Porcine epidemic diarrhea virus vaccine as well as related products and application thereof

Abstract

The invention discloses a porcine epidemic diarrhea virus vaccine as well as related products and application thereof, and relates to the field of vaccines. The invention provides the RNA vaccine of the porcine epidemic diarrhea virus, the vaccine adopts an optimized antigen sequence and a saRNA skeleton, and compared with the traditional porcine epidemic diarrhea virus vaccine, the RNA vaccine has the advantages of early antibody production period, higher antibody titer and challenge protection rate, good safety, simple immune procedure and the like.

Description

Technical Field

[0001] The present invention relates to the field of vaccines, and in particular to porcine epidemic diarrhea virus vaccines and related products and applications. Background Art

[0002] Porcine epidemic diarrhea (PED) is a major disease threatening the swine industry. It is caused by the porcine epidemic diarrhea virus (PEDV), an alpha-coronavirus. PED has a distinct seasonal pattern, with a higher incidence in winter and spring. Its primary clinical features are vomiting, diarrhea, and dehydration in piglets, and the mortality rate in suckling piglets can reach 100%. PED first broke out in Europe in the 1970s and subsequently became widespread globally. With the development of prevention and control technologies, PED has become less common. However, since 2010, PED incidence has been rapidly increasing, primarily affecting large-scale pig farms, causing significant economic losses to the swine industry.

[0003] Currently, vaccines for porcine infectious diarrhea mainly consist of inactivated and attenuated vaccines. The large number of inactivated components can cause severe side effects and excessive, meaningless nonspecific immune responses, wasting the body's immune potential and hindering disease prevention and control. Furthermore, inactivated vaccines require multiple immunizations to achieve a certain level of efficacy. While attenuated vaccines have some effectiveness, they carry the risk of reversion to virulence and are therefore unsafe. Therefore, the market urgently needs a high-performance and safe porcine epidemic diarrhea vaccine.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a porcine epidemic diarrhea virus vaccine and related products and applications.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the S protein of porcine epidemic diarrhea virus, wherein the nucleotide sequence of the S protein of porcine epidemic diarrhea virus has at least 80% identity with the sequence shown in SEQ ID NO: 10 or 11.

[0008] In a second aspect, an embodiment of the present invention provides an RNA molecule, wherein a DNA vector for transcribing the RNA molecule includes an antigen region; the antigen region contains a sequence encoding an antigen, and the nucleotide sequence of the antigen region is the nucleic acid molecule described in the above embodiment.

[0009] In a third aspect, an embodiment of the present invention provides an isolated nucleic acid or a vector containing the nucleic acid, wherein the isolated nucleic acid encodes the RNA molecule described in the above embodiment.

[0010] In a fourth aspect, the embodiments of the present invention provide the use of the nucleic acid molecule described in the preceding embodiments, or the RNA molecule described in the preceding embodiments, or the isolated nucleic acid described in the preceding embodiments, or the vector containing the nucleic acid in the preparation of a vaccine for preventing or treating porcine epidemic diarrhea virus infection.

[0011] In a fifth aspect, an embodiment of the present invention provides a composition, the active ingredient of which includes the RNA molecule described in the above embodiment.

[0012] In a sixth aspect, an embodiment of the present invention provides a vaccine comprising: the RNA molecule described in the preceding embodiment or the composition described in the preceding embodiment.

[0013] The present invention has the following beneficial effects:

[0014] The saRNA vaccine provided by the present invention has an early antibody production stage, higher antibody titer and protection rate against virus, good safety, simple immunization procedure, and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 is the basic structure of saRNA;

[0017] Figure 2 The expression intensity of the target protein uniquely targeted by saRNA.V1-9;

[0018] Figure 3 is the proportion of fluorescent cells that respond uniquely to saRNA.V1-9;

[0019] Figure 4 This is a fluorescence microscopy image of cells after K562 cells were transfected with saRNA.V3 and V1 RNA molecules;

[0020] Figure 5 The secretion amount of saRNA.V3-1~V3-10 target antigen;

[0021] Figure 6 The data of liposome-encapsulated saRNA are: particle size intensity distribution (A), particle size (B), dispersion index (C), and fitting error (D). DETAILED DESCRIPTION

[0022] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0023] Definition of noun

[0024] The term "saRNA" in this article stands for Self-Amplifying RNA, which encodes RNA replicase and can replicate itself within the host cell.

[0025] The term "mRNA vaccine" in this article broadly refers to a general term for vaccine technologies that use messenger RNA (mRNA) molecules as active ingredients, which are delivered to host cells and translated into target antigens to induce immune responses. These technologies include traditional non-replicating mRNA vaccines, self-amplifying RNA vaccines (saRNA), and circular RNA vaccines. In a narrow sense, it specifically refers to non-replicating mRNA vaccines, which contain only the mRNA sequence encoding the target antigen and do not carry any replication or amplification elements.

[0026] The "nrmRNA" in this article is the abbreviation of non-replicating mRNA vaccine.

[0027] The term "NSP1-4" in this article is synonymous with "nsp1-4." NSP stands for nonstructural proteins, which refers to viral nonstructural proteins. These proteins are encoded by the viral genome and play a role in viral replication or gene expression regulation, but ultimately do not form a mature virus and are not part of the viral structure. Viral nonstructural proteins play different roles in various stages of viral replication, including RNA replication, transcription, and translation.

[0028] The term "poly-A" herein refers to polyadenylic acid or poly(A), and is the same as "poly-A tail".

[0029] As used herein, the term "percent identity" refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. Comparison of amino acid sequence identity percentages can be performed using various methods known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA, software well known in the art. Appropriate parameters for aligning sequences can be determined by those skilled in the art.

[0030] As used herein, the term "treating" includes preventing or alleviating a condition, reducing the rate of onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination thereof.

[0031] On the one hand, an embodiment of the present invention provides a nucleic acid molecule comprising: a nucleotide sequence encoding the S protein of porcine epidemic diarrhea virus, wherein the nucleotide sequence of the S protein of porcine epidemic diarrhea virus has at least 80% identity with the sequence shown in SEQ ID NO: 10 or 11.

[0032] In the embodiments of the present invention, the S protein of PEDV is used as the target antigen. Combined with structural biology research, the antigen sequence shown in SEQ ID NO: 10 or 11 is obtained after optimization. It is applied to the preparation of RNA vaccines, which can support high expression of RNA in cells and achieve good animal immune effects.

[0033] In some embodiments, the nucleic acid molecule is a DNA molecule.

[0034] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a trimerization motif; the sequence encoding the trimerization motif is located upstream (5' end) or downstream (3' end) of the nucleotide sequence of the porcine epidemic diarrhea virus S protein. The trimerization motif can promote the trimer folding of the antigen. The porcine epidemic diarrhea virus S protein exists as a homotrimer on the surface of the virus. Adding the trimerization motif to the C-terminus of the antigen can promote the trimer folding of the antigen and enhance the immunogenicity of the antigen.

