Porcine reproductive and respiratory syndrome virus mRNA molecule and application thereof

Abstract

The invention discloses a porcine reproductive and respiratory syndrome virus mRNA (messenger Ribonucleic Acid) molecule and application thereof, and four mRNA vaccines GP35m-LNP, GP45m-LNP, GP345m-LNP and GP2345m-LNP are designed by combining structural protein GP2a, GP3, GP4 and GP5 genes of a PRRSV FJ1402 strain of NADC30-like. Mouse test results show that the GP345m-LNP can simultaneously induce strong humoral and cellular immune response, the GP2345m-LNP is secondary, and the effect is superior to that of an inactivated vaccine (only inducing humoral immunity). After immunization, the PRRSV FJ1402 strain is adopted for counteracting toxic substances, the virus load in blood and lungs of a GP345m-LNP group is obviously reduced, pathological injuries of the lungs are obviously relieved, the immune effect is superior to that of an inactivated vaccine group, and the vaccine has a relatively good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a porcine reproductive and respiratory syndrome virus mRNA molecule and an application thereof. Background Art

[0002] PRRS is one of the most significant diseases facing the global swine industry, causing significant economic losses worldwide. Caused by PRRSV, the disease primarily causes respiratory illness and growth retardation in pigs of all ages, and can lead to reproductive failure in sows. PRRSV can be transmitted through multiple routes, including air, feed, and semen, and increases susceptibility of pigs to a variety of viruses and bacteria. Currently, the most widely used PRRSV vaccines are MLV and inactivated vaccines, while other vaccine types, such as viral vector vaccines, subunit vaccines, gene deletion vaccines, and VLP vaccines, are under investigation. Inactivated vaccines have a good safety profile, but they are less immunogenic, require adjuvants, and induce limited immune responses. MLV vaccines can induce stronger immune responses and provide more effective immune protection, but they are less effective against heterologous strains and carry the risks of viral recombination, mutations in key antigenic sites, and reversion to virulence. Therefore, the development of safer and more effective PRRSV vaccines is urgent. In the development of PRRSV subunit vaccines and viral vector vaccines, the main target proteins are the viral structural proteins, including GP2a, GP3, GP4, GP5, and M proteins that can induce the production of neutralizing antibodies. Potential T cell epitopes in PRRSV are located in NSP2, NSP5, NSP9, NSP10, GP3, GP4, GP5, M, and N proteins.

[0003] Among various vaccine development platforms, the mRNA vaccine platform is renowned for its rapidity, efficiency, safety, and high flexibility. During the COVID-19 pandemic, mRNA vaccines garnered global attention due to their rapid development and robust efficacy, becoming the most widely used SARS-CoV-2 vaccine. Furthermore, mRNA vaccines have shown promise in preventing infections with viruses such as RSV, ZIKV, RV, and VZV. While mRNA vaccines can elicit strong humoral and cellular immune responses, research on their protective effects against viral infections in animals remains limited.

[0004] Porcine reproductive and respiratory syndrome virus (PRRSV) exhibits genetic variation, and existing commercial PRRSV vaccines are suboptimal and ineffective against prevalent NADC30-like strains. Currently, there is no effective vaccine against prevalent NADC30-like strains in my country, and most of the PRRSV isolated and identified in our laboratory in recent years are also of this type. Therefore, the development of a vaccine that can prevent and control NADC30-like strains of PRRSV is crucial. Previous research on subunit, viral vector, and nanoparticle vaccines targeting PRRSV has revealed that vaccines using a single PRRSV structural protein as a target protein have limited immunogenicity and protective efficacy. Therefore, the development of an mRNA vaccine targeting NADC30-like strains is the technical challenge addressed by this present invention. Summary of the Invention

[0005] The present invention aims at a porcine reproductive and respiratory syndrome virus mRNA molecule and its application.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention claims protection for a porcine reproductive and respiratory syndrome virus mRNA molecule, which is mRNA molecule GP345m or mRNA molecule GP2345m;

[0008] The mRNA molecule GP345m encodes the fusion protein GP345 of GP3, GP4 and GP5 whose amino acid sequence is shown in SEQ ID NO.3;

[0009] The mRNA molecule GP2345m encodes the fusion protein GP2345 of GP2a, GP3, GP4 and GP5 whose amino acid sequence is shown in SEQ ID NO.4.

[0010] Furthermore, the nucleotide sequences of the mRNA molecule GP345m and the mRNA molecule GP2345m are as described above.

[0011] In a second aspect, the present invention seeks to protect a liposome nanoparticle, which is loaded with the aforementioned porcine reproductive and respiratory syndrome virus mRNA molecule.

[0012] In a third aspect, the present invention seeks to protect the use of the above-mentioned mRNA molecules in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccines.

[0013] In a fourth aspect, the present invention seeks to protect the use of the above-mentioned liposome nanoparticles in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccine.

[0014] In a fifth aspect, the present invention seeks protection for a biological material related to the porcine reproductive and respiratory syndrome virus mRNA molecule, wherein the biological material is selected from any one of the following:

[0015] (A1) a DNA molecule encoding the aforementioned mRNA molecule;

[0016] (A2) an expression cassette containing the DNA molecule described in (A1);

[0017] (A3) a recombinant vector containing the DNA molecule described in (A1) or a recombinant vector containing the expression cassette described in (A2);

[0018] (A4) A recombinant microorganism containing the DNA molecule described in (A1), or a recombinant microorganism containing the expression cassette described in (A2), or a recombinant microorganism containing the recombinant vector described in (A3).

[0019] Furthermore, the nucleotide sequence of the DNA molecule encoding the mRNA molecule GP345m is shown in SEQ ID NO.11; the nucleotide sequence of the DNA molecule encoding the mRNA molecule GP2345m is shown in SEQ ID NO.12.

[0020] In a sixth aspect, the present invention seeks to protect the use of the above-mentioned biological materials in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccine.

[0021] In a seventh aspect, the present invention claims protection for a porcine reproductive and respiratory syndrome virus mRNA vaccine, wherein the mRNA vaccine comprises an mRNA molecule GP345m or an mRNA molecule GP2345m;

[0022] The mRNA molecule GP345m encodes the fusion protein GP345 of GP3, GP4 and GP5 whose amino acid sequence is shown in SEQ ID NO.3;

[0023] The mRNA molecule GP2345m encodes the fusion protein GP2345 of GP2a, GP3, GP4 and GP5 whose amino acid sequence is shown in SEQ ID NO.4.

[0024] Furthermore, the nucleotide sequence of the mRNA molecule GP345m is shown as SEQ ID NO.7; the nucleotide sequence of the mRNA molecule GP2345m is shown as SEQ ID NO.8.

[0025] Furthermore, the porcine reproductive and respiratory syndrome virus mRNA vaccine is an mRNA-LNP complex formed by encapsulating the mRNA molecule GP345m or the mRNA molecule GP2345m in liposome nanoparticles.

[0026] The room temperature of the present invention is 25±5° C., but is not limited thereto.

[0027] Beneficial effects of the present invention:

[0028] The present invention combines the NADC30-like PRRSV FJ1402 strain structural protein GP2a, GP3, GP4, and GP5 genes to design four mRNA vaccines, namely (1) GP35m-LNP expressing a fusion protein of GP3 and GP5; (2) GP45m-LNP expressing a fusion protein of GP4 and GP5; (3) GP345m-LNP expressing a fusion protein of GP3, GP4, and GP5; and (4) GP2345m-LNP expressing a fusion protein of GP2a, GP3, GP4, and GP5. Mouse experiments showed that GP35m-LNP and GP45m-LNP failed to induce an effective immune response, while GP345m-LNP induced a strong humoral and cellular immune response, followed by GP2345m-LNP. After two immunizations with GP345m-LNP and an inactivated PRRSV vaccine, GP345m-LNP induced only a significant cellular immune response, while the inactivated PRRSV vaccine induced only humoral immunity. Following immunization, challenge with the PRRSV FJ1402 strain significantly reduced viral loads in the blood and lungs, and significantly alleviated lung pathological lesions in the GP345m-LNP group. This demonstrated superior immune efficacy compared to the inactivated vaccine group, suggesting promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the molecular design of four mRNA vaccines.

[0030] Figure 2 This is the mouse immunization procedure and sample collection time points for mRNA vaccine.

[0031] Figure 3 Flow cytometry gating strategy for mouse spleen T cell subsets.

[0032] Figure 4 This is the piglet immunization-challenge procedure and sample collection time points for mRNA vaccine.

[0033] Figure 5 Flow cytometric gating strategy for T cell subsets in piglet PBMCs.

[0034] Figure 6 Predicted the three-dimensional structures of four fusion proteins;

[0035] Among them, A is GP35m protein; B is GP45m protein; C is GP345m protein; D is GP2345m protein; purple: GP2a protein; orange: GP3 protein; pink: GP4 protein; blue: GP5 protein.

[0036] Figure 7 For the linearization of four recombinant plasmids.

[0037] Figure 8 Denatured mRNA was analyzed by formaldehyde agarose gel electrophoresis.

[0038] Figure 9 Characterization of four types of mRNA-LNPs; A is the particle size, PDI and Zeta potential determination of GP35m-LNP; B is the particle size, PDI and Zeta potential determination of GP45m-LNP; C is the particle size, PDI and Zeta potential determination of GP345m-LNP; D is the particle size, PDI and Zeta potential determination of GP2345m-LNP; E. Cryo-electron microscopy photos of four types of mRNA-LNPs.

[0039] Figure 10 The protein expression of four mRNA-LNPs was identified by Western blotting; A shows the expression of four mRNA-LNPs (GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP) in PK-15 cell lysates identified by Western blotting; B shows the expression of four mRNA-LNPs in PAMs cell lysates identified by Western blotting; C shows the expression of four mRNA-LNPs in PK-15 cell culture supernatant identified by Western blotting; D shows the expression of four mRNA-LNPs in PAMs cell culture supernatant identified by Western blotting.

