Cat infectious peritonitis s protein mutant mRNA vaccine and preparation and application thereof

Abstract

The present application relates to the technical field of biological medicine, and discloses a feline infectious peritonitis S protein mutant mRNA vaccine and a preparation and application thereof, a mutant sequence is shown as SEQ ID NO: 14, a plurality of sites of a core antigen epitope prone to inducing an antibody-dependent enhancement (ADE) effect are mutated, a mutant S protein mRNA with high immunogenicity and eliminating the ADE effect is successfully screened, and a problem that the S protein is difficult to be practically applied to a vaccine is broken through. After the feline body is immunized by the mutant S protein mRNA of the present application, the ADE effect does not appear in the feline body, and the humoral immune response and the cellular immune response can be significantly induced; meanwhile, IFN-gamma is significantly expressed, further enhancing the cellular immune response and the antiviral activity of macrophages, synergistically neutralizing the antibody to play a protective role, simultaneously further inhibiting the ADE related pathological process from the cellular immune level, and significantly improving the clinical application value of the vaccine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a feline infectious peritonitis S protein mutant mRNA vaccine and its preparation and application. Background Technology

[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.

[0003] Feline infectious peritonitis (FIP) is a chronic, progressive, and fatal infectious disease caused by feline infectious peritonitis virus (FIPV). FIPV is the pathogenic biotype of feline coronavirus (FCoV), and its structure can induce antibody-dependent enhancement (ADE), making the host's immune system an accomplice to the virus, facilitating viral invasion. The spike protein (S protein) of FIPV is a key structure for viral invasion of host cells and a core target for inducing ADE. Studies have shown that there is a high degree of overlap between neutralizing epitopes and ADE epitopes on the FIPV S protein, making it difficult to accurately remove ADE epitopes while preserving neutralizing epitopes.

[0004] In current FIPV vaccine development, the core antigen widely used is the wild-type S protein. However, the wild-type S protein contains a complete ADE-inducing epitope, which easily triggers the ADE effect after immunization, accelerating viral infection. For example, in this study, the protective test showed that the mortality rate in the wild-type S mRNA vaccine group (group 2) was 100%, and the time to death was faster than in the PBS group (group 1). The IHC positive signal was also stronger in the wild-type S mRNA vaccine group (group 1), confirming that the ADE effect significantly aggravated the disease and cannot be used clinically. In traditional modified vaccines, due to the high overlap in position and amino acid sequence between the S protein neutralizing epitope and the ADE epitope, the high immunogenicity of the S protein and the ADE effect are highly bound, making it difficult to avoid the ADE effect from inducing a high immune response. This has long directly led to slow progress in FIPV vaccine development and poor clinical application results. While N proteins and others have a low ADE risk, they also have low immunogenicity and cannot induce high titers of neutralizing antibodies, resulting in poor protection.

[0005] Therefore, there is an urgent need for highly effective protective FIPV vaccines that can induce high-titer neutralizing antibody levels while precisely avoiding ADE-induced epitopes. Summary of the Invention

[0006] The purpose of this invention is to address the problem that current FIPV vaccines cannot simultaneously achieve immunogenicity and ADE safety risks, and to provide a feline infectious peritonitis S protein mutant mRNA vaccine, its preparation and application. By mutating the wild-type S protein, the ADE effect of the wild-type S protein is eliminated, improving its safety. At the same time, a large number of neutralizing antibodies against the S protein are successfully retained, and the ADE effect of the S protein is precisely eliminated.

[0007] The technical solution of the present invention is as follows: One aspect of the present invention provides a feline infectious peritonitis S protein mutant mRNA vaccine, comprising S protein mutant mRNA, the mutant sequence of which is shown in SEQ ID NO:14.

[0008] According to a preferred embodiment, the following mutation is specifically performed on the wild-type S protein: V569S: The V valine at position 569 is mutated to S serine; Y570S: The Y-tyrosine at position 570 is mutated to S-serine; F577A: The F-phenylalanine at position 577 is mutated to A-alanine; Y580T: The Y-tyrosine at position 580 is mutated to T-threonine; V581T: The V valine at position 581 is mutated to T threonine; ΔD591: Deletion of D-aspartic acid at position 591; A645S: The A alanine at position 645 is mutated to S serine; R647K: The R-arginine at position 647 is mutated to K-lysine; R649K: The R-arginine at position 649 is mutated to the K-lysine; V654A: The V valine at position 654 is mutated to A alanine.

