Fat calicivirus vaccine as well as related products and application thereof

Abstract

The invention discloses a feline calicivirus vaccine as well as a related product and application thereof, and relates to the field of vaccines. The invention provides the saRNA vaccine of the feline calicivirus, the saRNA vaccine adopts an optimized antigen sequence and a saRNA skeleton, and compared with a traditional feline calicivirus vaccine, the saRNA vaccine has the advantages of being early in antibody production period, good in safety, simple in immune procedure, higher in antibody titer and the like.

Description

Technical Field

[0001] The present invention relates to the field of vaccines, in particular to a feline calicivirus vaccine and related products and applications thereof. Background Art

[0002] Feline calicivirus (FCV) is a member of the Caliciviridae family and the genus Vesivirus. FCV is a single-stranded, positive-sense RNA virus lacking an envelope and possessing icosahedral symmetry. Its capsid is composed of 90 dimers of the major structural protein VP1, forming 32 cup-shaped depressions. FCV has only one serotype, and its antigens are highly variable.

[0003] Felines are generally susceptible to FCV, with kittens being the most susceptible. Cats carrying the virus are important sources of infection, excreting the virus through ocular and nasal secretions, feces, and saliva, and transmitting it through contact, foodborne infection, and the respiratory tract. Clinical symptoms caused by FCV can be categorized as ulcerative disease, periodontal stomatitis, pneumonia, lameness syndrome, and fatal systemic disease. The type and severity of symptoms depend primarily on the infecting FCV strain, the host's immune response, and co-infection with other pathogens.

[0004] FCV can maintain a relatively high prevalence in cat populations for a long time. On the one hand, this is because infected cats can excrete the virus for a long time. On the other hand, it is caused by multiple factors such as mixed infection of multiple strains and secondary infection. There may be multiple FCV strains circulating in a multi-cat population. Even with the widespread use of vaccines, the prevalence of FCV remains high, and the mortality rate is high for kittens with severe upper respiratory tract infections.

[0005] There is currently no specific treatment for FCV, and vaccination is the primary means of preventing and controlling FCV. Currently, the primary FCV vaccine is a triple inactivated vaccine. Numerous studies have confirmed the effectiveness of commercially available FCV vaccines, and while existing vaccines play an important role in clinical prevention and treatment, they lack the ability to completely prevent infection with wild-type strains, and vaccinated cats may develop subclinical and persistent infections.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The present invention aims to provide a feline calicivirus vaccine and related products and applications thereof.

[0008] In a first aspect, an embodiment of the present invention provides a nucleic acid molecule comprising: a nucleotide sequence of a feline calicivirus VP1 protein; the nucleotide sequence of the feline calicivirus VP1 protein has at least 80% identity with the sequence shown in SEQ ID NO: 10.

[0009] 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.

[0010] 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.

[0011] In a fourth aspect, 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 drug for preventing, treating, or assisting in the treatment of feline calicivirus infection.

[0012] 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.

[0013] 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.

[0014] The present invention has the following beneficial effects:

[0015] The saRNA vaccine provided by the present invention has the advantages of early antibody production, high safety, simple immunization procedure and higher antibody titer, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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.

[0017] Figure 1 is the basic structure of saRNA;

[0018] Figure 2 is the target protein expression intensity corresponding to saRNA.V1~9;

[0019] Figure 3 is the ratio of fluorescent cells corresponding to saRNA.V1-9;

[0020] Figure 4 This is a fluorescence microscopy image of cells after K562 cells were transfected with saRNA.V3 and V1 RNA molecules;

[0021] Figure 5The secretion amount of saRNA.V3-1~V3-7 target antigen. 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 do not ultimately integrate into the 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 of a feline calicivirus VP1 protein; the nucleotide sequence of the feline calicivirus VP1 protein has at least 80% identity with the sequence shown in SEQ ID NO: 10.

[0032] In the embodiment of the present invention, the VP1 protein of FCV is used as the target antigen, combined with structural biology research, and optimized to obtain the antigen sequence shown in SEQ ID NO: 10, which is applied to the preparation of RNA vaccines (such as non-replicating mRNA vaccines and saRNA vaccines), which can support high expression of RNA in cells and achieve good animal immune effects.