[0035] In some embodiments, the trimerization motif comprises either T4-Foldon or GCN-Foldon. T4-Foldon is the C-terminal domain of T4 bacteriophage fibritin. GCN-Foldon is an artificially designed trimerization motif formed by fusion of the leucine zipper domain of the yeast transcription factor GCN4 with the T4 bacteriophage Foldon domain, combining the trimerization properties of both.

[0036] In some embodiments, the amino acid sequence of the T4-Foldon is shown in SEQ ID NO: 12; in some embodiments, the amino acid sequence of the GCN-Foldon is shown in SEQ ID NO: 13.

[0037] In some embodiments, the nucleic acid molecule may further include a sequence encoding a lytic tag, the nucleotide sequence of the lytic tag being located downstream or upstream of the nucleotide sequence of the S protein of the porcine epidemic diarrhea virus. The lytic tag includes any one of maltose binding protein (MBP) and small ubiquitin-like modifier protein (SUMO). MBP is used to enhance the solubility and soluble expression of the target protein, and can be used in conjunction with Foldon to improve the expression and folding of the target protein. SUMO can enhance protein solubility and assist protein folding, and can be used in conjunction with foldon to optimize trimer assembly after protein expression.

[0038] In some embodiments, the amino acid sequence of the maltose binding protein is shown in SEQ ID NO: 14. In some embodiments, the amino acid sequence of the small ubiquitin-like modified protein is shown in SEQ ID NO: 15.

[0039] On the other hand, an embodiment of the present invention provides an RNA molecule, and the DNA vector that transcribes to generate the RNA molecule includes an antigen region; the antigen region contains a sequence encoding an antigen, and the nucleotide sequence of the antigen region is a nucleic acid molecule as described in any of the above embodiments.

[0040] In some embodiments, the RNA molecule comprises mRNA or saRNA.

[0041] In some embodiments, the DNA vector further comprises a non-structural protein region; the non-structural protein region comprises a sequence encoding a non-structural protein, and the non-structural protein is derived from an alphavirus.

[0042] In some embodiments, the alphavirus is selected from Venezuelan Equine Encephalitis Virus (VEEV), Sindbis virus (SINV), Eastern Equine Encephalitis Virus (EEEV), Everglades Virus (EVEV), Semliki Forest Virus (SFV), Chikungunya Virus (CHIKV), Ross River Virus (RRV), Western Equine Encephalitis Virus (WEEV), Highlands J Virus (HJV), Salmonid Alphavirus (SAV) and Beechey Ground Squirrel Virus (BCRV).

[0043] In some embodiments, the non-structural proteins include any one or more of NSP1, NSP2, NSP3, and NSP4. The function of non-structural proteins is to drive the self-replication of mRNA, thereby amplifying antigen expression. The specific functions are as follows: NSP1 is responsible for capping the 5' end of viral RNA, protecting RNA from nuclease degradation, and promoting ribosome recognition; NSP2 has helicase, protease, and RNA triphosphatase activities, responsible for cleaving polyprotein precursors (NSP1-4) and unwinding the RNA double-stranded structure; NSP3 participates in the interaction between the virus and host proteins and regulates the assembly of the viral replication complex; NSP4 is an RNA-dependent RNA polymerase (RdRp) that catalyzes the replication of RNA chains.

[0044] In some embodiments, the amino acid sequences of NSP1, NSP2, NSP3, and NSP4 are at least 80% identical to the sequences shown in SEQ ID NOs: 5 to 8, in sequence;

[0045] In some embodiments, the DNA vector further comprises poly-A (with a poly-A tail).

[0046] In some embodiments, the nucleotide sequence of the poly-A is at least 80% identical to the sequence shown in SEQ ID NO:9.

[0047] In some embodiments, at least 80% identity includes at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%.

[0048] In some embodiments, the DNA vector comprises, in order from 5' end to 3' end: a promoter, a 5'UTR, a nonstructural protein region, a subgenomic promoter region, the antigen region, a 3'UTR and poly-A.

[0049] In some embodiments, the promoter comprises a T7 promoter, a T3 promoter, or an SP6 promoter. In some embodiments, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 1.

[0050] In some embodiments, the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO:2.

[0051] In some embodiments, the nucleotide sequence of the subgenomic promoter region is shown in SEQ ID NO:3.

[0052] In some embodiments, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO:4.

[0053] In some embodiments, the sequence of the DNA vector is shown in SEQ ID NO:16.

[0054] On the other hand, an embodiment of the present invention provides an isolated nucleic acid or a vector containing the nucleic acid, wherein the isolated nucleic acid encodes the RNA molecule described in any of the above embodiments.

[0055] On the other hand, an embodiment of the present invention also provides the use of the nucleic acid molecule as described in any of the foregoing embodiments, or the RNA molecule as described in any of the foregoing embodiments, or the isolated nucleic acid as described in any of the foregoing embodiments, or the vector containing the nucleic acid in the preparation of a vaccine for preventing or treating porcine epidemic diarrhea virus infection.

[0056] In some embodiments, the vaccine comprises a veterinary vaccine. In some embodiments, the animal comprises a pig.

[0057] In some embodiments, the vaccine comprises a saRNA vaccine.

[0058] On the other hand, an embodiment of the present invention further provides a composition, the active ingredient of which includes the RNA molecule described in any embodiment.

[0059] In some embodiments, the composition further comprises: any one or more of a buffer, a carrier, an adjuvant, and a stabilizer.

[0060] In some embodiments, the buffer comprises: citric acid buffer, sodium acetate buffer, phosphate buffer, citrate-citric acid buffer or acetic acid-sodium acetate buffer, with a pH of 3.5-5.

[0061] In some embodiments, the carrier comprises any one of lipid nanoparticles (LNP), polymer nanoparticles (PNP), and virus-like particles (VLP).

[0062] In some embodiments, the components of the lipid nanoparticles include any one or a combination of cationic lipids, neutral lipids, cholesterol or cholesterol derivatives, pegylated lipids, chitosan and QS-21.

[0063] In some embodiments, the components of the lipid nanoparticles include, by molar percentage, 40% to 60% cationic lipids, 10% to 15% neutral lipids, 35% to 38.5% cholesterol or cholesterol derivatives, 0.1% to 5% PEGylated lipids, and 0.1% to 5% chitosan or QS-21;

[0064] In some embodiments, the cationic lipid comprises cationic lipid SM-102 or ALC-0315.

[0065] In some embodiments, the neutral lipids include any one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and sphingomyelin (SM);

[0066] In some embodiments, the cholesterol derivative comprises β-sitosterol.

[0067] In some embodiments, the PEGylated lipid comprises 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG). In some embodiments, the PEG-DMG comprises PEG-DMG 2000 (same as DMG-PEG 2000), which refers to a PEG chain with a molecular weight of about 2000 Da.

[0068] In addition, an embodiment of the present invention provides a vaccine, which includes: the RNA molecule described in any of the foregoing embodiments or the composition described in any of the foregoing embodiments.

[0069] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0070] Example 1: Screening of saRNA backbones and optimized sequences of porcine epidemic diarrhea virus antigens.

