037] LB broth and LB agar were purchased from Beijing Solarbio Co., Ltd.

[0038] Example 1

[0039] 1.1.1 Plasmid Preparation

[0040] The DNA template of this application was synthesized by Suzhou Hongxun Biotechnology Co., Ltd., and includes a T7 promoter, a 5' untranslated region (5'UTR), a HA antigen encoding gene, and a 3' untranslated region (3'UTR) connected in sequence.

[0041] The specific synthesis method is as follows:

[0042] The HA gene sequence in A / swine / Guangdong / A7 / 2016(H1N2) was amplified and codons were optimized to obtain the HA antigen encoding gene. Subsequently, using human β-globin as the 5'UTR and two tandem human β-globins as the 3'UTR, the sequence was synthesized by Suzhou Hongxun Biotechnology Co., Ltd. The synthesized sequence was ligated into the PUC57 plasmid to obtain IVT-β2-mRNA-CAIHA.

[0043] It should be noted that the nucleotide sequence of the HA antigen encoding gene in the above IVT-β2-mRNA-CAIHA is shown in SEQ ID NO: 4;

[0044] The nucleotide sequence of the 5'UTR in IVT-β2-mRNA-CAIHA is shown in SEQ ID NO: 5;

[0045] The nucleotide sequence of the 3'UTR in IVT-β2-mRNA-CAIHA is shown in SEQ ID NO: 6;

[0046] 1.1.2 Linearized plasmids

[0047] Since this application requires a small amount of template, PCR is used to obtain the template, and the PCR reaction system is shown in Table 1:

[0048] Table 1

[0049]

[0050]

[0051] The reaction procedure is shown in Table 2:

[0052] Table 2

[0053]

[0054] Add 6 μL of 10× loading buffer to the PCR product, perform electrophoresis on a 1% agarose gel, cut out the target band, put it into a 2 mL centrifuge tube, and perform gel recovery to obtain the linearized plasmid.

[0055] 1.1.3 In vitro transcription reaction

[0056] T7 High Yield RNA Transcription Kit (DD4201-01) and T7 High Yield RN ATranscription Kit (N 1 Both the -Me-Pseudo UTP (DD4202-01) kits were purchased from Novizan. The difference between the two kits lies in the UTP. All consumables must be free of RNase contamination. RNase and nucleic acid scavenging agents were sprayed into the environment to remove RNase. Before preparing the reaction mixture, all components except the T7 RNA Polymerase Mix were vortexed and briefly centrifuged. All reagents were placed on ice. The reaction mixture was prepared at room temperature. The reaction mixture is shown in Table 3.

[0057] Table 3

[0058]

[0059]

[0060] For the vortex reaction system, the components were briefly centrifuged to the bottom of the centrifuge tube, and the reaction system was incubated in a 37°C water bath for 3 hours. After incubation, 1 μL of DNase I was added to the reaction system, the system was mixed well, and then incubated in a 37°C water bath for 15 minutes. This step can remove the DNA template in the transcription system and prevent the presence of the DNA template from affecting the subsequent experimental results.

[0061] 1.1.4 Product Purification

[0062] After transcription, the transcription product needs to be purified before proceeding to the next step. This application uses lithium chloride (LiCl) precipitation to purify RNA. The procedure is as follows:

[0063] (1) Add 30 μL of DEPC-treated water to the reaction product and mix well;

[0064] (2) Add 30 μL of 7.5 M lithium chloride solution and gently mix by blowing and blowing (white turbidity may appear);

[0065] (3) Incubate the product from the previous step in a -20°C freezer for 30 minutes to help RNA precipitation. Turn on the high-speed refrigerated centrifuge and adjust the centrifuge temperature to 4°C.

[0066] (4) Take out the incubated mixture, centrifuge at 12000g for 15min at 4℃, and discard the supernatant;

[0067] (5) Add 500 μL of pre-cooled 70% ethanol (prepared with DEPC-treated water), invert the centrifuge tube to wash the sediment, centrifuge at 12000g for 5 min at 4℃, and discard the supernatant.

[0068] (6) Repeat step 5 twice;

[0069] (7) Use a 10 μL pipette tip to aspirate the residual liquid, dry the precipitate for 2 min after opening the cap, and add 30 μL RNa se-freeddH2O to dissolve the RNA precipitate;

[0070] (8) Measure and record the RNA concentration, and prepare for the next experiment or store it in a -80℃ refrigerator for later use.

[0071] In addition, it should be noted that its transcription product includes the T7 promoter, 5'UTR, nucleotide sequence shown in SE Q ID NO: 1, and 3'UTR connected in sequence;

[0072] The nucleotide sequence of the HA antigen encoding gene is shown in SEQ ID NO: 1;

[0073] The nucleotide sequence of the 5'UTR is shown in SEQ ID NO: 2;

[0074] The nucleotide sequence of the 3'UTR is shown in SEQ ID NO: 3;

[0075] 1.1.5 mRNA Capping Reaction

[0076] The capping reaction, performed using Novizan products, adds a Cap1 cap to RNA. The entire experiment requires RNase-free consumables and must be conducted in an RNase-free environment. The experimental steps are as follows:

[0077] (1) Take out the purified transcription product and the vaccinia virus capping enzyme kit, and place the items in the kit on ice to thaw.

[0078] (2) After thawing, mix all components thoroughly and collect the components at the bottom of the tube by instant centrifugation;

[0079] (3) Take 7 μL of DEPC-treated water and add it to a 1.5 mL centrifuge tube. Then take 1 μL of SAM (32 mM) and add it to the centrifuge tube. Mix well and dilute SAM to 4 mM.

[0080] (4) Place the RNA in a 65°C water bath for 5 minutes to denature it, and then place it on ice for 5 minutes to cool it down quickly. This step can open the higher-order structure of the 5' end of the RNA and improve the capping efficiency.

