Mrna-lnps vaccine for prevention of classical swine fever and method of preparation thereof

Abstract

The application discloses an mRNA-LNPs vaccine for preventing classical swine fever and a preparation method thereof. The E2 protein of a swine fever virus is fused with XCL1 and ubiquitin respectively to obtain two kinds of chimeric antigens, mRNA encoding the two kinds of chimeric antigens and single E2 is encapsulated in a lipid nanoparticle respectively to construct an mRNA-LNPs vaccine; the immunogenicity and protective effect of the three kinds of mRNA-LNPs and E2 subunit vaccines are systematically evaluated in a rabbit and piglet model, and the results show that, compared with the unmodified E2-mRNA-LNP and E2 subunit vaccine, the Ub-E2-mRNA-LNP modified by the N-terminal ubiquitin and the XCL1-E2 mRNA-LNP targeting dendritic cells can all induce strong humoral and cellular immunity, and can all provide complete protection for pigs infected with the swine fever virus virulent Shimen strain, and the vaccine provided by the application has application prospects in preventing classical swine fever.

Description

Technical Field

[0001] This invention relates to mRNA vaccines for animals, and more particularly to an mRNA-LNPs vaccine for the prevention of classical swine fever and its preparation method, belonging to the field of mRNA vaccines for the prevention of classical swine fever. Background Technology

[0002] Classical swine fever (CSF) is a highly contagious disease caused by classical swine fever virus (CSFV), resulting in significant economic losses to global pig production. The CSFV C strain (a rabbit-modified live attenuated vaccine) is considered one of the most effective vaccines for controlling CSF globally. However, the widely used CSFV C strain in China has failed to provide cross-protection against the newly emerging type 2.1 variant (Yoo SJ, Kwon T, Kang K, Kim H, Kang SC, Richt JA, Lyoo YS. 2018. Genetic evolution of classical swine fever virus under immune environments conditioned by genotype 1-based modified live virus vaccine. Transbound. Emerg. Dis. 65(3):735-745. doi: 10.1111 / tbed.12798). In addition, a novel, highly virulent CSFV 2.1c strain isolated from southern China can cause a mortality rate of up to 60% in pigs. Infected animals exhibit persistent fever, high viremia, and systemic inflammation, which differs from the low virulence usually associated with gene 2.1 mutations, complicating CSF control measures (Gao X, Wu Y, Song Y, Huang F, Lin L, Zhao H, Ren B, Li Q, Gong L. 2025. Isolation and pathogenicity of an emerging highly virulent CSFV 2.1c strain in southChina.Vet. Sci. 12(7):606. doi: 10.3390 / vetsci12070606.).Although modified live CSFV vaccines (MLV) can effectively induce neutralizing antibodies and cellular immune responses, the antibody response induced by MLV is serologically indistinguishable from that induced by natural infection, leading to the inability to distinguish between infected animals and vaccinated animals (Differentiation of infected from vaccinated animals, DIVA) (Huang YL, Deng MC, Wang FI, Huang CC, Chang CY. 2014. The challenges of classical swinefever control: modified live and E2 subunit vaccines. Virus Res.179:1-11.doi: 10.1016 / j.virusres.2013.10.025.).

[0003] E2 glycoprotein, as the main envelope protein of CSFV, is a major target of neutralizing antibodies and has been widely used in the development of subunit vaccines, including the research and development of novel marker E2 subunit vaccines. However, traditional E2 subunit vaccines and CSFV C strains have key limitations, including reliance on potent adjuvants to enhance immunogenicity, difficulty in inducing potent cellular immunity, and the need for multiple immunizations (Huang YL, Deng MC, Wang FI, Huang CC, Chang CY. 2014. Thechallenges of classical swine fever control: modified live and E2 subunitvaccines. Virus Res. 179: 1-11. doi: 10.1016 / j.virusres.2013.10.025.). Furthermore, while E2 subunit vaccines have the advantage of compatibility with DIVA vaccines, their protective efficacy is still lower than that of MLV vaccines.

[0004] As professional antigen-presenting cells (APCs), dendritic cells (DCs) are responsible for phagocytizing and processing antigens and presenting them to Major Histocompatibility Complex (MHC) class I or II molecules, thereby activating naive T cells and triggering antigen-specific adaptive immunity (Mellman I, Steinman RM. 2001. Dendritic cells: specialized and regulated antigen processing machines. Cell 106(3):255-258. doi: 10.1016 / s0092-8674(01)00449-4.). In fact, ubiquitin (Ub)-based antigen targeting strategies have been widely used to construct T-cell epitope vaccines against a variety of viral pathogens. By fusing the N-terminus of an antigen with a G76A-mutated Ub sequence, the proteasome can be degraded, rapidly generating immunogenic peptides and efficiently presenting them to MHC-I molecules, significantly enhancing APC-mediated antigen presentation. Furthermore, fusing the antigen with XC motif chemokine ligand 1 (XCL1) allows XC motif chemokine receptor 1 (XCR1) expressed on the surface of dendritic cells (DCs) to bind to XCL1, thereby promoting antigen capture by DCs. The XCR1-XCL1 interaction enhances the ability of DCs to capture and process antigens and present antigen peptides to CD8+ T cells via MHC-I molecules, thus inducing cellular immune responses. The XCR1-XCL1 axis plays a crucial role in antiviral immunity by enhancing cross-presentation and cytotoxic T lymphocyte (CTL) responses. Therefore, fusing the antigen with XCL1 can significantly improve vaccine efficacy by targeting the antigen to XCR1+ DCs.