[0071] The basic structure of the saRNA DNA vector includes: T7 promoter region (SEQ ID NO: 1), 5'UTR region (SEQ ID NO: 2), subgenomic promoter region (SEQ ID NO: 3), viral non-structural protein region (NSP1, NSP2, NSP3 and NSP4), antigen region, 3'UTR region (SEQ ID NO: 4) and poly A structural region, such as Figure 1As shown. In this embodiment, the non-structural protein regions NSP2, NSP3 and polyA structural regions are optimized to reduce cytotoxicity and improve the immunogenicity of the antigen, and a saRNA skeleton that efficiently and stably expresses the target protein is screened through cell experiments. The eGFP fluorescent reporter system is used to verify the application effects of different non-structural protein regions (NSP1-4) and polyA structural regions on the saRNA skeleton by expression at the cellular level. The sequence NCBI (L01443.1) of Venezuelan equine encephalitis virus (TC-83 Venezuelan Equine Encephalitis Virus, VEEV) is selected as the skeleton basis, and the specific experiments are shown below.

[0072] 1.1 Designing Different Backbones: Using eGFP as an indicator, the effectiveness of different backbones was verified by cell expression. In this example, the Venezuelan Equine Encephalitis Virus (VEEV) sequence was selected as the backbone. By screening different promoters, the vector expression level was optimized and the gene expression intensity was adjusted to meet the application requirements.

[0073] 1.2 Construction of saRNA-eGFP expression vector

[0074] Based on the groupings in Table 1, a VEEV backbone driven by the T7 promoter (SEQ ID NO: 1) was constructed. Primers were designed to amplify the eGFP gene, and restriction enzyme sites were added at both ends. The VEEV-eGFP plasmid, using the T7 promoter and eGFP gene, was generated by enzyme digestion, ligation, and transformation to obtain the T7-VEEV-eGFP plasmid.

[0075] A T7-VEEV-eGFP backbone containing NSP2 mutations and / or NSP3 mutations was constructed, and mutations were introduced into the coding regions of NSP2 and NSP3 in T7-VEEV-eGFP according to Table 1 using fusion PCR technology and multi-fragment homologous recombination technology. That is, the point mutation in NSP2, p.P1249S (P at position 1249 was mutated to S, and the amino acid sequence of the mutated NSP2 is shown in SEQ ID NO: 6), and NSP3 homologous recombination IDR-1018 (the amino acid sequence of the recombinant NSP3 is shown in SEQ ID NO: 7). By mutating the active site of the NSP2 protease, cytotoxicity and adverse reactions in the body are reduced by reducing the cleavage of host cell proteins. IDR-1018 enhances the anti-infection and anti-inflammatory capabilities of immune cells by activating the TLR4 and NOD2 signaling pathways, while reducing the risk of proinflammatory cytokine storms. By introducing IDR-1018 into the hypervariable region of NSP3, the antibacterial and antiviral immune responses of the backbone itself are enhanced without affecting the expression of the backbone non-structural proteins. The specific method is as follows: two pairs of primers were designed to perform PCR amplification on the T7-VEEV-eGFP vector fragment, thereby introducing nucleotide mutations leading to amino acid changes. The ligation products were transformed into DH5α competent cells, and positive colonies were selected for plasmid amplification and sequencing.

[0076] For the construction of insertion mutations: an upstream primer (FP) of the mutant sequence was used to introduce a restriction site (SacI / XhoI), a flexible linker (GGGGS×3) and a signal peptide to connect the immunostimulatory molecule IDR-1018 to NSP3, and the PCR product and the T7-VEEV-eGFP backbone were double-digested. After ligation, the linearized plasmid was digested with NotI. In addition, the amino acid sequence of NSP1 is shown in SEQ ID NO:5, and the amino acid sequence of NSP4 is shown in SEQ ID NO:8. Different non-structural proteins NSP2, NSP3 and poly-A were selected and homologously recombined with the VEEV-eGFP plasmid to design and synthesize 9 new backbones: saRNA.V1-eGFP, saRNA.V2-eGFP, saRNA.V3-eGFP, saRNA.V4-eGFP, saRNA.V5-eGFP, saRNA.V6-eGFP, saRNA.V7-eGFP, saRNA.V8-eGFP, and saRNA.V9-eGFP.

[0077] Table 1: Different skeleton groups

[0078] Group NSP2 NSP3 poly-A saRNA.V1 p.P1249S Homologous recombination IDR-1018 60A saRNA.V2 p.P1249S Homologous recombination IDR-1018 90A saRNA.V3 p.P1249S Homologous recombination IDR-1018 30A-G-69A saRNA.V4 p.P1249S Not reorganized 30A-G-69A saRNA.V5 No mutation Homologous recombination IDR-1018 30A-G-69A saRNA.V6 No mutation Not reorganized 30A-G-69A saRNA.V7 p.Q1275E Homologous recombination IDR-1018 60A saRNA.V8 p.Q1275E Homologous recombination IDR-1018 90A saRNA.V9 p.Q1275E Homologous recombination IDR-1018 30A-G-69A

[0079] Note: The sequence of 30A-G-69A is shown in SEQ ID NO: 9.

[0080] 1.3 Preparation of saRNA

[0081] The nine strains containing the target plasmids obtained above were fermented and cultured, and the fermented cells were then collected for plasmid preparation. Here, in this example, the QIAGEN endotoxin-free plasmid extraction kit was used for plasmid extraction, and the recovered plasmid was calibrated with Nanodrop for plasmid concentration.

[0082] The prepared plasmid was linearized using restriction endonuclease (BspQI), and the linearized DNA was recovered by ethanol precipitation. The recovered plasmid was calibrated with Nanodrop for linearized plasmid concentration.

[0083] Based on the template prepared above, an in vitro transcription reaction was prepared (as shown in Table 2), followed by incubation at 37°C for 2 h. After the IVT reaction was completed, 1 μL of DNase I was added to the reaction solution, and the reaction was again incubated at 37°C for 0.5 h.

[0084] Table 2: In vitro transcription system

[0085] Components Volume (μL) Linearized plasmid template 1 μg T7 RNA Polymerase 1 μL 10×Reaction buffer 2μL ATP 1.5 μL GTP 1.5 μL CTP 1.5 μL UTP 1.5 μL Hat analogues 1 μL Sterile enzyme-free water To 20μL

[0086] The reaction solution prepared above was purified using magnetic beads to obtain saRNA stock solution for subsequent studies. The saRNA concentration was measured using a Qubit4 fluorometer.

[0087] 1.4 ELISA quantitative determination of IDR-1018 concentration in cell supernatant and expression on cells

[0088] The Hycult Biotech HK321-02 kit was used to collect the transfected cell culture supernatant and filter it through a high-speed centrifugation (≥10,000 × g) or a 0.22 μm filter membrane to prevent impurities from clogging the microplate.

[0089] According to the kit instructions, the lyophilized standard was reconstituted with 500 μL of deionized water (final concentration 100 ng / mL).

[0090] Serial dilutions were performed to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0 ng / mL.

