A porcine reproductive and respiratory syndrome virus mRNA molecule and its application

Abstract

This invention discloses a porcine reproductive and respiratory syndrome virus (PRRSV) mRNA molecule and its applications. Four mRNA vaccines, GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP, were designed by combining the NADC30-like structural proteins GP2a, GP3, GP4, and GP5 of the PRRSV FJ1402 strain. Mouse experiments showed that GP345m-LNP induced both strong humoral and cellular immune responses, followed by GP2345m-LNP, with better efficacy than the inactivated vaccine (which only induced humoral immunity). After immunization, challenge with the PRRSV FJ1402 strain resulted in significantly reduced viral load in the blood and lungs of the GP345m-LNP group, with significantly less pathological damage to the lungs. The immunization effect was superior to the inactivated vaccine group, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a porcine reproductive and respiratory syndrome virus mRNA molecule and its applications. Background Technology

[0002] PRRS is one of the most significant diseases facing the global swine industry, causing enormous economic losses worldwide. Caused by PRRSV, it primarily leads to respiratory disease and growth retardation in pigs of all ages and triggers reproductive disorders in sows. PRRSV can be transmitted through multiple routes, including air, feed, and semen, and increases pigs' susceptibility to various viruses and bacteria. Currently, the most widely used PRRSV vaccines are MLV and inactivated vaccines, while other types of vaccines, such as viral vector vaccines, subunit vaccines, gene-deleted vaccines, and VLP vaccines, are still under investigation. Inactivated vaccines have good safety profiles, but their immunogenicity is low, requiring the addition of adjuvants, and the induced immune response is limited. MLV vaccines can induce a stronger immune response and provide more effective immune protection, but their protective effect against heterologous strains is poor, and there are risks of viral recombination, mutations at key antigenic sites, and virulence recovery. Therefore, the development of safer and more effective PRRSV vaccines is urgently needed. In the development of PRRSV subunit vaccines and viral vector vaccines, the main target proteins are the structural proteins of the virus, including GP2a, GP3, GP4, GP5, and M proteins, which 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, mRNA vaccine platforms are renowned for their speed, efficiency, safety, and flexibility. During the COVID-19 pandemic, mRNA vaccines garnered global attention due to their rapid development and potent efficacy, becoming the most widely used SARS-CoV-2 vaccine. Furthermore, mRNA vaccines have shown promising potential in preventing infections from viruses such as RSV, ZIKV, RV, and VZV. While mRNA vaccines can elicit strong humoral and cellular immune responses, research on their immunoprotective effects in animal viral infections remains relatively limited.

[0004] Porcine reproductive and respiratory syndrome virus (PRRSV) exhibits genetic mutations, and existing commercially available PRRSV vaccines are not entirely ideal, failing to effectively protect against NADC30-like dominant circulating strains. Currently, there are no effective vaccines in my country targeting circulating NADC30-like strains, and most of the PRRSV strains isolated and identified in our laboratory in recent years are of this type. Therefore, developing a vaccine to control PRRSV NADC30-like strains is essential. Previous studies on subunit vaccines, viral vector vaccines, and nanoparticle vaccines against PRRSV have found that vaccines prepared using a single PRRSV structural protein as a target protein have limited immunogenicity and protective efficacy. Therefore, developing an mRNA vaccine targeting NADC30-like strains is the technical problem this invention aims to solve. Summary of the Invention

[0005] The purpose of this invention is to provide a porcine reproductive and respiratory syndrome virus mRNA molecule and its application.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention seeks to protect a porcine reproductive and respiratory syndrome virus mRNA molecule, wherein the mRNA molecule is mRNA molecule GP345m or mRNA molecule GP2345m.

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

[0009] The mRNA molecule GP2345m encodes the fusion protein GP2345, which contains the amino acid sequences GP2a, GP3, GP4 and GP5 as shown in SEQ ID NO.4.

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

[0011] Secondly, the present invention claims protection for a liposome nanoparticle loaded with the aforementioned porcine reproductive and respiratory syndrome virus mRNA molecule.

[0012] Thirdly, the present invention seeks protection for the use of the above-mentioned mRNA molecule in the preparation of a porcine reproductive and respiratory syndrome virus mRNA vaccine.

[0013] Fourthly, the present invention seeks protection for the use of the above-described liposome nanoparticles in the preparation of a porcine reproductive and respiratory syndrome virus mRNA vaccine.

[0014] Fifthly, the present invention seeks protection for biological materials related to the aforementioned porcine reproductive and respiratory syndrome virus mRNA molecule, wherein the biological material is selected from any of the following:

[0015] (A1) The 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] Sixthly, the present invention seeks protection for the use of the above-mentioned biological materials in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccines.

[0021] Seventhly, the present invention claims protection for a porcine reproductive and respiratory syndrome virus mRNA vaccine, the mRNA vaccine containing mRNA molecule GP345m or mRNA molecule GP2345m;

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

[0023] The mRNA molecule GP2345m encodes the fusion protein GP2345, which contains the amino acid sequences GP2a, GP3, GP4 and GP5 as shown in SEQ ID NO.4.

[0024] Furthermore, 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.

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

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

[0027] The beneficial effects of this invention are:

[0028] This invention combines the NADC30-like PRRSV FJ1402 strain structural protein genes GP2a, GP3, GP4, and GP5 to design four mRNA vaccines: (1) GP35m-LNP expressing the GP3 and GP5 fusion protein; (2) GP45m-LNP expressing the GP4 and GP5 fusion protein; (3) GP345m-LNP expressing the GP3, GP4, and GP5 fusion protein; and (4) GP2345m-LNP expressing the GP2a, GP3, GP4, and GP5 fusion protein. 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 PRRSV inactivated vaccines, GP345m-LNP induced only a significant cellular immune response, while the PRRSV inactivated vaccine induced only humoral immunity. Following immunization, challenge with the PRRSV FJ1402 strain resulted in a significant reduction in viral load in the blood and lungs of the GP345m-LNP group, along with a significant reduction in lung pathological damage. The immunization effect was superior to that of the inactivated vaccine group, demonstrating promising application prospects. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the molecular design of four mRNA vaccines.

[0030] Figure 2 This refers to the mouse immunization program and sample collection time points for the mRNA vaccine.

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

[0032] Figure 4 This document outlines the immunization-challenge procedure and sample collection time points for mRNA vaccines in piglets.

[0033] Figure 5 A flow cytometry gating strategy for T cell subsets in piglet PBMCs.

[0034] Figure 6 Three-dimensional structure prediction for four fusion proteins;

[0035] In this diagram, A represents GP35m protein; B represents GP45m protein; C represents GP345m protein; D represents GP2345m protein; purple represents GP2a protein; orange represents GP3 protein; pink represents GP4 protein; and blue represents GP5 protein.

[0036] Figure 7 Linearization of four recombinant plasmids.

[0037] Figure 8 Formaldehyde agarose gel electrophoresis was performed on denatured mRNA.

[0038] Figure 9 Characterization of four mRNA-LNPs: A shows the particle size, PDI, and Zeta potential of GP35m-LNP; B shows the particle size, PDI, and Zeta potential of GP45m-LNP; C shows the particle size, PDI, and Zeta potential of GP345m-LNP; D shows the particle size, PDI, and Zeta potential of GP2345m-LNP; E shows cryo-electron microscopy images of the four mRNA-LNPs.