[0005] In recent years, mRNA vaccines have shown promise in overcoming the limitations of traditional CSF vaccines. mRNA vaccines utilize host translation mechanisms to synthesize pathogen-specific antigens, thereby inducing strong humoral and cellular immune responses (Chaudhary N, Weissman D, Whitehead KA. 2021. mRNA vaccines for infectious diseases:principles,delivery and clinical translation. Nat. Rev. Drug Discov. 20(11):817-838.doi: 10.1038 / s41573-021-00321-2.). Furthermore, the mRNA platform offers significant advantages in rapid vaccine development and large-scale production. The successful global launch of mRNA vaccines against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) demonstrates that this platform has been successfully applied to the development of mRNA vaccines against various infectious diseases, including COVID-19.

[0006] Lipid nanoparticles (LNPs) have emerged as a novel delivery platform for mRNA vaccines. Their hydrophobic core encapsulates and protects mRNA, enabling efficient cellular uptake and intracellular release. LNPs exhibit excellent safety, high transfection efficiency, and the ability to induce potent immune responses, making them a promising delivery system for vaccine strategies. Therefore, recent advances in mRNA technology offer new avenues for CSF vaccine design.

[0007] Traditional E2 subunit vaccines have limited ability to rapidly adapt to newly emerging strains and suffer from insufficient induction of cellular immune responses. Therefore, developing an mRNA-LNPs vaccine for the prevention of classical swine fever based on an E2 antigen fusion strategy and an mRNA lipid nanoparticle delivery platform will effectively improve the immunoprotective efficacy of vaccines against classical swine fever. Summary of the Invention

[0008] One of the objectives of this invention is to provide a chimeric antigen obtained by fusing classical swine fever virus E2 protein with XC motif chemokine ligand 1 (XCL1) or ubiquitin (Ub), respectively. The second objective of this invention is to provide an mRNA-LNPs vaccine for the prevention of swine fever; A third objective of this invention is to provide a method for preparing the mRNA-LNPs vaccine for preventing swine fever.

[0009] The above-mentioned objectives of the present invention are mainly achieved through the following technical solutions: One aspect of the present invention provides a chimeric antigen obtained by fusing classical swine fever virus E2 protein with XCL1 or Ub, wherein the amino acid sequence of the chimeric antigen obtained by fusing classical swine fever virus E2 protein with XCL1 is shown in SEQ ID No. 1, and the amino acid sequence of the chimeric antigen obtained by fusing classical swine fever virus E2 protein with Ub is shown in SEQ ID No. 2.

[0010] Another aspect of the present invention provides an mRNA-LNPs vaccine for the prevention of classical swine fever, comprising mRNA and lipid nanoparticles, wherein the mRNA is encapsulated in the lipid nanoparticles; wherein the mRNA contains a coding sequence encoding a chimeric antigen obtained by fusing classical swine fever virus E2 protein with XCL1 or Ub, respectively.

[0011] Another aspect of the present invention provides a method for preparing the aforementioned mRNA-LNPs vaccine, comprising: (1) The gene sequence encoding the E2 protein was cloned into the pLVX-XCL1 or pLVX-Ub plasmid to obtain a plasmid containing the gene encoding the E2 protein; the plasmid was used to synthesize mRNA by in vitro transcription using T7 RNA polymerase; (2) mRNA was encapsulated in lipid nanoparticles to obtain an mRNA-LNPs vaccine for the prevention of swine fever.

[0012] In a preferred embodiment of the present invention, the sequence of the plasmid containing the gene encoding the E2 protein includes the following elements from the T7 promoter to the 3' end: 5' UTR, Kozak sequence, tissue plasminogen activator (tPA) signal sequence, XCL1 or Ub gene, E2 gene, V5 tag, 3' UTR and polyadenylate tail.

[0013] The nucleotide sequence of the XCL1 gene is shown in SEQ ID No. 3, the nucleotide sequence of the Ub gene is shown in SEQ ID No. 4, the nucleotide sequence of the E2 gene is shown in SEQ ID No. 5, and the nucleotide sequence of the tissue plasminogen activator (tPA) signal sequence is shown in SEQ ID No. 6.

[0014] In a preferred embodiment of the present invention, uridine in the UTR region is completely replaced with N1-methylpseudouridine-5'-triphosphate (m1ΨTP) during the in vitro transcription synthesis of mRNA.

[0015] In a preferred embodiment of the present invention, in order to enhance mRNA stability and translation efficiency, a Cap1 structure is introduced at the 5' end of the mRNA.

[0016] In a preferred embodiment of the present invention, the method for preparing the lipid nanoparticles includes: (1) Dissolve ionizable lipids, DSPC, cholesterol and DMG-PEG2000 in ethanol to obtain a lipid-ethanol mixture; (2) Dissolve mRNA in sodium citrate buffer and mix with the lipid-ethanol mixture; (3) Collect the sample, remove ethanol by ultrafiltration (100 kDa ultrafiltration centrifuge tube), add sucrose as a protectant, and filter sterile through a 0.22 μm PTFE filter membrane to obtain the final product.

[0017] In a preferred embodiment of the present invention, in step (1), the ratio of ionizable lipids, DSPC, cholesterol and DMG-PEG2000 is 50:38.5:10:1.5 based on the molar ratio.

[0018] In a preferred embodiment of the present invention, in step (2), mRNA is dissolved in 50 mM acetate-sodium acetate buffer and mixed with lipid-ethanol mixture at a flow rate ratio of 3:1, with a total flow rate of 30 mL / min.