[0033] In some embodiments, the nucleic acid molecule further comprises a signal peptide and / or a folding tag; the signal peptide and / or folding tag is fused upstream (5' end) or downstream (3' end) of the nucleotide sequence of the feline calicivirus VP1 protein.

[0034] Signal peptide, also known as signal sequence, is a short peptide chain (usually 15-30 amino acids) located at the N-terminus of a protein (sometimes not necessarily at the N-terminus), responsible for guiding newly synthesized proteins into specific cellular transport pathways.

[0035] In some embodiments, the signal peptide comprises any one of CD5sp, gDsp, IgGsp, HSAsp and IL-2sp.

[0036] CD5sp (CD5 Signal Peptide) is a signal peptide of the human CD5 molecule. In some embodiments, the amino acid sequence of the CD5sp is shown in SEQ ID NO: 11.

[0037] gDsp (Herpes Simplex Virus Glycoprotein D Signal Peptide) is the signal peptide of glycoprotein D of herpes simplex virus (HSV). In some embodiments, the amino acid sequence of gDsp is shown in SEQ ID NO: 12.

[0038] IgGsp (Immunoglobulin G Signal Peptide) is the signal peptide of the immunoglobulin G (IgG) heavy chain or light chain. In some embodiments, the amino acid sequence of the IgGsp is shown in SEQ ID NO:13.

[0039] HSAsp (Human Serum Albumin Signal Peptide) is the signal peptide of human serum albumin (HSA). In some embodiments, the amino acid sequence of HSAsp is shown in SEQ ID NO: 14.

[0040] IL-2sp (Interleukin-2 Signal Peptide) refers to the N-terminal signal peptide sequence of the human interleukin-2 (IL-2) precursor, which is responsible for guiding the secretion of IL-2 protein. In some embodiments, the amino acid sequence of the IL-2sp is shown in SEQ ID NO: 15.

[0041] In some embodiments, the nucleic acid molecule further comprises a folding tag, wherein the folding tag comprises any one of a trimer motif, a solubility tag, and a DARPin.

[0042] Trimerization Motif, which can promote the trimeric folding of antigens.

[0043] In some embodiments, the trimerization motif comprises either T4-Foldon or GCN-Foldon. T4-Foldon is the trimerization motif 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.

[0044] In some embodiments, the amino acid sequence of T4-Foldon is shown in SEQ ID NO:16.

[0045] In some embodiments, the amino acid sequence of the GCN-Foldon is shown in SEQ ID NO:17.

[0046] The solubility-enhancing tag includes either maltose binding protein (MBP) or small ubiquitin-like modifier (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.

[0047] In some embodiments, the amino acid sequence of the maltose binding protein is shown in SEQ ID NO: 18.

[0048] In some embodiments, the amino acid sequence of the small ubiquitin-like modified protein is shown in SEQ ID NO:19.

[0049] DARPin (designed ankyrin repeat protein) binds to antigens, promoting antigen conformational stabilization and / or polymerization, thereby improving vaccine efficacy.

[0050] In some embodiments, the amino acid sequence of the DARPin is shown in SEQ ID NO: 20.

[0051] In some embodiments, the nucleic acid molecule is a DNA molecule.

[0052] 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.

[0053] In some embodiments, the RNA molecule comprises mRNA (vaccine) or saRNA (vaccine).

[0054] 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.

[0055] 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).

[0056] In some embodiments, the nonstructural protein comprises any one or more of NSP1, NSP2, NSP3, and NSP4.

[0057] The function of nonstructural proteins is to drive the self-replication of mRNA, thereby amplifying antigen expression. Their specific functions are as follows: NSP1 is responsible for capping the 5' end of viral RNA, protecting it from nuclease degradation and facilitating ribosome recognition; NSP2 possesses helicase, protease, and RNA triphosphatase activities, responsible for cleaving the polyprotein precursor (NSP1-4) and unwinding the RNA double-stranded structure; NSP3 participates in the interaction between the virus and host proteins, regulating the assembly of the viral replication complex; and NSP4 is an RNA-dependent RNA polymerase (RdRp), catalyzing the replication of the RNA chain.