[0091] Wash buffer, standard wells: Add 100 μL of standard of varying concentrations to each well. Sample wells: Add 100 μL of the sample to be tested to each well (repeated wells are recommended). Blank wells: Add only 100 μL of diluent. After adding reagents to each well, incubate at room temperature (25°C) for 2 hours. Discard the liquid and add 300 μL of 1× wash buffer to each well. Let stand for 1 minute, then discard. Repeat three times. Pat dry after the final wash. Add 100 μL of biotinylated detection antibody (diluted) to each well and incubate at room temperature for 1 hour. Wash. Add 100 μL of HRP-streptavidin solution (diluted) to each well and incubate at room temperature for 30 minutes in the dark. Wash: Repeat step 2. Add 100 μL of TMB substrate to each well. Incubate at room temperature in the dark for 15-30 minutes (observe the standard wells for blue coloration). Add 50 μL of stop solution to each well. Immediately read the OD value using a microplate reader at 450 nm (main wavelength) and 540 nm or 630 nm (reference wavelength). Use four-parameter fitting (4PL) or linear regression, with the standard concentration as the horizontal axis and the reference value as the vertical axis. Calculate the concentration: Substitute the sample OD value into the standard curve equation and multiply by the dilution factor to obtain the actual concentration.

[0092] Standard curve equation (4PL fitting): y = (AD) / (1+(x / C)B)+D;

[0093] The concentrations of IDR-1018 detected in saRNA.V1-eGFP, saRNA.V2-eGFP, saRNA.V3-eGFP, and saRNA.V5-eGFP were 16 ng / mL, 18 ng / mL, 30 ng / mL, and 10 ng / mL, respectively. The concentration of IDR-1018 secreted by saRNA.V3-eGFP was higher, indicating that simultaneous mutations in NSP2 and NSP3 have a synergistic effect, and combined with the 30A-G-69A PolyA tail, the expression of IDR-1018 can be further increased.

[0094] In order to screen out the better skeleton, further in vitro expression was performed on cells to detect the expression level. The saRNA was transfected into K562 cells in vitro. After 24 hours, the cells were lysed on ice and the supernatant was collected. Samples were taken on the 1st, 4th, 6th, 8th and 12th days for fluorescence detection and fluorescent cell ratio detection. The total fluorescence intensity was used to characterize the expression intensity of the target protein, and the fluorescent cell ratio was used to characterize the persistence and stability of saRNA expression. The results are shown in Figure 2. Figure 2 , Figure 3 , Figure 4As shown, only saRNA.V3-eGFP achieved a positive transfection rate exceeding 70%, demonstrating high transfection efficiency and efficient saRNA activation. Data analysis revealed that saRNA.V3-eGFP was preferred based on fluorescence intensity and the proportion of transfected fluorescent cells. Compared to the original, unoptimized backbone, saRNA.V3-eGFP mutates the nonstructural proteins NSP2 and NSP3. The NSP3 hypervariable region does not affect viral replication, while enabling efficient cell expression and secretion. The NSP3 hypervariable region incorporates immunostimulatory molecules, enhancing its role in macrophage differentiation compared to the original backbone. This promotes the differentiation of monocytes into a specialized macrophage phenotype, enhances the activation of antigen-presenting cells, and improves the vaccine's immunogenicity.

[0095] 1.5 Screening of porcine epidemic diarrhea virus antigen sequences

[0096] Porcine epidemic diarrhea virus is a coronavirus, and its main structural protein is the S protein, which is a glycoprotein. It has the ability to bind to host cell receptors and is the key to the virus invading cells. The S protein of the coronavirus has a trimer structure, and each monomer consists of an S1 subunit and an S2 subunit. The S1 subunit is responsible for recognizing and binding to the host cell receptor, while the S2 subunit mediates the fusion of the virus and the cell. The S1 subunit contains two domains: the receptor binding domain (RBD) at the N-terminus and the S1 membrane-proximal domain (S1-NTD) at the C-terminus. RBD is the region where the S protein interacts with the host cell receptor, while S1-NTD is involved in the conformational changes of RBD. In this example, the codons of the full-length PEDV-S protein were optimized; multiple different trimer motifs (T4-foldon or GCN-foldon) or lytic tags (MBP or SUMO) were selected and fused to the C-terminus of the antigen sequence to enhance the immunogenicity of the antigen, ultimately forming saRNA.V3-1, saRNA.V3-2, saRNA.V3-3, saRNA.V3-4, saRNA.V3-5, saRNA.V3-6, saRNA.V3-7, saRNA.V3-8, saRNA.V3-9, saRNA.V3-10, and saRNA.V3-11. As shown in the following table.

[0097] Table 3: Antigen sequence screening

[0098]

[0099]

[0100] Note: The nucleotide sequence of PEDV-S optimization 1 is shown in SEQ ID NO: 10, the nucleotide sequence of PEDV-S optimization 2 is shown in SEQ ID NO: 11, the amino acid sequence of T4-foldon is shown in SEQ ID NO: 12, the amino acid sequence of GCN-foldon is shown in SEQ ID NO: 13, the amino acid sequence of MBP is shown in SEQ ID NO: 14, and the amino acid sequence of SUMO is shown in SEQ ID NO: 15. The nucleotide sequence of the DNA vector for saRNA.V3-1 is shown in SEQ ID NO: 16.

[0101] After sequence synthesis, the reaction solution prepared using an in vitro transcription system was purified using magnetic beads to obtain saRNA stock solution for subsequent studies. The saRNA was transfected into cells in vitro, and 24 hours later, the cells were lysed on ice and the supernatant was collected. The PEDV-S protein expressed by the saRNA was identified by Western blot. After plasmid transfection, the supernatant was collected for identification and the protein sample was subjected to SDS-PAGE electrophoresis on a 10% SDS-PAGE gel at 80V for 30 minutes, followed by 120V for 60 minutes. Following electrophoresis, the protein was transferred to a PVDF membrane at a constant current of 250mA for 90 minutes. The membrane was blocked with 5% skim milk powder overnight at 4°C and washed three times with PBS. A mouse anti-His tag monoclonal antibody diluted 10,000-fold in PBS was added, incubated at room temperature for 1 hour, and washed three times. A 20,000-fold diluted horseradish peroxidase-conjugated goat anti-mouse IgG was added and incubated at room temperature for 1 hour. The membrane was then washed three times with PBST buffer. Finally, the cells were washed once with PBS and ECL luminescence was performed using SuperKineTM enhanced ECL luminescence solution.

[0102] By collecting different types of samples for 24 hours and measuring the gray value of WB experiment, the secretion amount of target antigens of different components was determined, and finally the better PEDV-S protein antigen was screened. Figure 5 ) showed that PEDV-S antigen saRNA.V3-1, saRNA.V3-2, saRNA.V3-3, and saRNA.V3-4 sequences can secrete higher amounts of antigen.

[0103] Western blot analysis of target protein solubility: HEK293 cells were cultured in 10% FBS at 37°C and 5% CO. HEK293 cells were seeded into 6-well plates at a density of 70%-90%. Preparation of liposome-DNA complexes: Tube A: Dilute DNA (1-4 μg) in 250 μL serum-free medium. Tube B: Dilute liposome reagent in 250 μL serum-free medium. Mix A and B and let stand at room temperature for 15-20 minutes to form a complex. Add the complex dropwise to the cell culture medium and mix gently. After 4-6 hours of incubation at 37°C and 5% CO, replace the culture medium with complete serum. Collect the supernatant and cell lysate 72 hours after transfection. Western blot analysis of target protein solubility.