[0081] (5) Add reagents according to the capped reaction system in Table 4;

[0082] Table 4

[0083] reagents Volume (μL) 10×CappingBuffer 2 VacciniaCappingEmzyme 1 mRNACap2'-O-Methyltransferase 1 GTPSolution (10mM) 1 SAM (4mM) 1 Murine RNase Inhibitor (40 U / μL) 2 RNA 10μg RNase-free ddH2O Add to 20μL

[0084] (6) Mix the above system evenly, place it in a 37°C water bath, and incubate for 1 hour;

[0085] The product was then purified according to the purification method in 1.1.4, and its concentration was measured for further testing, or it was stored in a -80°C freezer for later use.

[0086] 1.1.6 mRNA tailing reaction

[0087] The experimental steps for the tailing reaction are as follows:

[0088] (1) Take out the mRNA capping reaction product and the mRNA tailing kit, and place the items in the kit on ice to thaw.

[0089] (2) After thawing, mix all components thoroughly and collect the components at the bottom of the tube by instant centrifugation;

[0090] (3) Add reagents to the tailing reaction system according to Table 5;

[0091] Table 5

[0092] reagents Volume (μL) 10×PolymeraseBuffer 2 E.coliPoly(A)Polymerase(5U / μl) 1 ATP (10mM) 1 Murine RNase Inhibitor (40 U / μL) 2 Cap1 RNA 10μg RNase-free ddH2O Add to 20μL

[0093] (4) Mix the above system evenly, place it in a 37°C water bath, and incubate for 3 hours;

[0094] (5) Use lithium chloride precipitation to purify mRNA, measure the concentration, and proceed to the next step of the experiment, or store it in a -80℃ freezer for later use;

[0095] It should be noted that the nucleotide sequence of Poly A in 80% of the mRNAs obtained after tailing in this application is an adenine base sequence with a length of 93bp to 489bp.

[0096] In the actual preparation process, the use of E. coli Poly(A) polymerase for tailing makes it difficult to accurately determine the length of Poly A, which in turn makes it impossible to obtain the specific nucleotide sequence of Poly A.

[0097] 1.2 mRNA cell transfection assay

[0098] 1.2.1 mRNA cell transfection

[0099] Use Lipofectamine TM The following are the specific steps for transfecting mRNA into cells using Reagent 2000:

[0100] (1) Treat the required 6-well cell plates with poly-L-lysine solution;

[0101] (2) Digest the prepared HEK-293T cells and dilute them to 2×10⁻⁶ cells using DMEM medium containing 10% fetal bovine serum (FBS). 5 Cells / mL, and inoculated into cell plates;

[0102] (3) Place the cell plate in a 37°C cell culture incubator with 5% CO2 and culture until 80% cell confluence.

[0103] (4) Refer to Lipofectamine TM According to the 2000Reagent instructions, use Opti-MEM to dilute and treat liposomes and mRNA, mix at a ratio of 1.5 μL of liposomes per 1 μg of mRNA, and incubate at room temperature for 20 min;

[0104] (5) Carefully wash the cell plate twice with PBS, add Opti-MEM, and after the previous incubation is completed, add the mRNA-liposome complex into the cell plate and place the cell plate in a 37°C cell culture incubator with 5% CO2.

[0105] 1.2.2 Immunoblot (Western Blot, WB)

[0106] 24 hours after mRNA transfection, discard the culture medium, wash once with PBS, add RIPA cell lysis buffer, collect the lysate in a 1.5 mL centrifuge tube, centrifuge at 12000g for 5 min, and transfer the supernatant to a new centrifuge tube. Measure the protein concentration of the sample in preparation for Western blotting (WB) experiments, as follows:

[0107] (1) Take the same mass of protein, dilute it to the same volume, add 0.25 times the volume of 5×SDS loading buffer, mix well, incubate in a metal bath at 99℃ for 10 min, and centrifuge at 1200g for 1 min.

[0108] (2) Prepare for polyacrylamide electrophoresis, and add 30 μL of sample to each well;

[0109] (3) After electrophoresis at 80V for 30 minutes, adjust the voltage to 120V and continue electrophoresis for 1 hour;

[0110] (4) Use wet electrophoresis. Take out the polyacrylamide gel, cut it into appropriate sizes and perform electrophoresis. Use a current of 400mA for 1 hour.

[0111] (5) Take out the nitrocellulose membrane (NC membrane), put it into 5% skim milk powder and seal it on a shaker at room temperature for 2 hours;

[0112] (6) Wash with TBST three times, 5 minutes each time;

[0113] (7) Add the diluted primary antibody and incubate overnight at 4°C;

[0114] (8) Wash with TBST three times, 5 minutes each time;

[0115] (9) Incubate with diluted fluorescent secondary antibody at room temperature for 30 min;

[0116] (10) Wash with TBST three times, 5 minutes each time;

[0117] (11) On-machine scanning imaging.

[0118] 1.2.3 Results of Immunoblotting Assay

[0119] refer to Figure 1 M represents the protein marker; 1 represents the total cellular protein; 2 represents the virus; and 3 represents the negative control. A clear protein imprint was visible near the 70 kDa protein bar in lane 1, indicating that this system can express HA protein normally.

[0120] Example 2

[0121] 2.1 Relevant reagents and consumables:

[0122] FITC Anti-Mouse CD4 Antibody, PE Anti-Mouse CD3 Antibody, and APC Anti-Mouse CD8a Antibody were purchased from eLabscience.

[0123] The A / swine / Guangdong / A7 / 2016(H1N2) swine influenza virus was preserved by the Key Laboratory of Infectious Diseases, College of Veterinary Medicine, South China Agricultural University.

[0124] The mouse spleen lymphocyte separation kit and erythrocyte lysis buffer were purchased from Tianjin Haoyang Biological Products Technology Co., Ltd.

[0125] RPMI Medium 1640 culture medium was purchased from Gibco.