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

[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 12For the identification of recombinant proteins; where A represents the identification of GP2a protein expressed in prokaryotic cells of Escherichia coli using SDS-PAGE (left lane) and Western blotting (right lane); B represents the identification of GP3 protein expressed in prokaryotic cells of Escherichia coli using SDS-PAGE (left lane) and Western blotting (right lane); C represents the identification of GP4 protein expressed in prokaryotic cells of Escherichia coli using SDS-PAGE (left lane) and Western blotting (right lane); D represents the identification of GP5 protein expressed in prokaryotic cells of Escherichia coli using 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 The values ​​represent the ELISA antibody levels induced by four mRNA-LNPs in mice; where A represents the ELISA antibody levels against GP2a generated after the initial and booster immunizations of the four mRNA-LNPs; B represents the ELISA antibody levels against GP3 ​​generated after the initial and booster immunizations of the four mRNA-LNPs; C represents the ELISA antibody levels against GP4 generated after the initial and booster immunizations of the four mRNA-LNPs; and D represents the ELISA antibody levels against GP5 generated after the initial and booster immunizations of the four mRNA-LNPs.

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

[0044] Figure 15 This study describes the specific T-cell immune responses in mice immunized with four mRNA vaccines using an ICS assay. In this assay, A represents the CD4+ IL-2 production induced in mice after immunization with the four mRNA vaccines, as determined by the ICS assay. + The proportion of T cells; B represents the CD4+ IL-4 production induced in mice after immunization with four mRNA vaccines, as determined by the ICS experiment. + The proportion of T cells; C and D represent the CD4+ production of IFN-γ induced in mice after immunization with four mRNA vaccines, as determined by ICS assay. + T cells and CD8 + The proportion of T cells.

[0045] Figure 16 These represent the antigen-specific lymphocyte proliferation responses induced by four mRNA vaccines in mice.

[0046] Figure 17The values ​​represent the ELISA antibody levels induced by GP345m-LNP in piglets; where A represents the ELISA antibody titer against GP3 ​​after immunization with GP345m-LNP and a commercially available inactivated vaccine; B represents the ELISA antibody titer against GP4 after immunization with GP345m-LNP and a commercially available inactivated vaccine; and C represents the ELISA antibody titer against GP5 after immunization with GP345m-LNP and a commercially available inactivated vaccine.

[0047] Figure 18 The level of neutralizing antibodies induced by GP345m-LNP in piglets.

[0048] Figure 19 The study used an ICS assay to detect the specific T-cell immune response induced by GP345m-LNP in piglets; where A represents the CD4+ IFN-γ production induced in piglets after GP345m-LNP immunization, as determined by the ICS assay. + The proportion of T cells; B represents the CD8+ cells induced in piglets after GP345m-LNP immunization, as determined by ICS assay. + The proportion of T cells; C represents the γδ produced by piglets after immunization with GP345m-LNP, as determined by ICS assay. + The proportion of T cells.

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

[0050] Figure 21 To detect GP345m-LNP-induced antigen-specific lymphocyte responses using ELISPOT.

[0051] Figure 22 Clinical symptoms of piglets after viral challenge; where A is the daily rectal temperature and B is the average daily weight gain.

[0052] Figure 23 The viral load in the blood and lungs of piglets after challenge with the virus is: A represents the viral load in the blood, and B represents the viral load in the lungs.

[0053] Figure 24 The images show pathological changes in the lungs of piglets; A represents H&E staining of lung pathological sections from the PBS-LNP control group; B represents H&E staining of lung pathological sections from the inactivated vaccine immunization group; and C represents H&E staining of lung pathological sections from the GP345m-LNP immunization group.

[0054] Figure 25 For the assessment of pathological damage to lung tissue.

[0055] Figure 26This study describes the specific T-cell immune response in piglets 21 days after viral challenge using an ICS assay; where A represents the CD4+ IFN-γ production in piglets after viral challenge, as determined by the ICS assay. + The proportion of T cells; B represents the CD8+ IFN-γ production in piglets after challenge, as determined by the ICS assay. + The proportion of T cells; C represents the γδ of IFN-γ produced in piglets after challenge, as determined by the ICS assay. + The proportion of T cells. Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make equivalent changes to the disclosed technical content to create equivalent embodiments. Any modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the present invention fall within the protection scope of the present invention.

[0057] Example 1

[0058] Four mRNA vaccines expressing PRRSV antigen fusion proteins were designed in this study: (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 inducing the strongest immune response. Immunization of piglets with GP345m-LNP effectively induced humoral and cellular immune responses. In the challenge protection test, compared with commercial inactivated vaccines, 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 laboratory animals: PAMs, Marc-145, and PK-15 cells were prepared and preserved in our laboratory; PRRSV FJ1402 F7 generation virus solution and PRRSV-positive pig serum were prepared, identified, and preserved in our laboratory according to standard methods; GP3 (4H9) and GP4 (6E9) monoclonal antibodies were prepared and preserved in our laboratory according to standard methods; GP5 monoclonal antibody was a gift from the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; 4-week-old healthy piglets (PRRSV nucleic acid and antibody negative) were provided by a pig farm in Nantong City, Jiangsu Province; 6-8-week-old female BALB / c mice were purchased from the Experimental Animal Center of Yangzhou University and raised under suitable temperature and humidity conditions.

[0062] Reagents: Plasmid large-scale extraction kit (DP116) was purchased from Tiangen Biotech; 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), AceQ® qPCR SYBR® Master Mix (Q111-02), and CCK-8 (A311) were all purchased from Novizan; Restriction endonucleases EcoRI (FD0274) and AflII (FD0834), RNA marker RiboRuler High Range RNA Ladder (SM1821), and Quant-iT RiboGreen RNA quantification kit were all purchased from Novizan. Kit (R11490) was purchased from Thermo; cationic lipid ALC-0315 and PEGylated lipid ALC-0159 were purchased from Synobond; cholesterol and distearate phosphatidylcholine (DSPC) were purchased from Avetrol; 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 chromogenic solution was purchased from Beyotime; fixation / permeabilization kit, GolgiPlug, anti-CD16 / 32, PerCP-Cy5.5 Rat Anti-MouseCD8a (53-6.7), PerCP-Cy5.5 Rat Anti-MouseCD8a (53-6.7), and PerCP-Cy5.5 Rat Anti-MouseCD8a (53-6.7) were also used.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), APC / Fire 750 anti-mouse IL-2 (503841) was 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 routine chemical reagents were of analytical grade.