[0104] Table 4: Solubility ratio

[0105] Soluble ratio (%) WT-PEDV-S 19％ saRNA.V3-1 73％ saRNA.V3-2 70％ saRNA.V3-3 63％ saRNA.V3-4 65％ saRNA.V3-5 65％ saRNA.V3-6 0％

[0106] By collecting different samples for 72 hours and conducting Western blotting to measure solubility, the researchers determined the secretion levels of the target antigens in each component, ultimately identifying the optimal trimer motif. The results showed that the saRNA.V3-1 sequence, which binds to the T4-foldon, exhibited higher solubility. Compared to other wild-type porcine epidemic diarrhea (PED) S proteins, the addition of the trimer motif improved their solubility, facilitating subsequent packaging with delivery vectors.

[0107] Example 2. Screening of saRNA delivery vectors and components and preparation of saRNA vaccines

[0108] 2.1 saRNA Components

[0109] 2.1.1 Components of aqueous buffer

[0110] The screening of aqueous buffers for LNP (lipid nanoparticle) vaccine preparation is a complex and critical process involving multiple factors, including the pH value, ionic strength, composition of the buffer and its effect on the properties and stability of LNP. Screening an effective buffer plays an important role in the stability and transfection efficiency of saRNA. In this example, 15 aqueous buffers were selected, including citrate buffer (pH 3.5, 4 and 4.5), sodium acetate buffer (pH 3.5, 4 and 4.5), phosphate buffer (pH 3.5, 4 and 4.5), citrate-citric acid buffer (pH 3.5, 4 and 4.5) and acetic acid-sodium acetate buffer (pH 3.5, 4 and 4.5).

[0111] Table 5: Aqueous buffer grouping

[0112]

[0113] Citric Acid Buffer: pH 3.5 Buffer: Weigh 1.0689 g of citric acid (MW: 192.14), add 80 mL of purified water, initially 55.6 mM pH = 2.15, adjust pH to 3.5 with 2 M NaOH. pH 4 Buffer: Weigh 1.0689 g of citric acid (MW: 192.14), add 80 mL of purified water, initially 55.6 mM pH = 2.15, adjust pH to 4 with 2 M NaOH. pH 4.5 Buffer: Weigh 1.0689 g of citric acid (MW: 192.14), add 80 mL of purified water, initially 55.6 mM pH = 2.15, adjust pH to 4.5 with 2 M NaOH.

[0114] Sodium Acetate Buffer: pH 3.5 Buffer: Weigh 16.4g of anhydrous sodium acetate (CH3COONa, molecular weight 82.03g / mol) and dissolve in 800mL of deionized water. Add glacial acetic acid (or 1M HCl) dropwise to adjust to the desired pH. pH 4 Buffer: Dissolve 4.1g of 0.05M sodium acetate in 8.6mL of glacial acetic acid. pH 4.5 Buffer: Dissolve 8.2g of 0.1M anhydrous sodium acetate in 5.7mL of glacial acetic acid.

[0115] Phosphate Buffer: pH 3.5 Buffer: Weigh 27.6g NaH2PO4·H2O and dissolve in 800mL deionized water. Stir until completely dissolved. Add concentrated phosphoric acid (85% H3PO4) dropwise to adjust to the desired pH. pH 4 Buffer: Weigh 27.6g NaH2PO4·H2O and dissolve in 800mL deionized water. Stir until completely dissolved. Add concentrated phosphoric acid (85% H3PO4) dropwise to adjust to the desired pH. pH 4.5 Buffer: Weigh 27.6g NaH2PO4·H2O and dissolve in 800mL deionized water. Stir until completely dissolved. Add concentrated phosphoric acid (85% H3PO4) dropwise to adjust to the desired pH.

[0116] Citrate-Citric Acid Buffer: pH 3.5 Buffer: Add 81 mL of 0.2M citric acid solution and 19 mL of 0.2M sodium citrate solution, add water to 100 mL (final concentration 0.2M). pH 4.0 Buffer: Add 65 mL of 0.2M citric acid solution and 35 mL of 0.2M sodium citrate solution, add water to 100 mL (final concentration 0.2M). pH 4.5 Buffer: Add 55 mL of 0.2M citric acid solution and 45 mL of 0.2M sodium citrate solution, add water to 100 mL (final concentration 0.2M).

[0117] Acetic acid-sodium acetate buffer: pH 3.5 buffer: Add 95 mL of 0.2M acetic acid solution and 5 mL of 0.2M sodium acetate solution, and dilute to 200 mL with distilled water (final concentration 0.2M). pH 4 buffer: Add 82 mL of 0.2M acetic acid solution and 18 mL of 0.2M sodium acetate solution, and dilute to 200 mL with distilled water (final concentration 0.2M). pH 4.5 buffer: Add 59 mL of 0.2M acetic acid solution and 41 mL of 0.2M sodium acetate solution, and dilute to 200 mL with distilled water (final concentration 0.2M).

[0118] 2.1.2 Lipid mixture components

[0119] This embodiment also provides 6 groups of saRNA vaccine delivery carrier lipid mixtures, the preparation method of which is as follows.

[0120] Cationic lipid SM-102, distearoylphosphatidylcholine DSPC, cholesterol / β-sitosterol, and DMG-PEG 2000 were dissolved and mixed in ethanol at a molar ratio of 50:10:38.5:1.5 to obtain lipid mixture 1 and lipid mixture 2. Cationic lipid SM-102, distearoylphosphatidylcholine DSPC, cholesterol, DMG-PEG 2000, and chitosan / Qs-21 were dissolved and mixed at a molar ratio of 50:10:37:1.5:1.5 to obtain lipid mixture 3 and lipid mixture 5. Cationic lipid SM-102, distearoylphosphatidylcholine DSPC, β-sitosterol, DMG-PEG 2000, and chitosan (molecular weight 10-50 kDa) / Qs-21 were dissolved and mixed at a molar ratio of 47:15:35:1.5:1.5 to obtain lipid mixture 4 and lipid mixture 6. The groups are shown in Table 6.

[0121] Table 6: Liposome Grouping

[0122]

[0123] 2.2 Preparation of saRNA solution

[0124] The saRNA.V3-eGFP prepared in Example 1 was prepared with the 15 buffers in step 2.1.1 at a volume ratio of 1:30 to obtain an aqueous phase, which was adjusted to the same concentration (2 mg / mL) to form 15 groups of saRNA aqueous preparations.

[0125] 2.3saRNA-LNP Preparation

[0126] Lipid mixtures 1 to 6 were dissolved in ethanol solution to form a lipid phase (1 mL of ethanol solution was added for every 10 mg of total lipid, for a final concentration of 10 mg / mL). The saRNA aqueous phase formulation and the lipid phase were rapidly mixed using a microfluidic system and extruded simultaneously at a lipid:aqueous phase volume ratio of 1:3. The saRNA in the aqueous phase and the various lipid molecules in the oil phase solution were able to encapsulate the saRNA to form LNPs under electrostatic attraction and hydrophilic-hydrophobic interactions, thereby encapsulating the saRNA within the LNPs. A total of 90 saRNA-LNPs were formed.