[0126] The 70μm cell sieve was purchased from White Shark Biological Products Co., Ltd.

[0127] Cell counting chambers and AO / PI cell staining solution were purchased from Countstar.

[0128] The inactivated vaccine against the H1N1 subtype of swine influenza virus was purchased from Keqian Biotechnology.

[0129] The receptor-destroying enzyme RDE was purchased from Nippon Sanken Corporation; the ELISA kit was purchased from Guangdong Orida Biotechnology Co., Ltd.

[0130] 2.2 Related Instruments and Equipment:

[0131] The ST40R horizontal rotor centrifuge was purchased from Thermo Fisher Scientific.

[0132] The cell counter was purchased from Countstar.

[0133] The CytoFLEX flow cytometer was purchased from Beckman Coulter.

[0134] 2.3 Laboratory Animals

[0135] Six-week-old SPF BALB / c female mice were purchased from Beijing Spaford Biotechnology Co., Ltd.

[0136] 2.4 Solution Preparation

[0137] PBS buffer: Weigh 8g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 1.44g disodium hydrogen phosphate (Na2HPO4), and 0.24g potassium dihydrogen phosphate (KH2PO4), dissolve them in 800mL of deionized water, adjust the pH to 7.4 with dilute hydrochloric acid, add more deionized water, bring the volume to 1L, and autoclave at 121℃ for 15min.

[0138] Preparation of penicillin-streptomycin bispecific antibody stock solution: Weigh 10g penicillin powder and 22.2g streptomycin powder, add 120mL sterile PBS to dissolve, and after dissolution, add sterile PBS to make up to 160mL. Mix the solution well, filter it using a 0.22μm filter membrane, dispense the filtered bispecific antibody stock solution, and store it at -20℃ for later use.

[0139] 2.5 mRNA vaccine preparation

[0140] 2.5.1 Liposome Preparation

[0141] (1) The ionizable lipids D-Link-MC3-DMA and PEG-DMG were diluted to 100 mM with pharmaceutical grade anhydrous ethanol, respectively.

[0142] (2) Dissolve DSPC and cholesterol in pharmaceutical grade anhydrous ethanol to 10 mM;

[0143] (3) Mix them sequentially according to the molar ratio of D-Link-MC3:DSPC:cholesterol:PEG-DMG = 50:10:38.5:1.5, and finally add pharmaceutical grade anhydrous ethanol to dilute the mixture into solutions of 10mg / mL, 15mg / mL and 20mg / mL.

[0144] (4) Mix the above mixture thoroughly and dispense it into brown glass bottles, then store at -20℃ for later use.

[0145] 2.5.2 Preparation of mRNA-LNP complex

[0146] (1) Take the mRNA out of the -80℃ freezer and let it thaw. Then add 20× citrate-sodium citrate buffer and DEPC-treated water to dilute the mRNA to a solution with pH 4 and a concentration of 1 mg / mL.

[0147] (2) The mRNA-LNP complex was prepared by adjusting the microfluidic system to push the lipid complex and mRNA solution into a 100 μm diameter T-tube at a ratio of 1:1 for aqueous phase and ethanol phase.

[0148] (3) Dilute the mRNA-LNP complex prepared in the previous step with 10 times the volume of PBS;

[0149] (4) Add the diluted sample to the ultrafiltration tube and centrifuge it using a horizontal rotor centrifuge to reduce the volume to 1 / 10 of the original volume. The centrifugal force shall not exceed 4000g and the centrifugation time shall not exceed 10min.

[0150] (5) Add 9 times the volume of PBS, mix well, centrifuge again, repeat this cycle once more, collect the concentrate, and dilute appropriately.

[0151] 2.6 Preparation of penicillin-streptomycin dual antibody PBS

[0152] Add the bispecific antibody stock solution to sterile PBS at a ratio of 1:1000 and shake well. The PBS containing the bispecific antibody should be stored at 4°C.

[0153] 2.7 Total Clinical Infection Dose (TCID) 50 ) Measurement

[0154] (1) Inoculate MDCK into a 96-well plate in advance, and start the experiment when the cells reach 80% density;

[0155] (2) Take a tube of frozen virus solution and perform serial dilution using virus-inoculated cell culture medium;

[0156] (3) Wash the spread MDCK cells twice with PBS;

[0157] (4) Add the virus solution that has been serially diluted in the second step to a 96-well plate at a ratio of 100 μL per well for each dilution of 4 wells.

[0158] (5) Place the cell plate in a 37℃ CO2 incubator and incubate for 72 hours;

[0159] (6) Detect the HA of the virus and calculate the virus titer according to the Reed-Muench method.

[0160] 2.8 Production of Inactivated Virus Vaccines

[0161] Influenza virus inactivation was performed using β-propiolactone according to the national influenza laboratory standard operating procedures, and the virus solution was diluted to 10⁻⁶ using PBS. 7 TCID 50 / 100μL.

[0162] 2.9 Mouse Immunization and Challenge Experiment Procedure

[0163] The purchased mice were divided into five groups: PBS group, mRNA (10 μg) group, mRNA (20 μg) group, inactivated vaccine group, and commercial vaccine group. Mice were immunized via intramuscular injection in the hind limbs, with 50 μL injected into each hind limb, for a total of 100 μL. Challenge was performed via intranasal instillation. The specific immunization procedure, dosage, challenge time, and dose are shown in Table 6.

[0164] Table 6

[0165]

[0166]

[0167] Spleens were harvested from mice on days 3, 7, and 14 after immunization and challenge for lymphocyte isolation and T cell subset detection. Lungs were harvested from mice on days 1, 3, 5, and 7 after challenge to determine the viral titer in the lungs. Partial lung sections were harvested from mice challenged after secondary immunization on days 1, 3, 5, and 7 for pathological sectioning and scanning to observe changes in the lungs.