[0040] Figure 11 IFA was used to identify the protein expression of four mRNA-LNPs; IFA was used to identify the expression of GP35m-LNP, GP45m-LNP, GP345m-LNP and GP2345m-LNP in PK-15 cells.

[0041] Figure 12Identification of recombinant proteins; A is the identification of GP2a protein expressed in E. coli prokaryotes by SDS-PAGE (left lane) and Western blotting (right lane); B is the identification of GP3 protein expressed in E. coli prokaryotes by SDS-PAGE (left lane) and Western blotting (right lane); C is the identification of GP4 protein expressed in E. coli prokaryotes by SDS-PAGE (left lane) and Western blotting (right lane); D is the identification of GP5 protein expressed in E. coli prokaryotes by SDS-PAGE (left lane) and Western blotting (right lane); M: protein molecular weight standard, 1: purified GP2a protein, 2: purified GP3 protein, 3: purified GP4 protein, 4: purified GP5 protein.

[0042] Figure 13 are the ELISA antibody levels induced by the four mRNA-LNPs in mice; among them, A is the ELISA antibody level against GP2a produced after the first and booster immunizations of the four mRNA-LNPs; B is the ELISA antibody level against GP3 ​​produced after the first and booster immunizations of the four mRNA-LNPs; C is the ELISA antibody level against GP4 produced after the first and booster immunizations of the four mRNA-LNPs; D is the ELISA antibody level against GP5 produced after the first and booster immunizations of the four mRNA-LNPs.

[0043] Figure 14 The neutralizing antibody levels induced by the four mRNA-LNPs in mice.

[0044] Figure 15 The ICS test detects the specific T cell immune response of mice after immunization with four mRNA vaccines; A is the CD4 T cell immune response that induces mice to produce IL-2 after immunization with four mRNA vaccines by ICS experiment. + B is the ratio of CD4 T cells to produce IL-4 in mice after immunization with four mRNA vaccines determined by ICS experiment. + C, D are the proportions of CD4 T cells that induced IFN-γ production in mice after immunization with four mRNA vaccines determined by ICS experiments. + T cells and CD8 + The proportion of T cells.

[0045] Figure 16 Antigen-specific lymphocyte proliferation responses induced by four mRNA vaccines in mice.

[0046] Figure 17is the ELISA antibody level induced by GP345m-LNP in piglets; wherein, A is the ELISA antibody titer against GP3 ​​produced after immunization with GP345m-LNP and a commercial inactivated vaccine; B is the ELISA antibody titer against GP4 produced after immunization with GP345m-LNP and a commercial inactivated vaccine; C is the ELISA antibody titer against GP5 produced after immunization with GP345m-LNP and a commercial inactivated vaccine.

[0047] Figure 18 is the neutralizing antibody level induced by GP345m-LNP in piglets.

[0048] Figure 19 The ICS test was used to detect the specific T cell immune response induced by GP345m-LNP in piglets; A is the CD4 T cells that induced IFN-γ in piglets after immunization with GP345m-LNP by ICS test. + B is the proportion of CD8 T cells that induced IFN-γ in piglets after GP345m-LNP immunization determined by ICS experiment. + The proportion of T cells; C is the γδ of IFN-γ produced by piglets after GP345m-LNP immunization determined by ICS experiment + The proportion of T cells.

[0049] Figure 20 This is the antigen-specific lymphocyte proliferation response induced by GP345m-LNP in piglets.

[0050] Figure 21 ELISPOT was used to detect the antigen-specific lymphocyte response induced by GP345m-LNP.

[0051] Figure 22 These are the clinical symptoms of piglets after infection; A is the daily rectal temperature; B is the average daily weight gain.

[0052] Figure 23 It is the viral load of blood and lungs of piglets after virus infection; A is the viral load of blood; B is the viral load of lungs.

[0053] Figure 24 Pathological changes in the piglet lungs; A is the H&E staining of the lung pathological section of the PBS-LNP control group; B is the H&E staining of the lung pathological section of the inactivated vaccine immunization group; C is the H&E staining of the lung pathological section of the GP345m-LNP immunization group.

[0054] Figure 25 To evaluate lung tissue pathological damage.

[0055] Figure 26The ICS test was used to detect the specific T cell immune response of piglets 21 days after the virus challenge; A is the CD4 T cells that produce IFN-γ in piglets after the virus challenge, which was determined by the ICS experiment. + B is the proportion of CD8 T cells that produce IFN-γ in piglets after challenge with ICS experiment. + The proportion of T cells; C is the γδ of IFN-γ produced by piglets after challenge by ICS experiment + The proportion of T cells. DETAILED DESCRIPTION

[0056] The present invention will be further described below with reference to the following examples. The following description is merely a preferred embodiment of the present invention and does not limit the present invention in any other form. Any person skilled in the art may utilize the above disclosed technical content to make equivalent embodiments with equivalent variations. Any modification or equivalent variation of the following examples made in accordance with the technical essence of the present invention without departing from the content of the present invention shall fall within the scope of protection of the present invention.

[0057] Example 1

[0058] In this study, four mRNA vaccines expressing PRRSV antigen fusion proteins were designed: (1) GP35m-LNP expressing GP3 and GP5 fusion proteins; (2) GP45m-LNP expressing GP4 and GP5 fusion proteins; (3) GP345m-LNP expressing GP3, GP4, and GP5 fusion proteins; and (4) GP2345m-LNP expressing GP2a, GP3, GP4, and GP5 fusion proteins. After immunization of mice with the four mRNA vaccines, GP345m-LNP and GP2345m-LNP effectively induced humoral and cellular immune responses, with GP345m-LNP induced the strongest immune response. Immunization of piglets with GP345m-LNP effectively induced both humoral and cellular immune responses. In the challenge protection test, compared with the commercial inactivated vaccine, GP345m-LNP immunization can effectively reduce the viral load in the blood and lungs of piglets and alleviate lung pathological damage, indicating that GP345m-LNP can be used as a candidate vaccine to prevent and control PRRS.

[0059] 1 Materials and Methods

[0060] 1.1 Experimental Materials

[0061] Cells, viruses, and experimental animals: PAMs, Marc-145, and PK-15 cells were prepared and maintained in our laboratory. PRRSV FJ1402 F7 virus fluid and PRRSV-positive porcine serum were prepared, identified, and maintained in our laboratory according to conventional methods. GP3 (4H9) and GP4 (6E9) monoclonal antibodies were prepared and maintained in our laboratory according to conventional methods. GP5 monoclonal antibody was a gift from the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Four-week-old healthy piglets (PRRSV nucleic acid and antibody negative) were provided by a pig farm in Nantong, Jiangsu Province. 6- to 8-week-old BALB / c female mice were purchased from the Experimental Animal Center of Yangzhou University and housed under appropriate temperature and humidity conditions.

[0062] Reagents: Plasmid extraction kit (DP116) was purchased from Tiangen Company; DNA recovery kit (CW2302M) was purchased from Kangwei Century; RNA extraction kit (R6834) was purchased from OMEGA; CAG Trimer (DD4118-PC), T7 High Yield RNA Transcription Kit (DD4202), VAHTS RNA Clean Beads (N412), VAHTS DNA Clean Beads (N411), HiScript qRT SuperMix (R223-01), real-time fluorescence quantitative PCR reagent AceQ® qPCR SYBR® Master Mix (Q111-02), CCK-8 (A311) were all purchased from Norvegian; restriction endonucleases EcoRI (FD0274) and AflII (FD0834), RNA marker RiboRuler High Range RNA Ladder (SM1821), RNA quantification kit Quant-iT RiboGreen RNA Kit (R11490) was purchased from Thermo Fisher Scientific; cationic lipid ALC-0315 and PEGylated lipid ALC-0159 were purchased from Sinopharm; cholesterol and distearoylphosphatidylcholine (DSPC) were purchased from Avitor; fetal bovine serum (FBS), DMEM, and RPMI 1640 were purchased from Gibco; porcine peripheral blood lymphocyte isolation kit (LTS1110) was purchased from Tianjin Haoyang; porcine IFN-γ ELISpot kit (3130-2A) was purchased from MABTECH; concanavalin A (ConA) was purchased from Sigma; TMB colorimetric solution was purchased from Beyotime; fixation / permeabilization kit, GolgiPlug, anti-CD16 / 32, PerCP-Cy5.5, rat anti-mouse CD8a (53-6.7), PerCP-Cy5.5 Mouse Anti-Pig CD3ε (BB23-8E6-8C8), PE-Cy7 Mouse Anti-Pig CD4a (74-12-4), FITC Mouse Anti-Pig CD8a (76-2-11), PE Rat Anti-Pig γδT Lymphocytes (MAC320) and Alexa Fluor 647 Mouse Anti-Pig IFN-γ (P2G10) was purchased from BDBiosciences; Zombie NIR™ Fixable Viability Kit (423105), FITC anti-mouse CD3 (100203), PE / Cyanine7 anti-mouse CD4 (100421), PE anti-mouse / human CD44 (103007), APC anti-mouse CD62L (104411), PE anti-mouse IL-4 (504103), APC anti-mouse IFN-γ (505810), and APC / Fire 750 anti-mouse IL-2 (503841) were purchased from BioLegend; HRP-conjugated goat anti-mouse IgG (H+L) and HRP-conjugated goat anti-swine IgG (H+L) were purchased from Jackson Immunoresearch; all other conventional chemical reagents were of analytical grade.