[0058] 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;

[0059] In some embodiments, the DNA vector further comprises poly-A (same as poly-A tail, poly(A));

[0060] In some embodiments, the nucleotide sequence of the poly-A is at least 80% identical to the sequence shown in SEQ ID NO:9.

[0061] In some embodiments, at least 80% identity includes at least 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%.

[0062] 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.

[0063] 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.

[0064] In some embodiments, the nucleotide sequence of the 5'UTR is as shown in SEQ ID NO:2.

[0065] In some embodiments, the nucleotide sequence of the subgenomic promoter region is shown in SEQ ID NO:3.

[0066] In some embodiments, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO:4.

[0067] In some embodiments, the sequence of the DNA vector is shown in SEQ ID NO:21.

[0068] 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.

[0069] In some embodiments, the isolated nucleic acid is a DNA molecule.

[0070] 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 preceding embodiments, or the RNA molecule as described in any of the preceding embodiments, or the isolated nucleic acid as described in any of the preceding embodiments, or the vector containing the nucleic acid in the preparation of a vaccine for preventing or treating feline calicivirus infection.

[0071] In some embodiments, the vaccine comprises a veterinary vaccine. In some embodiments, the veterinary vaccine comprises a feline (e.g., cat, lion, leopard cat) and / or a canine (dog).

[0072] In some embodiments, the vaccine comprises a non-replicating mRNA vaccine or a saRNA vaccine.

[0073] 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.

[0074] In some embodiments, the composition further comprises: any one or more of a buffer, a carrier, an adjuvant, and a stabilizer;

[0075] 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 to 5, specifically any one of 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5 or a range between any two of them.

[0076] In some embodiments, the carrier comprises any one of lipid nanoparticles (LNP), polymer nanoparticles (PNP), and virus-like particles (VLP).

[0077] 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.

[0078] In some embodiments, the components of the lipid nanoparticles include, by mole 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.

[0079] In some embodiments, the molar percentage of the cationic lipid can be any one of 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, or any range between any two thereof. The molar percentage of the neutral lipid can be any one of 10%, 11%, 12%, 13%, 14%, 15%, or any range between any two thereof. The molar percentage of cholesterol or a cholesterol derivative can be any one of 35%, 36%, 37%, 38%, 38.5%, or any range between any two thereof. The molar percentage of the pegylated lipid can be any one of 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%, or any range between any two thereof. The molar percentage of chitosan or QS-21 can be any one of 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5%, or any range between any two thereof.

[0080] In some embodiments, the cationic lipid comprises cationic lipid SM-102 or ALC-0315.

[0081] 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);

[0082] In some embodiments, the cholesterol derivative comprises β-sitosterol.

[0083] 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.

[0084] 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.

[0085] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0086] Example 1: Screening of saRNA backbones and optimized sequences of feline calicivirus antigens.

[0087] The basic structure of the DNA vector of saRNA includes: T7 promoter (SEQ ID NO: 1), 5'UTR (SEQ ID NO: 2), subgenomic promoter region (SEQ ID NO: 3), non-structural protein region (NSP1, NSP2, NSP3 and NSP4), antigen region, 3'UTR (SEQ ID NO: 4) and poly-A, such as Figure 1As shown. In this example, the non-structural protein regions NSP2, NSP3 and poly-A 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 was used to verify the application effects of different non-structural protein regions (NSP1-4) and poly-A on the saRNA skeleton by expression at the cellular level. The sequence NCBI (L01443.1) of Venezuelan equine encephalitis virus (TC-83Venezuelan Equine Encephalitis Virus, VEEV) was selected as the skeleton basis, and the specific experiments are shown below.

[0088] 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.

[0089] 1.2 Construction of saRNA-eGFP expression vector

[0090] 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.

[0091] 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.

[0092] 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 and then linearized with NotI after ligation. 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.

[0093] Table 1: Different skeleton groups

[0094] 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

[0095] Note: The sequence of 30A-G-69A is shown in SEQ ID NO: 9.

[0096] 1.3 Preparation of saRNA

[0097] The nine strains containing the target plasmid obtained above were fermented and amplified, 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.