[0168] 2.10 Lymphocyte staining

[0169] (1) Take 10 6 One cell was added to 1 mL of PBS containing 2% FBS to resuspend the cells;

[0170] (2) Centrifuge the cells at 300g for 5 minutes;

[0171] (3) Each sample was prepared into a 100 μL staining solution using 75 μL of PBS containing 2% FBS and 5 μL each of FITC Anti-Mouse CD4 Antibody, PE Anti-Mouse CD3 Antibody and APC Anti-Mouse CD8a Antibody.

[0172] (4) Remove the cells from step 2, discard the supernatant, add 100 μL of staining solution, and incubate at 4°C in the dark for 30 min.

[0173] (5) After staining, centrifuge at 300g for 5min, discard the supernatant, and resuspend the cells in 1mL of PBS containing 2% FBS.

[0174] (6) Centrifuge at 300g for 5min, discard the supernatant, and then add 1mL of PBS containing 2% FBS to wash the cells once;

[0175] (7) Add 200 μL of PBS containing 2% FBS to resuspend the cells and wait for flow cytometry detection.

[0176] 2.11 Pathological sections of mouse lung tissue

[0177] After the second immunization challenge, mice were euthanized by cervical dislocation on days 1, 3, 5, and 7. The lungs of the mice were removed, cleaned in PBS, and then transferred to 4% paraformaldehyde fixative for pathological sectioning.

[0178] 2.12 Determination of viral titer in mouse lungs

[0179] (1) Weigh and record the weight of mouse lung tissue, and transfer the tissue into a 2mL centrifuge tube;

[0180] (2) Add two steel balls to the centrifuge tube and grind them using a liquid nitrogen cryogenic grinder;

[0181] (3) Add DMEM medium at a ratio of 0.1g to 0.9mL according to weight, and vortex to mix well;

[0182] (4) Measure the viral titer in the lungs according to the steps in 4.3.6.

[0183] 2.13 Determination of HI titer in mouse serum

[0184] Blood was collected by enucleation of the mouse eyeball. The mouse was fixed in one hand, and one eyeball was removed using ophthalmic surgical forceps. The outflowing blood was collected into a centrifuge tube and placed in a 37°C incubator for 30 minutes. After that, the tube was removed and placed in a 4°C refrigerator for 2 hours. The centrifuge tube was then removed, and the serum was carefully drawn into a new centrifuge tube. The collected serum was centrifuged at 3000 rpm for 5 minutes, the supernatant was collected, and the red blood cells at the bottom were discarded.

[0185] (1) Take 30 μL of mouse serum, add 90 μL of receptor-destroying enzyme (RDE), mix well, and incubate at 37°C for 18-24 hours.

[0186] (2) After incubation, place the serum in a 56°C water bath for 30 min, remove the serum, add 180 μL of PBS, and mix well.

[0187] (3) Measure the viral HA titer and dilute the virus according to the result until the virus is diluted to the point that it can only agglutinate the first three columns of red blood cells, thus obtaining 4 units of antigen.

[0188] (4) Add the diluted antibody to the first column of a 96-well V plate, 50 μL per well, 4 wells for each sample, and add 25 μL of PBS to the remaining wells;

[0189] (5) Use a multi-channel pipette to draw 25 μL of the first column of serum and add it to the second column. Mix well. Draw 25 μL of the second column of liquid and add it to the third column. Mix well. Continue diluting until the last column.

[0190] (6) Take 25 μL of 4 units of antigen, add it to the diluted serum, mix well, and incubate at 37°C for 30 min;

[0191] (7) Add 50 μL of 1% red blood cells to each well, mix well, incubate at room temperature for 30 min, observe and record the results.

[0192] 2.14 Mouse serum micro-neutralization test (MN)

[0193] (1) Incubate the serum in a 56°C water bath for 30 min and then dilute it 10-fold with Opti-MEM;

[0194] (2) Dilute the virus with Opti-MEM to 200 TCID 50 / 100μL;

[0195] (3) Take a new 96-well cell culture plate, add diluted serum to the first column, add 120 μL of serum to each well, add 4 wells for each sample, and add 60 μL of Opti-MEM to the remaining columns;

[0196] (4) Perform serial dilutions from the first to the eighth column, discarding the excess 60 μL of serum from the eighth column;

[0197] (5) Add diluted virus solution to diluted serum, add 60 μL to each well, mix well, and incubate at 37°C for 2 h;

[0198] (6) Wash the 96-well plate with pre-coated cells twice with PBS;

[0199] (7) Take 100 μL of the incubated mixture from step 5 and add it to the cell plate. Incubate in a cell culture incubator at 37°C and 5% CO2 for 2 hours.

[0200] (8) After incubation, discard the mixture and wash the cells twice with PBS;

[0201] (9) Add 200 μL of Opti-MEM containing TPCK-treated trypsin and incubate in a cell culture incubator at 37°C and 5% CO2 for 72 h;

[0202] (10) Take 25 μL of culture medium to test HA and record the results.

[0203] 2.15 Detection of total IgG, IgG1 and IgG2a antibody levels in mouse serum

[0204] Serum levels of IgG, IgG1, and IgG2a antibodies were detected at 2, 3, and 4 weeks after the initial immunization using a one-step sandwich enzyme-linked immunosorbent assay (ELISA). The procedure was performed according to the instructions from Orida Pharmaceutical Co., Ltd.

[0205] (1) Set up standard wells and sample wells, and add 50 μL of different concentrations of standard products to the standard wells;

[0206] (2) Add 10 μL of the serum to be tested to the sample well first, then add 40 μL of sample diluent, and keep the blank well;

[0207] (3) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to the standard and the sample wells to be tested;

[0208] (4) Seal the reaction wells with a sealing plate and incubate at 37°C for 60 min;

[0209] (5) Discard the liquid, pat dry on absorbent paper, add 300 μL of washing solution to each well, let stand for 1 min, discard and pat dry, wash a total of five times.

