High-pathogenicity porcine reproductive and respiratory syndrome virus mRNA vaccine composition and application thereof

Abstract

The invention discloses a high-pathogenicity porcine reproductive and respiratory syndrome virus mRNA vaccine composition and application thereof. The vaccine composition is selected from any one of the following combinations: (1) an mRNA vaccine composition consisting of a PRRSV GP3 mRNA (messenger ribonucleic acid), a PRRSV GP4 mRNA (messenger ribonucleic acid) and a PRRSV GP5 mRNA (messenger ribonucleic acid); or (2) an mRNA vaccine composition consisting of a PRRSV GP5 mRNA, a PRRSV membrane protein (M) mRNA and a PRRSV nucleocapsid protein (N) mRNA. According to the invention, a high-pathogenicity PRRSV JX-A1 strain is used as a template, and mRNAs capable of synthesizing a plurality of fusion proteins GP345, GP5M and GP5MN and single structural proteins GP3, GP4, GP5, M and N are respectively constructed. After liposome encapsulation, mice are inoculated for immunological evaluation, and results show that humoral immunity and cellular immunity levels of a single mRNA-LNPs mixed group are higher than those of recombinant mRNA-LNPs, and GP5 + M + N and GP3 + 4 + 5 have relatively best effects. The invention provides a new technical means for preventing PRRSV (Porcine Reproductive and Respiratory Syndrome Virus).

Description

Technical Field

[0001] The present invention relates to an mRNA vaccine composition and its application, and in particular to a highly pathogenic PRRSV mRNA vaccine composition and its application. The present invention belongs to the field of biomedicine technology. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) first appeared in North America in the 1980s and then spread to Europe. Since then, the disease has spread worldwide and evolved into one of the most devastating diseases in the pig industry, characterized by reproductive failure and respiratory diseases. PRRS virus (PRRSV) is a single-stranded positive-sense, enveloped RNA virus belonging to the order Nidovirales of the family Arteriviridae, with a full-length genome of approximately 15 kb. Its genome contains at least 10 open reading frames (ORFs), ORF1a and ORF1b account for more than two-thirds of the viral genome, encoding 14 non-structural proteins, including Nsp1a, Nsp1b, Nsp2-6, Nsp7a, Nsp7b, and Nsp8-12, which are essential for viral replication; while ORFs 2-7 encode 8 structural proteins, including glycoprotein (GP2), envelope protein (E), GP3, GP4, GP5, ORF5a, membrane protein (M), and nucleocapsid protein (N). PRRSV mainly includes two genotypes, genotype I (European type) and type II (North American type). The whole genome difference between different genotypes is as high as 40%, and the difference between different strains of the same genotype is as high as 20%. PRRSV is susceptible to monocytes and macrophages, and after infection, it inhibits the host's cellular immune response and delays the production of neutralizing antibodies. Its high genetic variation and complex pathogenic mechanism make the prevention and control of PRRSV difficult. Although great efforts have been made to mitigate the impact of PRRSV, PRRSV is still not under adequate control. Currently available commercial vaccines are only inactivated vaccines and attenuated vaccines, but inactivated vaccines can only alleviate some clinical symptoms and cannot provide protection, and attenuated vaccines can only provide protection against homologous strains and cannot resist attacks from heterologous strains, so there is an urgent need to develop a new and effective vaccine.

[0003] mRNA vaccines have received widespread attention due to their efficient development speed and potential to provide protection against viral attacks. Compared with traditional vaccines such as live attenuated vaccines and inactivated vaccines, mRNA vaccines have superior advantages in safety, effectiveness and industrial production. Although antigens are encoded by mRNA, consistent technology in vaccine production simplifies the process and ensures timely and scalable vaccine development. At present, mRNA vaccines for a variety of infectious diseases have made good research progress. Summary of the invention

[0004] The purpose of the present invention is to provide a highly pathogenic PRRSV mRNA vaccine composition and its application.