[0098] 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.

[0099] 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.

[0100] Table 2: In vitro transcription system

[0101] 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

[0102] 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.

[0103] 1.4 ELISA quantitative determination of IDR-1018 concentration in cell supernatant and expression on cells

[0104] 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.

[0105] According to the kit instructions, the lyophilized standard was reconstituted with 500 μL of deionized water (final concentration 100 ng / mL).

[0106] Serial dilutions were performed to the following concentrations: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, and 0 ng / mL.

[0107] 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.

[0108] Standard curve equation (4PL fitting): y = (AD) / (1+(x / C)B)+D;

[0109] 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 poly-A tail, the expression of IDR-1018 can be further increased.

[0110] In order to screen out the better skeleton, further in vitro expression was performed on cells to observe 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 taken. 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 of over 70%, indicating 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 mutated the nonstructural proteins NSP2 and NSP3. The NSP3 hypervariable region did not affect the viral replication ability, while enabling effective expression and secretion on cells. The NSP3 hypervariable region incorporates immunostimulatory molecules, enhancing its role in macrophage differentiation compared to the original backbone, promoting the differentiation of monocytes into a functionally specific macrophage phenotype, enhancing the activation of antigen-presenting cells, and improving the immunogenicity of the vaccine.

[0111] 1.5 Screening of optimized feline calicivirus antigen sequences

[0112] Feline calicivirus (FCV), belonging to the Caliciviridae family and the genus Vesivirus, is a significant pathogen causing oral and upper respiratory tract illness in felines. The FCV gene encodes three open reading frames: ORF1, ORF2, and ORF3. ORF2 encodes the VP1 protein, which consists of six regions, each with specific structure and function. The VP1 protein is the primary site of interaction between the virus and host cells and is a key molecule for studying its pathogenicity and developing diagnostic tools.

[0113] In the embodiment of the present invention, the amino acid sequence of the VP1 protein is converted synonymously, and the optimized VP1 protein is screened for a suitable signal peptide, so that the target antigen is better secreted in the animal body. The signal peptides mainly include CD5sp, gDsp, IgGsp, HSAsp and IL-2sp. A signal peptide is added to the N-terminus of the antigen. The function of the signal peptide is to guide the target protein to the subcellular organelles containing different membrane structures and to cut the signal peptide under the action of a signal peptidase. It can also be used to monitor whether the protein is secreted. Finally, saRNA.V3-1, saRNA.V3-2, saRNA.V3-3, saRNA.V3-4, saRNA.V3-5, saRNA.V3-6 and saRNA.V3-7 were designed, of which the unoptimized antigen was saRNA.V3-6 and saRNA.V3-7 was the control.

[0114] Cell transfection

[0115] Culture HEK293 cells with 10% FBS at 37°C and 5% CO. Seed HEK293 cells into 6-well plates to a confluence of 70%-90%. Prepare 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 the complex. Add the complex dropwise to the cell culture medium and mix gently. Incubate at 37°C and 5% CO for 4-6 hours, then replace the medium with complete serum. Collect the supernatant and cell lysate 72 hours after transfection. Assess the secretion efficiency of the target protein by Western blotting.

[0116] Table 3: Signal peptide screening

[0117] Serial number Target antigen signal peptide skeleton saRNA.V3-1 Optimized VP1 CD5sp saRNA.V3 saRNA.V3-2 Optimized VP1 gDsp saRNA.V3 saRNA.V3-3 Optimized VP1 IgGsp saRNA.V3 saRNA.V3-4 Optimized VP1 hsasp saRNA.V3 saRNA.V3-5 Optimized VP1 IL-2sp saRNA.V3 saRNA.V3-6 VP1 - saRNA.V3 saRNA.V3-7 - - saRNA.V3

[0118] Note: The nucleotide sequence of the optimized VP1 is shown in SEQ ID NO: 10, the amino acid sequence of CD5sp is shown in SEQ ID NO: 11; the amino acid sequence of gDsp is shown in SEQ ID NO: 12; the amino acid sequence of IgGsp is shown in SEQ ID NO: 13; the amino acid sequence of HSAsp is shown in SEQ ID NO: 14; and the amino acid sequence of IL-2sp is shown in SEQ ID NO: 15.