[0063] 1.2 mRNA preparation

[0064] 1.2.1 Design of mRNA vaccines

[0065] The envelope glycoproteins GP2a, GP3, GP4, and GP5 of PRRSV were selected as target proteins to construct mRNAs encoding fusion proteins. Using the 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 the GP3 and GP5 fusion protein; (2) GP45m-LNP expressing the GP4 and GP5 fusion protein; (3) GP345m-LNP expressing the GP3, GP4, and GP5 fusion protein; and (4) GP2345m-LNP expressing the GP2a, GP3, GP4, and GP5 fusion protein. To ensure effective expression and secretion of the fusion protein, the original signal peptide and transmembrane region were removed from the protein. A flexible linker (GGGGS) 3 linker was used to connect the target protein to the fusion protein. A tissue plasminogen activator (tPA) signal peptide was added to the N-terminus of the fusion protein to induce secretion (e.g., ...). Figure 1 (As 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] After optimizing the codons of the DNA sequence encoding the above fusion protein, 5′UTR, 3′UTR and polyA sequences were added. The gene was synthesized by Nanjing GenScript and cloned into the pUC57 and pCDNA3.1 plasmids to synthesize the 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 plasmids

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

[0071] 1.2.3 Extraction of mRNA vaccine template plasmid

[0072] The top 10 single colonies in the plate were selected, and after sequencing confirmed to be correct, they were amplified and cultured. Plasmids were extracted in large quantities using a plasmid extraction kit.

[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 6-8 times to fully lyse the cells, and let it stand at room temperature for 5 min;

[0076] (4) Add 10 mL of solution P4 to the centrifuge tube, gently invert 6-8 times until a white dispersed precipitate appears, and let 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 the volume of isopropanol to the filtrate, and mix by inverting the container.

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

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

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

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

[0082] (10) Determine the plasmid concentration.

[0083] 1.2.4 Linearization of mRNA vaccine template plasmids

[0084] Linearized plasmids were used as templates for in vitro transcription to prepare mRNA. The obtained recombinant plasmids pUC57-GP35m, pUC57-GP45m, and pUC57-GP2345m were digested with EcoRI, and the recombinant plasmid pCDNA3.1-GP345m was linearized by AflII digestion. The steps were as follows: 1 μg plasmid, 1 μL restriction endonuclease EcoRI or AflII, 2 μL 10× buffer, and ddH2O to a final volume of 20 μL. Digestion was performed at 37℃ for 30 min.

[0085] 1.2.5 Purification of linearized plasmids

[0086] The following steps were taken to recover linearized plasmids using DNA purification magnetic beads:

[0087] (1) Remove the magnetic bead solution from 2-8℃ 30 min in advance and allow it to equilibrate to room temperature;

[0088] (2) Vortex mix the magnetic beads. Add 0.5 times the volume of magnetic bead liquid to the linearized plasmid solution and mix by blowing.

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

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

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

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

[0093] (7) Dry with the lid off at room temperature for 5-10 minutes;

[0094] (8) Remove the sample from the magnetic rack, add an appropriate amount of enzyme-free water, mix by blowing and stirring, and let stand for 2 min;

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

[0096] 1.2.6 In vitro transcription of mRNA

[0097] In vitro transcription of mRNA was performed using the T7 High Yield RNA Transcription Kit and CAGTrimer, following these steps:

[0098] (1) Preparation of the reaction system:

[0099] 2 μL of T7 RNA polymerase premix

[0100] 10 × Transcription Buffer 2 μL

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

[0102] 2 μL of ATP solution

[0103] 2 μL of CTP solution

[0104] 2 μL GTP solution

[0105] 1 μL of CAG solution

[0106] 1 μg of linearized plasmid

[0107] Add enzyme-free water to a final volume of 20 μL

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

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

[0110] 1.2.7 mRNA purification

[0111] The following steps are taken to purify mRNA using RNA purification magnetic beads:

[0112] (1) Remove the RNA purification magnetic beads from 2-8℃ 30 min in advance, allow them to 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 min;

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

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

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

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

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

[0120] (9) Place the sample on a magnetic rack for 5 min, take the supernatant into a new Nuclease-free centrifuge tube to obtain purified mRNA, and determine the concentration.

[0121] 1.2.8 mRNA formaldehyde denaturing agarose gel electrophoresis

[0122] All equipment was treated with RNase removal reagent. 0.8 g of agarose powder was added to 72 mL of enzyme-free water and dissolved. Then, 10 mL of 10×MOPS buffer was added. When the agarose solution cooled to 60°C, 18 mL of formaldehyde was added and the mixture was thoroughly mixed to prepare an agarose gel. Two volumes of 2X RNA loading dye were added to 1 μg of mRNA solution and 2 μL of RNA marker. The mixture was heated at 70°C for 10 min and then placed on ice. Electrophoresis was performed using 1×MOPS buffer at 130V, and images were taken and stored using a gel imaging system.

[0123] 1.3 Preparation and Validation of mRNA-LNP

[0124] 1.3.1 Preparation of mRNA-LNP

[0125] The mRNA is delivered by encapsulating it with LNPs, and the steps are as follows:

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

[0127] (2) Dissolve ALC-0315, ALC-0159, DSPC and cholesterol 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 mRNA-LNP, use a 100 kDa ultrafiltration tube, centrifuge at 2000 g / min at 4℃ to reduce the ethanol concentration to below 1%;

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

[0131] 1.3.2 Characterization of mRNA-LNP

[0132] The particle size, polydispersity index (PDI), and zeta potential of mRNA-LNP were determined using a nanoparticle size potentiostat. The encapsulation efficiency and drug loading were determined using the Quant-iT RiboGreen RNA Kit. 3.0 µL of mRNA-LNP was added to the treated grid and vitrified using a Vitrobot Mark IV (Thermo) at 4°C and 100% humidity in liquid nitrogen-cooled liquid ethane. The samples 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, following the steps outlined below:

[0135] (1) PK-15 cells were seeded into 6-well plates, and the density was approximately 80% after 8 hours;

[0136] (2) Discard 10% (v / v) FBS-DMED nutrient solution before transfection and add 2% (v / v) FBS-DMED nutrient solution;

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

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

[0139] (5) Transfect PK-15 cells with mRNA-LNP to identify protein expression, as follows:

[0140] (6) PAMs cells were seeded into 6-well plates, and the density was approximately 80% after 8 hours;

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

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

[0143] (9) Incubate at 37°C 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 the concentrated cell nutrient solution supernatant with 5× Loading Buffer;

[0146] (2) Boil the sample at 100℃ for 10 min;

[0147] (3) Electrophoresis was performed using 10% SDS-PAGE;

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

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

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

[0151] (7) Wash with PBST 3 times, 10 min each time;

[0152] (8) Use HRP-Goat Anti-Mouse IgG (H+L) diluted 1:5000 with 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 3 times, 10 min each time;

[0154] (10) Apply the color developing solution evenly to the surface of the NC film and take a picture using a chemical exposure instrument.

[0155] 1.3.5 IFA identification of mRNA-LNP expression

[0156] (1) Seed PK-15 cells into 12-well plates. After 8 hours, add 2 μg mRNA-LNP to each well and gently shake to mix. Also, set up negative control wells.

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

[0158] (3) Wash 3 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 for 10 min. Wash 3 times with 1 mL 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 PBS;

[0161] (6) Primary antibody binding: Add 400 µL of PRRSV GP3, GP4 and GP5 monoclonal antibodies diluted 1:400 respectively, incubate at 37℃ for 2 h, and wash 3 times with 1 mL PBS;

[0162] (7) Secondary antibody binding: Under light-protected conditions, add 400 µL of 1:400 diluted HRP-Goat Anti-Mouse IgG (H+L) to each well, FITC conjugate, incubate at 37℃ for 50 min, and wash 3 times with 1 mL PBS;

[0163] (8) Staining cell nuclei: Add 400 µL DAPI to each well, incubate in the dark for 5 min, and wash 3 times with 1 mL PBS;

[0164] (9) Observe the fluorescence.