[0005] In order to achieve the above object, the present invention adopts the following technical means:

[0006] The present invention uses the highly pathogenic PRRSV JX-A1 strain as a template to develop a PRRSV mRNA vaccine, selects GP3, GP4, GP5, M and N proteins of the PRRSV JX-A1 strain as antigens, and mutates the glycosylation site of the GP5 protein to improve the production of neutralizing antibodies. The mRNAs of the fusion proteins GP345, GP5M, GP5MN and the single structural proteins GP3, GP4, GP5, M and N are constructed and synthesized. After liposome encapsulation, the mRNA-LNPs vaccine is obtained, and mice are mixed with the fusion protein mRNA-LNPs and the mRNA-LNPs of the corresponding single proteins to immunize mice, and the immune effect in mice is evaluated. The results show that the humoral immunity and cellular immunity levels of the single mRNA-LNPs mixed group are higher than those of the recombinant mRNA-LNPs, among which GP5+M+N and GP3+4+5 have the best effects.

[0007] Therefore, based on the above research, the present invention proposes a highly pathogenic porcine reproductive and respiratory syndrome virus mRNA vaccine composition, wherein the vaccine composition is selected from any one of the following combinations:

[0008] (1) an mRNA vaccine composition consisting of PRRSV GP3 mRNA, PRRSV GP4 mRNA and PRRSV GP5 mRNA; or

[0009] (2) An mRNA vaccine composition consisting of PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA.

[0010] Among them, preferably, the PRRSV GP3 mRNA, PRRSV GP4 mRNA, PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA are N1-methylpseudouracil-modified mRNAs obtained by transcription of the sequences shown in SEQ ID NO.4-8, respectively.

[0011] Among them, preferably, the PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA are mixed in an equimolar ratio.

[0012] Preferably, the PRRSV GP3 mRNA, PRRSV GP4 mRNA and PRRSV GP5 mRNA are mixed in an equimolar ratio.

[0013] Furthermore, the present invention also proposes the use of the mRNA vaccine composition in the preparation of an mRNA vaccine for preventing highly pathogenic porcine reproductive and respiratory syndrome virus infection.

[0014] Furthermore, the present invention also proposes an mRNA vaccine for preventing highly pathogenic porcine reproductive and respiratory syndrome virus infection, wherein the vaccine contains the mRNA vaccine composition.

[0015] Preferably, the vaccine further contains lipids, and the lipids include ionizable lipid ALC-0315, distearoylphosphatidylcholine (DSPC), cholesterol and polylipid DMG-PEG2000.

[0016] Wherein, preferably, the mRNA vaccine is prepared by the following method:

[0017] (1) dissolving ionizable lipid ALC-0315, distearoylphosphatidylcholine (DSPC), cholesterol, and polylipid DMG-PEG2000 in ethanol to obtain lipids;

[0018] (2) dissolving a mixture of the PRRSV GP3 mRNA, PRRSV GP4 mRNA and PRRSV GP5 mRNA or a mixture of PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA in 20 mM, pH 4.5 citric acid buffer to obtain an mRNA solution;

[0019] (3) preparing lipid nanoparticles (LNPs) by mixing the lipid obtained in step (1) and the mRNA solution obtained in step (2) with the lipid and mRNA in a microfluidic mixer;

[0020] (4) LNPs were dialyzed in RNase-free pH 7.4 phosphate buffer for 24 h to remove ethanol. Finally, LNPs were filtered through a 0.22 μm filter and stored at 4 °C until use.

[0021] Among them, preferably, in step (1), the molar ratio of ionizable lipid ALC-0315, distearoylphosphatidylcholine (DSPC), cholesterol, and polylipid DMG-PEG2000 is 46.3:9.4:42.7:1.6, and the total lipid concentration is 19.1mM; in step (2), the PRRSVGP3 mRNA, PRRSV GP4 mRNA and PRRSV GP5 mRNA are mixed in an equimolar ratio, or the PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA are mixed in an equimolar ratio, the amount of mRNA is calculated according to the nitrogen-phosphorus ratio of 1:6, and the mRNA is dissolved in 20mM, pH 4.5 citric acid buffer.

[0022] Preferably, in step (3), the lipid obtained in step (1) and the mRNA solution obtained in step (2) are mixed in a microfluidic mixer at a volume ratio of 1:3 to prepare lipid nanoparticles (LNPs).