[0119] By collecting different types of samples for 72 hours and measuring the gray value by WB experiment, the secretion amount of target antigens of different components was determined, and finally the better signal peptide was screened. Figure 5 ) showed that the saRNA.V3-1 sequence bound to the CD5 signal peptide can secrete a high amount of antigen. CD5 is suitable for expression as a signal peptide in mammalian systems. Since CD5 is mainly expressed in T cells and B1 cell subsets, it participates in lymphocyte activation and promotes the secretion of recombinant proteins. Compared with other signal peptides, the CD5 signal peptide has the advantage of high hydrophobicity, which makes it more efficient in secretion in the present invention.

[0120] 1.5.2VP1 protein is a key protein on the surface of the virus responsible for virus particle assembly, host cell receptor binding and immunogenicity. The natural folding form of the mimic protein links the VP1 protein to the trimer, thereby improving the immunogenicity of the VP1 protein. Selecting a suitable folding tag has a promoting effect on the expression of VP1 protein and the production of neutralizing antibodies. According to the conformation of the VP1 protein, folding tags such as the T4 phage fiber protein trimer motif (T4-folden) and yeast (GCN-folden) (signal peptide + antigen + folding tag) are introduced at the C-terminus of the antigen to form saRNA.V3-1-1, saRNA.V3-1-2, saRNA.V3-1-3, saRNA.V3-1-4, and saRNA.V3-1-5 new sequences, respectively. The one without the trimer motif is saRNA.V3-1-6, and saRNA.V3-1-7 is the control.

[0121] Table 4: Trimeric motif screening

[0122] Serial number Target antigen Trimer motif skeleton saRNA.V3-1-1 Optimized VP1 T4-foldon saRNA.V3 saRNA.V3-1-2 Optimized VP1 GCN-foldon saRNA.V3 saRNA.V3-1-3 Optimized VP1 MBP saRNA.V3 saRNA.V3-1-4 Optimized VP1 SUMO saRNA.V3 saRNA.V3-1-5 Optimized VP1 DARPin saRNA.V3 saRNA.V3-1-6 Optimized VP1 - saRNA.V3 saRNA.V3-1-7 - - saRNA.V3

[0123] Note: The optimized VP1 nucleotide sequence is shown in SEQ ID NO: 10, the amino acid sequence of T4-Foldon is shown in SEQ ID NO: 16, the amino acid sequence of GCN-Foldon is shown in SEQ ID NO: 17, and the amino acid sequence of maltose binding protein is shown in SEQ ID NO: 18. The amino acid sequence of small ubiquitin-like modifier protein is shown in SEQ ID NO: 19. The amino acid sequence of DARPin is shown in SEQ ID NO: 20. The DNA vector sequence of saRNA.V3-1-2 is shown in SEQ ID NO: 21.

[0124] 1.5.3. Competitive ELISA for quantitative detection of antigens

[0125] Dilute VS-FCV to 1-5 μg / mL with coating buffer, add 100 μL to each well, and incubate at 4°C overnight. Wash 3 times (PBST, 5 minutes each time). Add 200 μL blocking solution to each well and block at 37°C for 1 hour. Wash 3 times. Pre-incubation: Add FCV1-43

[0126] (Purchased from Abcam) Antibody 1 μg / mL was mixed with serially diluted competing antigens (saRNA.V3-1-1, saRNA.V3-1-2, saRNA.V3-1-3, saRNA.V3-1-4, saRNA.V3-1-5, saRNA.V3-1-6, saRNA.V3-1-7, VS-FCV, concentration range: 0.1, 1, 10, 100 μg / mL) in an EP tube (total volume 100 μL) and incubated at 37°C for 1 hour. Transfer to ELISA plate: Add the pre-incubation mixture to the wells coated with antigen and incubate at 37°C for 1 hour. Wash 5 times. Add HRP-labeled secondary antibody (1:5000 dilution) and incubate at 37°C for 1 hour. Wash 5 times. Add 100 μL of TMB color development solution to each well and react at room temperature in the dark for 10-15 minutes. Add 50 μL of stop solution and immediately read the absorbance (OD value) at 450 nm using a microplate reader. Inhibition rate (%) = (1-(OD of competition group-OD of blank) / (OD of non-competition group-OD of blank)) × 100.