[0165] 1.4 Immunological evaluation of mRNA-LNP 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 with PBS to a final concentration of 150 ng / μL before administration. The mice were divided into 5 groups of 5 mice each, with 4 vaccine immunization groups: GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP. Each group of mice was intramuscularly injected with 0.1 mL / mouse (15 µg / mouse) of GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP, respectively. PBS-LNP (using PBS instead of mRNA) served as the control group. A booster immunization with the same dose of vaccine was administered three weeks later. Blood and / or spleen were collected on days 0, 21, and 42 after the initial immunization. Serum was separated from the blood, inactivated at 56℃ for 30 min, and stored at -20℃ for later use. Spleen single-cell suspensions were prepared for use in intracellular cytokine staining (ICS) experiments and lymphocyte proliferation experiments (such as...). Figure 2 (As shown). The animal experiment protocol was approved by the Animal Welfare and Ethics Committee of Nanjing Agricultural University and was conducted in accordance with the "Guiding Principles for Biomedical Research Involving Animals" (PT2020023).

[0168] 1.4.2 Protein Expression and Purification

[0169] Predicted transmembrane regions in PRRSV GP2a, GP3, GP4, and GP5 were deleted. The gene sequences were optimized using E. coli codons and sent to Nanjing GenScript Biotech Co., Ltd. for gene synthesis, and then cloned into the pET-28a plasmid. Following our laboratory's protein expression and purification methods, the main steps are as follows (Zhang Lujie. Development of Monoclonal Antibodies for African Swine Fever Virus p72 and p30 Proteins and Establishment of Blocking ELISA Antibody Detection Method [D]; Nanjing Agricultural University, 2021):

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

[0171] (2) Take 30 μL of bacterial culture into 3 mL of LB liquid medium containing kanamycin resistance, and culture 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 culture and sonicate the bacteria;

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

[0174] (5) Proteins were purified by affinity chromatography using a nickel column-His tag;

[0175] (6) The precipitate after 200 mL of bacterial culture was broken up was resuspended in 15 mL of 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) Blood was collected from mice via the submandibular vein. After standing at 37°C for 30 min, the mice were centrifuged at 5,000g for 5 min at 4°C, and the supernatant was collected. The supernatant was serially diluted with PBST twice to serve as the primary antibody. The antibody titer in mice was detected by indirect ELISA, and the steps are as follows:

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

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

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

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

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

[0186] (9) Add 200 μL of 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 serum endpoint dilution titer.

[0189] 1.4.4 Neutralization Test

[0190] PRRSV FJ1402 F7 generation virus solution was diluted to 200 TCID using 4% (v / v) FBS-DMEM. 50 The inactivated serum was serially diluted twofold using DMEM. The diluted virus solution was added to the diluted serum at a 1:1 volume ratio, mixed thoroughly, and incubated at 37°C for 1 hour. The nutrient solution in the 96-well plate was discarded, and the virus-serum mixture was seeded into Marc-145 cells in the 96-well plate. Positive serum control, negative serum control, virus control, and normal cell control were also included, with four parallel wells for each dilution. The cell culture plates were incubated at 37°C for 4 days. The endpoint titer was determined by IFA, defined as the ability to neutralize 100 TCID in the well. 50 PRRSV is the reciprocal of the highest serum dilution factor of 50%.

[0191] 1.4.5 ICS

[0192] The antigen-specific T-cell immune response in mice immunized with mRNA vaccine was measured using flow cytometry, and the steps are as follows:

[0193] (1) Under sterile conditions, mouse spleens were taken, ground into a single-cell suspension, and red blood cells were lysed using red blood cell lysis buffer;

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

[0195] (3) Add 500 μL of 1×10 to each well of the 24-well plate. 7 Spleen cell suspensions of cells / mL were supplemented with purified PRRSV FJ1402 at a final concentration of 10 μg / mL, while the positive control group was supplemented with ConA at a final concentration of 10 μg / mL.

[0196] (4) After incubating at 37℃ for 12 h, add the protein transport inhibitor GolgiPlug to each well and continue incubation for 5 h;

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

[0198] (6) Add anti-CD16 / 32 to block, and incubate at 4℃ for 10 min;

[0199] (7) Add extracellular staining antibodies PerCP-Cy5.5 Rat Anti-Mouse CD8a, FITC anti-mouse CD3 and PE / Cyanine7 anti-mouse CD4, incubate at 4℃ in the dark for 30 min, centrifuge at 4℃ and 400 g for 3 min, and discard the supernatant;

[0200] (8) Add 1 mL of 0.9% BSA-PBS to wash the cells twice, centrifuge at 400 g for 3 min at 4℃, and discard the supernatant;

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

[0202] (10) Add 0.5 mL of 1×Perm / Wash™ Buffer to wash the cells twice, centrifuge at 400 g for 3 min at 4℃, 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℃ in the dark for 30 min, centrifuge at 4℃ 400 g for 3 min, and discard the supernatant;

[0204] (12) Add 1 mL of 0.9% BSA-PBS to wash the cells twice, centrifuge at 400 g for 3 min at 4℃, and discard the supernatant;

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

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

[0207] 1.4.6 Lymphocyte proliferation experiment

[0208] (1) Add 100 μL of 1×10 to each well of a 96-well plate. 6 Spleen cell suspensions of cells / mL were supplemented with purified PRRSV FJ1402 at a final concentration of 10 μg / mL, while the positive control group was supplemented with ConA at a final concentration of 10 μg / mL.

[0209] (2) After incubating at 37℃ for 72 h, add CCK-8 solution and continue incubation for 4 h;

[0210] (3) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance (OD value) at 450 nm;

[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 piglets each. Group 1 received an intramuscular injection of GP345m-LNP (2 mL / pig, 200 µg / pig) in the neck; Group 2 received an intramuscular injection of commercially available inactivated PRRSV vaccine (CH-1a strain, product of Zhejiang Hangzhou YB Company) in the neck (1 mL / pig); and Group 3 served as a control group, receiving an intramuscular injection of PBS-LNP in the neck. Three weeks later, piglets were boosted with the same dose of vaccine. Three weeks after the booster immunization, each group of piglets received 2 mL of 10 mg / mL vaccine via intramuscular injection and intranasal administration. 5 TCID 50 / mL of PRRSV FJ1402 virus solution was administered to each group of piglets in isolation, and they were continuously monitored for 21 days. Blood was collected on days 5, 10, 15, and 21 (dpc) after challenge, and the piglets' condition was observed daily, rectal temperature was measured, and body weight was measured every 5 days. Blood was collected via the anterior vena cava at the designated time points. Serum was separated from a portion of the blood and inactivated at 56℃ for 30 min, then stored at -20℃ for later use. A portion of the blood was used to isolate peripheral blood mononuclear cells (PBMCs) for ICS assays, lymphocyte proliferation assays, and ELISpot assays (e.g., ...). Figure 4 (As shown). The animal experiment protocol was approved by the Animal Welfare and Ethics Committee of Nanjing Agricultural University and was conducted in accordance with the "Guiding Principles for Biomedical Research Involving Animals" (PT2020023).