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention designed several PRRSV mRNA vaccines based on the HP-PRRSV JX-A1 strain and conducted detailed immune evaluation in mice, demonstrating the potential of mRNA vaccines in preventing PRRSV infection;

[0025] 2. The present invention constructs and prepares mRNA-LNPs of PRRSV fusion protein and single protein, and immunizes mice with a mixed group of mRNA-LNPs of fusion protein and corresponding single protein mRNA-LNPs. It is found that the cellular immunity level and humoral immunity level of the mixed group of single protein mRNA-LNPs are higher than those of the fusion protein group. This may be due to the change in conformation of multiple proteins caused by fusion, which can not fully expose the epitope of each protein. This also reminds us that for pathogens like PRRSV that do not have obvious protective advantage proteins but have protective epitopes in multiple proteins, vaccines developed by multi-protein mixing will play a better role than multi-protein fusion vaccines. Among them, GP3+4+5 also showed a relatively good cellular immunity level. In addition, in the detection of neutralizing antibodies, the neutralizing antibodies of GP3+4+5 and GP5+M+N have been greater than 1 to 10 since the second week after immunization, and the highest can reach 1 to 34;

[0026] 3. Those skilled in the art know that the level of neutralizing antibodies produced by PRRSV is very low. Studies have reported that the infusion of a neutralizing antibody titer of 1:16 into pregnant sows can protect sows from reproductive disorders and placental infection, and the infusion of antibodies with a titer of 1:8 or higher can prevent the occurrence of piglet viremia. When the titer reaches 1:32, the viral infection can be cleared. The results of neutralizing antibodies show that the PRRSV mRNA vaccine can quickly induce relatively high levels of antibody titers, while the currently licensed MLV vaccine mainly induces non-neutralizing antibodies in the first 4 weeks, which creates a "window period" and triggers an antibody-dependent enhancement effect (ADE), promotes viral infection, and increases the risk of vaccinated herds. This delayed neutralizing antibody response only provides partial protection for vaccinated piglets and limits the effectiveness of the MLV vaccine. The PRRSV mRNA vaccine in the present invention produces neutralizing antibodies greater than 1 to 8 starting from the second week of immunization, which may alleviate the ADE problem and effectively clear PRRSV infection;

[0027] 4. The present invention evaluates the IgG subclass distribution of sera immuned with these PRRSV mRNA vaccines. Among them, half of the IgG induced by GP3+4+5 and GP345, GP5+M and GP5M mRNA vaccines were IgG2b, indicating the presence of a potential Th1 immune response; in contrast, the sera of mice immunized with GP5+M+N and GP5MN mRNA vaccines had significant IgG1 and IgG2b responses, indicating that Th1 and Th2 immune responses were induced simultaneously. N protein, which does not contain neutralizing antibody epitopes, is often overlooked. However, in the present invention, the incorporation of N protein mRNA can significantly increase antibody levels and cellular immune response levels, and significantly change the IgG subclass distribution. In the study of universal influenza vaccines, the conservation of NP protein can be used as a target antigen for universal influenza vaccines because the antibody has the potential for cross-protection and humoral longevity, which further indicates that antibodies targeting enveloped viral nucleoproteins can protect hosts from viral attacks in vivo. In addition, the N protein is not expressed on the surface of PRRSV virus particles, so it cannot cause ADE. Current research on PRRSV vaccines focuses on the structural proteins on its surface, which can theoretically induce effective neutralizing antibodies; however, these antibodies can also induce ADE, affecting the effective elimination of PRRSV. Reducing ADE is also an important goal in the design of PRRSV vaccines, so the N protein can be used as a component of the vaccine.

[0028] In summary, we designed several PRRSV mRNA vaccines and studied their immunogenicity and specific T cell immunity by immunizing mice with different combinations. The results showed that GP3+4+5, GP5MN and GP5+M+N could induce PRRSV-specific humoral and cellular immune responses, among which GP5+M+N performed best. mRNA vaccines can be synthesized in vitro in a cell-free manner, thus avoiding the manufacturing complications associated with viral vectors and subunit vaccines. mRNA does not need to enter the cell nucleus and can be translated in large quantities in the cytoplasm, overcoming the obstacles posed by nuclear transport. Unlike DNA vaccines, mRNA is non-infectious and non-integrating, which minimizes the potential risks of infection and insertional mutagenesis. These characteristics together suggest that mRNA vaccines have the potential to be a new and promising vaccine platform against PRRS with diverse genetic variations. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Design and synthesis characterization of PRRSV-mRNA vaccine;