[0009] According to a preferred embodiment, it also includes a pharmaceutically acceptable excipient or delivery system.

[0010] According to a preferred embodiment, the delivery system is a lipid nanoparticle (LNP) comprising ionizable lipids, cofactor lipids, cholesterol, or polyethylene glycol-modified lipids.

[0011] Preferably, the auxiliary lipid is a PEG-modified lipid or an auxiliary structural phospholipid.

[0012] According to a preferred embodiment, the excipients include at least one of a buffer salt, an osmotic pressure regulator, a stabilizer, and a preservative; the buffer salt is a phosphate buffer or a Tris-HCl buffer, and the osmotic pressure regulator is sucrose, trehalose, or mannitol.

[0013] According to a preferred embodiment, the vaccine is administered via intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, or nebulized inhalation.

[0014] According to a preferred embodiment, the vaccine can be prepared in combination as a bivalent or multivalent vaccine.

[0015] According to a preferred embodiment, the vaccine further includes an adjuvant, such as IL-12, IL-2, IL-15, IL-18, IFN-γ, etc.

[0016] Another aspect of the present invention provides a method for preparing feline infectious peritonitis S protein mutant mRNA as described above, comprising the following steps: (1) Synthesize a DNA template encoding the mRNA of the S protein mutant as shown in SEQ ID NO:14; (2) Using the DNA template as raw material, prepare an amplification plasmid vector, and generate a recombinant DNA sequence through in vivo amplification in microorganisms; (3) Synthesize modified recombinant mRNA molecules through in vitro transcription reaction; (4) Purify the obtained recombinant mRNA molecules to remove residual DNA template, free nucleotides and extraneous proteins; (5) The purified recombinant mRNA molecule is compounded with a pharmaceutical carrier, and pharmaceutical excipients are added to prepare a mutant mRNA vaccine formulation, which is then sterilely filtered, dispensed and stored.

[0017] According to a preferred embodiment, the amplification plasmid vector in step (2) is selected from: pIVT-D1-Kan-BsaI, pUC19, pUC18, pGEM-3Z, pGEM-4Z or pET-28a plasmid vector.

[0018] According to a preferred embodiment, step (3) further includes the following sub-steps: Step (3.1): Plasmid extraction; Step (3.2): Plasmid linearization; Step (3.3): Linear plasmid purification; Step (3.4): In vitro transcription.

[0019] Another aspect of the present invention provides the application of a feline infectious peritonitis (FIP) S protein mutant mRNA vaccine as described above in the prevention of FIP, wherein the FIP includes FIPV Type I strains and FIPV Type II strains.

[0020] Compared with existing technologies, the advantages of this invention are: 1. By mutating multiple sites of the core antigenic epitope that is prone to inducing antibody-dependent enhancement (ADE) effect, we successfully screened out the mRNA of mutant S protein that both eliminates the ADE effect and has high immunogenicity, thus overcoming the difficulty of applying S protein to vaccines in practice. 2. The screened mRNA vaccines can induce high levels of humoral and cellular immunity, balancing safety with humoral immune efficacy.

[0021] 3. Significantly induces IFN-γ expression. Increased IFN-γ further enhances cellular immune responses and macrophage antiviral activity, synergistically strengthening neutralizing antibodies for protection. Simultaneously, it further inhibits ADE-related pathological processes at the cellular immune level, improving vaccine safety and efficacy. It protects vaccinated cats from FIPV virus infection, maintaining a 100% survival rate; significantly enhancing the clinical protective value of the vaccine. Attached Figure Description