[0063] 1.2 mRNA preparation

[0064] 1.2.1 Design of mRNA vaccines

[0065] The PRRSV envelope glycoproteins GP2a, GP3, GP4, and GP5 were selected as target proteins, and mRNA encoding fusion proteins was constructed. Using GP2a, GP3, GP4, and GP5 of the PRRSV NADC30-like strain FJ1402 (GenBank number: KX169191) isolated and identified in our laboratory as target proteins, four mRNA vaccines were designed: (1) GP35m-LNP expressing a fusion protein of GP3 and GP5; (2) GP45m-LNP expressing a fusion protein of GP4 and GP5; (3) GP345m-LNP expressing a fusion protein of GP3, GP4, and GP5; and (4) GP2345m-LNP expressing a fusion protein of GP2a, GP3, GP4, and GP5. To ensure the effective expression and secretion of the fusion protein, the original signal peptide and transmembrane region in the protein were deleted, the target protein was connected to the fusion protein using a flexible linker (GGGGS) 3 linker, and the tissue plasminogen activator (tPA) signal peptide was added to the N-terminus of the fusion protein to enable protein secretion (e.g. Figure 1 shown).

[0066] The amino acid sequence of the GP3 and GP5 fusion protein GP35 is shown in SEQ ID NO.1, the amino acid sequence of the GP4 and GP5 fusion protein GP45 is shown in SEQ ID NO.2, the amino acid sequence of the GP3, GP4 and GP5 fusion protein GP345 is shown in SEQ ID NO.3, and the amino acid sequence of the GP2a, GP3, GP4 and GP5 fusion protein GP2345 is shown in SEQ ID NO.4.

[0067] The DNA sequence encoding the above-mentioned fusion protein was codon-optimized and 5′UTR, 3′UTR and polyA sequences were added. The gene was synthesized by Nanjing GenScript Co., Ltd. and cloned into pUC57 and pCDNA3.1 plasmids to synthesize mRNA vaccine template plasmids pUC57-GP35m, pUC57-GP45m, pCDNA3.1-GP345m and pUC57-GP2345m.

[0068] The DNA sequence encoding the fusion protein GP35 is shown in SEQ ID NO.9, the DNA sequence encoding the fusion protein GP45 is shown in SEQ ID NO.10, the DNA sequence encoding the fusion protein GP345 is shown in SEQ ID NO.11, and the DNA sequence encoding the fusion protein GP2345 is shown in SEQ ID NO.12.

[0069] 1.2.2 Transformation of mRNA vaccine template plasmid

[0070] The synthesized plasmids pUC57-GP35m, pUC57-GP45m, pCDNA3.1-GP345m and pUC57-GP2345m were transformed into TOP10 competent Escherichia coli.

[0071] 1.2.3 Extraction of mRNA vaccine template plasmid

[0072] The TOP10 single colonies on the plate were picked, and after sequencing, they were amplified and cultured, and a plasmid extraction kit was used to extract a large amount of plasmids.

[0073] (1) Collect bacteria by centrifugation at 10,000 rpm for 3 min at room temperature;

[0074] (2) Add 10 mL of solution P1 to the bacterial pellet and resuspend the cell pellet;

[0075] (3) Add 10 mL of solution P2 to the centrifuge tube, invert it upside down 6-8 times to fully lyse the bacteria, and let it stand at room temperature for 5 minutes;

[0076] (4) Add 10 mL of solution P4 to the centrifuge tube and gently invert 6-8 times until a white dispersed precipitate appears. Let it stand at room temperature for 10 min. Centrifuge at 10,000 rpm for 10 min. Pour the supernatant into filter CS1 and collect the filtrate in a 50 mL tube.

[0077] (5) Add 0.35 times the volume of isopropanol and 1 / 2 times the volume of isopropanol to the filtrate and mix thoroughly by inverting.

[0078] (6) Centrifuge at 10,000 rpm for 30 min at 4°C and discard the supernatant;

[0079] (7) Add 6 mL of 70% (v / v) ethanol to rinse the precipitate, centrifuge at 10,000 rpm for 10 min at 4°C, and discard the supernatant;

[0080] (8) Repeat step (7);

[0081] (9) Leave the centrifuge tube open for 15 min to evaporate the ethanol, then add 1 mL of eluent to dissolve the precipitate;

[0082] (10) Determine the plasmid concentration.

[0083] 1.2.4 Linearization of mRNA vaccine template plasmid

[0084] Using the linearized plasmid as a template, in vitro transcription was performed to produce mRNA. The resulting recombinant plasmids pUC57-GP35m, pUC57-GP45m, and pUC57-GP2345m were digested with EcoRI, and the recombinant plasmid pCDNA3.1-GP345m was linearized with AflII. The steps were as follows: 1 μg of plasmid, 1 μL of restriction enzyme EcoRI or AflII, 2 μL of 10× buffer, and ddH2O to 20 μL. Enzyme digestion was incubated at 37°C for 30 min.

[0085] 1.2.5 Purification of linearized plasmid

[0086] Use DNA purification magnetic beads to recover the linearized plasmid as follows:

[0087] (1) Take out the magnetic bead solution from 2-8°C 30 min in advance and equilibrate it to room temperature;

[0088] (2) Vortex the magnetic beads and add 0.5 times the volume of magnetic bead solution to the linearized plasmid solution and pipette to mix thoroughly;

[0089] (3) Let it stand at room temperature for 10 min to allow the DNA to bind to the magnetic beads;

[0090] (4) Place the sample on a magnetic rack and remove the supernatant after the solution is clarified;

[0091] (5) Keep the sample on the magnetic stand, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and remove the supernatant;

[0092] (6) Repeat step (5);

[0093] (7) Open the lid and dry at room temperature for 5-10 minutes;

[0094] (8) Remove the sample from the magnetic rack, add an appropriate amount of enzyme-free water, pipette to mix, and let it stand for 2 minutes;

[0095] (9) After standing on the magnetic rack for 5 min, aspirate the supernatant into a new centrifuge tube to obtain the purified linearized plasmid and measure the concentration.

[0096] 1.2.6 mRNA in vitro transcription

[0097] In vitro transcription of mRNA was performed using the T7 High Yield RNA Transcription Kit and CAG Trimer as follows:

[0098] (1) Prepare the reaction system:

[0099] 2 μL T7 RNA polymerase premix

[0100] 10 × transcription buffer 2 μL

[0101] 2 μL N1-methylpseudouridine triphosphate (N 1 -Me-Pseudo UTP) solution

[0102] ATP solution 2 μL

[0103] CTP solution 2 μL

[0104] 2 μL GTP solution

[0105] CAG solution 1 μL

[0106] 1 μg of linearized plasmid

[0107] Add enzyme-free water to 20 μL

[0108] (2) Mix thoroughly and incubate at 37°C for 2 h;

[0109] (3) Add 1 μL DNase I and incubate at 37°C for 15 min.

[0110] 1.2.7 mRNA purification

[0111] To purify mRNA using RNA purification magnetic beads, follow these steps:

[0112] (1) Remove the RNA purification magnetic beads from 2-8°C 30 min in advance, return to room temperature, and vortex to mix;

[0113] (2) Add 1.8 times the volume of RNA purification magnetic beads to the mRNA solution and mix thoroughly;

[0114] (3) Let stand at room temperature for 5 minutes;

[0115] (4) Place the sample on a magnetic rack for 5 minutes and remove the supernatant;

[0116] (5) Keep the sample on the magnetic stand, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, let it stand for 30 seconds, and remove the supernatant;

[0117] (6) Repeat step (5);

[0118] (7) Keep the sample in the magnetic rack and dry it for 5 minutes with the lid open;

[0119] (8) Remove the sample from the magnetic rack, add an appropriate volume of enzyme-free water, mix thoroughly, and let it stand for 5 minutes;

[0120] (9) Place the sample on a magnetic rack for 5 minutes, transfer the supernatant to a new nuclease-free centrifuge tube to obtain purified mRNA, and measure the concentration.

[0121] 1.2.8 mRNA formaldehyde denaturation agarose gel electrophoresis

[0122] All instruments were treated with RNase removal reagent. Add 0.8 g of agarose powder to 72 mL of enzyme-free water and allow to melt. Then, add 10 mL of 10× MOPS buffer. When the agarose solution has cooled to 60°C, add 18 mL of formaldehyde and mix thoroughly to prepare an agarose gel. Add two volumes of 2× RNA loading dye to a 1 μg mRNA solution and 2 μL of RNA marker. Heat at 70°C for 10 minutes and place on ice. Perform electrophoresis at 130 V using 1× MOPS buffer and photograph the gel using a gel imaging system.

[0123] 1.3 Preparation and validation of mRNA-LNPs

[0124] 1.3.1 Preparation of mRNA-LNPs

[0125] The steps for delivering mRNA using LNP encapsulation are as follows:

[0126] (1) Dilute mRNA to 127 ng / μL using 50 mM pH 4.0 citrate buffer;

[0127] (2) ALC-0315, ALC-0159, DSPC, and cholesterol were dissolved in ethanol at a molar ratio of 46.3:1.6:9.4:42.7;

[0128] (3) The aqueous phase and lipid phase were mixed at a volume ratio of 3:1 using a microfluidic device to form mRNA-LNP;

[0129] (4) Add PBS to dilute the ethanol in the mRNA-LNP and centrifuge using a 100 kDa ultrafiltration tube at 2000 g / min at 4°C to reduce the ethanol concentration to below 1%;

[0130] (5) Filter the mRNA-LNP using a 0.22 μm filter and store at 4°C.

[0131] 1.3.2 Characterization of mRNA-LNPs

[0132] The particle size, polydispersity index (PDI), and zeta potential of the mRNA-LNPs were measured using a nanometer size analyzer, and the encapsulation efficiency and drug loading were determined using the Quant-iT RiboGreen RNA Kit. 3.0 µL of mRNA-LNPs were dropwise added to the treated grid and vitrified in liquid ethane cooled by liquid nitrogen at 4°C and 100% humidity using a Vitrobot Mark IV (Thermo). The resulting particles were then observed and photographed using a 200 kV Glacios transmission electron microscope (Thermo).

[0133] 1.3.3 mRNA-LNP transfection

[0134] PK-15 cells were transfected with mRNA-LNP to identify protein expression as follows:

[0135] (1) PK-15 cells were plated in a 6-well plate, and the density was about 80% after 8 h;

[0136] (2) Before transfection, discard the 10% (v / v) FBS-DMED nutrient solution and add 2% (v / v) FBS-DMED nutrient solution;

[0137] (3) Add 3 μg of mRNA-LNP to each well and gently shake the cell plate to mix;

[0138] (4) Culture the cells in a 37°C incubator for 24 h, and collect the cell nutrient solution supernatant and whole cell lysate.