[0210] (6) Add 50 μL of substrate A and B to each well and incubate at 37°C for 15 min;

[0211] (7) Add 20 μL of stop solution and measure the OD value at a wavelength of 450 nm using an ELISA reader;

[0212] (8) Calculate the sample concentration based on the standard curve.

[0213] 2.16 Detection of IFN-γ, TNF-α, IL-2 and IL-6 levels in mouse serum

[0214] The serum levels of IFN-γ, TNF-α, IL-2, and IL-6 were detected at week 3 after the first immunization using a one-step sandwich enzyme-linked immunosorbent assay (ELISA) with double antibodies, as described in 4.15.

[0215] Example 3

[0216] 3.1 Mouse serum HI titer

[0217] Serum from mice 14 days after single and booster immunizations was used to measure the hemagglutination inhibition titer (HItiter) and evaluate the level of hemagglutination inhibition antibodies in the serum. Results are as follows: Figure 2 As shown in a, the HI titers in the serum of mice after a single immunization with the mRNA vaccine were all higher than the protective titer of 1:40, with an average titer higher than 1:80. Furthermore, the serum HI titers of mice immunized with the 20 μg mRNA vaccine were superior to those of the 10 μg group. However, this phenomenon was not observed in the HI titers after booster immunization. The average serum titer of mice immunized with the inactivated vaccine was lower than that of the mRNA vaccine group, but the average titer reached the protective titer. The HI titers of mice immunized with the commercial vaccine did not reach the protective titer. The serum HI titers of mice immunized twice were as follows: Figure 2As shown in b, the average serum HI titer in the mRNA vaccine group reached 1:2560, with some mice exhibiting a serum HI titer as high as 1:5120. The average serum HI titer in the inactivated vaccine group was 1:160, while the serum titer in mice using the commercial vaccine reached 1:320. The serum HI titer results showed that, regardless of whether it was a single or double immunization, the serum HI titer in the mRNA vaccine group was higher than that in other vaccine groups. There was no significant difference in serum HI titer after booster immunization with different doses of the mRNA vaccine. After two immunizations, serum titers significantly increased in both the mRNA vaccine group and other groups, especially in the mRNA vaccine group.

[0218] 3.2 Changes in T cell subsets in mouse spleen

[0219] Lymphocytes were isolated from mice on days 3, 7, and 14 post-immunization. The cell surface antigens CD3, CD4, and CD8 were labeled with fluorescent antibodies. CD3 surface antigen was present on almost all T cells. T cell subsets were detected by flow cytometry, and cell gating patterns were as follows: Figure 3 First, select lymphocyte populations based on FSC-H and SS-CH. Then, select single-cell populations from the lymphocyte populations. Since the single-cell populations FSC-H and FSC-A show a basically linear relationship, select cells near the diagonal line. Finally, select CD3 cells. + CD 4 + and CD3 + CD8a + Positive group.

[0220] The results after the first immunization were as follows Figure 4 As shown, comparing the vaccine groups and the PBS group after the first immunization, it was found that on day 3, CD3+ cells in the spleen lymphocytes... + CD4 + The number of cells decreased, and the difference between the 10 μg mRNA vaccine group and the PBS group was highly significant (P<0.001), while the difference between the 20 μg mRNA vaccine group and the PBS group was extremely significant (P<0.01). Furthermore, CD3+ levels in the spleen decreased. + CD8a + Cell counts also decreased, with a significant difference between the 10 μg mRNA vaccine group and the inactivated vaccine group (P < 0.05); on day 7, compared with the PBS group, the inactivated vaccine group had significantly lower CD3 counts. + CD4 + The number of cells increased significantly compared to PBS (P<0.05) in the commercial vaccine group. + CD4 + The number of cells increased significantly compared to PBS (P<0.001), and the CD3 count in the commercial vaccine group was significantly higher.+ CD8a + Cell counts also increased significantly (P<0.01); by day fourteen, CD3 counts varied significantly among groups. + CD4 + and CD3 + CD8a + The positive groups were relatively consistent, with no significant differences between the groups (P>0.05).

[0221] To observe the effects of booster immunization on mice, we also administered a second immunization to each group of mice and observed the differences in spleen lymphocytes after the second immunization compared to the first immunization. Figure 5 The changes in spleen T cell populations in mice after the second immunization are shown below. Specific results are as follows: On day 3 after the second immunization, the CD3 cell population in the group receiving the 10μg mRNA vaccine was... + CD4 + The number of cells was significantly increased in the spleen of mice in the 10 μg mRNA vaccine group compared to the PBS group (P<0.05). + CD8a + The cells in the 20 μg mRNA vaccine group showed a highly significant difference compared to the PBS group (P < 0.01). + CD8a + The difference in cell count between the mRNA vaccine group and the PBS group was very significant (P<0.001); on day 7, although there were significant differences in spleen cells between the mRNA vaccine group and the PBS group, the trends were exactly opposite between the 10 μg mRNA vaccine group and the 20 μg mRNA vaccine group; on day 14, the only spleen lymphocyte population in all groups was CD3 in the 20 μg mRNA vaccine group. + CD8a + The PBS group showed a significant increase (P<0.01), while the other groups showed no significant difference compared to the PBS group. Figure 5 In this context, ns represents P > 0.1; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; and **** represents P < 0.0001.

[0222] 3.3 Neutralizing antibody titer in mouse serum

[0223] Neutralizing antibodies can bind to viral surface antigens, preventing pathogens from binding to cell surface receptors and thus preventing viral invasion of cells. Therefore, the level of neutralizing antibodies is closely related to the protective effect. The levels of neutralizing antibodies in mouse serum were measured 14 days after single and double immunizations. The results are shown in Table 7.