[0022] Figure 1 The vector is pIVT-D1-Kan-BsaI empty plasmid; Figure 2 It is a pIVT-FIPV-S-mut recombinant vector; Figure 3 Serum IgG antibody expression levels in mice of the A1 mutation group; Figure 4 Serum IgG antibody expression levels in mice of the A2 mutation group; Figure 5 Serum IgG antibody expression levels in mice of the A1 / A2 mutation groups; Figure 6 Survival curves of cats after viral infection in each group; Figure 7 Neutralizing antibodies in the serum of cats in each group after immunization; Figure 8 The expression levels of IFN-γ in the serum of cats in each group after immunization; Figure 9 Immunohistochemical images of the duodenum of cats in each group after viral challenge. Detailed Implementation

[0023] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0025] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0026] Example 1: Site-directed mutagenesis of the S protein Based on the wild-type S protein amino acid sequence (SEQ ID NO:1) from GenBank accession number AGZ84526.1, mutations were performed on A1 and A2 in the ADE region. Different mutations were made to the epitopes A1 (AA568-591) and A2 (AA643-656), and the mutation sites and protein fragment sequences are shown below: Protein WT: AA499-683 (SEQ ID NO: 5); ProteinA1-1: Y570T, F577A, Y580T, L589A, ΔD591 (SEQ ID NO: 6); ProteinA1-2: V569S, Y570S, F577A, Y580T, V581T, ΔD591 (SEQ ID NO: 7); ProteinA1-3: Y570A, F577A, Y580S, L589S, ΔD591 (SEQ ID NO: 8); ProteinA2-1: A645S, R647K, R649K, V654A (SEQ ID NO: 9); ProteinA2-2: V644T, R647K, E652Q (SEQ ID NO: 10); ProteinA2-3: R649K, E652Q (SEQ ID NO: 11); The above-mentioned wild-type and mutant protein sequences were sent to a biotechnology company for synthesis and prokaryotic expression and purification to obtain a high-purity target protein with a purity >90%, sterility, and low endotoxin.

[0027] (II) Immunogenicity Study and Screening To verify the immunogenicity of each mutant protein, SPF-grade BALB / c mouse animal experiments were conducted.

[0028] 1. Screening of A1 epitope mutation sequences Forty 6-8 week old female BALB / c mice were randomly divided into 5 groups. Group 1 was injected with PBS as a blank control group, Group 2 was injected with Protein WT protein, Group 3 with Protein A1-1 protein, Group 4 with Protein A1-2 protein, and Group 5 with Protein A1-3 protein. The protein concentration was 1 mg / mL, and the injection volume was 100 µL. The blank control group was injected with 100 µL of PBS. Mice were immunized twice, on day 0 and day 14. Serum was collected 14 days after the second immunization, and the IgG level in the serum was detected by ELISA to analyze its immunogenicity. The mutation sequence with the lowest immunogenicity was screened out.

[0029] The specific information is as follows: Table 1. Vaccination regimen and immunogenicity for A1 mutant protein.

[0030] As shown in Table 1 and Figure 3 As shown. Figure 3 The changes in serum IgG antibody levels in mice after inoculation with each A1-mutated protein were investigated. The absorbance at OD450 nm was 0.53 in the ProteinA1-2 group (group 4), 0.76 in the PtoteinA1-3 group (group 5), 0.94 in the PtoteinA1-1 group (group 3), 1.30 in the Protein WT group (group 2), and 0.12 in the PBS group (group 1). Among the three mutations in the A1 region, the ProteinA1-2 group showed the lowest serum IgG antibody expression level and the lowest immunogenicity.

[0031] 2. Screening of A2 epitope mutation sequences Forty 6-8 week old female BALB / c mice were randomly divided into 5 groups. Group 1 was injected with PBS as a blank control group, Group 2 was injected with Protein WT protein, Group 3 with Protein A2-1 protein, Group 4 with Protein A2-2 protein, and Group 5 with Protein A2-3 protein. The protein concentration was 1 mg / mL, and the injection volume was 100 µL. The blank control group was injected with 100 µL of PBS. Mice were immunized twice, on day 0 and day 14. Serum was collected 14 days after the second immunization, and the IgG level in the serum was detected by ELISA to analyze its immunogenicity. The mutation sequence with the lowest immunogenicity was screened out.

[0032] The specific information is as follows: Table 2. Vaccination regimen and immunogenicity for A2 mutant protein.