[0139] (5) Use mRNA-LNP to transfect PK-15 cells to identify protein expression. The steps are as follows:

[0140] (6) PAMs cells were plated in 6-well plates, and the density was about 80% after 8 h;

[0141] (7) Replace the medium with fresh 10% (v / v) FBS-RPMI 1640 nutrient solution before transfection;

[0142] (8) Add 3 μg of mRNA-LNP to each well and gently shake the cell plate to mix;

[0143] (9) Culture the cells in a 37°C incubator for 24 h, and collect the cell nutrient solution supernatant and whole cell lysate.

[0144] 1.3.4 Western blotting detection of mRNA-LNP expression

[0145] (1) Mix the whole cell lysate and concentrated cell nutrient solution supernatant with 5× Loading Buffer;

[0146] (2) Cook the sample at 100°C for 10 min;

[0147] (3) Electrophoresis on 10% SDS-PAGE;

[0148] (4) Transfer the separation gel to a nitrocellulose membrane (NC membrane) using a semi-dry transfer apparatus (23 V, 45 min);

[0149] (5) Place the NC membrane in 5% (w / v, g / 100 ml) skim milk and block it on a shaker at room temperature for 2 h;

[0150] (6) Use PRRSV GP3, GP4, and GP5 monoclonal antibodies diluted 1:1000 in PBST containing 3% (w / v, g / 100 ml) BSA as primary antibodies and incubate overnight at 4°C;

[0151] (7) Wash with PBST three times, 10 min each time;

[0152] (8) Use HRP-Goat Anti-Mouse IgG (H+L) diluted 1:5000 in PBST containing 5% skim milk as the secondary antibody and incubate on a shaker at room temperature for 45 min;

[0153] (9) Wash with PBST three times, 10 min each time;

[0154] (10) Apply the color developing solution evenly to the surface of the NC membrane and take a photo using a chemical exposure device.

[0155] 1.3.5 IFA identification of mRNA-LNP expression

[0156] (1) PK-15 cells were plated onto a 12-well plate. After 8 hours, 2 μg of mRNA-LNP was added to each well and gently shaken to mix. A negative control well was also set up.

[0157] (2) Fixation: 24 h after transfection, add 1 mL PBS to wash twice, add 400 μL paraformaldehyde to each well, and fix at 37°C for 15 min.

[0158] (3) Wash three times with 1 mL PBS;

[0159] (4) Permeabilization: Add 400 μL of 0.1% (v / v) Triton-100 solution diluted in PBS to each well and permeabilize the membrane for 10 min. Wash three times with 1 mL of PBS.

[0160] (5) Blocking: Add 400 μL of 3% BSA solution to each well, block at 37 °C for 1 h, and wash twice with 1 mL of PBS;

[0161] (6) Primary antibody binding: add 400 μL of 1:400 diluted PRRSV GP3, GP4, and GP5 monoclonal antibodies respectively, incubate at 37°C for 2 h, and wash three times with 1 mL PBS;

[0162] (7) Secondary antibody binding: Add 400 μL of 1:400 diluted HRP-Goat Anti-Mouse IgG (H+L) conjugated with FITC to each well under light-protected conditions, incubate at 37°C for 50 min, and wash three times with 1 mL of PBS;

[0163] (8) Staining cell nuclei: Add 400 μL DAPI to each well, incubate in the dark for 5 min, and wash three times with 1 mL PBS;

[0164] (9) Observe fluorescence.

[0165] 1.4 Immunological evaluation of mRNA-LNP in mice

[0166] 1.4.1 Mouse Experimental Protocol

[0167] To evaluate the immunogenicity of the mRNA vaccine, 6-8-week-old female BALB / c mice were immunized. The vaccine was diluted in PBS to a final concentration of 150 ng / μL before use. The mice were divided into five groups of five mice each, each receiving four vaccines: GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP. Each group received an intramuscular injection of 0.1 mL (15 μg) of GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP, respectively. A PBS-LNP control group (PBS instead of mRNA) was used. Three-week booster immunizations were performed with the same vaccine dose. Blood and / or spleen samples were collected 0, 21, and 42 days after the initial immunization. Serum was separated from the blood, inactivated at 56°C for 30 minutes, and stored at -20°C until use. Single cell suspensions were prepared from spleen for intracellular cytokine staining (ICS) and lymphocyte proliferation assays (e.g. Figure 2 The animal experiment protocol was approved by the Animal Care and Ethics Committee of Nanjing Agricultural University and was performed in accordance with the Guiding Principles for Biomedical Research Involving Animals (PT2020023).

[0168] 1.4.2 Protein expression and purification

[0169] The predicted transmembrane regions of PRRSV GP2a, GP3, GP4, and GP5 were deleted. The gene sequences were codon-optimized in E. coli and sent to Nanjing GenScript for gene synthesis and cloning into the pET-28a plasmid. The protein expression and purification methods used in our laboratory were referenced and the main steps are as follows (Zhang Lujie. Development of monoclonal antibodies against African swine fever virus p72 and p30 proteins and establishment of a blocking ELISA antibody detection method [D]; Nanjing Agricultural University, 2021):

[0170] (1) Transform the recombinant plasmid into BL21 competent cells, plate and pick single colonies for amplification and culture;

[0171] (2) Take 30 μL of bacterial solution and add it to 3 mL of LB liquid medium containing kanamycin resistance. Incubate at 37°C with shaking for about 2 h. When the OD value is in the range of 0.4-0.6, add 3 μL of 1 M IPTG and induce at 16°C for 20 h.

[0172] (3) Collect the induced bacterial solution and disrupt the bacteria by ultrasonication;

[0173] (4) Take the whole bacteria, supernatant and precipitate after ultrasonic lysis, resuspend them in 400 μL PBS, prepare protein samples, perform SDS-PAGE electrophoresis and Coomassie brilliant blue staining to identify the expression and form of the protein;

[0174] (5) Purify the protein by affinity chromatography using a nickel column-His tag;

[0175] (6) The precipitate after the 200 mL bacterial suspension was resuspended in 15 mL lysis buffer (containing 8 M urea) and loaded into a protein purification column for protein purification;

[0176] (7) The collected protein solution was subjected to SDS-PAGE and Western blotting to determine the purification effect.

[0177] 1.4.3 ELISA

[0178] (1) Collect blood from the submandibular vein of mice, incubate at 37°C for 30 minutes, centrifuge at 5,000g for 5 minutes at 4°C, and collect the supernatant. Dilute the supernatant twice with PBST and use it as the primary antibody to detect the antibody titer of mice by indirect ELISA method. The steps are as follows:

[0179] (2) Dilute the purified protein to 1 μg / mL using antigen coating solution, add 100 μL to each well, and incubate at 37°C for 1.5 h;

[0180] (3) Add 200 μL PBST to each well and wash three times;

[0181] (4) Add 200 μL of 5% skim milk (dissolved in PBST) to each well and block overnight at 4°C;

[0182] (5) Add 200 μL PBST to each well and wash three times;

[0183] (6) Add 100 μL of serum diluted two-fold to each well and incubate at 37°C for 1 h;

[0184] (7) Add 200 μL PBST to each well and wash three times;

[0185] (8) Add 100 μL of HRP-Goat Anti-Mouse IgG (H+L) diluted 1:5,000 in PBST to each well and incubate at 37°C for 45 min;

[0186] (9) Add 200 μL PBST to each well and wash three times;

[0187] (10) Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 10 min;

[0188] (11) Add 50 μL of stop solution to each well and read the OD450nm value. When the OD value of the highest dilution is 2.1 times higher than that of the negative control at the same dilution, it is taken as the endpoint dilution titer of the serum.

[0189] 1.4.4 Neutralization test

[0190] PRRSV FJ1402 F7 virus solution was diluted to 200 TCID using 4% (v / v) FBS-DMEM. 50 The inactivated serum was diluted twice with DMEM. The diluted virus solution was added to the diluted serum at a volume ratio of 1:1, mixed thoroughly, and incubated in a 37°C incubator for 1 hour. The nutrient solution in the 96-well plate was discarded, and the virus-serum mixture was inoculated into Marc-145 cells in the 96-well plate. At the same time, positive serum control, negative serum control, virus control and normal cell control were set up, and 4 parallel wells were set up for each dilution. The cell plate was cultured in a 37°C incubator for 4 days, and the endpoint titer was determined by IFA, which was defined as the ability to neutralize 100 TCID in the well. 50 The reciprocal of the highest serum dilution that produces 50% of PRRSV.

[0191] 1.4.5 ICS

[0192] Flow cytometry was used to measure the antigen-specific T cell immune response of mice after mRNA vaccine immunization. The steps are as follows:

[0193] (1) Under sterile conditions, remove the mouse spleen, grind it into a single cell suspension, and lyse the red blood cells using red blood cell lysis buffer;

[0194] (2) Resuspend the spleen cells in 10% (v / v) FBS-RPMI 1640 nutrient solution and count them;

[0195] (3) Add 500 μL of 1×10 7 cells / mL spleen cell suspension, added with a final concentration of 10 μg / mL purified PRRSV FJ1402, and the positive control group added with a final concentration of 10 μg / mL ConA;

[0196] (4) After incubation at 37°C for 12 h, the protein transport inhibitor GolgiPlug was added to each well and incubated for another 5 h.

[0197] (5) Collect the cells, add 1 mL of 0.9% (w / v, g / 100 ml) BSA-PBS to wash the cells, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0198] (6) Add anti-CD16 / 32 for blocking and incubate at 4°C for 10 min;

[0199] (7) Add extracellular staining antibodies PerCP-Cy5.5 Rat Anti-Mouse CD8a, FITC anti-mouseCD3, and PE / Cyanine7 anti-mouse CD4, incubate at 4°C in the dark for 30 min, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0200] (8) Wash the cells twice with 1 mL of 0.9% BSA-PBS, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant.