[0127] Table 5: Inhibition rate of different antigens

[0128]

[0129]

[0130] The results showed that the saRNA.V3-1-2 sequence bound to GCN-foldon had a higher inhibition rate, and was close to the inhibition rate of the VP1 protein of the epidemic strain VS-FCV, indicating that the addition of the folding tag had little effect on the binding epitope of the VP1 protein neutralizing antibody, and could stimulate the body to produce better immune protection, further testing the immunogenicity of saRNA.V3-1-2 in animal bodies.

[0131] Example 2. Screening of saRNA delivery vectors and components and preparation of saRNA vaccines

[0132] 2.1 saRNA Components

[0133] 2.1.1 Components of aqueous buffer

[0134] 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).

[0135] Table 6: Aqueous buffer grouping

[0136]

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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).

[0141] 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).

[0142] 2.1.2 Lipid mixture components

[0143] This embodiment also provides 6 groups of saRNA vaccine delivery carrier lipid mixtures, the preparation method of which is as follows.

[0144] 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 7.

[0145] Table 7: Liposome Grouping

[0146]

[0147] 2.2 Preparation of saRNA solution

[0148] 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.

[0149] 2.3saRNA-LNP Preparation

[0150] Lipid mixtures 1-6 were dissolved in ethanol solution to form lipid phases (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 lipid phase were rapidly mixed using a microfluidic system and extruded simultaneously at a lipid:aqueous phase volume ratio of 1:3. Electrostatic attraction and hydrophilic-hydrophobic interactions allowed the plasmids in the aqueous phase and the various lipid molecules in the oil phase to encapsulate the plasmids to form LNPs, allowing the saRNA to be encapsulated within the LNPs. A total of 90 saRNA-LNPs were produced.

[0151] 2.3.1saRNA-LNP Particle Size and Dispersion Coefficient Detection

[0152] 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 (1g sucrose was dissolved in 9ml 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 105nm were screened out as saRNA-LNP-1, saRNA-LNP-2, saRNA-LNP-3, saRNA-LNP-4, saRNA-LNP-5, and saRNA-LNP-6.

[0153] Table 8: Particle size and dispersion coefficient of saRNA-LNP in each group

[0154] 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

[0155] 2.3.2saRNA-LNP Encapsulation Efficiency Detection

[0156] 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:

[0157] Table 9: Standard Dilutions

[0158] 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

[0159] To release RNA using Triton-100, lyse LNPs using Triton-100. Prepare a 2% Triton-100 solution (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 concentrated, centrifuged samples, dilute them 20-fold before proceeding with the above procedure.

[0160] Dilute Ribo Green RNA Reagent 500-fold (5 μL Ribo Green RNA Reagent + 2.495 mL 1× TE buffer). For the test, add 150 μL of the newly prepared RiboGreen RNA Reagent to each tube, mix well, and incubate in the dark for 5 minutes. Then, transfer 200 μL of the solution to a 96-well black plate. Detect 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 the table below.

[0161] Table 10: Sample parameters

[0162] 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％

[0163] 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.

[0164] 2.4 Ultrafiltration

[0165] 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.

[0166] Table 11: Particle size of saRNA-LNP in each group

[0167] 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

[0168] 2.5 Calcein-AM / PI double staining assay to detect the effect of saRNA-LNP on cells

[0169] 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.

[0170] Staining working solution: Calcein-AM (final concentration 4 μM) and PI (final concentration 5 μg / mL) were diluted in PBS or serum-free medium.

[0171] 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.

[0172] Table 12: HEK293T cell mortality (%)

[0173]

[0174] 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.

[0175] While saRNA-LNP-5's aqueous citric acid buffer provides an acidic environment for cationic lipid ionization, the use of a strongly acidic buffer may have caused oxidative side reactions 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 citrate-citric acid buffer and β-sitosterol in saRNA-LNP-4 resulted in stable encapsulation efficiency and safety, while the addition of chitosan enhanced the immune response.