[0127] 2.3.1saRNA-LNP Particle Size and Dispersion Coefficient Detection

[0128] The particle size and dispersion coefficient of the 90 prepared saRNA-LNPs were detected, and the instrument was preheated for 20-30 minutes; 1 mL of the diluted saRNA.V3-eGFP vaccine sample (sample diluted 40 times) was taken and added to the particle size analyzer sample pool; the parameters were set - the dispersant was a 10% sucrose solution (1 g sucrose was dissolved in 9 mL of water), the dispersoid was liposome particles, and the above six lipid mixtures were selected for encapsulation. The encapsulated liposome sample was placed in the instrument for measurement three times and the average value of the results was automatically calculated. The measurement results were collected, and finally 6 groups with PDI less than 0.20 and particle size less than 105 nm were screened out as saRNA-LNP-1, saRNA-LNP-2, saRNA-LNP-3, saRNA-LNP-4, saRNA-LNP-5, and saRNA-LNP-6.

[0129] Table 7: Particle size and dispersion coefficient of saRNA-LNP in each group

[0130] serial number liposomes Aqueous buffer Particle size / nm PDI saRNA-LNP-1 Lipid mixture 1 Citrate-citric acid buffer (pH = 4.5) 89.89 0.1567 saRNA-LNP-2 Lipid mixture 2 Citrate-citric acid buffer (pH = 4.5) 100.02 0.1910 saRNA-LNP-3 Lipid mixture 3 Citrate-citric acid buffer (pH = 4.5) 103.45 0.1645 saRNA-LNP-4 Lipid mixture 4 Citrate-citric acid buffer (pH = 4.5) 84.56 0.1010 saRNA-LNP-5 Lipid mixture 4 Citric acid buffer (pH = 4.5) 85.56 0.1967 saRNA-LNP-6 Lipid mixture 5 Citrate-citric acid buffer (pH = 4.5) 86.89 0.1543

[0131] 2.3.2saRNA-LNP Encapsulation Efficiency Detection

[0132] Use Invitrogen TM Quant-iTRiboGreenRNA Kit (Cat. No. R11491) was used to detect encapsulation efficiency. Dilute 20× TE buffer to 1× TE buffer: Use DEPC water for this process. Store the diluted 1× TE buffer at ≤30°C (0.5 mL 20× TE buffer + 9.5 mL DEPC water). Prepare a 400 ng / mL RNA standard sample: Dilute the stock solution 250-fold. (1 μL original RNA + 249 μL 1× TE buffer; prepare a 2 μg / mL stock solution. Dilute the diluted 400 ng / mL RNA standard to the following concentrations according to Table 6:

[0133] Table 8: Standard Dilutions

[0134] 1×TE buffer 400 ng / mL RNA concentration 60 90 240 ng / mL 105 45 120 ng / mL 127.5 22.5 60 ng / mL 138.75 11.25 30 ng / mL 150 0 0

[0135] To release RNA using Triton-100, lyse LNPs using Triton-100 (120 μL Triton-100 + 480 μL 1× TE buffer). Prepare a solution containing 2% Triton-100 (120 μL Triton-100 + 480 μL 1× TE buffer). Lyse the sample with 24 μL of 1× TE buffer containing 2% Triton-100 plus 6 μL of sample. Vortex and lyse for 5 minutes. Add 120 μL of 1× TE buffer to each sample. For a control, add 30 μL of sample (no lysis) plus 120 μL of 1× TE buffer. For samples that have been concentrated and centrifuged, dilute them 20-fold before proceeding with the above steps.

[0136] Dilute Ribo Green RNA Reagent 500-fold (5 μL Ribo Green RNA Reagent + 2.495 mL 1× TE buffer). For testing, add 150 μL of the newly prepared Ribo Green RNA Reagent to each tube, mix thoroughly, and incubate in the dark for 5 minutes. Then, transfer 200 μL of the solution to a 96-well black plate. Measure fluorescence intensity at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The concentration of the standard curve is then half of the previous concentration, and the dye prepared with the new RNA detection reagent is added to the corresponding wells. The results are shown in Table 9 below.

[0137] Table 9: Sample parameters

[0138] serial number liposomes Encapsulation efficiency saRNA-LNP-1 Lipid mixture 1 95.51％ saRNA-LNP-2 Lipid mixture 2 96.42％ saRNA-LNP-3 Lipid mixture 3 96.52％ saRNA-LNP-4 Lipid mixture 4 99.11％ saRNA-LNP-5 Lipid mixture 4 98.72％ saRNA-LNP-6 Lipid mixture 5 98.51％

[0139] β-Sitosterol was used to replace cholesterol and different proportions of fusion lipid DSPC to improve the encapsulation efficiency of the lipid delivery system and optimize the saRNA delivery efficiency. The preferred delivery systems were saRNA-LNP-4, saRNA-LNP-5, and saRNA-LNP-6.

[0140] 2.4 Ultrafiltration

[0141] The prepared LNP solution was ultrafiltered, and RC ultrafiltration tubes were prepared and rinsed with Tris-sucrose in advance. The prepared saRNA-LNPs were concentrated by centrifugation at 2500g to less than 10mL. The filtered solution and the concentrate were collected for subsequent testing, and 1mL of each permeate was taken. 10μL of the concentrated sample plus 990μL of sucrose buffer was taken for particle size and dispersion coefficient testing. The experimental procedures were the same as step 2.3.1 in Example 2.

[0142] Table 10: Particle size of saRNA-LNP in each group

[0143] serial number liposomes Aqueous buffer Particle size / nm PDI saRNA-LNP-4 Lipid mixture 4 Citrate-citric acid buffer (pH = 4.5) 90.15 0.1423 saRNA-LNP-5 Lipid mixture 4 Citric acid buffer (pH = 4.5) 95.21 0.1915 saRNA-LNP-6 Lipid mixture 5 Citrate-citric acid buffer (pH = 4.5) 100.01 0.1854

[0144] 2.5 Calcein-AM / PI double staining assay to detect the effect of saRNA-LNP on cells

[0145] 5×10 HEK293T cells per well were seeded in 24-well plates and cultured for 24 hours to 70% confluence. A negative control group of untreated cells and a positive control group of cells treated with 1% (v / v) TritonX-100 were set up. Three experimental groups were set up with each of the three LNP vaccines, at concentrations of 1, 10, and 50 μg / mL, respectively, for 24 hours.

[0146] Staining working solution: Calcein-AM (final concentration 4 μM) and PI (final concentration 5 μg / mL) were diluted in PBS or serum-free medium.

[0147] Remove the culture medium and gently wash the cells 1-2 times with PBS. Add the staining solution (500 μL / well) and incubate at 37°C in the dark for 15-30 minutes. Remove the staining solution and wash once with PBS. Observe under a fluorescence microscope using the green channel (Calcein): excitation filter 470-495 nm, emission filter 510-550 nm; red channel (PI): excitation filter 530-550 nm, emission filter 590-650 nm. Flow cytometry: Collect cells, resuspend in PBS, and analyze on the flow cytometer (Calcein-FITC channel, PI-PE channel). Calculate the viable cell percentage (Calcein+PI-) and dead cell percentage (Calcein-PI+) for flow cytometry data. ImageJ analysis: Count the ratio of green to red fluorescent cells in a random field of view. Use the formula: Cell death rate (%) = total cell number, number of PI-positive cells / total cell number × 100.