[0215] 1.5.2 ELISA

[0216] The method was the same as described in 1.4.3, using HRP-Goat Anti-Pig IgG (H+L) as the secondary antibody.

[0217] 1.5.3 Neutralization Test

[0218] The method is the same as described in 1.4.4.

[0219] 1.5.4 PBMCs Separation

[0220] PBMCs were isolated using a porcine peripheral blood lymphocyte isolation kit, and the steps are as follows:

[0221] (1) Collect piglet blood using negative pressure blood collection tubes containing anticoagulant, and separate PBMCs within 2 hours;

[0222] (2) Add 5 mL of separation solution to a 15 mL sterile centrifuge tube, and slowly add 5 mL of fresh anticoagulated blood to the interface of the separation solution. Centrifuge at 20℃ and 600 g 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 clear separation fluid layer, and the fourth layer is the red blood cell layer. Discard the plasma layer;

[0224] (4) Take the ring-shaped milky white lymphocyte layer and transfer it 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] The antigen-specific T-cell immune response in piglets after immunization with mRNA vaccine was measured using flow cytometry, and the steps are as follows:

[0228] (1) Add 500 μL of 1×10 to each well of a 24-well plate. 7 PBMCs cell suspensions were prepared at a concentration of 10 μg / mL, with purified PRRSV FJ1402 added to a final concentration of 10 μg / mL. The positive control group was prepared with ConA at a final concentration of 10 μg / mL.

[0229] (2) After incubating at 37℃ for 12 h, add the protein transport inhibitor GolgiPlug to each well and continue incubation for 5 h;

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

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

[0232] (5) Add anti-CD16 / 32 to block, and incubate at 4℃ 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℃, 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℃ in the dark for 30 min, centrifuge at 4℃ 400 g for 3 min, 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℃, and discard the supernatant;

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

[0237] (10) Add 0.5 mL of 1×Perm / Wash™ Buffer to wash the cells twice, centrifuge at 400 g for 3 min at 4℃, and discard the supernatant;

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

[0239] (12) Add 1 mL of 0.9% BSA-PBS to wash the cells twice, centrifuge at 400 g for 3 min at 4℃, and discard the supernatant;

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

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

[0242] 1.5.6 Lymphocyte proliferation experiment

[0243] Add 100 μL of 1×10 to each well of a 96-well plate. 6 PBMCs cell suspensions were prepared at a concentration of 10 μg / mL, with purified PRRSV FJ1402 added to the final concentration. The positive control group was supplemented with ConA at a final concentration of 10 μg / mL. The method was as described in 1.4.6.

[0244] 1.5.7 ELISpot

[0245] The ELISpot experiment was performed using a porcine IFN-γ ELISpot kit, and the steps are as follows:

[0246] (1) Add 20 µL of 35% ethanol (EtOH) to each well of the MSIP plate, let stand for 1 min, and then add 200 µL of sterile water to each well to 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 seal at room temperature for 30 min;

[0250] (5) Add 100 μL of 5×10 to each well. 5PBMCs cell suspensions were prepared with cells / mL, and purified PRRSV FJ1402 was added to a final concentration of 10 μg / mL. The positive control group was supplemented with ConA to a final concentration of 10 μg / mL, and the negative control group was supplemented with 10% FBS-RPMI 1640 nutrient solution.

[0251] (6) Incubate at 37℃ 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) Wash the plate 5 times with 200 µL PBS in each well;

[0255] (10) Add 100 µL of streptavidin-alkaline phosphatase (Streptavidin-ALP) diluted 1:1000 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 for 5-30 minutes until the spots are clearly visible;

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

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

[0260] 1.6 Virus Challenge Protection Experiment in Piglets

[0261] Three weeks after booster immunization, each group of piglets received 2 mL of 10 ml ... 5 TCID 50 / mL of PRRSV FJ1402 viral fluid was continuously monitored for 21 days (e.g., ...). Figure 4 (As shown).

[0262] 1.6.1 Measurement of viral load in blood and lungs

[0263] Blood samples were collected from piglets on days 5, 10, 15, and 21 post-infection, and serum was separated to determine the PRRSV viral load in the blood. Piglets were euthanized on day 21 post-infection, and lungs were collected to determine the PRRSV viral load in the lungs. The PRRSV genomic cDNA copy number was quantitatively detected by qRT-PCR, with the main steps as follows (Zhang Jie. Genetic Variation of Porcine Reproductive and Respiratory Syndrome Virus GP5 Gene and Establishment of Dual-Fluorescent Quantitative PCR Detection Method [D]. Nanjing Agricultural University, 2020).

[0264] (1) Take 5 g of lung tissue from two parts of each lung lobe, add 10 mL of PBS for homogenization and disruption, freeze and thaw three times, centrifuge, take 200 μL of supernatant to extract RNA and reverse transcribe;

[0265] (2) Primer sequence:

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

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

[0268] (3) Using the laboratory-preserved standard plasmid pClone007-ORF7 (Zhang Jie. Establishment of genetic variation of porcine reproductive and respiratory syndrome virus GP5 gene and dual fluorescence quantitative PCR detection method [D]. Nanjing Agricultural University, 2020), according to 10 1 -10 9 Perform gradient dilution to construct a standard curve;

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

[0270] 10 μL of SBYR Green premix

[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℃ for 2 min, 95℃ for 2 min, 95℃ for 15 s, 60℃ for 1 min, collect fluorescence signal, 40 cycles;

[0277] 1.6.2 Histopathological observation

[0278] Lung tissue was fixed with 4% paraformaldehyde, and pathological sections were prepared. Hematoxylin-eosin (H&E) staining was used to observe the pathological changes in lung tissue. The lung pathological sections were scored on a 5-point scale: 0-no lesions; 1-mild lesions, local or scattered interstitial pneumonia (lesion ratio <50%); 2-moderate lesions, lesions are locally clustered (lesion ratio 50–75%); 3-severe lesions, lesions are patchy clusters or widely distributed (lesion ratio 75–90%); 4-very severe lesions (lesion ratio >90%). (Guo BQ, Lager KM, Henningson JN, et al. Experimental infection of United Statesswine 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] The method is the same as described in 1.4.4.

[0281] 1.6.4 ICS

[0282] The method is the same as described in 1.5.5.

[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 was used to compare differences between 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-LNP

[0287] 2.1.1 Structural prediction of fusion proteins

[0288] Protein structure analysis of four mRNA-encoded fusion proteins, GP35m, GP45m, GP345m, and GP2345m, was performed using AlphaFold 3 and PyMol. The predicted three-dimensional structures revealed the folding states of the four fusion proteins, such as... Figure 6 As 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 linearized product templates for in vitro mRNA transcription: pUC57-GP35m-EcoRI, pUC57-GP45m-EcoRI, pUC57-GP2345m-EcoRI, and pCDNA3.1-GP345m-AflII. Figure 7 As shown.