[0224] Table 7

[0225]

[0226]

[0227] The PBS group showed no neutralizing antibody titer, and even with a single immunization using a commercial vaccine, no neutralizing antibody titer was produced. The groups using inactivated vaccines and mRNA vaccines also showed poor efficacy with the first immunization, and neutralizing antibody titers were undetectable in the serum of some mice. However, after the second immunization, the neutralizing antibody titers increased significantly, especially in the mRNA vaccine group, whose serum, even after being diluted 1280 times, still had a neutralizing effect on the virus, with some samples showing an MN titer of 1:20480.

[0228] 3.4 Detection of serum total IgG, IgG1 and IgG2a antibody levels in mice

[0229] We used a one-step sandwich ELISA with double antibodies to detect the levels of total IgG, IgG1, and IgG2a antibodies in mouse serum at weeks 2, 3, and 4 after primary immunization. The results are as follows: Figure 6 As shown, a represents the total IgG concentration in serum, b represents the total IgG1 concentration, and c represents the total IgG2a concentration. Two weeks after the first immunization, there was a significant difference in total IgG between the mRNA vaccine group and the inactivated vaccine group. Both IgG1 and IgG2a showed significant differences two weeks after immunization. In the first and second weeks after the second immunization, all vaccine groups showed significant differences compared to PBS. Figure 6 In this context, ns represents P > 0.1; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; and **** represents P < 0.0001.

[0230] At 2, 3, and 4 weeks post-immunization, the concentrations of total IgG, IgG1, and IgG2a in the mRNA vaccine group differed significantly from the other two vaccine groups. At two weeks, the total IgG in the mRNA vaccine group was 1.33 times that of the inactivated vaccine group and 1.72 times that of the commercial vaccine group; the total IgG1 in the mRNA vaccine group was 1.25 times that of the inactivated vaccine group and 1.41 times that of the commercial vaccine group; and the total IgG2a in the mRNA vaccine group was 1.19 times that of the inactivated vaccine group and 1.32 times that of the commercial vaccine group. At three weeks, the total IgG in the mRNA vaccine group was 1.21 times that of the inactivated vaccine group and 1.57 times that of the commercial vaccine group; the total IgG1 in the mRNA vaccine group was 1.19 times that of the inactivated vaccine group and 1.49 times that of the commercial vaccine group; and the total IgG2a in the mRNA vaccine group was 1.18 times that of the inactivated vaccine group and 1.35 times that of the commercial vaccine group. At four weeks, the total IgG in the mRNA vaccine group was 1.27 times that of the inactivated vaccine group and 1.26 times that of the commercial vaccine group. The total IgG1 in the NA vaccine group was 1.14 times that of the inactivated vaccine group and 1.24 times that of the commercial vaccine group. The total IgG2a in the mRNA vaccine group was 1.30 times that of the inactivated vaccine group and 1.55 times that of the commercial vaccine group.

[0231] 3.5 Detection of serum IFN-γ, TNF-α, IL-2 and IL-6 cytokine levels in mice

[0232] We used a one-step sandwich ELISA with double antibodies to detect the levels of IFN-γ, TNF-α, IL-2, and IL-6 cytokines in the serum of mice one week after secondary immunization. The results are as follows: Figure 7 As shown, a represents serum IFN-γ concentration, b represents TNF-α concentration, c represents IL-2 concentration, and d represents IL-6 concentration. One week after secondary immunization, serum IFN-γ and IL-2 levels in the 10 μg mRNA vaccine group and the inactivated vaccine group were significantly different from those in the PBS group, while no significant difference was observed in the commercial vaccine group. One week after secondary immunization, serum TNF-α and IL-6 levels in all three vaccine groups were significantly different from those in the PBS group. Figure 7 In this context, ns represents P > 0.1; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; and **** represents P < 0.0001.

[0233] One week after booster immunization, the serum concentrations of IFN-γ, TNF-α, IL-2, and IL-6 were measured. The IFN-γ concentration in the mRNA vaccine group was 1.48 times that of the inactivated vaccine group and 1.79 times that of the commercial vaccine group, respectively; the TNF-α concentration in the mRNA vaccine group was 1.51 times that of the inactivated vaccine group and 1.56 times that of the commercial vaccine group; the IL-2 concentration in the mRNA vaccine group was 1.57 times that of the inactivated vaccine group and 2.08 times that of the commercial vaccine group; and the IL-6 concentration in the mRNA vaccine group was 1.30 times that of the inactivated vaccine group and 1.78 times that of the commercial vaccine group.

[0234] IFN-γ, TNF-α, and IL-2, as Th1 cytokines, can promote the development of cytotoxic lymphocytes (CTLs and NK cells), mediate the proliferation of activated T cells, and play an important role in cellular immune responses. IL-6, a Th2 cytokine, is a pleiotropic cytokine with broad functions. It not only induces B cell proliferation, differentiation, and antibody production, but also acts as a terminal helper factor for cytotoxic T lymphocytes (CTLs), inducing CTL activity and promoting the development of immature thymocytes into CTLs. These results indicate that the mRNA vaccine in this study can effectively induce the secretion of Th1 and Th2 cytokines, combined with... Figure 7 The data results indicate that mRNA vaccines have good effects in inducing both cellular and humoral immunity.

[0235] 3.6 Pulmonary virus titer

[0236] On day 14 after immunization, we used 10 6 TCID 50Mice were challenged with the virus by inhaling 50 μL of viral fluid through the nasal cavity. Five mice were dissected on days 1, 3, 5, and 7 post-challenge. Lungs were removed, ground, mixed with PBS, centrifuged, and the supernatant was serially diluted and inoculated into cells. Viral titers in the organs were measured, and the results are as follows: Figure 8 a represents a single immunization, and b represents two immunizations. Vaccines can effectively reduce the viral titer in the lungs of mice after challenge.