[0019] In a preferred embodiment of the present invention, the ultrafiltration in step (3) is performed using a 100 kDa ultrafiltration centrifuge tube; the protective agent is sucrose.

[0020] This invention fuses the classical swine fever virus E2 protein with XC motif chemokine ligand 1 (a class C chemokine, XC motif chemokine ligand, XCL1, which targets dendritic cells via XCL1 fusion) and ubiquitination (Ub, which enhances antigen presentation through ubiquitination-mediated degradation) to obtain two chimeric antigens; further, it fuses antigens encoding a transmembrane domain-free protein. Three novel mRNA-LNP vaccines were constructed from the mRNA of the classical swine fever virus (CSFV) E2 protein and the mRNA encoding two chimeric antigens: E2-mRNA-LNP, XCL1-E2-mRNA-LNP, and Ub-E2-mRNA-LNP. This invention systematically evaluated the immunogenicity and protective efficacy of different mRNA-LNPs and E2 subunit vaccines in rabbit and piglet models. The results showed that, compared with unmodified E2 mRNA-LNP and E2 subunit vaccines, both N-terminal ubiquitin-modified Ub-E2-mRNA-LNP and XCL1-E2 mRNA-LNP targeting dendritic cells (DCs) could induce strong humoral and cellular immunity. Furthermore, Ub-E2-mRNA-LNP and XCL1-E2... Both mRNA-LNPs can provide complete protection for pigs infected with CSFV-Shimen (CSFV-SM). The Ub-E2-mRNA-LNP and XCL1-E2-mRNA-LNP provided by this invention are promising candidate vaccines for the prevention of classical swine fever. Attached Figure Description

[0021] Figure 1 Design and expression validation of different E2 protein mRNA vaccines; (A) Schematic diagram of mRNA vaccine design; The genes encoding E2 protein, XCL1-E2 chimeric protein, or ubiquitinated E2 chimeric protein were cloned into template plasmid vectors containing 5'Cap, 5'UTR, 3'UTR, and 3'poly (A), respectively; (BG) Expression of pLVX-E2, pLVX-XCL1-E2, and pLVX-Ub-E2 in HEK293T cells; The expression of E2 (B and C), XCL1-E2 (D and E), and ubiquitinated E2 (F and G) was detected using anti-V5 tag antibody or anti-E2 antibody; (H) Ubiquitination modification of Ub-E2; The ubiquitination modification of Ub-E2 was analyzed by Western blotting using anti-ubiquitination antibody.

[0022] Figure 2The preparation and characterization results of different CSFV E2-mRNA-LNPs are as follows: (A) Physicochemical characterization of different CSFV E2-mRNA-LNPs; the zeta potential and particle size of different CSFV E2-mRNA-LNPs were determined by zeta potential analysis and DLS, respectively; (B) and (C) E2 expression detection of different CSFV E2-mRNA-LNPs; the protein expression of different CSFV E2-mRNA-LNPs was detected by anti-V5 tag antibody and anti-E2 antibody.

[0023] Figure 3 The following are the results of efficacy evaluation of different CSFV E2-mRNA-LNPs in rabbits; (A) Schematic diagram of rabbits immunized with different CSFV E2-mRNA-LNPs; (B) Blocking rate of CSFV-specific antibodies in the serum of immunized rabbits; (C) Rectal temperature of rabbits immunized after challenge with CSFV strain C; (D) Viral genome copy number / mL in the spleen of immunized rabbits 72 hours after challenge (Hours postchallenge, hpc); Data are expressed as mean ± standard deviation. ns indicates no statistical difference (P=0.05); * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001.

[0024] Figure 4 Results of a study on the complete protection of Ub-E2-mRNA-LNP against lethal CSFV challenge in piglets; (A) Vaccination regimens for piglets using different E2-based mRNA-LNP constructs; (B)-(D) Evaluation of 10 vaccinations 5.0 TCID 50 The efficacy of the CSFV-SM challenge vaccine in pigs was assessed using parameters including survival rate (B), rectal temperature (C), and clinical scores (D) and anatomical damage scores based on typical CSF clinical symptoms (E); (F) representative gross lesions of immunized piglets after CSFV challenge, including spleen, submandibular lymph nodes, kidneys, inguinal lymph nodes, and tonsils.

[0025] Figure 5 The results of the assessment of viral replication levels in immunized piglets after CSFV challenge include: viral replication levels in blood samples (A), oral swabs (B), nasal swabs (C), and rectal swabs (D).

[0026] Figure 6Results of humoral and cellular immune responses in piglets induced by CSFV E2-mRNA-LNPs; (A) Antibody response induced by CSFV E2-mRNA-LNPs, the blocking rate of anti-CSFV antibodies in the serum of immunized piglets was detected by blocking ELISA; (B) Cellular immune response induced by CSFV E2-mRNA-LNPs, the quantitative analysis of CSFV-specific IFN-γ secreted by peripheral blood mononuclear cells of immunized piglets by enzyme-linked immunospot assay (ELISpot). γ The number of PBMCs. Detailed Implementation

[0027] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, these experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0028] Biomaterials and testing methods 1. Cells and Viruses HEK293T and PK-15 cells were cultured in DMEM medium (catalog number D6429, Gibco) containing 5% antibiotics (10,000 IU / mL penicillin, 10,000 μg / mL streptomycin; catalog number 15240-062, Gibco) and 10% heat-inactivated fetal bovine serum (FBS; catalog number 10270106, Gibco). CSFV C strain (GenBank accession number AY805221) and CSFV-SM strain (GenBank accession number AF092448.2) were passaged in PK-15 cells.