[0148] Table 11: HEK293T cell mortality (%)

[0149]

[0150] Calcein-AM / PI double staining experiments show that saRNA-LNP-4 results in low cell mortality after culture in HEK293T cells, maintaining good metabolic activity and membrane integrity. Compared to saRNA-LNP-5 and saRNA-LNP-6, saRNA-LNP-4 has lower cytotoxicity, is safer, and induces a safer immune response.

[0151] While saRNA-LNP-5's aqueous citric acid buffer provides an acidic environment to assist in the ionization of cationic lipids, the use of a strongly acidic buffer may have caused an oxidative side reaction with β-sitosterol. While saRNA-LNP-6's particle size and dispersion coefficient were comparable to those of saRNA-LNP-4 and saRNA-LNP-5, the addition of QS-21 to lipid mixture 5 triggered cell membrane rupture, leading to a relatively high cell mortality rate and low safety, requiring further optimization. The combination of the citrate-citric acid buffer and the replacement of cholesterol with β-sitosterol in saRNA-LNP-4 resulted in stable encapsulation efficiency and safety, while the addition of chitosan enhanced the immune response.

[0152] Based on the above data, saRNA-LNP-4 was finally selected as the most preferred saRNA-LNP formulation.

[0153] 2.6 Preparation of porcine epidemic diarrhea virus saRNA-LNP vaccine

[0154] The lipid mixture 4 was dissolved in an ethanol solution to form a lipid phase (1 mL of ethanol solution was added for every 10 mg of total lipid, with a final concentration of 10 mg / mL). The saRNA.V3-1 aqueous phase preparation (the aqueous phase buffer was a citrate-citric acid buffer (pH = 4.5)) was rapidly mixed with the lipid phase through a microfluidic system and extruded simultaneously with a volume ratio of lipid phase: aqueous phase = 1:3. The plasmid in the aqueous phase and the various lipid molecules in the oil phase solution can encapsulate the plasmid to form LNP under electrostatic attraction and hydrophilic-hydrophobic forces, thereby allowing the saRNA to be encapsulated into the LNP. The saRNA-LNP preparation of saRNA.V3-1 was formed with a particle size of 99.15 nm and a PDI of 0.09042. Figure 6 .

[0155] Example 3: Safety and efficacy evaluation of porcine epidemic diarrhea virus saRNA vaccine

[0156] 1. Experimental Immunity and Detection of Specific Neutralizing Antibodies

[0157] In order to verify the immune effect of the porcine epidemic diarrhea virus saRNA vaccine (the saRNA-LNP preparation of saRNA.V3-1 in Example 2, abbreviated as saRNA-PEDV), a piglet immunization test was carried out. The experimental pigs were provided by pig farms around Huzhou, Zhejiang. The neutralizing antibodies of porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine delta coronavirus of all pigs were not higher than 1:4, and the PCR detection of porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine delta coronavirus were all negative. 20 healthy susceptible piglets aged 3 to 5 days were randomly divided into 4 groups, and the piglets were immunized in the neck muscle according to the grouping in Table 12 below. Among them, the saRNA-PEDV vaccine was used for a single immunization, and the commercial inactivated vaccine was used for a single immunization and a secondary immunization respectively. The secondary immunization procedure is to boost the immunization with the same dose once after a single immunization 2 weeks later. The groups are as follows:

[0158] Table 12: Piglet Immunization Schedule

[0159] Group number Grouping Inoculation site Immunization dose Immunization program 1 saRNA-PEDV muscle 50 μg / dose One immunization 2 Commercial inactivated vaccines muscle 2mL / dose One immunization 3 Commercial inactivated vaccines muscle 2mL / dose Secondary immunization 4 control group / / /

[0160] The immune serum, control positive serum and negative control serum were inactivated in a 56°C water bath for 30 minutes. The inactivated samples, positive control and negative control serum were serially diluted 2-fold (1:2, 1:4, 1:8, 1:16, 1:32...1:256) in a 96-well plate with DMEM (containing 10 μg / mL trypsin). Each dilution was 4 wells, 100 μL per well. The (standard strain) was diluted with DMEM (containing 10 μg / mL trypsin) to a virus content of 200 TCID 50 / 0.1mL virus solution. Add 100μL of the diluted virus solution to each well of a 96-well U-shaped plate containing serum and neutralize in a 37℃ 5% CO2 incubator for 1 hour. Take the cells in the 96-well culture plate that have been cultured for 24-48 hours, discard the culture medium, transfer the virus and serum mixture that has been neutralized for 1 hour to the corresponding wells of the 96-well culture plate, add 100μL to each well, incubate in a 37℃ 5% CO2 incubator and observe for 5 days. Record the number of wells with CPE in the sample to be tested, and calculate the neutralizing antibody titer according to the Reed-Müench method.

[0161] Table 13: Neutralizing antibodies in piglets

[0162]

[0163]

[0164] The above experiments demonstrate that the saRNA-PEDV mRNA vaccine provided by the present invention can effectively induce humoral immunity and produce high levels of neutralizing antibodies against PEDV strains in piglets after immunization. Furthermore, a single immunization with the saRNA-PEDV vaccine produces slightly higher antibody levels than a secondary immunization with the inactivated vaccine, thereby simplifying the immunization procedure while maintaining the original immune level.

[0165] 3. Safety trials of saRNA vaccines

[0166] To evaluate the safety of saRNA vaccines, a piglet animal study was conducted. Fifteen healthy, susceptible piglets aged 3-5 days were randomly divided into three groups and vaccinated intramuscularly with the saRNA vaccine. The immunization schedule was as follows.

[0167] Table 14: Piglet safety vaccination program

[0168] Group number Grouping Inoculation site dose Immunization program 1 saRNA-PEDV muscle 50 μg / dose One immunization 2 saRNA-PEDV muscle 100 μg / dose One immunization 3 Blank control / / /

[0169] A single immunization was performed. Observe for 2 days before vaccination, measure body temperature daily, and take the average value as basal body temperature. After vaccination, measure body temperature daily for 14 consecutive days. Compared to pre-vaccination levels, there should be no significant changes in spirits and appetite, and the body temperature should not rise more than 1°C. The results are shown in Table 15.

[0170] Table 15: Safety test results of piglet vaccination

[0171] Group number Clinical manifestations injection site Average body temperature changes 1 Mental state, appetite and body temperature are normal The inoculation sites were normal and no abnormalities were found. <1℃ 2 Mental state, appetite and body temperature are normal The inoculation sites were normal and no abnormalities were found. <1℃ 3 Mental state, appetite and body temperature are normal The inoculation sites were normal and no abnormalities were found. <1℃

[0172] The results showed that after piglets were immunized with the saRNA vaccine (LNP process) expressing PEDVS protein provided by the present invention, the piglets in the normal immunization group, the overdose immunization group and the blank control group showed normal clinical manifestations and vaccination sites, demonstrating the safety of the saRNA vaccine.