[0291] 2.1.3 Characterization of in vitro transcribed mRNA

[0292] Linearized recombinant plasmids were used as templates for in vitro transcription to prepare mRNA. After purification, the mRNA underwent quality analysis and was identified by RNA denaturing formaldehyde agarose gel electrophoresis. The OD ratios of the four mRNAs were all within the ranges of 1.9 < A260 / A280 < 2.1 and 2.0 < A260 / A230, indicating high mRNA purity and no significant degradation, protein, or impurity residues (see Table 1). RNA denaturing formaldehyde agarose gel electrophoresis results showed that all four mRNAs exhibited a single band, with no significant degradation, and were consistent with the expected size (e.g., ...). Figure 8 (As shown).

[0293] Table 1 mRNA quality analysis

[0294]

[0295] 2.1.4 Preparation and Characterization of mRNA-LNP

[0296] Because naked mRNA is highly unstable and can be rapidly degraded in the environment and in vivo, in order to effectively deliver mRNA to host cells, efficiently express target proteins and induce immune responses, 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 84.38 nm, 85.8 nm, 89.47 nm, and 90.66 nm, respectively, and their polydispersity indices (PDI) were 0.048, 0.062, 0.06, and 0.071, respectively, indicating that mRNA-LNPs exhibited good homogeneity in solution. Changes in zeta potential demonstrated the responsiveness of mRNA-LNPs to pH changes (e.g., ...). Figure 9 (As shown in AD).

[0298] Cryo-electron microscopy results showed that the four mRNA-LNPs appeared as uniform spheres in solution (e.g., Figure 9 (As shown in E in the table). The encapsulation efficiency of the four mRNA-LNPs was good, all greater than 94% (see Table 2). The above results indicate that the four prepared mRNA-LNPs all meet the standard physicochemical properties and satisfy the requirements for subsequent experiments.

[0299] Table 2 Encapsulation efficiency analysis of four mRNA-LNPs

[0300]

[0301] 2.1.5 Validation of mRNA-LNP expression

[0302] To validate in vitro vaccine expression, four mRNA-LNPs were transfected into PK-15 and PAM cells. Western blotting analysis demonstrated that effective expression of GP35m (approximately 43 kDa), GP45m (approximately 38 kDa), GP345m (approximately 70 kDa), and GP2345m proteins (approximately 90 kDa) could be detected in PK-15 and PAM cell lysates using monoclonal antibodies against GP3, GP4, and GP5. Figure 10 As shown in A and B in the diagram), GP345m-LNP showed 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 the fusion proteins in the cell lysates. GP35m protein was not effectively secreted. None of the four fusion proteins could be secreted in PAMs (e.g., ...). Figure 10 (As shown in C and D in the figure). IFA results showed that GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP were all effectively expressed in PK-15 cells (e.g., ...). Figure 11 (As shown).

[0303] 2.2 Determination of mRNA-LNP mouse immune characteristics

[0304] BALB / c mice were immunized with 15ug each of GP35m-LNP, GP45m-LNP, GP345m-LNP and GP2345m-LNP. Humoral and cellular immune responses of the mice were measured at specified time points to evaluate the immunogenicity 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 prokaryotic cells of *E. coli*, and ELISA antibody titers were determined. The SDS-PAGE and Western blotting results of the four recombinant proteins are shown below. Figure 12 This proves that they all have good antigenicity.

[0307] No GP2a or GP4 protein antibodies were detected 21 days after the initial immunization of mice with GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP. 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 of GP5 protein antibodies were approximately 152 and 132, respectively. At 21 days after the booster immunization, the GP2a protein antibody level was relatively low in the GP2345m-LNP group, 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; the GMTs of GP4 protein antibodies were all relatively low (approximately 4.78-66.1); and the GMTs of GP5 protein antibodies were approximately 23, 10, 16,900, and 8,450, respectively. This indicates that booster immunization with GP345m-LNP and GP2345m-LNP significantly increased the antibody levels induced in mice, while booster immunization with GP35m-LNP and GP45m-LNP still only induced low levels of binding antibodies. This may be because GP35m-LNP and GP45m-LNP have lower translation efficiency in mice (e.g., ...). Figure 13 (As shown).

[0308] 2.2.2 Serum neutralizing antibody titer

[0309] Twenty-one days after booster immunization in mice, serum samples were collected to measure PRRSV neutralizing antibodies. The results showed that none of the four mRNA vaccines produced detectable neutralizing antibodies (e.g., ...). Figure 14 (As shown).

[0310] 2.2.3 Cellular immune response

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

[0312] 2.3 Determination of GP345m-LNP Immune Characteristics in Piglets

[0313] Given that GP345m-LNP induced the strongest immune response, GP345m-LNP was selected for an immune challenge protection experiment in piglets at a dose of 200 μg / pig. A commercially available inactivated vaccine immunization group was simultaneously established for comparison, with PBS-LNP injected as a negative control. Blood samples were collected from piglets at specified 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] Twenty-one days after the initial immunization of piglets, the GP345m-LNP immunization group failed to produce measurable antibodies against GP3 ​​and GP4 proteins, while the commercially available inactivated vaccine immunization group produced GMTs of approximately 5,570 and 3,200 against GP3 ​​and GP4 proteins, respectively. The GMTs against GP5 protein in the GP345m-LNP immunization group and the commercially available inactivated vaccine immunization group were approximately 91 and 4,230, respectively. Antibody levels further increased after booster immunization. At 21 days post-booster, the GMTs against GP3 ​​protein in the commercially available inactivated vaccine immunization group and the GP345m-LNP immunization group were approximately 25,600 and 4,230, against GP4 protein were approximately 16,900 and 229, and against GP5 protein were approximately 33,800 and 6,400, respectively (e.g., 4,570 and 4,230). Figure 17 (As shown). Both GP345m-LNP and commercially available inactivated vaccines can significantly induce antibody production in piglets after immunization, with the commercially available inactivated vaccine producing higher antibody levels.

[0316] 2.3.2 Serum neutralizing antibody titer

[0317] Serum neutralizing antibody titers measured 21 days after booster immunization revealed that GP345m-LNP could not induce the production of PRRSV neutralizing antibodies, while the commercially available inactivated vaccine could produce neutralizing antibodies against the PRRSV FJ1402 strain, with GMTs of approximately 3.8 (e.g., ...). Figure 18 (As shown).

[0318] 2.3.3 Cellular Immune Response

[0319] PBMCs were isolated from the blood of piglets 21 days after booster immunization. After stimulation with purified PRRSV FJ1402, vaccine-induced cellular immune responses were measured. ICS assay results showed that the GP345m-LNP immunization group significantly increased IFN-γ-producing CD4+ cells after two immunizations. + T cells and CD8 + The proportion of T cells was not different between the commercially available inactivated vaccine group and the PBS-LNP immunization group (e.g., Figure 19 (As shown in A and B). GP345m-LNP immunization failed to induce a response in γδ+ T cells to produce IFN-γ (e.g., ...). Figure 19 (As shown in C). Lymphocyte proliferation assays showed that, compared to the commercially available inactivated vaccine group and the PBS-LNP immunization group, the GP345m-LNP immunization group produced a PRRSV-specific lymphocyte proliferation response after PRRSV stimulation (e.g., ...). Figure 20(As shown). ELISpot assay results indicate that GP345m-LNP immunization can induce antigen-specific lymphocytes to secrete IFN-γ, while inactivated vaccines cannot effectively induce the production of IFN-γ-secreting lymphocytes (e.g., Figure 21 The above results indicate that, compared to commercially available 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 cellular immune responses.