[0237] 3.7 Observation of histopathological sections of mouse lung tissue after challenge

[0238] Mice were challenged with the virus after two immunizations. Lungs were collected on days 1, 3, 5, and 7 post-challenge, paraffin sections were prepared, and hematoxylin and eosin (HE) staining was performed. The case sections were scanned and magnified 20 times to compare and analyze the pathological changes in the lungs of the vaccine group and the control group on different dates.

[0239] Lung results in each group after virus challenge are as follows Figure 9 On day 1 post-challenge, abnormalities were observed in the lung tissue of the PBS group, with significant alveolar atrophy (as indicated by the yellow arrows), marked thickening of some alveolar walls, slight consolidation, and minimal interstitial congestion and inflammatory cell infiltration (as indicated by the red and black arrows, respectively). The groups using the inactivated vaccine and the commercial vaccine also exhibited overall lung tissue structural abnormalities, with significant alveolar collapse. The inactivated vaccine group showed extensive lung consolidation (as indicated by the yellow and black arrows), with abundant inflammatory cell infiltration in the interstitial space. In the commercial vaccine group, significant necrosis of numerous alveolar epithelial cells was observed, with pyknosis and fragmentation of cell nuclei (as indicated by the green arrows), and slight congestion was also visible. In both groups using the mRNA vaccine, the lung tissue structure was largely normal, with only a small amount of lymphocyte infiltration observed.

[0240] On day 3 post-challenge, except for mice using the 20μg mRNA vaccine, all other groups showed more severe lung damage than on day 1. In the PBS group, the overall lung structure was moderately abnormal, with significant alveolar atrophy and noticeable thickening of some alveolar walls (as shown by the yellow arrows). No significant vascular congestion or dilation was observed in the interstitial tissue, but a small amount of inflammatory cell infiltration was visible (as shown by the black arrows). In the 10μg mRNA vaccine group, the overall lung structure was mildly abnormal, with clear alveolar structure and no signs of alveolar fusion, dilation, or atrophy. No significant loosening, edema, or sloughing of alveolar epithelial cells was observed, and no significant interstitial congestion was seen (as indicated by the black arrows). A small amount of inflammatory cell infiltration was visible. In the inactivated vaccine and commercial vaccine groups, the overall lung structure was moderately abnormal, with significant alveolar atrophy (as shown by the yellow arrows), noticeable thickening of some alveolar walls, slight consolidation (as shown by the black arrows), and a large amount of lymphocyte infiltration. The inactivated vaccine group also showed a small amount of vascular congestion (as shown by the red arrows).

[0241] On day 5 post-challenge, lung tissue in the mRNA vaccine group also began to show lesions, while the lesions in the other three groups continued to worsen. In the PBS group, a large number of lung epithelial cells sloughed off and died, as indicated by the yellow arrows, and there was obvious inflammatory cell infiltration between the tissues. Mice using the mRNA vaccine also showed mild structural abnormalities in their lungs, with the alveolar structure remaining basically normal, as indicated by the yellow arrows, and obvious inflammatory cell infiltration between the tissues, as shown by the black arrows. In the two groups using the inactivated vaccine and the commercial vaccine, the lungs showed overall structural abnormalities, with alveolar atrophy and collapse, complete lung consolidation, and a large number of inflammatory cell infiltrations.

[0242] On day 7 post-challenge, mice in the PBS group continued to show severe lung damage, with abnormal overall lung structure, alveolar atrophy and collapse, thickened alveolar walls, and significant consolidation, as shown by the yellow arrows. Large areas of congestion and dilation were observed in the interstitial tissue, as shown by the red arrows, and a small number of inflammatory cells were infiltrated, as shown by the black arrows. The lungs of the other four vaccine-treated groups also showed damage. The mRNA vaccine group showed mild lung tissue damage, as shown by the black arrows, with inflammatory cell infiltration. The lung damage in mice treated with inactivated vaccines and commercial vaccines did not worsen further, and their performance was similar to that on day 5.

[0243] In summary, after viral challenge, the lungs of the PBS group continued to be attacked by the virus, causing lung damage to worsen continuously. Vaccines can protect the lungs from damage to some extent, but they cannot completely prevent lung damage. Of course, the challenge method and dosage used in our experiment are unlikely to occur in reality, so in reality, the protective effect of vaccines should be better than the results of this experiment. In the experiment, the groups using inactivated vaccines and commercial vaccines showed worse protective effects than the group using mRNA vaccines. On day seven after challenge, intact alveolar tissue was still visible in the mRNA vaccine group, with only mild lung damage. In contrast, the groups using inactivated vaccines and commercial vaccines showed severe lung consolidation and significant lung damage by day five. These results indicate that mRNA vaccines are more effective than conventional vaccines in protecting the lungs from damage.

[0244] 3.8 Changes in body weight and mortality rate of mice after challenge

[0245] In this experiment, the body weight of mice was measured daily after challenge with the vaccine to observe changes in body weight and evaluate the protective effect of the vaccine on mice. The experimental design was the same as in Table 4, using intranasal drops, with 10 6 TCID 50 Mice were challenged with the virus, and changes in body weight and mortality were measured 14 days post-challenge. Results are as follows: Figure 10As shown, groups a and c represent mice challenged 14 days after a single immunization. After challenge, the body weight of mice in all groups decreased. In the 10μg (mRNA-LNP) and 20μg (mRNA-LNP) groups, body weight stopped decreasing on day 4 after challenge and began to gradually recover on day 5. The group using the self-made inactivated vaccine showed the same trend as the two mRNA groups, beginning to recover on day 5. Mice in the commercial vaccine group continued to lose weight on day 5 and only began to recover on day 6. According to... Figure 10 As shown in 'c', mice using the mRNA vaccine and the homemade inactivated vaccine did not die, indicating that the vaccines in these three groups can provide complete protection after a single immunization. The commercial vaccine group provides 80% protection, while the control group has a 40% mortality rate.