[0029] 2 Elispot

[0030] The secretion of IFN-γ was detected using the ELIspot kit (catalog number 3130-4APW-2, Mabtech). γ The number of T cells was determined according to the manufacturer's instructions. In short, the pre-coated culture plate was placed in RPMI-1640 medium (200 μL / well) containing 10% FBS and incubated at room temperature for 30 min. Afterwards, the medium was discarded, and 2.5 × 10⁶ cells were added to each well. 5.0 10 cells and 10 5.0 TCID 50CSFV was used as the experimental group, with culture medium added as the negative control and 5 μg phytohemagglutinin (PHA) added to each well as the positive control. The culture plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Afterward, the cells were discarded, and the plates were washed five times with 200 μL / well of PBS. The detection antibody (P2C11-biotin) was diluted to 0.5 μg / mL with PBS containing 0.5% FBS (PBS-0.5% FCS), and 100 μL of the diluted antibody was added to each well. After incubation at room temperature for 2 h, the plates were washed five times with PBS. 100 μL of streptavidin-HRP (1:1000) diluted in PBS-0.5% fetal bovine serum (PBS-0.5% FCS) was added to each well, and after incubation at room temperature for 1 h, the plates were washed five times with 200 μL of PBS. 100 μL of TMB substrate solution was then added to each well until distinct spots appeared, and the reaction was terminated by thorough washing with deionized water. After drying, the spots were quantitatively analyzed using an ELIspot microplate reader and AID software. The number of IFN-γ-secreting T cells per million PBMCs was expressed as the spot-forming cell count. A negative control was subtracted from the peptide mimicry set as background, and each set was configured in triplicate.

[0031] 3 ELISA

[0032] All reagents in the CSFV ELISA kit (catalog number 99-43220, IDEXX) were equilibrated to room temperature. Then, 50 μL of standards at different concentrations were added to the standard wells, and 50 μL of serum samples were added to the sample wells. Next, 100 μL of detection antibody was added to each well, and the mixture was incubated at 37°C for 2 h. After washing five times with washing buffer, 50 μL of substrate A and substrate B were added to each well, and the mixture was incubated at 37°C in the dark for 15 min. Finally, 50 μL of stop solution was added to each well to terminate the reaction, and the absorbance (OD) was immediately measured at 450 nm. 450nm Using standards of known concentrations and their corresponding OD values. 450nm A standard curve was plotted, and the concentration of cytokines in the serum of each sample was then calculated from the curve. Based on the cutoff value of the ELISA kit, a result was considered positive if the blocking rate was ≥40%.

[0033] 4 RT-qPCR

[0034] Total RNA was extracted using the RNASimply Total RNA Kit (catalog number DP419, Tiangen). RNA was reverse transcribed into cDNA in vitro using the Tiangen FastKinggDNA Dissociation RT Supermix Kit (catalog number KR118-03, Tiangen). The CSFV genome copy number in the samples was quantified by RT-qPCR as described above.

[0035] Experiment 1: Preparation and Physicochemical Property Determination of CSFV E2-mRNA-LNPs 1 Experimental Methods 1.1 Vaccine Design The gene sequence encoding the E2 protein (GenBank accession number KC597187.1) was cloned into the pLVX-Tev plasmid (its nucleotide sequence is shown in SEQ ID No. 7). The resulting plasmid sequence, from the T7 promoter to the 3' end, contains the following elements: 5' UTR, Kozak sequence, tPA signal sequence, XCL1 or Ub gene, E2 gene, V5 tag, 3' UTR, and polyadenylated tail. mRNA was synthesized via in vitro transcription using T7 RNA polymerase, with the uridine in the UTR region completely replaced by N1-methylpseudouridine-5'-triphosphate (m1ΨTP). A Cap1 structure was introduced at the 5' end using Cleancap reagent AG (catalog number N-7113-10, TriLink) to enhance mRNA stability and translation efficiency.

[0036] 1.2 Preparation of lipid nanoparticles In vitro transcription protocol: Take out the circular plasmids (eukaryotic expression plasmids pLVX-E2, pLVX-XCL1-E2, and pLVX-Ub-E2) to be digested, dilute the concentration to 500 ng / μL, select the amount of circular plasmid to be digested, and perform the digestion. The digestion system and the amount of each component are shown in Table 1.

[0037] Table 1 Plasmid digestion system

[0038] Place the prepared enzyme digestion system in a constant temperature incubator at 37℃ for 1 h. Prepare a 0.8% agarose gel. Take 1 μL of the enzyme digestion system sample and add 1 μL + 1 μL 10× Loading Buffer + 8 μL pure water. Load the same volume of 1 μL (circular plasmid) + 1 μL 10× Loading Buffer + 8 μL pure water into adjacent wells. Load the non-linearized plasmid as the digestion control. Next to the two plasmids, load 6 μL of DNA Marker as a fragment size reference control. Then perform electrophoresis at 150 V for 30 min. During electrophoresis, the enzyme digestion system can be placed at room temperature to wait for the results.

[0039] Gel imaging: The 0.8% agarose gel was examined using a gel imaging system. The original gel image was saved, and the linearized plasmid was compared with the circular plasmid.

[0040] Enzyme digestion plasmid recovery: Since the linearization efficiency of different plasmids is inconsistent, the linearization time needs to be adjusted according to the results of simultaneous gel electrophoresis. After the plasmid is completely linearized, phenol-chloroform is used for recovery.