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

[0321] 21 days after the booster immunization, each group of piglets received 2 mL of 10 ml ... 5 The protective efficacy of GP345m-LNP against piglets was determined and analyzed by continuously monitoring PRRSV FJ1402 virus solution with TCID50 / mL for 21 days.

[0322] 2.4.1 Clinical symptoms

[0323] All three groups of piglets developed fever symptoms (body temperature > 40℃) on the fourth day after viral challenge, and their body temperature returned to normal by the 10th day (e.g., Figure 22 As shown in A). There was no significant difference in average daily weight gain among the three groups of piglets within 21 days after viral challenge (e.g., ...). Figure 22 (As shown in B in the diagram).

[0324] 2.4.2 Viral load in blood and lungs

[0325] Blood samples were collected from piglets via the anterior vena cava at 5, 10, 15, and 21 days post-challenge (dpc). Serum was separated, and viral load in the blood was measured. Results showed no significant difference between the GP345m-LNP immunization group and the inactivated vaccine immunization group and the PBS-LNP control group at 5 dpc. However, starting at 10 dpc, the viral load in the blood of the GP345m-LNP immunization group was significantly lower than that of the PBS-LNP control group, and significantly lower than that of the inactivated vaccine group at 15 and 21 dpc, until PRRSV RNA was undetectable in the blood at 21 dpc. Throughout the challenge period, there was no significant difference in viral load between the blood of the inactivated vaccine immunization group and the PBS-LNP control group (e.g., ...). Figure 23 (As shown in A in the diagram).

[0326] Piglets were euthanized at 21 days post-contraction (dpc), and lung samples were collected to determine viral load. Results showed that the viral load in the lungs of the GP345m-LNP immunized group was significantly lower than that of the PBS-LNP control group and the inactivated vaccine immunized group. In three piglets, PRRSV RNA was undetectable in the lungs (e.g., Figure 23 (As shown in B in the diagram).

[0327] 2.4.3 Pathological changes in lung tissue

[0328] Pathological analysis of lung tissue sections from piglets in each group revealed that both the inactivated vaccine immunization group and the PBS-LNP control group exhibited alveolar wall thickening, alveolar cavities containing exudate, and extensive inflammatory cell infiltration, presenting as interstitial pneumonia (e.g., Figure 24 (As shown in A and B in the diagram). In contrast, the GP345m-LNP immunized group had thinner alveolar septa, alveolar morphology that was nearly normal, with only a few lesions and significantly reduced inflammatory cell infiltration (e.g., ...). Figure 24 (As shown in C). Statistical analysis of lung pathological damage is as follows: Figure 25 As shown.

[0329] 2.5 Cellular immune response in piglets after viral challenge

[0330] To further investigate the immune status of piglets in different groups after viral challenge, blood samples were collected from piglets at 21 days post-continuation (dpc) to analyze the production of cellular immunity. ICS analysis showed that the GP345m-LNP immunized group produced IFN-γ-mediated CD4+. + and CD8 + The proportion of T cells remained significantly higher than that of the PBS-LNP control group and the inactivated vaccine immunization group, while γδ cells that can produce IFN-γ were significantly higher. + There was no significant difference in the proportion of T cells (e.g. Figure 26 (As 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. Modified nucleoside mRNA vaccines delivered via LNP have shown reliable efficacy in the prevention and control of SARS-CoV-2 and RSV. mRNA vaccine technology has not only shown 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 nucleoside: (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; (4) GP2345m-LNP expressing GP2a, GP3, GP4 and GP5 fusion proteins (e.g., GP2a, GP3, GP4 and GP5 fusion proteins). Figure 1(As shown). Animal experiments showed that GP345m-LNP can efficiently induce humoral and cellular immune responses in mice and piglets. Although GP345m-LNP and the PRRSV inactivated vaccine failed to completely prevent PRRSV infection in immunized piglets, GP345m-LNP effectively reduced viral load in blood and lung tissue and reduced pathological damage to lung tissue, indicating that the GP345m-LNP vaccine has good immunoprotective effects and significant research and development potential.

[0333] PRRSV encodes multiple structural proteins, among which GP2a, as a structural protein, participates in the binding of PRRSV to cell receptors, indicating the presence of neutralizing epitopes. Several B-cell epitopes have been identified, but few T-cell epitopes have been found. GP3 is one of the major structural and immunogenic proteins of PRRSV, capable of stimulating humoral and cellular immune responses and playing an important role in viral replication, assembly, and pathogenicity. Even in the absence of neutralizing antibody responses, GP3 can provide protection for piglets. GP4 is a minor envelope glycoprotein containing B-cell and T-cell epitopes and is commonly used in vaccine design. GP5 is the most prominent envelope glycoprotein of PRRSV and is considered a major 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 all exhibit good immunogenicity and can effectively induce humoral and cellular immune responses. Based on the NADC30-like strain FJ1402 isolated and identified in our laboratory, this study selected GP2, GP3, GP4, and GP5 proteins as the basic antigens for the vaccine and designed four different combinations of fusion protein genes to construct mRNA vaccine target antigens. To ensure that the target protein translated from the mRNA can be effectively secreted extracellularly after expression within the cell and to promote CD4+ expression... + T cell response involved the deletion of the original signal peptides and transmembrane regions of GP2a, GP3, GP4, and GP5 proteins. The modified structural proteins were linked via a flexible linker (GGGGS)3 to form fusion proteins GP35, GP45, GP345, and GP2345. A tPA signal peptide was added to the N-terminus of the fusion proteins to guide them into the endoplasmic reticulum, followed by Golgi apparatus processing and secretion into the extracellular space. After encapsulating mRNA with LNPs, all four mRNA vaccines were effectively expressed in PK-15 and PAMs cells, with GP345m-LNP showing the highest expression level (e.g., ...). Figure 10(As shown in A and B in the diagram). GP45m-LNP, GP345m-LNP, and GP2345m-LNP can achieve effective secretory expression of the fusion protein in PK-15 cells. Similarly, GP345m-LNP has the highest secretory expression level, while GP35m-LNP cannot be effectively secreted. This may be because the fused GP35 protein is not properly folded or modified, thus being trapped in the cell, or triggering a protein degradation mechanism that prevents effective secretion (e.g., ...). Figure 10 (As shown in C). Although all four mRNA vaccines were effectively expressed in PAMs, none were secreted into the cell supernatant, which may be due to the characteristics of macrophages (e.g., Figure 10 (As shown in D in the diagram). Primary macrophages typically exhibit strong membrane stability and resistance, resisting the entry of exogenous nucleic acids into the cell, thus limiting the secretion of exogenous proteins. Furthermore, although macrophages have the ability to secrete cytokines and chemokines, their efficiency in secreting non-natural exogenous proteins may be low, leading to protein accumulation within the cell rather than efficient secretion. One of the main functions of macrophages is phagocytosis and degradation of exogenous substances. Even if exogenous proteins are successfully overexpressed, these proteins may be recognized as abnormal substances by macrophages and degraded through endocytosis rather than secreted extracellularly.