[0201] (9) Add cell fixation and permeabilization solution, incubate at 4°C in the dark for 20 min, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0202] (10) Wash the cells twice with 0.5 mL of 1× Perm / Wash™ Buffer, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant.

[0203] (11) Add intracellular staining antibodies PE anti-mouse IL-4, APC / Fire™ 750 anti-mouse IL-2, and APC anti-mouse IFN-γ, incubate at 4°C in the dark for 30 min, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0204] (12) Wash the cells twice with 1 mL of 0.9% BSA-PBS, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant.

[0205] (13) Add 300 μL of 0.9% BSA-PBS to resuspend the cells and test on the microscope;

[0206] (14) ICS gating strategies such as Figure 3 shown.

[0207] 1.4.6 Lymphocyte proliferation assay

[0208] (1) Add 100 μL of 1×10 6 cells / mL spleen cell suspension, added with a final concentration of 10 μg / mL purified PRRSV FJ1402, and the positive control group added with a final concentration of 10 μg / mL ConA;

[0209] (2) After 72 h of incubation at 37°C, CCK-8 solution was added and incubated for another 4 h.

[0210] (3) Measure the absorbance (OD value) at 450 nm using a microplate reader;

[0211] (4) Stimulation index = (OD450 of experimental group - OD450 of blank control) / (OD450 of negative control group - OD450 of blank control)

[0212] 1.5 Immunological evaluation of mRNA-LNP in piglets

[0213] 1.5.1 Piglet Experimental Protocol

[0214] Before use, the vaccine was diluted with PBS to a final concentration of 100 ng / μL. Fifteen healthy piglets aged 28-30 days (negative for Classical Swine Fever, African Swine Fever, PRRSV, and PCV2) were randomly divided into three groups of five. Group 1 received a neck intramuscular injection of GP345m-LNP (200 μg / pig) at 2 mL / pig; Group 2 received a neck intramuscular injection of a commercial inactivated PRRSV vaccine (CH-1a strain, produced by YB Company, Hangzhou, Zhejiang) at 1 mL / pig; Group 3 received a neck intramuscular injection of PBS-LNP as a control group. Three weeks after the booster immunization, the piglets in each group received 2 mL of the vaccine via neck intramuscular injection and intranasal drops. 5 TCID 50 / mL of PRRSV FJ1402 virus solution, each group of piglets was isolated and kept under continuous monitoring for 21 days. Blood was collected on the 5th, 10th, 15th and 21st days (dpc) after infection, and the piglets were observed daily, rectal temperature was measured, and body weight was weighed every 5 days. Blood was collected through the anterior vena cava at designated time points, part of the blood was separated from the serum and inactivated at 56℃ for 30 minutes, and stored at -20℃ for use. Part of the blood was used to separate peripheral blood mononuclear cells (PBMCs) for ICS experiments, lymphocyte proliferation experiments and ELISpot experiments (such as Figure 4 The animal experiment protocol was approved by the Animal Care and Ethics Committee of Nanjing Agricultural University and was performed in accordance with the Guiding Principles for Biomedical Research Involving Animals (PT2020023).

[0215] 1.5.2 ELISA

[0216] The method is similar to 1.4.3, using HRP-Goat Anti-Pig IgG (H+L) as the secondary antibody.

[0217] 1.5.3 Neutralization test

[0218] Refer to 1.4.4 for the method.

[0219] 1.5.4 PBMCs Isolation

[0220] PBMCs were isolated using the porcine peripheral blood lymphocyte isolation kit as follows:

[0221] (1) Collect piglet blood using a negative pressure blood collection tube containing anticoagulant and isolate PBMCs within 2 hours;

[0222] (2) Add 5 mL of separation solution to a 15 mL sterile centrifuge tube, slowly add 5 mL of fresh anticoagulated blood to the separation solution interface, and centrifuge at 600 g at 20°C for 25 min.

[0223] (3) After centrifugation, the first layer is the plasma layer, the second layer is the ring-shaped milky white lymphocyte layer, the third layer is the transparent separation fluid layer, and the fourth layer is the red blood cell layer. Discard the plasma layer;

[0224] (4) Transfer the circular milky white lymphocyte layer to a new 15 mL centrifuge tube, add 10 mL of washing solution, centrifuge at 250 g for 10 min, discard the supernatant, and wash the cells twice;

[0225] (5) Add 1 mL of 10% FBS-RPMI 1640 to resuspend the cells and count them. The isolated PBMCs were used for subsequent ICS experiments, lymphocyte proliferation experiments, and ELISpot experiments.

[0226] 1.5.5 ICS

[0227] Flow cytometry was used to measure the antigen-specific T cell immune response in piglets after immunization with mRNA vaccines. The steps were as follows:

[0228] (1) Add 500 μL of 1×10 7 cells / mL of PBMCs suspension, added with purified PRRSV FJ1402 at a final concentration of 10 μg / mL, and the positive control group added with ConA at a final concentration of 10 μg / mL;

[0229] (2) After incubation at 37°C for 12 h, the protein transport inhibitor GolgiPlug was added to each well and incubated for another 5 h.

[0230] (3) Collect the cells, add 1 mL of 0.9% BSA-PBS to wash the cells, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0231] (4) Add Zombie NIR™ Fixable Viability Kit to perform cell viability staining and incubate at room temperature for 20 minutes;

[0232] (5) Add anti-CD16 / 32 for blocking and incubate at 4°C for 10 min;

[0233] (6) Add 1 mL of 0.9% BSA-PBS to wash the cells, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0234] (7) Add extracellular staining antibodies PerCP-Cy5.5 Mouse Anti-Pig CD3ε, PE-Cy7 Mouse Anti-Pig CD4a, FITC Mouse Anti-Pig CD8a, and PE Rat Anti-Pig γδ T Lymphocytes, incubate at 4°C in the dark for 30 min, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0235] (8) Add 1 mL of 0.9% BSA-PBS to wash the cells twice, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0236] (9) Add fixation / permeabilization solution, incubate at 4°C in the dark for 20 min, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0237] (10) Wash the cells twice by adding 0.5 mL of 1× Perm / Wash™ Buffer, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant.

[0238] (11) Add intracellular staining antibody Alexa Fluor 647 Mouse Anti-Pig IFN-γ, incubate at 4°C in the dark for 30 min, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant;

[0239] (12) Wash the cells twice with 1 mL of 0.9% BSA-PBS, centrifuge at 400 g for 3 min at 4°C, and discard the supernatant.

[0240] (13) Add 300 μL of 0.9% BSA-PBS to resuspend the cells and test on the microscope;

[0241] (14) ICS gating strategies such as Figure 5 shown.

[0242] 1.5.6 Lymphocyte proliferation assay

[0243] 100 μL of 1×10 6 To a PBMCs suspension of 10 cells / mL, add purified PRRSV FJ1402 to a final concentration of 10 μg / mL. For the positive control group, add ConA to a final concentration of 10 μg / mL. Refer to 1.4.6 for the method.

[0244] 1.5.7 ELISpot

[0245] Perform an ELISpot assay using the porcine IFN-γ ELISpot kit as follows:

[0246] (1) Add 20 μL of 35% ethanol (EtOH) to each well of the MSIP plate, let it stand for 1 min, and then add 200 μL of sterile water to each well and wash the plate 5 times;

[0247] (2) Add 100 μL of 10 μg / mL capture antibody to each well and incubate overnight at 4°C;

[0248] (3) Add 200 μL of sterile water to each well and wash the plate 5 times;

[0249] (4) Add 200 μL of 10% FBS-RPMI 1640 nutrient solution to each well and block at room temperature for 30 min;

[0250] (5) Add 100 μL of 5×10 5cells / mL of PBMCs suspension, added with purified PRRSV FJ1402 at a final concentration of 10 μg / mL, the positive control group added with ConA at a final concentration of 10 μg / mL, and the negative control added with 10% FBS-RPMI 1640 nutrient solution;

[0251] (6) Incubate at 37°C for 36 h;

[0252] (7) Add 200 μL PBS to each well and wash the plate 5 times;

[0253] (8) Add 100 μL of 0.5 μg / mL biotinylated detection antibody to each well and incubate at room temperature for 2 h;

[0254] (9) Add 200 μL PBS to each well and wash the plate 5 times;

[0255] (10) Add 100 μL of 1:1000 diluted Streptavidin-ALP to each well and incubate at room temperature for 1 h;

[0256] (11) Add 200 μL PBS to each well and wash the plate 5 times;

[0257] (12) Add 100 μL of BCIP / NBT-plus substrate to each well and develop the color for 5-30 minutes until the spots are clearly visible;

[0258] (13) Rinse with tap water to terminate the reaction;

[0259] (14) After natural drying, use an ELISpot reader to take photos and read the results.

[0260] 1.6 Piglet protection against infection

[0261] Three weeks after the booster immunization, each group of piglets received 2 mL of 10 5 TCID 50 / mL of PRRSV FJ1402 virus solution, and continue monitoring for 21 days (e.g. Figure 4 shown).

[0262] 1.6.1 Blood and lung viral load determination

[0263] Blood was collected from piglets on days 5, 10, 15, and 21 after challenge, and serum was isolated for determination of PRRSV viral load in the blood. Piglets were sacrificed on day 21 after challenge, and lungs were collected for determination of PRRSV viral load in the lungs. Quantitative qRT-PCR was used to quantify PRRSV genomic cDNA copy number. The main steps are as follows (Zhang Jie. Genetic variation of the porcine reproductive and respiratory syndrome virus GP5 gene and establishment of a dual fluorescence quantitative PCR detection method [D]. Nanjing Agricultural University, 2020):

[0264] (1) 5 g of lung tissue was collected from two locations in each lung lobe, added to 10 mL of PBS for homogenization, and then frozen and thawed three times before centrifugation. 200 μL of the supernatant was used to extract RNA and perform reverse transcription.