[0176] Based on the above data, saRNA-LNP-4 was finally selected as the most preferred saRNA-LNP formulation.

[0177] 2.6 Preparation of Feline Calicivirus saRNA-LNP Vaccine

[0178] 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.V3-1-2 aqueous phase formulation (the aqueous phase buffer was a citrate-citric acid buffer (pH = 4.5)) and the lipid phase were rapidly mixed using a microfluidic system and extruded simultaneously at a lipid phase:aqueous phase volume ratio of 1:3. The mRNA in the aqueous phase and the various lipid molecules in the oil phase solution were able to encapsulate the mRNA to form LNPs under electrostatic attraction and hydrophilic-hydrophobic forces, thereby encapsulating the saRNA within the LNPs. This formed the saRNA-LNP formulation of saRNA.V3-1-2.

[0179] Example 3 Safety and Efficacy Evaluation of Feline Calicivirus mRNA Vaccine in Cats

[0180] 3.1. Vaccine safety trials

[0181] To evaluate the vaccine's safety, a feline animal study was conducted. Twenty healthy, susceptible cats aged 8-9 weeks were randomly divided into four groups and administered the mRNA vaccine intramuscularly in the thigh. The immunization schedule was as follows.

[0182] Table 13: Safe vaccination schedule for cats

[0183] Group number Grouping Inoculation site dose Immunization program 1 saRNA.V3-1-2 vaccine muscle 20 μg / dose One immunization 2 saRNA.V3-1-2 vaccine muscle 20 μg / dose Repeat vaccination after 14 days 3 saRNA.V3-1-2 vaccine muscle 40 μg / dose One immunization 4 Blank control / / /

[0184] A single immunization is performed. Observe for 2 days before vaccination, measure body temperature regularly every day, and take the average value as the basal body temperature. After vaccination, measure body temperature once a day regularly for 14 consecutive days of observation. Compared with pre-vaccination, there should be no significant changes in spirit and appetite, and the temperature should not rise by more than 1°C. Repeat single-dose vaccination, with an interval of 14 days between immunizations. 14 days after the second immunization and compared with the two days before the first vaccination, there should be no significant changes in spirit and appetite, and the temperature should not rise by more than 1°C. Observe for 14 days, observing spirit, appetite, and injection site reactions every day. The experimental results are shown in the table.

[0185] Table 14: Safety trial results of cat vaccination

[0186] 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℃ 4 Mental state, appetite and body temperature are normal The inoculation sites were normal and no abnormalities were found. <1℃

[0187] 3.2. Evaluation of the effectiveness of feline calicivirus mRNA vaccine in cats

[0188] To verify the immune effect of the feline calicivirus saRNA vaccine (saRNA-LNP formulation of saRNA.V3-1-2 of Example 2, abbreviated as saRNA.V3-1-2 vaccine), a cat immunization test was carried out. The experimental cats were provided by a cat breeding base in the vicinity of Huzhou, Zhejiang Province, and all cats tested negative for feline calicivirus by PCR. Fifteen healthy cats aged 8-9 weeks were randomly divided into three groups and intramuscularly immunized according to the grouping in the table below.

[0189] Inactivate the immune serum, positive control serum, and negative control serum in a 56°C water bath for 30 min. Serially dilute the inactivated test sample, positive control, and negative control serum in a 96-well plate in two-fold increments using DMEM. 4 wells per dilution, 100 μL of diluted serum per well. Dilute FCV virus to 100 TCID using DMEM containing 2% FBS. 50 / 50μL. 100μL of the diluted virus solution was added to each well of a 96-well plate containing serum and neutralized in a 37°C, 5% CO2 incubator for 1 hour. Cells cultured for 24 hours in a 96-well plate were transferred to the corresponding wells of the 96-well plate, with 100μL of the virus-serum mixture transferred to each well. The cells were incubated in a 37°C, 5% CO2 incubator for 72 hours. The culture medium was discarded, and the cells were fixed with pre-cooled 80% acetone for 10 minutes and dried at room temperature. Antibody specific for FCV (GenBank: XCO35655.1) diluted 1:1000 in PBS was added to each well and incubated in a 37°C incubator for 1 hour. The primary antibody was discarded, and the plate was washed three times with PBS. The PBS was discarded, and goat anti-mouse IgG-AlexaFluor-594 diluted 1:1000 in PBS was added to each well. The cells were incubated in a humidified chamber at 37°C for 30 minutes in the dark. Unbound antibody was removed by washing three times with PBS. The number of fluorescent foci in each well was observed using a fluorescence microscope (10× objective lens). The titer of FCV neutralizing antibodies in the tested serum was calculated using the Reed-Muench method under the condition that both the virus control and the positive serum control groups were established.