[0334] Following PRRSV infection, humoral immunity develops rapidly, but the production of neutralizing antibodies is relatively slow. Previous studies have shown that neutralizing antibody titers of 1:8 or higher can prevent PRRSV viremia and reduce viral load in tissues. In our mouse experiments, booster immunization with GP345m-LNP and GP2345m-LNP for 21 days resulted in the production of ELISA antibodies against GP3 ​​and GP5 proteins. The GMTs in the GP345m-LNP immunization group were 19,380 and 16,900, respectively, while those in the GP2345m-LNP immunization group were 12,800 and 8,450, indicating higher antibody titers in the GP345m-LNP immunization group. In piglet experiments, the GMTs of ELISA antibodies against GP3 ​​and GP5 proteins induced 21 days after booster immunization with GP345m-LNP were 4,230 and 6,400, respectively, lower than those in the inactivated vaccine immunization group. This may be related to mRNA expression levels and immunization dose. In the future, increasing the immunization dose of GP345m-LNP or optimizing the 5′UTR, 3′UTR, codons, and delivery system can enhance the protein expression level of mRNA vaccines, thereby improving the humoral immune response induced by mRNA vaccines. PRRSV may exhibit antibody-dependent enhancement (ADE), where non-neutralizing or sub-neutralizing antibody concentrations can enhance viral infection and replication. In this study, although IgG antibodies were produced after vaccination without the generation of neutralizing antibodies, no ADE-related enhancement of viral replication or pathological damage was observed. Although our GP345m-LNP antigen design integrated previously reported linear neutralizing epitopes of GP3 and GP5, it did not induce the production of neutralizing antibodies, whereas inactivated vaccines can induce the production of 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 PRRSV protein structural data hinders structure-based rational antigen design.

[0335] T-cell immunity is considered 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 clearing the virus from lymphoid tissues in the later stages of infection. In this study, GP345m-LNP induced mice to produce IFN-γ... + IL-2 + and IL-4 +Characterized by specific T-cell immune responses and lymphocyte proliferation. In the piglet immune challenge protection experiment, the GP345m-LNP immunization group significantly produced IFN-γ both before and after challenge. + CD4 + T cells and CD8 + T-cell response, while inactivated vaccines failed to induce cellular immune responses. Even 21 days after challenge, IFN-γ was not detected in piglets in the PBS-LNP control group and the inactivated vaccine immunization group. + T cell response indicates that GP345m-LNP immunization induced a PRRSV-specific cellular immune response.

[0336] It is noteworthy that the antibody levels induced by mRNA vaccines against GP2a and GP4 proteins were low in both mice and piglets, 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 be correctly formed in the constructed fusion protein, while GP3 and GP5 proteins contain more linear 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) The T cell epitopes in GP2a and GP4 proteins are insufficient, which cannot effectively induce the activation of follicular helper T (Tfh) cells, thereby affecting the production of high-affinity antibodies by Tfh cells to help B cells, resulting in low antibody titers against GP2a and GP4 proteins; (4) The host immune system may be more inclined to process and deliver the peptides of GP3 and GP5 in the fusion protein, preferentially recognizing the immunodominant epitopes in GP3 and GP5 in the fusion protein, thus suppressing the immune response against GP2a and GP4 proteins. In the future, fusion proteins can be optimized from the perspective of protein structure, adjusting the sequence and structure of each protein component, identifying and adding immune epitopes, and optimizing the design of fusion proteins to enhance the immune effect of mRNA vaccines.

[0337] LNPs used for mRNA delivery have potent adjuvant effects, primarily relying on their ionizable lipid components. These components stimulate innate immune system receptors such as TLR, MDA5, and NLRP3, inducing the production of IFN-γ, IL-6, and IL-1β, and promoting Tfh and germinal center B cell responses, thus enhancing adaptive immune responses. Adjusting the components of LNPs allows for flexible modification of mRNA vaccine properties, enabling organ-targeted delivery. PRRSV can spread rapidly through the respiratory system via the air; establishing effective mucosal immunity in the respiratory tract could effectively prevent PRRSV infection. Using lung-targeting mRNA vaccines to establish robust T-cell immunity in the lungs can play a crucial role in viral clearance and symptom relief. Induced memory T cells can provide long-term protection; therefore, developing mRNA vaccines capable of inducing local mucosal immunity in the respiratory tract is essential for future development.

[0338] PRRSV comprises multiple lineages and exhibits rapid genetic variation, with several prevalent strains posing a significant challenge to China's swine industry. Currently, the main prevalent strains in China belong to lineage 1, specifically NADC30-like and NADC34-like strains. Existing commercial vaccines offer limited protection against heterologous strains. This study evaluated the protective efficacy of GP345m-LNP and a commercially available inactivated vaccine against the NADC30-like FJ1402 strain. Future research should focus on mRNA vaccines that can induce broad-spectrum protective immunity, assessing cross-protective effects against different PRRSV lineages. Broad-spectrum protection is a key objective in vaccine development. Given the flexibility and speed of mRNA vaccine design, they provide a promising platform for addressing the variable PRRSV and developing broad-spectrum vaccines. Continued exploration of viral proteins capable of inducing protective immunity, combined with further antigen modification, holds promise for achieving broad-spectrum immune protection against PRRSV.

[0339] In summary, we have designed and constructed, for the first time, mRNA vaccines expressing GP35m-LNP, GP45m-LNP, GP345m-LNP, and GP2345m-LNP fusion proteins of PRRSV GP2a, GP3, GP4, and GP5, and evaluated humoral and cellular immune responses in mice and piglets. GP345m-LNP effectively induced humoral and cellular immune responses in mice and piglets, and provided effective protection for piglets after challenge, laying a research foundation for further development of broad-spectrum and 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, which encodes the fusion protein GP345 of GP3, GP4 and GP5 with the amino acid sequence shown in SEQ ID NO.3; the nucleotide sequence of the mRNA molecule GP345m is shown in SEQ ID NO.

7.

2. A liposome nanoparticle, characterized in that, The liposome nanoparticles are loaded with the porcine reproductive and respiratory syndrome virus mRNA molecule as described in claim 1.

3. The use of the mRNA molecule according to claim 1 in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccine.

4. The use of the liposome nanoparticles according to claim 2 in the preparation of porcine reproductive and respiratory syndrome virus mRNA vaccine.

5. A biomaterial associated with the porcine reproductive and respiratory syndrome virus mRNA molecule of claim 1, characterized in that, The biomaterial is selected from any of the following: (A1) A DNA molecule encoding the mRNA molecule of claim 1; (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).

6. The use of the biomaterial described in claim 5 in the preparation of a porcine reproductive and respiratory syndrome virus mRNA vaccine.

7. A porcine reproductive and respiratory syndrome virus mRNA vaccine, characterized in that, The mRNA vaccine contains the mRNA molecule GP345m; the mRNA molecule GP345m encodes the fusion protein GP345 of GP3, GP4 and GP5 with the amino acid sequence shown in SEQ ID NO.3; the nucleotide sequence of the mRNA molecule GP345m is shown in SEQ ID NO.

7.

8. The porcine reproductive and respiratory syndrome virus mRNA vaccine according to claim 7, characterized in that, The porcine reproductive and respiratory syndrome virus mRNA vaccine is an mRNA-LNP complex formed by encapsulating the mRNA molecule GP345m in liposome nanoparticles.

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