[0041] Phenol-chloroform recovery steps: Add an equal volume (500 μL) of phenol:chloroform:isoamyl alcohol (phenol:chloroform:isoamyl alcohol = 25:24:1) to a 1.5 mL EP tube; invert the EP tube to mix, then centrifuge at 13500 rpm for 1 min; transfer the supernatant to a new 1.5 mL EP tube using a pipette. Add an equal volume of chloroform, mix, and centrifuge at 12000 rpm for 1 min. Transfer the supernatant to a new 1.5 mL EP tube using a pipette. Add 2 volumes of anhydrous ethanol and 0.1 volumes of NaAc solution (3 M concentration), and freeze at -20°C for 30 min. After freezing, centrifuge at 12000 rpm for 15 min, remove the supernatant, add 600 μL of 70% ethanol to the precipitate, centrifuge at 12000 rpm for 5 min, remove the supernatant, and repeat twice. Place the EP tube at room temperature and allow the ethanol to evaporate completely. Then, add 100 μL of enzyme-free water to the precipitate to dissolve it and bring the volume up to 0.5 μg / μL.

[0042] mRNA in vitro transcription (IVT): Before the experiment, thaw all components. Thaw the enzyme mixture on ice and thaw other reagents at room temperature. Before adding each reagent, shake well to mix, briefly incubate, and then place on ice for later use (for the enzyme mixture, incubate briefly and then place on ice for later use). The IVT system is shown in Table 2.

[0043] Table 2 IVT Reaction System

[0044] IVT experimental operation steps: Add the components to the eight-tube array in the order shown in Table 2. After adding the components, cap the eight-tube array, vortex for 5 seconds, and centrifuge briefly for 3 seconds. If air bubbles are present, tap the tube lightly with your finger and centrifuge briefly again. Place the eight-tube array in an incubator and incubate at 37°C for 2 hours. Remove the sample, carefully open the cap of the eight-tube array, add 0.1 times the volume of DNase I (2 μL) to each well, cap the eight-tube array, vortex for 5 seconds, and centrifuge briefly for 3 seconds. If air bubbles are present, tap the tube lightly with your finger and centrifuge briefly again. Place the eight-tube array in an incubator and incubate at 37°C for 30 minutes. Remove the sample, carefully open the cap of the eight-tube array, add 22 μL of 5M LiCl to each well, vortex for 5 seconds, centrifuge briefly for 3 seconds, and precipitate at -20°C for 30 minutes. Remove the eight-tube array, centrifuge for 15 minutes, and discard the supernatant. Add 100 μL of pre-chilled 70% ethanol, centrifuge for 5 min, and discard the supernatant. Repeat the step, discarding the supernatant and centrifuging again for 10 s to remove as much residual ethanol as possible. Place the sample tubes in a 37°C incubator and dry for 5 min. Remove the samples, add 100 μL of purified water to each, place on ice for 30 min, and gently reconstitute using a 100 μL pipette. Detect the concentration using a micro-volume spectrophotometer, selecting RNA-40 for concentration detection, perform the test twice, and record the results.

[0045] Encapsulation protocol: 1-octylnonyl8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate (SM-102, catalog number SDP13, Shengdi), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypoly(ethylene glycol)-2000 (DMG-PEG2000) were dissolved in anhydrous ethanol at a molar ratio of 50:38.5:10:1.5 to a total concentration of 30 mM; mRNA was dissolved in 10 mM acetate-sodium acetate buffer. Using a microfluidic preparation system, the lipid phase and mRNA solution were mixed at a flow rate ratio of 1:3 (FRR = 3), with a total flow rate of 30 mL / min. The nitrogen-phosphorus molar ratio (N / P) was 6 (N / P = 6). The sample was collected, and after removing ethanol by ultrafiltration (100 kDa ultrafiltration centrifuge tubes) (replacement buffer was 20 mM Tris-HCl buffer (pH 7.5)), sucrose was added as a preservative, and the sample was sterilized by filtration through a 0.22 μm PTFE membrane to obtain the final product.

[0046] Detection of the physicochemical properties of mRNA-LNP: 1) Visual inspection to detect mRNA-LNP characteristics; 2) pH value: Take an appropriate amount of sample and measure the pH value using a pH meter; 3) Particle size and polydispersity index (PDI): Take an appropriate amount of sample and measure and record the Z-Average particle size using the Zetasizer Nano Pro (Malvern Instruments, UK) at 25℃ using the dynamic light scattering (DLS) method. The particle size range is 40~180 nm, and the PDI is <0.3; 4) Apparent potential: Measure the ζ-potential using the Zetasizer Nano Pro (Malvern Instruments, UK); 5) mRNA content and encapsulation efficiency: Measure the total mRNA content and free mRNA content using the Quant-iT Ribogreen RNA Quantification Assay (ThermoFisher Scientific) according to the product instructions (external standard curve method), and calculate the mRNA encapsulation efficiency. The calculation formula is: Encapsulation efficiency (%) = [(total mRNA−free mRNA) / total mRNA] × 100%.

[0047] 1.3 Transfection and Western blotting HEK293T cells encoded by XCL1-E2 (pLVX-XCL1-E2), Ub-E2 (pLVX-Ub-E2), or E2 alone (pLVX-E2) were transfected into 24-well cell culture plates. Cells were then incubated at 37°C with 5% CO2 for 48 h. Transfected and untransfected HEK 293T cells were collected and lysed, and protein samples were analyzed by Western blotting. PVDF membranes were incubated at room temperature for 2 h with the corresponding primary antibody (anti-V5 tag antibody, rabbit polyclonal antibody, Abcam, 1:1000 dilution) or anti-GAPDH antibody (rabbit polyclonal antibody, Abcam, 1:5000 dilution). Subsequently, the PVDF membranes were incubated at room temperature for 1 h with horseradish peroxidase-labeled anti-rabbit secondary antibody (donkey polyclonal antibody, Jackson Immuno Research, 1:20000 dilution). The signal was developed using a tetramethylbenzidine substrate solution (Beyotime) and imaged using an Azure Biosystems imaging system.