[0246] Figure 10 b and d in the results are from a challenge administered 14 days after the second immunization, according to... Figure 10 As shown in b, in both groups using the mRNA vaccine, the mice experienced a slight decrease in body weight. The group using the 20μg mRNA vaccine performed slightly better, with weight loss only occurring on day 1 after challenge and ceasing on day 2. In the group using the 10μg mRNA vaccine, mouse weight began to recover on day 3. The group using the self-made inactivated vaccine began to regain weight on day 5, consistent with the results of a single immunization. Mice using the commercial vaccine also began to gradually regain weight on day 6. Figure 10 As shown in d, after the second immunization, all mice in the vaccine group survived, and all groups provided 100% protection; the control group still had a mortality rate of 40%.

[0247] After reducing the dosage of mRNA vaccines, such as Figure 10 As shown in figures b and d, mice in both the 5 μg and 2.5 μg groups survived after the second immunization. Based on weight changes, reducing the dosage of the mRNA vaccine resulted in greater weight loss in the mice. When the dosage of the mRNA vaccine was reduced to 2.5 μg, its protective effect was comparable to that of the self-made inactivated vaccine. The decrease in mouse weight was greater with decreasing mRNA vaccine dosage, indicating a positive correlation between the protective effect of the mRNA vaccine and its dosage. Exploring an appropriate mRNA vaccine dosage is beneficial for reducing vaccine costs while maintaining effective protection.

[0248] In summary, 14 days after the first immunization, the serum HI antibody titer in mice ranged from 1:80 to 1:160. 14 days after the second immunization, the serum HI antibody titer in mice immunized with the 10 μg mRNA vaccine ranged from 1:2560 to 1:10240; the serum HI antibody titer in mice immunized with the 20 μg mRNA vaccine ranged from 1:1280 to 1:5120, with a neutralizing titer of 1:1280 to 1:20480.

[0249] (2) Changes in splenic lymphocyte populations were detected by flow cytometry. The results showed that 3 days after the first immunization, the CD4 count in the mRNA vaccine-immunized group was significantly lower. + Lymphocyte count was significantly higher in the PBS control group (p≤0.005), and CD8 count was also higher in the 10μg mRNA vaccine immunization group. + The CD4 count was significantly lower than that in the PBS control group (p≤0.05); 3 days after the second immunization, the CD4 count in the 10μg mRNA vaccine immunization group was significantly lower. + Lymphocyte count was significantly higher in the mRNA vaccine immunization group than in the PBS control group (p≤0.05). + Lymphocyte count was significantly higher in the PBS control group than in the control group (p≤0.005); 7 days after the second immunization, the CD4 count in the 10μg mRNA vaccine immunization group was significantly higher. + and CD8 + Lymphocyte count was significantly higher in the PBS control group (p≤0.05), and CD4 count was also higher in the 20μg mRNA vaccine immunization group. + and CD8 + The number of lymphocytes was significantly lower than that in the PBS control group (p≤0.005).

[0250] (3) The levels of total IgG, IgG1, and IgG2a antibodies in mouse serum were detected at weeks 2, 3, and 4 after the first immunization. The levels of IgG, IgG1, and IgG2a antibodies in the mRNA vaccine group were significantly better than those in other vaccine groups at all three time points. One week after the booster immunization, the concentrations of IFN-γ, TNF-α, IL-2, and IL-6 in serum were detected. The IFN-γ concentration in the mRNA vaccine group was 1.48 times that of the inactivated vaccine group and 1.79 times that of the commercial vaccine group, respectively. The TNF-α concentration in the mRNA vaccine group was 1.51 times that of the inactivated vaccine group and 1.56 times that of the commercial vaccine group. The concentration of IL-2 in the mRNA vaccine group was 1.57 times that of the inactivated vaccine group and 2.08 times that of the commercial vaccine group. The IL-6 concentration in the mRNA vaccine group was 1.30 times that of the inactivated vaccine group and 1.78 times that of the commercial vaccine group.

[0251] (4) A challenge protection test was conducted 14 days after the first immunization. The results showed that the maximum weight loss of mice in each group was as follows: PBS group 19.56%, commercial vaccine group 23.43%, 10μg mRNA vaccine group 16.98%, 20μg mRNA vaccine group 20.37%, and inactivated vaccine group 16.03%. The mortality rates of mice in the PBS group and commercial vaccine group were 40% and 20%, respectively, with no deaths in the other groups. After the second immunization, the maximum weight loss of mice in each group was as follows: PBS group 25.63%, 10μg mRNA vaccine group 4.42%, 20μg mRNA vaccine group 1.64%, inactivated vaccine group 14.51%, and commercial vaccine group 18.76%, with no deaths in any of the vaccine groups.

[0252] (5) Lung pathological sections showed that the lung tissue of mice in the mRNA vaccine group suffered mild damage, while the lung tissue damage in mice in the PBS group, inactivated vaccine group, and commercial vaccine group gradually worsened, eventually developing into severe damage. In conclusion, the 10 μg mRNA vaccine can effectively activate humoral and cellular immunity in mice.

Claims

1. An mRNA, characterized in that, It includes the Cap1 cap, T7 promoter, 5'UTR, nucleotide sequence of the HA antigen encoding gene as shown in SEQ ID NO: 1, 3'UTR, and ployA tail connected in sequence.

2. The mRNA according to claim 1, characterized in that, The nucleotide sequence shown in SEQ ID NO: 1 was designed based on the gene sequence of swine influenza virus H1N2.

3. The mRNA according to claim 2, characterized in that, The swine influenza virus H1N2 is strain A / swine / Guangdong / A7 / 2016.

4. Use of mRNA for preparing swine influenza vaccine as described in any one of claims 1-3.

5. The use according to claim 4, characterized in that, The vaccine in question is a multivalent swine flu vaccine.

6. A vaccine, characterized in that, It contains the mRNA as described in any one of claims 1-3.

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