[0030] Among them, A: mRNA design diagram of PRRSV fusion protein and single protein; fusion protein genes GP345, GP5M, GP5MN and single structural protein genes (GP3, GP4, GP5, M, N) were inserted into the 5' and 3' non-coding regions of the linear mRNA framework through BamH I and Not I, and EcoRV that can produce blunt ends was added after the poly (A) tail of 120 A to linearize the plasmid; fusion proteins were connected with (G4S)3, and a Flag tag was added to the end of the fusion target gene for subsequent identification; B: particle size of PRRSV mRNA-LNPs; C: PDI of PRRSV mRNA-LNPs; D: encapsulation efficiency of PRRSV mRNA-LNPs; E: electron microscopy observation of PRRSV mRNA-LNPs; F: PRRSV Expression verification of mRNA-LNPs; lanes 1-9 are GP3, GP4, GP5, M, N, HEK293T cells, GP345, GP5M, and GP5MN, respectively;

[0031] Figure 2 Antigen-specific immune responses were induced in mice immunized with PRRSV-mRNA-LNPs;

[0032] Among them, A: mouse immunization program, primary immunization on day 0, booster immunization on days 14 and 28, mice were killed 2 weeks after the second immunization, lymphocytes were isolated for cellular immunity evaluation. Blood samples were collected from the orbital venous plexus of mice every week to separate serum and detect its humoral immunity level; B: immunization grouping, a total of 8 groups were designed, 3 fusion protein mRNA groups and 3 corresponding single mRNA mixed groups, and negative control groups of PBS and LNP. Leg muscle injection, the immunization dose was 20μg; C and D: specific antibody levels after PRRSV-mRNA-LNPs immunization; E: neutralizing antibody levels after PRRSV-mRNA-LNPs immunization; F: subclass distribution of serum IgG antibodies after PRRSV-mRNA-LNPs immunization;

[0033] Figure 3 The cellular immune response induced by immunizing mice with PRRSV-mRNA-LNPs;

[0034] Among them, A: the proliferation level of mouse splenic lymphocytes after immunization with PRRSV-mRNA-LNPs; B and C: the ratios of CD3+CD4+ and CD3+CD8+ of mouse splenic lymphocytes after immunization with PRRSV-mRNA-LNPs; D: the secretion level of IFN-γ of mouse splenic lymphocytes after immunization with PRRSV-mRNA-LNPs; E: the secretion level of IL-2 of mouse splenic lymphocytes after immunization with PRRSV-mRNA-LNPs. DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0036] Example 1

[0037] 1 Materials

[0038] 1.1 Ethical statement

[0039] This study was performed in accordance with the legal and recommended guidelines for the care and use of laboratory animals of the Ministry of Science and Technology of the People's Republic of China. All animal experiments were approved by the Animal Care and Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, with approval number LVRIAEC-2023-088.

[0040] 1.2 Cells, viruses, and animals

[0041] African green monkey kidney epithelial cells (Marc-145 cells) and human embryonic kidney 293T cells (HEK293T cells) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum and penicillin (100 U / mL)-streptomycin (100 mg / mL) (Thermo Fisher Science, USA). 6-8 week old BALB / c mice were purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences and raised under appropriate temperature and humidity conditions.

[0042] 2 Methods and Results

[0043] 2.1 mRNA synthesis

[0044] The sequences of GP3, GP4, GP5, M and N were referenced from the prototype PRRSV-2 strain JX-A1 (Genbank ID: EF112445.1). We designed and synthesized nucleotide sequences encoding three fusion proteins, namely GP345, GP5M and GP5MN. At the same time, GP3, GP4, GP5, M and N were amplified using the synthesized fusion proteins as templates. The three glycosylation sites of GP5 were mutated (N35A, N44A, N51A). The designed sequence was inserted between the 5' and 3' untranslated regions (UTRs), with 120 A at the 3' end, as shown in the schematic diagram. Figure 1 A, the nucleotide sequences of GP345, GP5M and GP5MN are shown in SEQ ID NO.1-3, and the nucleotide sequences of GP3, GP4, GP5, M, and N are shown in SEQ ID NO.4-8. The plasmid was linearized by a restriction endonuclease that produces a blunt end, and the linearized plasmid template was in vitro transcribed using T7 Co-transcription RNA Synthesis Kit (Shenji, China) to synthesize N1-methyl pseudouracil-modified mRNA, which was purified by lithium chloride precipitation and resuspended in RNAse-free water. The concentration of the purified mRNA was determined using a NanoDrop ultra-micro spectrophotometer (ThermoFisher Science, USA), and the purified mRNA was analyzed by denaturing agarose gel electrophoresis, and then Lipofectamine TM HEK293T cells were transfected with 2000 Reagent.