[0033] As shown in Table 2 and Figure 4 As shown. Figure 4 The changes in serum IgG antibody levels in mice after inoculation with each of the A2-mutated proteins were investigated. The absorbance at OD450 nm was 0.46 in the ProteinA2-1 group (groups 3), 0.88 in the PtoteinA2-2 group (groups 4), 1.04 in the PtoteinA2-3 group (groups 5), 1.31 in the Protein WT group (groups 2), and 0.12 in the PBS group (group 1). Among the three mutations in the A2 region, the ProteinA2-1 group showed the lowest serum IgG antibody expression level and the lowest immunogenicity.

[0034] 3. Mutation analysis of A1 and A2 combinations Based on the aforementioned analysis results, the two mutant sequences ProteinA1-2 and ProteinA2-1 with the lowest immunogenicity in A1 and A2 were combined to generate the mutant sequence ProteinA1-2 / 2-1 (SEQ ID NO:12). The protein was then sent to a biotechnology company for synthesis and prokaryotic expression and purification to obtain a high-purity target protein with a purity >90%, sterility, and low endotoxin.

[0035] Twenty-four 6-8 week old female BALB / c mice were randomly divided into three groups. Group 1 was injected with PBS as a blank control group, Group 2 was injected with Protein WT protein, and Group 3 was injected with Protein A1-2 / 2-1 protein. The protein concentration was 1 mg / mL for all groups, and the injection volume was 100 µL. The blank control group was injected with 100 µL of PBS. Mice were immunized twice, on day 0 and day 14. Serum was collected 14 days after the second immunization, and the IgG level in the serum was detected by ELISA to analyze its immunogenicity. The mutation sequence with the lowest immunogenicity was screened out.

[0036] The specific information is as follows: Table 3. A1 / A2 Mutant Inoculation Protocol

[0037] As shown in Table 3 and Figure 5 As shown. Figure 5Changes in serum IgG antibody levels in mice after inoculation with A1 / A2 mutant proteins were investigated. The absorbance at OD450 nm was 0.36 in the Protein A1-2 / 2-1 group (groups 3), 1.30 in the Protein WT group (groups 2), and 0.11 in the PBS group (group 1). Following combined mutations of A1 and A2, the expression level of serum IgG antibodies in mice decreased again, further reducing immunogenicity.

[0038] Example 2 I. Preparation of S-mut mRNA 1. S-mut sequence optimization and vector construction ① Based on the screening results of Example 1, the S protein A1 and A2 epitopes were combined and mutated to generate the S protein mutant sequence S-mut (SEQ ID NO:13). The specific mutation scheme is as follows: 1: V569S: The V valine at position 569 is mutated to S serine; 2: Y570S: The Y tyrosine at position 570 is mutated to S serine; 3: F577A: The F phenylalanine at position 577 is mutated to A alanine; 4: Y580T: The Y tyrosine at position 580 is mutated to T threonine; 5: V581T: The V valine at position 581 is mutated to a T threonine; 6: ΔD591: Deletion of D-aspartic acid at position 591; 7: A645S: The alanine at position 645 is mutated to a serine; 8: R647K: The R-arginine at position 647 is mutated to K-lysine; 9: R649K: The R-arginine at position 649 is mutated to the K-lysine; 10: V654A: The V valine at position 654 is mutated to A alanine.

[0039] The mutated S-mut sequence was optimized with feline codons and then synthesized into pcDNA3.1 by a biotechnology company. After confirming its correct sequencing, it was transformed into stbl3 competent cells to amplify the recombinant plasmid. Single clones were selected for preservation.

[0040] ② Extract plasmids: Amplified recombinant plasmids were extracted from stbl3 competent cells and the target S-mut sequence was isolated and purified.

[0041] The S-mut gene template sequence and the pIVT-D1-Kan-BsaI empty vector will be optimized. Figure 1The recombinant vector pIVT-FIPV-S-mut was generated by double digestion with XbaI and SalI restriction endonucleases and ligation overnight at 16°C using T4 ligase. Figure 2 ), and were transformed into stbl3 competent cells to select single clones for preservation.