[0190] Table 15: Cat Immunization Schedule

[0191] Group number Grouping Inoculation site Immunization dose Immunization program 1 saRNA.V3-1-2 vaccine muscle 20 μg / dose One immunization 2 Commercialized triple inactivated vaccine muscle 1mL / dose One immunization 3 Blank control / / /

[0192] The above tests demonstrate that cat antiserum protection is achieved when its neutralization titer is 1:90 or higher. Immunization of cats with the saRNA.V3-1-2 vaccine provided by the present invention effectively induces humoral immunity, producing high levels of neutralizing antibodies against the FCV strain (GenBank: XCO35655.1). Antibody levels produced 15 days after immunization are twice those of commercial vaccines and remain stable for up to 45 days.

[0193] Table 16: Average neutralizing antibodies in cats

[0194]

[0195] The above tests show that the saRNA.V3-1-2 vaccine provided by the present invention induces a more sustained neutralization level after immunization of cats, and the level of neutralizing antibodies produced 30 days after immunization is much higher than that of the commercial control group, stimulating a better immune response in the body. Not only that, the mRNA vaccine has good safety, avoids the risk of virulence reversion of live vaccines, reduces immunization costs and simplifies the immunization procedure. The experimental results of the saRNA backbone screened by the present invention and the saRNA.V3-1-2 vaccine prepared by the preferred delivery vector system have demonstrated the feasibility and good application prospects of the feline calicivirus vaccine.

[0196] The sequences involved in the present invention are as follows.

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] 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: The nucleotide sequence of the feline calicivirus VP1 protein; the nucleotide sequence of the feline calicivirus VP1 protein has at least 80% identity with the sequence shown in SEQ ID NO:

10.

2. The nucleic acid molecule according to claim 1, characterized in that The nucleic acid molecule further comprises: a signal peptide and / or a folding tag; the signal peptide and / or the folding tag are fused upstream or downstream of the nucleotide sequence of the feline calicivirus VP1 protein; Optionally, the signal peptide includes any one of CD5sp, gDsp, IgGsp, HSAsp and IL-2sp; Optionally, the amino acid sequence of the CD5sp is shown in SEQ ID NO: 11; Optionally, the amino acid sequence of gDsp is shown in SEQ ID NO: 12; Optionally, the amino acid sequence of the IgGsp is shown in SEQ ID NO: 13; Optionally, the amino acid sequence of the HSAsp is shown in SEQ ID NO: 14; Optionally, the amino acid sequence of the IL-2sp is shown in SEQ ID NO: 15; Optionally, the folding tag comprises any one of: T4-Foldon, GCN-Foldon, maltose binding protein, small ubiquitin-like modifier protein and DARPin; Optionally, the amino acid sequence of the T4-Foldon is shown in SEQ ID NO: 16; Optionally, the amino acid sequence of the GCN-Foldon is shown in SEQ ID NO: 17; Optionally, the amino acid sequence of the maltose binding protein is shown in SEQ ID NO:

18. Optionally, the amino acid sequence of the small ubiquitin-like modified protein is shown in SEQ ID NO:

19. Optionally, the amino acid sequence of the DARPin is shown in SEQ ID NO:

20.

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 sequence of 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:

21.

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 3 to 5.

7. Use of the nucleic acid molecule according to claim 1, 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 feline calicivirus infection; Optionally, the vaccine comprises a non-replicating mRNA vaccine or 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 pH is 4.4 to 4.6; Optionally, the pH is 4.5; 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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