[0045] 2.2 Preparation of lipid nanoparticles

[0046] Ionizable lipid ALC-0315, distearoylphosphatidylcholine (DSPC), cholesterol, and polylipid PEG2000-DMG were dissolved in ethanol at a molar ratio of 46.3:9.4:42.7:1.6, and the total lipid concentration was 19.1 mM. The amount of mRNA was calculated based on the nitrogen-phosphorus ratio of 1:6, and the mRNA was dissolved in citric acid buffer (20 mM, pH 4.5). GP3+4+5 was a mixture of PRRSVGP3mRNA, PRRSVGP4mRNA, and PRRSVGP5mRNA in an equal molar ratio, and GP5+M+N was a mixture of PRRSVGP5mRNA, PRRSV membrane protein mRNA, and PRRSV nucleocapsid protein mRNA in an equal molar ratio. The lipid and mRNA solutions were prepared in a microfluidic mixer (Nanomedicine Manufacturing System, Myana) at a volume ratio of 1:3 (lipid / mRNA). LNPs were dialyzed in RNase-free phosphate buffer (pH 7.4) for 24 h to remove ethanol. Finally, LNPs were filtered through a 0.22 μm filter and stored at 4 °C until use. The particle size and zeta potential of LNPs were measured by Malvern Nanosizer (Malvern, UK), the morphology was observed by transmission electron microscopy, and the encapsulation efficiency was measured by Quant-iT TM RiboGreen TM The RNA was determined using an RNA kit (Thermo Fisher Scientific, USA).

[0047] The results are as follows Figure 1 As shown, the average particle size of the obtained mRNA-LNPs was within 100 nm ( Figure 1 B), the distribution is narrow, and the PDI is less than 0.1 ( Figure 1 C), packaging efficiency exceeds 93% ( Figure 1 D). PDI < 0.1, indicating that the size of mRNA-LNP particles is more uniform. Transmission electron microscopy observation shows that the actual diameter of the synthesized nanoparticles is about 70 nm ( Figure 1 E), which is one of the optimal sizes to facilitate its internalization and biodistribution.

[0048] 2.3 Western blot

[0049] Western blotting was used to detect mRNA expression. HEK293T cells cultured in 24-well plates were incubated with liposome-encapsulated mRNA (1 μg per well) for 24 h. The cells were washed with cold PBS and lysed with RIPA lysis buffer (Beyotime, China). The whole cell lysate was mixed with 5×Loading, boiled for 10 min, separated by 12% SDS-PAGE, and the protein gel was transferred to an NC membrane (Millipore, USA), which was blocked with 5% skim milk for 1 h at room temperature. Washed three times with PBS-T. Anti-Flag mouse monoclonal antibody diluted 1:1000 and PRRSV high immune serum (1 to 500) were used as primary antibodies, incubated at room temperature for 1 h, and washed three times with PBS-T. The membrane was incubated with HRP-labeled goat anti-mouse / pig antibody (Beyotime, China) at room temperature for 1 h. Washed three times with PBS-T, and the image was captured by the ECL system (CLINX, China). The results are shown in Figure 2. Figure 1 As shown in F, all encapsulated mRNAs can be expressed, among which GP3 and GP4 have tails, which may be related to their glycosylation modification in the cell.