[0042] 2. mRNA in vitro transcription and purification ① Plasmid extraction: The strain containing the pIVT plasmid was cultured for expansion, and plasmid extraction and purification were performed using a mini-prep kit. Elution was performed with 100 μL of solution, resulting in a concentration of approximately 200–300 μg / μL, OD... 260 / 280 =1.8~2.0.

[0043] ② Linearization: The extracted pIVT-FIPV-S-mut plasmid was digested with BsaI for 3 hours. The reaction system is shown in Table 4. Table 4 Linearized Enzyme Digestion Reaction System

[0044] ③ Linear plasmid purification: The linearized harvest was purified using DNA magnetic beads. The DNA magnetic beads were equilibrated at room temperature for 30 min, then vortexed to mix. 700 μL of DNA magnetic beads were added at a DNA:magnetic bead ratio of 1:1, and gently mixed by pipetting. The mixture was incubated at room temperature for 10 min. The DNA then bound to the magnetic beads. The sample was placed on a magnetic rack for 5 min, and the supernatant was carefully removed after the solution became clear. 700 μL of freshly prepared 80% ethanol solution was added, and the mixture was incubated at room temperature for 30 s. The supernatant was carefully removed, and this process was repeated once. The sample was then allowed to dry at room temperature for 5 min until the magnetic bead slant was no longer wet. The sample was removed from the magnetic rack, and 100 μL of RNase-free ddH2O was added along the magnetic bead slant. The mixture was mixed by pipetting and allowed to stand for 2 min. The sample was then placed on a magnetic rack and allowed to stand for 5 min. After clarification, the supernatant was carefully aspirated to obtain the purified linearized plasmid template.

[0045] ④ In vitro transcription: 15 μg of linearized vector DNA was mixed with T7 transcriptase, ATP, CTP, GTP, Pseudo-UTP, and a cap-like external cap transcription system, and incubated at 37°C for 3 h; the reaction system is shown in Table 5. Table 5 In vitro transcription system

[0046] ⑤ Purification: Digest with DNase I for 30 min, then elute with RNA magnetic beads. Equilibrate the RNA magnetic beads at room temperature for 30 min. Add 540 μL of magnetic beads at an RNA:magnetic bead ratio of 1:8, gently mix by pipetting, and let stand at room temperature for 5 min to allow RNA to bind to the magnetic beads. Place the sample on a magnetic rack and let stand for another 5 min until the solution is clear, then carefully discard the supernatant. Slowly add 800 μL of freshly prepared 80% ethanol solution, let stand for 30 s, carefully discard the supernatant, and repeat once. Open the cap and dry at room temperature for 5 min until the magnetic bead slant is no longer wet. Remove the sample, add 1 mL of RNase-free ddH2O to elute, gently mix, let stand for 2 min, then place on a magnetic rack for adsorption for 5 min until the solution is clear; the supernatant is the purified mRNA solution. Capillary electrophoresis analysis showed that the mRNA purity was over 95%, and Nanodrop detection showed a purity of (A260 / A280 = 1.92).

[0047] ⑥ Ultrafiltration medium change: Use a 30kDa ultrafiltration tube and change the medium with 25mM citrate buffer (pH 4.0±0.3). Rinse the ultrafiltration tube once with 0.1M NaOH, and then rinse three times with RNase-free ddH2O. Add 500μL of RNA solution to each tube, press 14000g, and ultrafilter for 3-5 minutes until 100μL remains. Then, add sodium citrate solution to bring the total to 500μL. Repeat the ultrafiltration concentration and medium change three times. Finally, add sodium citrate solution to bring the total to 500μL, gently pipette to mix, and aspirate the solution completely. This is the mRNA after medium change.