[0048] 2. Experimental Results 2.1 Expression of CSFV E2 eukaryotic expression plasmid To enhance the ability of mRNA vaccines to stimulate immune responses, this experiment, for the first time, fused XCL1 or Ub with E2 proteins to construct chimeric antigens XCL1-E2 or Ub-E2, aiming to improve the efficiency of antigen recognition, processing, and handling by antigen-presenting cells. Therefore, this experiment constructed eukaryotic expression plasmids expressing three different proteins, including: pLVX-E2 (E2 protein without TMDs), Ub-E2 (Ub-E2 chimeric protein), and XCL1-E2 (XCL1-E2 chimeric protein). Figure 1 A).

[0049] In this experiment, eukaryotic expression plasmids pLVX-E2, pLVX-XCL1-E2, and pLVX-Ub-E2 were transfected into HEK293T cells. The expression of E2 protein was analyzed using either an anti-V5 tag antibody or an anti-E2 antibody. The results showed that pLVX-E2, pLVX-XCL1-E2, or pLVX-Ub-E2 expressed the target proteins E2, XCL1-E2, and Ub-E2, respectively. Figure 1 BG).

[0050] To further verify the ubiquitination modification of Ub-E2 protein, the pLVX-Ub-E2 plasmid was transfected into HEK293T cells, and the cells were treated with or without the proteasome inhibitor MG132 8 h post-transfection. Compared with the sample treated with MG132, the sample without MG132 treatment produced a large amount of ubiquitinated protein, which is consistent with proteasome inhibition. Figure 1 H). The results of this experiment indicate that the Ub-E2 protein can undergo polyubiquitination modification via the proteasome pathway.

[0051] 2.2 Physicochemical properties of different CSFV E2-mRNA-LNPs Eukaryotic expression plasmids pLVX-E2, pLVX-XCL1-E2, and pLVX-Ub-E2 were synthesized into E2-mRNA-LNP, XCL1-E2-mRNA-LNP, and Ub-E2-mRNA-LNP, respectively, through in vitro transcription and LNP encapsulation. Zeta potential analysis showed that the surface charge range of the three CSFV E2-mRNA-LNPs was -1.90 to 0.223 mV. DLS analysis showed that the particle size of the three CSFV E2-mRNA-LNPs was between 82.80 and 91.68 nm. In summary, the three different CSFV E2-mRNA-LNPs all exhibited lipid nanoparticles with uniform particle size and stable charge. Figure 2 A).

[0052] To verify the in vitro expression of E2-mRNA-LNP, XCL1-E2-mRNA-LNP, or Ub-E2-mRNA-LNP, HEK293T cells were transfected with these three CSFV E2-mRNA-LNPs, and protein samples were collected for Western blot analysis. The results showed that HEK293T cells transfected with E2-mRNA-LNP, XCL1-E2-mRNA-LNP, or Ub-E2-mRNA-LNP successfully expressed E2, XCL1-E2, and Ub-E2 proteins, respectively. Figure 2 B and Figure 2 C).

[0053] Experiment Example 2: Immunization and Rabbit Challenge Experiments with Different CSFV E2-mRNA-LNPs 1 Experimental Methods To evaluate the immunogenicity of different CSFV E2-mRNA-LNPs, rabbits were immunized by intramuscular injection of 30 μg of E2 subunit vaccine, E2-mRNA-LNP, XCL1-E2-mRNA-LNP, Ub-E2-mRNA-LNP, or 1 mL of PBS in the right hind limb. The immunization schedule was as follows: Figure 3 A. The specific antibodies against E2 in the post-immunization serum were detected using a blocking ELISA. The specific method is as follows: Nineteen 12-week-old New Zealand white rabbits were randomly divided into five groups and immunized intramuscularly with 30 μg E2-mRNA-LNP (n=4), XCL1-E2-mRNA-LNP (n=4), Ub-E2-mRNA-LNP (n=4), E2 subunit vaccine (positive control, n=4), or 1 mL PBS (negative control, n=3), respectively. Each group received two immunizations on days 0 and 14. Serum samples were collected at days 0, 7, 14, and 21 post-immunization (dpv). Serum anti-CSFV E2 antibody levels were quantified using an enzyme-linked immunosorbent assay (ELISA). At 21 dpv, all rabbits received an intravenous injection of 10 μg E2-mRNA-LNP via the marginal ear vein. 5.0 TCID 50 CSFV strain C virus was detected. Rectal temperature was recorded every 6 hours from 24 to 72 hpc to monitor typing-related heat response in rabbits. At 72 hpc, all rabbits were euthanized by intravenous injection of sodium pentobarbital (20 mg / kg), and spleen tissue was collected for RT-qPCR to detect the copy number of the CSFV genome.

[0054] 2. Experimental Results Experimental results showed that the serum antibody blocking rates of the E2 subunit vaccine, XCL1-E2-mRNA-LNP, or Ub-E2-mRNA-LNP were significantly higher than those of the E2-mRNA-LNP and PBS groups. Furthermore, at 21 dpv, the serum of rabbits in the E2-mRNA-LNP group began to turn seropositive, but its antibody blocking rate was significantly lower than that of the E2 subunit vaccine, XCL1-E2-mRNA-LNP, and Ub-E2-mRNA-LNP groups. Figure 3 B). The above experimental results show that, compared with the E2-mRNA-LNP group, both XCL1-E2-mRNA-LNP and Ub-E2-mRNA-LNP, which fuse XCL1 and Ub with E2, can significantly enhance antibody production by effectively improving antigen capture and processing. Furthermore, the ability of E2 modified with XCL1 and Ub to induce antibody production is significantly enhanced.