[0050] 2.4 Animal experiments

[0051] To evaluate the immunogenicity of PRRSV mRNA-LNPs vaccine, we immunized mice with LNP-encapsulated fusion proteins of PRRSV mRNA GP345, GP5M, GP5MN and mRNA-LNPs of single proteins corresponding to the fusion proteins in mixed groups GP3+4+5, GP5+M, and GP5+M+N. Female BALB / c mice aged 6-8 weeks (n=8) were selected and the immunization schedule was as follows: Figure 2 As shown in A. The mice were grouped as follows Figure 2 As shown in B. 20 μg was injected into each thigh using an insulin syringe (BD, USA) at a volume of 50 μL, with PBS and blank LNP as placebo controls. Vaccinated mice received booster doses on days 14 and 28 after vaccination. Blood was collected from the orbital sinus after anesthesia, and serum was collected after centrifugation for detection of binding antibodies. To evaluate T cell responses, we removed the spleen and prepared single-cell suspensions of splenic lymphocytes for IFN-γ and IL-2 detection.

[0052] 2.5 ELISA

[0053] ELISA was used to detect specific antibody responses induced by PRRSV mRNA vaccines. The detection method was as previously reported. Purified PRRSV virions were diluted in bicarbonate buffer (100 mM, pH 9.6) and incubated overnight at 4°C in a 96-well plate at 100 ng / well. After discarding the coating solution, the microplate was washed three times with PBST buffer (PBS supplemented with 0.05% Tween20, pH 7.4), blocked with 1% BSA at 37°C for 1 h, washed three times with PBST, and then the diluted serum samples (1:100 or 1:1000) were added to the ELISA plate (100 μL / well). After incubation at 37°C for 1 h, the plates were washed three times with PBST buffer. Then, 100 μL of goat anti-mouse IgG-HRP was added to each well and incubated at 37°C for 1 h. Subsequently, 100 μL of TMB substrate solution was added to each well and incubated at 37°C for 15 min. Finally, 100 μL of 2M sulfuric acid was added to each well to terminate the reaction and the absorbance at 450 nm was measured. In order to analyze the type of antibody, ELISA was performed according to the product instructions (Biodragon, China) using purified PRRSV virions (100 ng / well) as the capture antigen. The concentration of IgG subtypes was evaluated according to the OD450 value, and the relative percentages of IgG1, IgG2a, IgG2b, IgG3, and IgGA were normalized to total IgG.

[0054] The results are as follows Figure 2 As shown in C and 2D, when the dilution ratio of mouse serum was 1:100, compared with the control group LNP and PBS, the PRRSV mRNA-LNPs vaccine group could produce specific binding antibodies, and when the dilution ratio of mouse serum was 1:1000, the specific antibody levels of GP5MN and GP5+M+N groups were significantly higher than those of other PRRSV mRNA-LNPs groups, indicating that GP5MN and GP5+M+N groups had good antibody production capabilities, among which the effect of GP5+M+N mixed group was better than that of recombinant GP5MN group, which shows that the mixing of single structural proteins is more conducive to the display of their respective antigenic epitopes and facilitates the production of antibodies. At the same time, the antibody subtypes of the PRRSV mRNA-LNPs vaccine group were further detected, and the immune type of the vaccine was further analyzed. It can be seen that the antibody subtypes of GP3+4+5, GP5+M, GP345, and GP5M are mainly IgG2b, which tends to be Th-1 type immunity, while the antibody subtypes of GP5+M+N and GP5MN are mainly IgG2b and IgG1, indicating that this form of mRNA vaccine focuses on both humoral immunity and cellular immunity ( Figure 2 F).

[0055] 2.6 Neutralization test

[0056] The separated serum samples were heat-inactivated at 56°C for 30 min and diluted in a 2-fold gradient. The diluted samples were mixed with an equal volume of PRRSV (200 TCID 50 ) were mixed and incubated at 37°C for 1 h, and then the serum virus mixture was added to the Marc-145 monolayer cells in a 96-well plate and incubated for 5 days at 37°C and 5% CO2. Using the Spearman-Karber method, the dilution of each serum sample was calculated to provide a neutralizing antibody titer that can protect 50% of the cells from cytopathic effect (CPE).

[0057] The results are as follows Figure 2 As shown in E, the levels of GP3+4+5 and GP5+M+N neutralizing antibodies have been greater than 1:10 since the second week after vaccination, and can reach a maximum of 1:34 ( Figure 2 E).