[0265] (2) Primer sequence:

[0266] Upstream primer F: 5'-AAACCAGTCCAGAGGCAAGG-3'

[0267] Downstream primer R: 5'-TCAGTCCGAAGAGGAAAATG-3'

[0268] (3) Use the standard plasmid pClone007-ORF7 stored in the laboratory (Zhang Jie. Genetic variation of porcine reproductive and respiratory syndrome virus GP5 gene and establishment of dual fluorescence quantitative PCR detection method [D]. Nanjing Agricultural University, 2020) according to 10 1 -10 9 Perform serial dilutions to construct a standard curve;

[0269] (4) qRT-PCR assay was performed using AceQ® qPCR SYBR® Master Mix. The reaction system was as follows:

[0270] SBYR Green premix 10 μL

[0271] ddH2O 7.8 μL

[0272] Upstream primer F 0.4 μL

[0273] Downstream primer R 0.4 μL

[0274] Template 1 μL

[0275] ROX 0.4 μL

[0276] (5) Reaction conditions: 50°C for 2 min, 95°C for 2 min, 95°C for 15 s, 60°C for 1 min, fluorescence signal collection, 40 cycles;

[0277] 1.6.2 Histopathological observation

[0278] Lung tissue was fixed with 4% paraformaldehyde, and pathological sections were prepared and stained with hematoxylin-eosin (H&E) to observe the pathological changes of lung tissue. Lung pathological sections were scored on a 5-point scale: 0-no lesions; 1-mild lesions, localized or scattered interstitial pneumonia (lesion ratio <50%); 2-moderate lesions, localized aggregation of lesions (lesion ratio 50–75%); 3-severe lesions, patchy aggregation of lesions or widespread distribution (lesion ratio 75–90%); 4-very severe lesions (lesion ratio >90%) (Guo BQ, Lager KM, Henningson JN, et al. Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus [J]. Virology, 2013, 435(2): 372-384.).

[0279] 1.6.3 Neutralization experiment

[0280] Refer to 1.4.4 for the method.

[0281] 1.6.4 ICS

[0282] Refer to 1.5.5 for the method.

[0283] 1.7 Statistical Analysis

[0284] Statistical analysis was performed using GraphPad Prism 9.5.0. Data are expressed as mean ± SEM. One-way ANOVA or two-way ANOVA were used to compare differences among the groups (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns indicates no significant difference).

[0285] 2 Results

[0286] 2.1 Preparation, characterization, and identification of mRNA-LNPs

[0287] 2.1.1 Structure prediction of fusion proteins

[0288] AlphaFold 3 and PyMol were used to analyze the protein structure of four mRNA-encoded fusion proteins GP35m, GP45m, GP345m, and GP2345m. The predicted three-dimensional structures revealed the folding states of the four fusion proteins, such as Figure 6 shown.

[0289] 2.1.2 Construction and linearization of recombinant plasmids

[0290] The constructed recombinant plasmids pUC57-GP35m, pUC57-GP45m, and pUC57-GP2345m were digested with EcoRI, and the recombinant plasmid pCDNA3.1-GP345m was digested with AflⅡ to obtain the linearized product templates pUC57-GP35m-EcoRI, pUC57-GP45m-EcoRI, pUC57-GP2345m-EcoRI, and pCDNA3.1-GP345m-AflⅡ for in vitro transcription of mRNA. Figure 7 shown.

[0291] 2.1.3 Characterization of in vitro transcribed mRNA

[0292] The linearized recombinant plasmid was used as a template for in vitro transcription to prepare mRNA. After purification, the mRNA was subjected to quality analysis and RNA denaturing formaldehyde agarose gel electrophoresis for identification. The OD value ratios of the four mRNAs were all within the range of 1.9<A260 / A280<2.1 and 2.0<A260 / A230, indicating that the mRNA purity was high, with no obvious degradation and residual proteins and impurities (see Table 1). The results of RNA denaturing formaldehyde agarose gel electrophoresis showed that the four mRNAs all showed a single band, no obvious degradation, and were in line with the expected size (such as Figure 8 shown).

[0293] Table 1 mRNA quality analysis

[0294]

[0295] 2.1.4 Preparation and Characterization of mRNA-LNPs

[0296] Because naked mRNA is highly unstable and will be rapidly degraded in the outside world and in the body, in order to effectively deliver mRNA into host cells, efficiently express the target protein and induce an immune response, mRNA is encapsulated in lipid nanoparticles through a microfluidic system to generate mRNA vaccines, named GP35m-LNP, GP45m-LNP, GP345m-LNP and GP2345m-LNP.

[0297] Dynamic light scattering results showed that the particle sizes of the four prepared mRNA vaccines were 84.38nm, 85.8nm, 89.47nm and 90.66nm, and the polydispersity index (PDI) was 0.048, 0.062, 0.06 and 0.071, respectively, indicating that the mRNA-LNPs showed good uniformity in the solution. The change in Zeta potential showed the responsiveness of mRNA-LNPs to pH changes (such as Figure 9 (as shown in AD in the figure).

[0298] The results of cryo-electron microscopy showed that the four mRNA-LNPs were uniformly spherical in solution (e.g. Figure 9 The encapsulation efficiencies of the four mRNA-LNPs were excellent, all exceeding 94% (see Table 2). These results demonstrate that the four prepared mRNA-LNPs meet the required physicochemical properties and meet the requirements of subsequent experiments.

[0299] Table 2 Analysis of the encapsulation efficiency of four mRNA-LNPs

[0300]

[0301] 2.1.5 Expression Verification of mRNA-LNP

[0302] To verify the in vitro expression of the vaccine, the four mRNA-LNPs were transfected into PK-15 and PAMs cells. Western blotting analysis demonstrated that GP35m (approximately 43 kDa), GP45m (approximately 38 kDa), GP345m (approximately 70 kDa), and GP2345m proteins (approximately 90 kDa) were effectively expressed in PK-15 and PAMs cell lysates using GP3, GP4, and GP5 monoclonal antibodies (e.g., Figure 10 ), among which GP345m-LNP had the highest expression level. In PK-15 cells, GP45m, GP345m, and GP2345m proteins were effectively secreted into the cell culture supernatant, and their molecular weights were slightly larger than those of the fusion proteins in the cell lysate. GP35m protein was not effectively secreted and expressed. None of the four fusion proteins could be secreted and expressed in PAMs (e.g. Figure 10 IFA results showed that GP35m-LNP, GP45m-LNP, GP345m-LNP and GP2345m-LNP were all effectively expressed in PK-15 cells (as shown in C and D). Figure 11 shown).

[0303] 2.2 Determination of the immune characteristics of mRNA-LNP mice

[0304] BALB / c mice were immunized with 15ug of GP35m-LNP, GP45m-LNP, GP345m-LNP and GP2345m-LNP. The humoral and cellular immune responses of the mice were measured at designated time points to evaluate the immune characteristics of the four mRNA vaccines.

[0305] 2.2.1 Serum ELISA antibody titer

[0306] To detect the humoral immune response induced by GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP in mice, ELISA plates were coated with GP2a, GP3, GP4, and GP5 proteins expressed in E. coli prokaryotically, and the ELISA antibody titers were determined. The results of SDS-PAGE and Western blotting identification of the four recombinant proteins are shown in Figure 12 , proving that they all have good antigenicity.

[0307] Twenty-one days after the first immunization with GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP, no antibodies to the GP2a and GP4 proteins were detected in mice. The geometric mean titers (GMTs) of GP3 protein ELISA antibodies produced in the GP345m-LNP and GP2345m-LNP immunization groups were approximately 174, while the GMTs for GP5 protein antibodies were approximately 152 and 132, respectively. Twenty-one days after the booster immunization, the GP2a protein antibody level in the GP2345m-LNP group was relatively low, with a GMT of approximately 30. The GMTs of GP3 protein antibodies in the GP35m-LNP, GP45m-LNP, GP345m-LNP and GP2345m-LNP groups were approximately 132, 4.8, 19,380 and 12,800, respectively, while the GMTs of GP4 protein antibodies were all lower (approximately 4.78-66.1). The GMTs of GP5 protein antibodies were approximately 23, 10, 16,900 and 8,450, respectively. This indicates that boosting immunization with GP345m-LNP and GP2345m-LNP can significantly increase the level of antibodies induced in mice, while boosting immunization with GP35m-LNP and GP45m-LNP can only induce low levels of binding antibodies, which may be due to the low translation efficiency of GP35m-LNP and GP45m-LNP in mice (e.g. Figure 13 shown).

[0308] 2.2.2 Serum neutralizing antibody titer

[0309] 21 days after the booster immunization, the serum of mice was collected to measure the PRRSV neutralizing antibodies. The results showed that none of the four mRNA vaccines could produce detectable neutralizing antibodies (such as Figure 14 shown).

[0310] 2.2.3 Cellular immune response

[0311] To evaluate the cellular immune response induced by the four mRNA vaccines in mice, spleens of the immunized mice were collected 21 days after the second immunization, and spleen cells were isolated and evaluated to test their ability to produce cytokines after in vitro stimulation with purified virus PRRSV FJ1402. The ICS assay results showed that spleen cells from mice immunized with GP345m-LNP could effectively stimulate CD4 + IL-2, IL-4, IFN-γ and CD8 in T cells + IFN-γ production in T cells (e.g. Figure 15 Lymphocyte proliferation experiments showed that spleen cells from mice immunized with GP345m-LNP and GP2345m-LNP could produce antigen-specific lymphocyte proliferation responses after protein stimulation (as shown in Figure 2). Figure 16 The above results indicate that GP345m-LNP and GP2345m-LNP can induce antigen-specific lymphocyte immune responses in addition to humoral immune responses in mice, and that GP345m-LNP induces stronger humoral and cellular immune responses.