[0055] At 21 dpv, all rabbits were infected with 100 MIDs. 50 CSFV strain C virus was used. Rectal temperature was monitored continuously every 6 hours starting at 24 hpc until 72 hpc. Results showed that rabbits in the E2 subunit vaccine, XCL1-E2-mRNA-LNP, or Ub-E2-mRNA-LNP groups did not exhibit a typing fever response. In contrast, one rabbit in the E2-mRNA-LNP group showed a fever similar to that in the PBS group. Figure 3 C). Furthermore, RT-qPCR detection revealed that CSFV was undetectable in rabbits immunized with the E2 subunit vaccine, XCL1-E2-mRNA-LNP, and Ub-E2-mRNA-LNP. Notably, viral RNA was detected in one rabbit in the E2-mRNA-LNP group and all rabbits in the PBS group, demonstrating that E2-mRNA-LNP only provides partial protection against CSFV C strain infection in rabbits. Figure 3 (D) In ​​contrast, rabbits in the E2 subunit vaccine group, the XCL1-E2-mRNA-LNP group, or the Ub-E2-mRNA-LNP group provided complete protection against CSFV strain C infection. Different CSFVE2-mRNA-LNPs provided complete protection against challenge with rabbit strain C.

[0056] Experiment Example 3: Immune challenge experiment on piglets with different CSFV E2-mRNA-LNPs 1 Experimental Methods Nineteen healthy, specific pathogen-free (SPF) piglets, aged 4 weeks and weighing 11.5–13.5 kg, were purchased from the Experimental Animal Center of Harbin Veterinary Research Institute. The piglets were randomly divided into 5 groups and administered the following vaccines via intramuscular injection: E2 subunit vaccine (n=4, one dose / piglet), XCL1-E2-mRNA-LNP (n=4, 100 μg / piglet), Ub-E2-mRNA-LNP (n=4, 100 μg / piglet), E2-mRNA-LNP (n=4, 100 μg / piglet), or PBS (n=3, 2 mL / piglet). Each group was immunized twice, at 0 and 14 days. Serum samples were collected from all piglets at 0, 3, 7, 10, 14, 21 and 28 days post-vaccination (dpv). Peripheral blood mononuclear cells (PBMCs) were isolated, and the number of IFN-γ secreting cells was measured using ELIspot. The level of anti-CSFVE2 antibody was quantified by blocking ELISA.

[0057] At 28 dpv, with 10 5.0 TCID 50 All piglets were infected with CSFV-SM virus via intramuscular injection. Anticoagulated blood, serum, oral, nasal, and anal swabs were collected at 0, 3, 7, and 14 days post-challenge (dpc). CSFV genome copy number was detected using RT-qPCR. At 14 dpc, piglets were anesthetized and euthanized by intravenous injection of zoletil (6 mg / kg). Spleen, kidney, tonsils, and lymph nodes were collected from the euthanized piglets.

[0058] 2. Experimental Results 2.1 Piglets immunized with antigen-modified CSFV E2-mRNA-LNPs were completely resistant to CSFV infection. To further evaluate the immunoprotective efficacy of different CSFV E2-mRNA-LNPs, piglets were vaccinated with Ub-E2-mRNA-LNP, XCL1-E2-mRNA-LNP, E2-mRNA-LNP, E2 subunit vaccine, or PBS, respectively. Subsequently, all piglets were infected with 10 dpv vaccines at 28 days. 5.0 TCID 50 CSFV-SM ( Figure 4A). Survival rate, rectal temperature, and clinical scores were recorded daily for 14 consecutive days. Simultaneously, whole blood, oral swabs, nasal swabs, and anal swabs were collected at 0, 3, 7, 10, and 14 dpc, and CSFV genome copy number was detected by RT-qPCR. The results showed that all piglets vaccinated with Ub-E2-mRNA-LNP and XCL1-E2-mRNA-LNP survived after challenge with a lethal dose of CSFV-SM. At 13 dpc, one piglet died in each of the E2-mRNA-LNP group and the E2 subunit vaccine group. Notably, all piglets vaccinated with PBS died between 5 and 9 dpc. Figure 4 B). Meanwhile, piglets in the Ub-E2-mRNA-LNP group did not exhibit fever. In contrast, piglets vaccinated with XCL1-E2-mRNA-LNP, E2-mRNA-LNP, E2 subunit vaccine, or PBS all experienced transient fever (B). Figure 4 C). At 3 dpc, pigs in the E2-mRNA-LNP group, E2 subunit vaccine group, and PBS group began to show typical clinical symptoms of CSF, such as fever, weakness, anorexia, unsteady gait, and erythema on the limbs. Figure 4 D).

[0059] Furthermore, no significant changes were observed in the tissues and organs of the Ub-E2-mRNA-LNP and XCL1-E2-mRNA-LNP groups. Swine immunized with E2-mRNA-LNP and E2 subunit vaccines showed mild lymph node hemorrhage. Notably, pigs in the simulant immunization group exhibited severe clinical lesions, including splenic infarction, renal hemorrhage, lymph node hemorrhage, and tonsillar hemorrhage. Figure 4 E and 4F).