[0058] 2.7 Lymphocyte proliferation

[0059] Mice were killed 42 days after immunization, and spleen lymphocytes were isolated under sterile conditions and suspended in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg / mL penicillin, and 100 μg / mL streptomycin (Invitrogen, USA). The cells were counted and diluted to 1×10 6 cells / mL. Add the diluted spleen lymphocytes to a 96-well plate at 50 μL / well and culture in a 37°C, 5% CO2 incubator. Each mouse was set up with a specific stimulation group, a non-specific stimulation group, and a non-stimulation control group, each with 3 replicates. The specific stimulation group was stimulated with purified PRRSV (10 μg / mL), the non-specific stimulation group was added with LPS (10 μg / mL), and the non-stimulation group was replaced with PBS. Each sample was repeated 4 times. Place in a 37°C 5% CO2 incubator and culture for 72 hours. Afterwards, add 10 μL MTS to each well and culture in a 37°C 5% CO2 incubator for 3.5-4 hours. Take out the cells and use an enzyme marker to measure the absorbance value of OD490 to calculate the stimulation index SI.

[0060]

[0061] The results are as follows Figure 3 As shown in A, it can be seen that the lymphocyte proliferation index of the experimental group was significantly higher than that of the PBS and LNP groups (P<0.01), among which the lymphocyte proliferation index of the GP5MN and GP5+M+N groups was higher than that of the GP3+4+5, GP5+M, GP345, and GP5M groups (P<0.05).

[0062] 2.8 T cell typing

[0063] The isolated lymphocytes were stimulated with purified PRRSV particles (10 μg / mL) at 37°C, 5% CO2 for 24 h, and the collected lymphocytes were centrifuged (1000 rpm, 10 min) and resuspended in sterile PBS containing 2% FBS, 100 μg / mL penicillin and 100 μg / mL streptomycin. The cells were stained with the following fluorescent antibodies: FITC Hamster Anti-Mouse CD3e, PE RatAnti-Mouse CD8a, APC RatAnti-Mouse CD4 (BD, USA), at 4°C, protected from light, for 30 min. Centrifuged at 1500 rpm for 5 min; the supernatant was taken and washed twice with PBS containing 2% FBS. The cell microspheres were resuspended in 500 μL PBS containing 2% FBS. The cells were detected by flow cytometry (BD, USA), and at least 10,000 events were recorded for each sample. The analysis was performed using FlowJo software.

[0064] The results are as follows Figure 3 As shown in B, it can be seen that the proportion of CD4+T cells in each experimental group was significantly higher than that in the PBS and LNP groups (P<0.01), except that there was no difference between the GP5M and GP5+M groups, and there were significant differences between GP3+4+5 and GP345, GP5+M+N and GP5MN, indicating that the proportion of CD4+T cells in the mixed group was significantly higher than that in the fusion protein group. Figure 3 C is the result of CD8+T lymphocyte subsets. It can be seen that except for the GP5M and GP5+M groups with the control groups (PBS, LNP), the CD8+T cell proportions of other experimental groups were significantly higher than those of the PBS and LNP groups (P<0.01). Among them, there were significant differences between GP3+4+5 and GP345, and between GP5+M+N and GP5MN. Similar to the results of CD4+T cells, the CD8+T cell proportion of the mixed group was also significantly higher than that of the fusion protein group.

[0065] 2.9 ELISpot assay

[0066] To further detect specific T lymphocyte responses, IFN-γ and IL-2 ELISpot assays were performed. 5 / well) and purified PRRSV particles (10 μg / mL) were added to 96-well plates pre-coated with IFN-γ antibody and IL-2 antibody (DAKEWE, China), and PAM was added as a positive control. Cells cultured without stimulation were used as negative controls. After incubation for 18 h, biotinylated antibodies, streptavidin-HRP and fresh substrate were added to the culture dish. Finally, the plate was rinsed with deionized water to stop the reaction. The data were analyzed using an ELISpot scanner for spot technology (AIDAutoimmun Diagnostika, Germany) and ELISpot analysis software.