[0312] 2.3 Determination of immune characteristics of GP345m-LNP piglets

[0313] Given that GP345m-LNP induced the strongest immune response, we selected it for a challenge and protection trial in piglets at a dose of 200 μg / pig. A commercial inactivated vaccine group was also administered for simultaneous comparison, along with a negative control group injected with PBS-LNP. Blood was collected from the piglets at designated time points to measure humoral and cellular immune responses and evaluate the immunogenicity of GP345m-LNP.

[0314] 2.3.1 Serum ELISA antibody titer

[0315] 21 days after the first vaccination, the GP345m-LNP vaccination group was unable to produce detectable antibodies against GP3 ​​and GP4 proteins, while the commercial inactivated vaccine vaccination group produced GMTs of approximately 5,570 and 3,200 antibodies against GP3 ​​and GP4 proteins. The GP345m-LNP vaccination group and the commercial inactivated vaccine vaccination group produced GMTs of approximately 91 and 4,230 against GP5 protein. Antibody levels were further improved after booster vaccination. 21 days after the booster vaccination, the commercial inactivated vaccine vaccination group and the GP345m-LNP vaccination group produced GMTs of approximately 25,600 and 4,230 against GP3 ​​protein, approximately 16,900 and 229 against GP4 protein, and approximately 33,800 and 6,400 against GP5 protein (e.g. Figure 17 Both GP345m-LNP and commercial inactivated vaccine significantly induced antibody production in piglets after immunization, with the commercial inactivated vaccine producing a higher level of antibodies.

[0316] 2.3.2 Serum neutralizing antibody titer

[0317] By measuring the serum neutralizing antibody titer 21 days after booster immunization, it was found that GP345m-LNP could not induce the production of PRRSV neutralizing antibodies, while the commercial inactivated vaccine could produce neutralizing antibodies against PRRSV FJ1402 strain with GMTs of about 3.8 (e.g. Figure 18 shown).

[0318] 2.3.3 Cellular immune response

[0319] PBMC cells were isolated from the blood of piglets 21 days after booster vaccination and stimulated with purified PRRSV FJ1402 to measure the vaccine-induced cellular immune response. The ICS experimental results showed that the GP345m-LNP immunization group could significantly increase the number of CD4 T cells that can produce IFN-γ after two immunizations. + T cells and CD8 + The proportion of T cells was not different between the commercial inactivated vaccine group and the PBS-LNP immunization group (e.g. Figure 19 GP345m-LNP immunization failed to induce γδ+ T cells to produce IFN-γ (as shown in A and B). Figure 19 Lymphocyte proliferation experiments showed that the GP345m-LNP immunization group could produce PRRSV-specific lymphocyte proliferation responses after PRRSV stimulation compared with the commercial inactivated vaccine group and the PBS-LNP immunization group (as shown in C). Figure 20ELISpot test results showed that GP345m-LNP immunization could induce antigen-specific lymphocytes to secrete IFN-γ, while inactivated vaccines could not effectively induce the production of lymphocytes that secrete IFN-γ (as shown in Figure 2). Figure 21 The above results show that compared with commercial inactivated vaccines, GP345m-LNP induces lower antibody levels in piglets, but can significantly induce antigen-specific cellular immune responses, while inactivated vaccines cannot effectively induce the production of cellular immune responses.

[0320] 2.4 Determination of the protective effect of GP345m-LNP in piglets against challenge

[0321] After 21 days of booster immunization, each group of piglets was injected with 2 mL of 10 5 The TCID50 / mL PRRSV FJ1402 virus solution was continuously monitored for 21 days to determine and analyze the protective efficacy of GP345m-LNP on piglets.

[0322] 2.4.1 Clinical symptoms

[0323] All three groups of piglets began to show fever symptoms (body temperature > 40°C) on the fourth day after infection, and returned to normal body temperature on the 10th day (e.g. Figure 22 There was no significant difference in the average daily weight gain of the three groups of piglets within 21 days after the challenge (as shown in A). Figure 22 (as shown in B in the figure).

[0324] 2.4.2 Blood and lung viral load

[0325] Piglet blood was collected through the anterior vena cava at 5, 10, 15 and 21 days (dpc) after the virus attack, and the serum was separated to determine the viral load in the blood. The results showed that there was no significant difference between the GP345m-LNP immunization group and the inactivated vaccine immunization group and the PBS-LNP control group at 5 dpc. Starting from 10 dpc, the blood viral load of the GP345m-LNP immunization group was significantly lower than that of the PBS-LNP control group, and was significantly lower than that of the inactivated vaccine phase at 15 dpc and 21 dpc. PRRSV RNA could not be detected in the blood until 21 dpc. There was no significant difference in the blood viral load between the inactivated vaccine immunization group and the PBS-LNP control group during the virus attack (such as Figure 23 (as shown in A in the figure).

[0326] Piglets were killed on 21 dpc and lungs were collected to measure viral load. The results showed that the lung viral load of the GP345m-LNP immunization group was significantly lower than that of the PBS-LNP control group and the inactivated vaccine immunization group. PRRSV RNA could not be detected in the lungs of 3 piglets (such as Figure 23 (as shown in B in the figure).

[0327] 2.4.3 Pathological changes in lung tissue

[0328] The analysis of lung tissue pathology of piglets in each group showed that both the inactivated vaccine immunization group and the PBS-LNP control group had thickening of the alveolar wall, exudate in the alveolar cavity, and infiltration of a large number of inflammatory cells, showing interstitial pneumonia (such as Figure 24 In contrast, the alveolar septa in the GP345m-LNP immunization group were thinner, the alveolar morphology was close to normal, with only a small amount of lesions, and inflammatory cell infiltration was significantly reduced (as shown in Figure 1). Figure 24 Lung pathological injury statistics are shown in Figure C. Figure 25 shown.

[0329] 2.5 Cellular immune response in piglets after challenge

[0330] To further investigate the immune status of piglets in each group after challenge, blood samples were collected from piglets on 21 dpc to analyze the production of cellular immunity. ICS analysis showed that the GP345m-LNP immunization group could produce IFN-γ in CD4 + and CD8 + The proportion of T cells was still significantly higher than that of the PBS-LNP control group and the inactivated vaccine immunization group, and the γδ cells that could produce IFN-γ + There was no significant difference in the proportion of T cells (e.g. Figure 26 shown).

[0331] 3 Discussion

[0332] PRRSV has caused huge economic losses to the global pig industry. Given the insufficient protective efficacy and safety of existing vaccines, the development of vaccines that can produce good protective effects against PRRSV is an urgent problem to be solved. mRNA vaccines containing modified nucleosides delivered by LNP have shown their reliable efficacy in preventing and controlling SARS-CoV-2 and RSV. mRNA vaccine technology not only shows great potential in the prevention and control of infectious diseases, but also shows broad prospects in the treatment of cancer and rare diseases through mRNA therapy. At present, research on the role of mRNA vaccines in the control of animal infectious diseases is relatively limited. This study designed and studied four mRNA vaccines encoding modified nucleosides: (1) GP35m-LNP expressing GP3 and GP5 fusion protein; (2) GP45m-LNP expressing GP4 and GP5 fusion protein; (3) GP345m-LNP expressing GP3, GP4 and GP5 fusion protein; (4) GP2345m-LNP expressing GP2a, GP3, GP4 and GP5 fusion protein (such as Figure 1Animal studies have shown that GP345m-LNP effectively induces both humoral and cellular immune responses in mice and piglets. Although GP345m-LNP and an inactivated PRRSV vaccine failed to completely prevent PRRSV infection in immunized piglets, GP345m-LNP effectively reduced viral loads in the blood and lung tissues and reduced lung pathological damage, demonstrating that the GP345m-LNP vaccine possesses significant immunoprotective properties and holds significant research and development potential.

[0333] PRRSV encodes multiple structural proteins. GP2a, as a structural protein, is involved in PRRSV binding to cellular receptors, suggesting the presence of neutralizing epitopes. Multiple B-cell epitopes have been identified, but few T-cell epitopes have been found. GP3 is one of the primary structural and immunogenic proteins of PRRSV, stimulating both humoral and cellular immune responses and playing a crucial role in viral replication, assembly, and pathogenicity. GP3 can provide protection in piglets even in the absence of neutralizing antibodies. GP4, a minor envelope glycoprotein, contains both B-cell and T-cell epitopes and is frequently used in vaccine design. GP5, the most prominent PRRSV envelope glycoprotein, is considered the primary target for neutralizing antibodies. Although GP5 alone induces only a weak neutralizing antibody response, it remains the most widely used protein in PRRSV vaccine development. GP2a, GP3, GP4, and GP5 of the virus all exhibit good immunogenicity, effectively inducing both humoral and cellular immune responses. In this study, based on the NADC30-like strain FJ1402 isolated and identified by our laboratory, GP2, GP3, GP4 and GP5 proteins were selected as vaccine basic antigens, and 4 different combinations of fusion protein genes were designed to construct mRNA vaccine target antigens. In order to ensure that the target protein translated by mRNA can be effectively secreted into the extracellular space after expression in the cell and promote CD4 + The T cell response deleted the original signal peptide and transmembrane region of the GP2a, GP3, GP4, and GP5 proteins. The modified structural proteins were connected by a flexible linker (GGGGS)3 to form fusion proteins GP35, GP45, GP345, and GP2345. The tPA signal peptide was added to the N-terminus of the fusion protein to guide the fusion protein into the endoplasmic reticulum, and then processed by the Golgi apparatus and secreted out of the cell. After using LNP to encapsulate mRNA, all four mRNA vaccines were effectively expressed in PK-15 and PAMs cells, with GP345m-LNP having the highest expression level (e.g., Figure 10GP45m-LNP, GP345m-LNP, and GP2345m-LNP can effectively secrete and express the fusion protein in PK-15 cells. Similarly, GP345m-LNP has the highest secretion expression, while GP35m-LNP cannot be effectively secreted. This may be because the fusion GP35 protein is not properly folded or modified, is trapped in the cell, or triggers protein degradation mechanisms, resulting in ineffective secretion (e.g. Figure 10 Although all four mRNA vaccines were effectively expressed in PAMs, they were not secreted into the cell supernatant, which may be due to the characteristics of macrophages (e.g. Figure 10 (As shown in Figure D). Primary macrophages typically exhibit strong membrane stability and resistance, making them resistant to the entry of exogenous nucleic acids into the cells, thereby limiting the secretion of exogenous proteins. Furthermore, while macrophages have the ability to secrete cytokines and chemokines, they may be less efficient at secreting non-native exogenous proteins, resulting in intracellular accumulation rather than effective secretion. One of the primary functions of macrophages is to phagocytose and degrade exogenous substances. Even if exogenous proteins are successfully overexpressed, they may be recognized by macrophages as abnormal substances and degraded through endocytosis rather than secreted outside the cell.