[0060] No viremia was detected in piglets in the Ub-E2-mRNA-LNP or XCL1-E2-mRNA-LNP groups. In contrast, piglets in the PBS group showed high viral loads at 3 days post-conception (dpc), reaching 10 viral genome copies by 7 days post-conception (dpc). 5.0 / mL, indicating rapid viral replication in pigs. Conversely, piglets in the E2 subunit vaccine group or the E2-mRNA-LNP group showed lower levels of viremia. At 10 dpc, only one pig in the E2 subunit vaccine group showed viremia ( Figure 5 A). Furthermore, viral RNA was not detected in oral, nasal, or anal swabs from pigs in the Ub-E2-mRNA-LNP group. However, CSFV nucleic acid was present in all swab types from both the PBS and E2-mRNA-LNP groups. Figure 5These results indicate that after infection with virulent CSFV-SM, CSFV was rapidly cleared from piglets in the Ub-E2-mRNA-LNP and XCL1-E2-mRNA-LNP groups, and no virus transmission occurred.

[0061] 2.2 Immunization: Antigen-modified CSFV E2-mRNA-LNPs can induce a strong immune response in piglets. To assess the immune response induced by different CSFV E2-mRNA-LNPs, the blocking rate of anti-CSFV E2 antibodies was detected using a blocking ELISA kit. At 10 dpv, one piglet in the Ub-E2-mRNA-LNP group tested positive for CSFV-specific antibodies. Notably, at 14 dpv, the seropositivity rate in the Ub-E2-mRNA-LNP group reached 100%, while it was 50% in the XCL1-E2-mRNA-LNP group and 75% in the E2 subunit vaccine group. Furthermore, the antibody blocking rate in the Ub-E2-mRNA-LNP group was significantly higher than that in the other groups. At 21 and 28 dpv, the XCL1-E2-mRNA-LNP group exhibited higher late immunogenicity. The seropositivity rates of antibody blocking in both the Ub-E2-mRNA-LNP and XCL1-E2-mRNA-LNP groups were higher than those in the E2 subunit vaccine group. In contrast, no specific CSFV E2 antibody was detected in the E2-mRNA-LNP group at any time point. Figure 6 A).

[0062] To further evaluate the CSFV E2-mRNA-LNPs-induced CSFV-specific cell-mediated immune response, peripheral blood cells (PBMCs) were isolated from the peripheral blood of piglets in each experimental group. After PBMCs were stimulated with CSFV, the secretion of IFN-γ was detected by ELIspot assay. γ The number of cells secreted was significantly higher in the Ub-E2-mRNA-LNP group and the XCL1-E2-mRNA-LNP group than in the E2 subunit vaccine group and the E2-mRNA-LNP group. Figure 6 B).

Claims

1. A chimeric antigen for preparing a preventive agent against classical swine fever, characterized in that, The classical swine fever virus E2 protein was fused with either XC motif chemokine ligand 1 or ubiquitin.

2. The chimeric antigen according to claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID No. 1 or SEQ ID No.

2.

3. An mRNA-LNPs vaccine for the prevention of classical swine fever, comprising mRNA and lipid nanoparticles, wherein the mRNA is encapsulated in the lipid nanoparticles; characterized in that, The mRNA contains a coding sequence that encodes the chimeric antigen of claim 1.

4. The method for preparing the mRNA-LNPs vaccine according to claim 3, characterized in that, include: (1) The gene sequence encoding the E2 protein of classical swine fever virus was cloned into the pLVX-XCL1 or pLVX-Ub plasmid to obtain a plasmid containing the gene encoding the E2 protein; the plasmid was used to synthesize mRNA by in vitro transcription using T7 RNA polymerase; (2) The synthesized mRNA was encapsulated in lipid nanoparticles to obtain an mRNA-LNPs vaccine for the prevention of classical swine fever.

5. The preparation method according to claim 4, characterized in that, The sequence of the plasmid containing the gene encoding the E2 protein, from the T7 promoter to the 3' end, includes the following elements: 5' UTR, Kozak sequence, tissue plasminogen activator signal sequence, XCL1 or Ub gene, classical swine fever virus E2 gene, V5 tag, 3' UTR, and polyadenylate tail.

6. The preparation method according to claim 4, characterized in that, During the in vitro transcription and synthesis of mRNA, uridine in the UTR region is completely replaced with N1-methylpseudouridine-5'-triphosphate.

7. The preparation method according to claim 4, characterized in that, A Cap1 structure is introduced at the 5' end of the mRNA.

8. The preparation method according to claim 4, characterized in that, The method for preparing mRNA encapsulated in lipid nanoparticles includes: (1) Dissolve ionizable lipids, cholesterol, DSPC and DMG-PEG2000 in ethanol to obtain a lipid-ethanol mixture; (2) Dissolve mRNA in acetate-sodium acetate buffer and mix it with the lipid-ethanol mixture; (3) Collect the sample, remove ethanol by ultrafiltration, add sucrose as a protectant, and then filter it through a filter membrane for sterilization to obtain the final product.

9. The preparation method according to claim 8, characterized in that, In step (1), the molar ratio of ionizable lipids, DSPC, cholesterol and DMG-PEG2000 is 50:38.5:10:1.

5.

10. The preparation method according to claim 8, characterized in that, In step (2), the mRNA was dissolved in 50 mM acetate-sodium acetate buffer and mixed with the lipid-ethanol mixture at a flow rate ratio of 3:1, with a total flow rate of 30 mL / min; The ultrafiltration described in step (3) is performed using a 100 kDa ultrafiltration centrifuge tube; the protective agent is sucrose.

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