[0173] 4. Porcine epidemic diarrhea virus saRNA vaccine challenge test in pigs

[0174] Thirty healthy susceptible piglets aged 3-5 years (neutralizing antibodies to porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine delta coronavirus were all less than 1:4) were selected and divided into 6 groups, with 5 piglets in each group. The piglets were immunized according to the immunization schedule in Table 16. 21 days after a single immunization or 7 days after a second immunization, each piglet was orally administered with 10.0 mL of PEDV cytotoxin (virus content of 10 6.0 TCID 50 The piglets were observed for 10 consecutive days after challenge. See Table 16.

[0175] Table 16: Immunization schedule for piglets before challenge

[0176]

[0177] Results showed that piglets in the blank control group developed diarrhea symptoms, including unformed and watery stools, with a protection rate of 0 / 5. Piglets in the saRNA-PEDV vaccine group maintained normal spirits, appetite, and body temperature, with a protection rate of 5 / 5. These results demonstrate that the saRNA-PEDV vaccine provides excellent protection against the virus. Some piglets developed symptoms after a single and secondary immunization with the commercial inactivated vaccine. Piglets were then challenged with the virus after a secondary immunization with both vaccines. Data indicate that the commercial inactivated vaccine provided slightly less protection than the saRNA-PEDV vaccine. All piglets in the blank control group developed disease.

[0178] Table 17: Results of protective efficacy test after piglet immunization with porcine epidemic diarrhea virus

[0179]

[0180]

[0181] From the experimental results of Example 3, it can be found that the present invention evaluates the immune efficacy of porcine epidemic diarrhea virus saRNA vaccine by implementing piglet animal experiments, which is more convincing for application. The vaccine effectiveness test shows that, judging from the neutralizing antibody level data, the neutralizing antibody values ​​at the third week of immunization at a single immunization dose of 50 μg are all above the single immunization commercial inactivated vaccine; and slightly better than the neutralizing antibody value of the secondary immunization commercial inactivated vaccine, and the corresponding neutralizing antibody level is also sufficient to protect the body. This proves that the saRNA-PEDV vaccine has good immunogenicity while reducing the number of immunizations and doses. The single immunization program of the liposome-encapsulated saRNA-PEDV vaccine induces high neutralizing antibody levels, can induce high levels of PEDV S protein-specific neutralizing antibodies in piglets, and shows good protection effects in the challenge protection experiment, proving that a single immunization of the saRNA-PEDV vaccine can resist the attack of porcine epidemic diarrhea virus and has good application prospects.

[0182] The sequences involved in the present invention are as follows.

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A nucleic acid molecule, characterized in that It includes a nucleotide sequence encoding the S protein of porcine epidemic diarrhea virus, and the nucleotide sequence of the S protein of porcine epidemic diarrhea virus has at least 80% identity with the sequence shown in SEQ ID NO: 10 or 11.

2. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule further comprises a sequence encoding a trimer motif; the sequence encoding the trimer motif is located upstream or downstream of the nucleotide sequence of the S protein of the porcine epidemic diarrhea virus; Optionally, the trimer motif includes: any one of T4-Foldon and GCN-Foldon; Optionally, the amino acid sequence of the T4-Foldon is shown in SEQ ID NO: 12; Optionally, the amino acid sequence of the GCN-Foldon is shown in SEQ ID NO:

13.

3. An RNA molecule, characterized in that The DNA vector that transcribes and generates the RNA molecule includes an antigen region; the antigen region contains a sequence encoding an antigen, and the nucleotide sequence of the antigen region is the nucleic acid molecule according to claim 1 or 2.

4. The RNA molecule according to claim 3, characterized in that The RNA molecule includes mRNA or saRNA; Optionally, the DNA vector further comprises a non-structural protein region; the non-structural protein region comprises a sequence encoding a non-structural protein, and the non-structural protein is derived from an alphavirus; Optionally, the non-structural protein includes any one or more of NSP1, NSP2, NSP3 and NSP4; Optionally, the amino acid sequences of NSP1, NSP2, NSP3 and NSP4 are at least 80% identical to the sequences shown in SEQ ID NOs: 5 to 8, in sequence; Optionally, the DNA vector further comprises poly-A; Optionally, the nucleotide sequence of the poly-A is at least 80% identical to the sequence shown in SEQ ID NO:

9.

5. The RNA molecule according to claim 3 or 4, characterized in that In order from the 5' end to the 3' end, the DNA vector includes: a promoter, a 5' UTR, a non-structural protein region, a subgenomic promoter region, the antigen region, a 3' UTR and poly-A; Optionally, the promoter includes a T7 promoter, a T3 promoter or an SP6 promoter; Optionally, the nucleotide sequence of the T7 promoter is shown in SEQ ID NO: 1; Optionally, the nucleotide sequence of the 5'UTR is shown in SEQ ID NO: 2; Optionally, the nucleotide sequence of the subgenomic promoter region is shown in SEQ ID NO: 3; Optionally, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO: 4; Optionally, the sequence of the DNA vector is shown as SEQ ID NO:

16.

6. An isolated nucleic acid or a vector containing the nucleic acid, characterized in that The isolated nucleic acid encodes the RNA molecule according to any one of claims 2 to 5.

7. Use of the nucleic acid molecule according to claim 1 or 2, the RNA molecule according to any one of claims 3 to 5, or the isolated nucleic acid according to claim 6, or a vector containing the nucleic acid, in the preparation of a vaccine for preventing or treating porcine epidemic diarrhea virus infection; Optionally, the vaccine comprises a saRNA vaccine.

8. A composition, characterized in that The active ingredient comprises the RNA molecule according to any one of claims 3 to 5.

9. The composition according to claim 8, characterized in that The composition further comprises: any one or more of a buffer, a carrier, an adjuvant and a stabilizer; Optionally, the buffer comprises: citric acid buffer, sodium acetate buffer, phosphate buffer, citrate-citric acid buffer or acetic acid-sodium acetate buffer, with a pH of 3.5 to 5; Optionally, the carrier comprises: any one of lipid nanoparticles, polymer nanoparticles and virus-like particles; Optionally, the components of the lipid nanoparticles include: any one or a combination of cationic lipids, neutral lipids, cholesterol or cholesterol derivatives, pegylated lipids, chitosan and QS-21; Optionally, the components of the lipid nanoparticles include, by molar percentage, 40% to 60% cationic lipids, 10% to 15% neutral lipids, 35% to 38.5% cholesterol or cholesterol derivatives, 0.1% to 5% PEGylated lipids, and 0.1% to 5% chitosan or QS-21; Optionally, the cationic lipid comprises cationic lipid SM-102 or ALC-0315; Optionally, the neutral lipids include any one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and sphingomyelin; Optionally, the cholesterol derivative comprises β-sitosterol; Optionally, the PEGylated lipid comprises 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol.

10. A vaccine, characterized in that It includes: The RNA molecule according to any one of claims 3 to 5 or the composition according to claim 8 or 9.

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