[0334] Humoral immunity rapidly develops after PRRSV infection, but neutralizing antibody production is slower. Previous studies have shown that neutralizing antibody titers of 1:8 or higher can prevent PRRSV viremia and reduce viral loads in tissues. This study demonstrated in mice that 21 days after booster immunization with GP345m-LNP and GP2345m-LNP, induced the production of ELISA antibodies against GP3 ​​and GP5 proteins. The GMTs for GP345m-LNP-immunized mice were 19,380 and 16,900, respectively, compared to 12,800 and 8,450 for the GP345m-LNP-immunized mice. The GP345m-LNP-immunized mice exhibited higher antibody titers. Piglet studies demonstrated that 21 days after booster immunization with GP345m-LNP-induced ELISA antibody production with GMTs of 4,230 and 6,400 for GP3 and GP5 proteins, respectively, lower than those induced by inactivated vaccine immunization. This may be related to mRNA expression levels and immunization dose. In the future, mRNA vaccine-induced humoral immune responses could be enhanced by increasing the immunization dose of GP345m-LNP or by optimizing the 5′UTR, 3′UTR, codons, and delivery system to improve protein expression levels. PRRSV may exhibit antibody-dependent enhancement (ADE), whereby non-neutralizing or subneutralizing concentrations of antibodies can enhance viral infection and replication. In this study, despite the production of IgG antibodies and the absence of neutralizing antibodies after vaccination, no ADE-related enhancement of viral replication or pathological damage was observed. Although our GP345m-LNP antigen design incorporates previously reported linear neutralizing epitopes from GP3 and GP5, it did not induce neutralizing antibodies, whereas inactivated vaccines induced neutralizing antibodies (nAbs). We speculate that the linear epitopes of the GP345 fusion protein may be underexposed, or that the generation of PRRSV-specific neutralizing antibodies may require conformational epitopes formed by multiple structural proteins rather than linear epitopes. The current lack of structural data for PRRSV proteins hinders rational structure-based antigen design.

[0335] T cell immunity is believed to play a key role in the control and clearance of PRRSV. αβ T cell-mediated cellular immunity, including CD4 + T cells and CD8 + T cells play a role in the early clearance of PRRSV, while γδ + T cell-mediated immunity is responsible for viral clearance in lymphoid tissues in the late stage of infection. In this study, GP345m-LNP can induce mice to produce IFN-γ + IL-2 + and IL-4 +In the piglet immune protection test, the GP345m-LNP immunization group significantly produced IFN-γ before and after the challenge. + CD4 + T cells and CD8 + T cell response, while the inactivated vaccine could not induce cellular immune response. Even 21 days after the challenge, IFN-γ was not detected in the piglets of the PBS-LNP control group and the inactivated vaccine immunization group. + T cell responses, indicating that GP345m-LNP immunization induced PRRSV-specific cellular immune responses.

[0336] It is worth noting that in mice and piglets, the antibody levels induced by mRNA vaccines against GP2a and GP4 proteins were low, which may be due to the following reasons: (1) The main B cell epitopes of GP2a and GP4 proteins may be mainly conformational epitopes, which failed to form correctly in the constructed fusion protein, while GP3 and GP5 proteins contain more linear epitopes or conformational epitopes in the fusion protein; (2) The key antigenic epitopes of GP2a and GP4 proteins may be masked and insufficiently exposed in the fusion protein, reducing their immunogenicity; (3) There are insufficient T cell epitopes in GP2a and GP4 proteins, which cannot effectively induce the activation of follicular helper T (Tfh) cells, thereby affecting the ability of Tfh cells to assist B cells in producing high-affinity antibodies, resulting in low antibody titers against GP2a and GP4 proteins; (4) The host immune system may be more inclined to process and deliver the peptide segments of GP3 and GP5 in the fusion protein, preferentially recognizing the immunodominant epitopes in GP3 and GP5 in the fusion protein, resulting in the suppression of the immune response against GP2a and GP4 proteins. In the future, fusion proteins can be optimized from the perspective of protein structure, the order and structure of each part of the protein can be adjusted, immune epitopes can be determined and added, and the design of fusion proteins can be optimized to enhance the immune effect of mRNA vaccines.

[0337] LNPs used for mRNA delivery have potent adjuvant properties, primarily due to their ionizable lipid components. These components stimulate innate immune system receptors such as TLRs, MDA5, and NLRP3, leading to the production of IFN-γ, IL-6, and IL-1β. These components also promote Tfh and germinal center B cell responses, enhancing the adaptive immune response. Adjusting LNP composition allows for flexible modification of mRNA vaccine properties, enabling organ-targeted delivery. PRRSV can spread rapidly through the respiratory system via air, and establishing effective mucosal immunity in the respiratory tract may be effective in preventing PRRSV infection. Lung-targeted mRNA vaccines can establish robust T cell immunity in the lungs, which plays a key role in viral clearance and symptom relief. The induced memory T cells can provide long-term protection, making the future development of mRNA vaccines that can induce local mucosal immunity in the respiratory tract highly desirable.

[0338] PRRSV comprises multiple lineages characterized by rapid genetic variation, and several circulating strains pose a significant challenge to China's swine industry. Currently, the predominant circulating strains in China belong to lineage 1, the NADC30-like and NADC34-like strains. Existing commercial vaccines offer limited protection against heterologous strains. In this study, we evaluated the protective efficacy of GP345m-LNP and a commercial inactivated vaccine against the NADC30-like FJ1402 strain. Future research should focus on developing mRNA vaccines that can generate broadly protective immunity and assess cross-protection against different lineages of PRRSV. Broad-spectrum protection is a key goal of vaccine development. Given their flexibility and rapid design, mRNA vaccines offer a promising platform for addressing the volatile PRRSV landscape and developing broad-spectrum vaccines. Continued exploration of viral proteins capable of eliciting protective immunity, combined with further antigenic modification, holds promise for achieving broad-spectrum immunity against PRRSV.

[0339] In summary, we designed and constructed, for the first time, mRNA vaccines expressing fusion proteins of PRRSV GP2a, GP3, GP4, and GP5, GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP. We evaluated humoral and cellular immune responses in mice and piglets. GP345m-LNP effectively induced both humoral and cellular immune responses in mice and piglets and provided effective protection after challenge, providing a foundation for the development of broad-spectrum, highly effective PRRSV vaccines.

Claims

1. A porcine reproductive and respiratory syndrome virus mRNA molecule, characterized in that: The mRNA molecule is mRNA molecule GP345m or mRNA molecule GP2345m; The mRNA molecule GP345m encodes the fusion protein GP345 of GP3, GP4 and GP5 whose amino acid sequence is shown in SEQ ID NO.3; The mRNA molecule GP2345m encodes the fusion protein GP2345 of GP2a, GP3, GP4 and GP5 whose amino acid sequence is shown in SEQ ID NO.

4.

2. The porcine reproductive and respiratory syndrome virus mRNA molecule according to claim 1, characterized in that The nucleotide sequence of the mRNA molecule GP345m is shown in SEQ ID NO.7; The nucleotide sequence of the mRNA molecule GP2345m is shown in SEQ ID NO.

8.

3. A liposome nanoparticle, characterized in that: The liposome nanoparticles are loaded with the porcine reproductive and respiratory syndrome virus mRNA molecule according to claim 1 or 2.

4. Use of the mRNA molecule according to claim 1 or 2 in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccine.

5. Use of the liposome nanoparticles according to claim 3 in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccine.

6. The biological material related to the porcine reproductive and respiratory syndrome virus mRNA molecule according to claim 1 or 2, characterized in that: The biological material is selected from any one of the following: (A1) a DNA molecule encoding the mRNA molecule according to claim 1 or 2; (A2) an expression cassette containing the DNA molecule described in (A1); (A3) a recombinant vector containing the DNA molecule described in (A1) or a recombinant vector containing the expression cassette described in (A2); (A4) A recombinant microorganism containing the DNA molecule described in (A1), or a recombinant microorganism containing the expression cassette described in (A2), or a recombinant microorganism containing the recombinant vector described in (A3).

7. Use of the biomaterial according to claim 6 in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccine.

8. A porcine reproductive and respiratory syndrome virus mRNA vaccine, characterized in that: The mRNA vaccine contains the mRNA molecule GP345m or the mRNA molecule GP2345m; The mRNA molecule GP345m encodes the fusion protein GP345 of GP3, GP4 and GP5 whose amino acid sequence is shown in SEQ ID NO.3; The mRNA molecule GP2345m encodes the fusion protein GP2345 of GP2a, GP3, GP4 and GP5 whose amino acid sequence is shown in SEQ ID NO.

4.

9. The porcine reproductive and respiratory syndrome virus mRNA vaccine according to claim 8, characterized in that The nucleotide sequence of the mRNA molecule GP345m is shown in SEQ ID NO.7; The nucleotide sequence of the mRNA molecule GP2345m is shown in SEQ ID NO.

8.

10. The porcine reproductive and respiratory syndrome virus mRNA vaccine according to claim 8 or 9, characterized in that The porcine reproductive and respiratory syndrome virus mRNA vaccine is an mRNA-LNP complex formed by encapsulating the mRNA molecule GP345m or the mRNA molecule GP2345m in liposome nanoparticles.

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