[0067] The results are as follows Figure 3 D and Figure 3 As shown in E, the IFN-γ results showed that the IFN-γ secretion level of each experimental group was significantly higher than that of the control group. Except for the GP3+4+5 and GP345 groups, there was no difference between GP5M and GP5+M, GP5+M+N and GP5MN, and the IFN-γ secretion capacity of the mixed group was significantly higher than that of the fusion protein group. The IL-2 results showed that the IL-2 secretion level of each experimental group was significantly higher than that of the control group. Except for the GP3+4+5 and GP345 groups, there was no difference between GP5M and GP5+M, GP5+M+N and GP5MN, and the IL-2 secretion capacity of the mixed group was significantly higher than that of the fusion protein group.

Claims

1. A highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) mRNA vaccine composition, characterized in that: The vaccine composition is selected from any one of the following combinations: (1) an mRNA vaccine composition consisting of PRRSV GP3 mRNA, PRRSV GP4 mRNA and PRRSV GP5 mRNA; or (2) An mRNA vaccine composition consisting of PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA.

2. The mRNA vaccine composition according to claim 1, characterized in that The PRRSV GP3 mRNA, PRRSV GGP4 mRNA, PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA are respectively obtained by transcribing the sequences shown in SEQ ID NO.4-8 to obtain N1-methylpseudouracil-modified mRNA.

3. The mRNA vaccine composition according to claim 1 or 2, characterized in that The PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA are mixed in an equal molar ratio.

4. The mRNA vaccine composition according to claim 1 or 2, characterized in that The PRRSV GP3 mRNA, PRRSV GP4 mRNA and PRRSV GP5 mRNA are mixed in an equal molar ratio.

5. Use of the mRNA vaccine composition according to any one of claims 1 to 4 in the preparation of an mRNA vaccine for preventing highly pathogenic porcine reproductive and respiratory syndrome virus infection.

6. An mRNA vaccine for preventing highly pathogenic porcine reproductive and respiratory syndrome virus infection, characterized in that: The vaccine contains the mRNA vaccine composition described in any one of claims 1-4.

7. The mRNA vaccine according to claim 6, characterized in that The vaccine also contains lipids, which include ionizable lipid ALC-0315, distearoylphosphatidylcholine (DSPC), cholesterol and polymer lipid DMG-PEG2000.

8. The mRNA vaccine according to claim 7, characterized in that The mRNA vaccine is prepared by the following method: (1) dissolving ionizable lipid ALC-0315, distearoylphosphatidylcholine (DSPC), cholesterol, and polylipid DMG-PEG2000 in ethanol to obtain lipids; (2) dissolving the mixture of PRRSV GP3 mRNA, PRRSV GP4 mRNA and PRRSV GP5 mRNA or the mixture of PRRSV GP5 mRNA, PRRSV membrane protein mRNA and PRRSV nucleocapsid protein mRNA in 20 mM citric acid buffer at pH 4.5 to obtain an mRNA solution; (3) preparing lipid nanoparticles (LNPs) by mixing the lipid obtained in step (1) and the mRNA solution obtained in step (2) with the lipid and mRNA in a microfluidic mixer; (4) LNPs were dialyzed in RNase-free pH 7.4 phosphate buffer for 24 h to remove ethanol. Finally, LNPs were filtered through a 0.22 μm filter and stored at 4 °C until use.

9. The mRNA vaccine according to claim 8, characterized in that In step (1), the molar ratio of ionizable lipid ALC-0315, distearoylphosphatidylcholine (DSPC), cholesterol, and polylipid DMG-PEG2000 is 46.3:9.4:42.7:1.6, and the total lipid concentration is 19.1 mM; in step (2), the PRRSV GP3 mRNA, PRRSV GP4 mRNA, and PRRSV GP5 mRNA are mixed in an equal molar ratio, or the PRRSV GP5 mRNA, PRRSV membrane protein mRNA, and PRRSV nucleocapsid protein mRNA are mixed in an equal molar ratio, the amount of mRNA is calculated according to a nitrogen-phosphorus ratio of 1:6, and the mRNA is dissolved in a 20 mM, pH 4.5 citric acid buffer.

10. The mRNA vaccine according to claim 9, characterized in that In step (3), the lipid obtained in step (1) and the mRNA solution obtained in step (2) are mixed in a microfluidic mixer at a volume ratio of 1:3 to prepare lipid nanoparticles (LNPs).

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