[0048] II. Preparation of S-mut vaccine (LNP-mRNA) 1. mRNA solution: Dilute the mRNA prepared in step one with 25mM citrate buffer to a final concentration of 266.7 μg / mL. 2. LNP preparation: ① Lipid solution: SM-102 (192mg), PEG-DMG (25mg), DSPC (52mg), and cholesterol (83mg) were dissolved in 20mL of anhydrous ethanol and filtered through a 0.22μm filter membrane. The molar ratio was SM-102:DSPC:PEG-DMG:cholesterol = 50:10:1.5:38.5. Table 6 LNP lipid preparation system

[0049] ② Mixed preparation: The lipid solution prepared in ① and each mRNA solution were injected into the rapid nano-preparation system at a volume ratio of 1:3, with a flow rate of 20 mL / min. The products were collected to obtain LNP stock solution. ③ Concentration and preservation: The LNP stock solution was diluted approximately 5 times with PBS, centrifuged at 2000g for 10 min using a 100kDa ultrafiltration tube, and concentrated back to the original volume. Then, it was diluted with 20mM Tris-HCl buffer and ultrafiltered back to the original volume. This process was repeated once. The ultrafiltered solution was diluted with sucrose solution to a final sucrose concentration of 5% and stored at -20℃. The LNP particle size was measured to be 100±10nm, the encapsulation efficiency was >90%, and the Zeta potential was <0.3.

[0050] III. Validation through protective studies 1. Grouping and Immunization of Laboratory Animals: ① Fifteen FIPV-negative cats aged 9-12 months (detected negative by feline coronavirus (FcoV) antibody ELISA kit) were randomly divided into 3 groups (n=5): Group 1 (PBS), Group 2 (wild-type S mRNA), and Group 3 (S-mut mRNA); the mRNA concentration was adjusted to 1 mg / mL, the injection volume was 50 µL, and the PBS group was injected with 50 µL of PBS.

[0051] Table 7 S-mut mRNA Immunization Regimen

[0052] ②Immunization program: Mice were immunized twice by subcutaneous injection on day 0 and day 14, with each immunization containing 50 μg of antigen mRNA.

[0053] 2. Invasive treatment and observation: ① Challenge with the virus: 14 days after the completion of immunization, all cats were given 10 doses of 10 drops of virgin drug via intranasal administration. 6 TCID 50 FIPV Type II 79-1146 strains; ② Clinical observation: Observe clinical symptoms (fever, loss of appetite, ascites, etc.) every other day and record the time of death; ③Indicator detection: Serum neutralizing antibody and IFN-γ concentration were detected 14 days after the second immunization. The virus was euthanized 28 days after challenge. The tissue lesions were observed by autopsy and IHC immunohistochemical sections were prepared. The antibody was FIPV3-70.

[0054] The results are as follows:

[0055] Figure 6The survival curves for cats in each group within 28 days after challenge are shown. The horizontal axis represents "time after challenge (days)" and the vertical axis represents "survival rate %". The survival rate of the S-mut mRNA vaccine group (groups 3) was 100%, the survival rate of the wild-type S mRNA group (groups 2) was 0%, and the time of death was 7, 13, 13, 16, and 18 days after challenge. The survival rate of the PBS group (group 1) was 0%, and the time of death was 10, 17, 23, 26, and 27 days after challenge, confirming the ADE effect.

[0056] Figure 7 Neutralizing antibody titers in feline serum 14 days after secondary immunization with the S-mut mRNA vaccine. The geometric mean neutralizing antibody titer in the S-mut mRNA vaccine group (group 3) was 1:2346, and the geometric mean neutralizing antibody titer in the wild-type S mRNA group (group 2) was 1:4069. The geometric mean neutralizing antibody titer in the PBS group (group 1) was 1:4.

[0057] Figure 8 The serum IFN-γ concentrations of cats 14 days after the second immunization were 610.342 pg / mL in the S-mut mRNA vaccine group (groups 3), 371.933 pg / mL in the wild-type S mRNA group (groups 2), and 80.650 pg / mL in the PBS group (group 1). The data are “mean ± SD”, p < 0.001.

[0058] Figure 9 Immunohistochemical staining (IHC) of the duodenum of cats 28 days after challenge (×200) showed no obvious positive signal in the S-mut mRNA vaccine group (groups 3), strong positive in the wild-type S mRNA group (groups 2), and moderate positive in the PBS group (group 1).

[0059] like Figures 6-9 As shown, the survival rate of the wild-type S mRNA group (groups 2) was 0%, with death times at days 7, 13, 13, 16, and 18 post-infection, respectively. The survival rate of the PBS group (group 1) was also 0%, with death times at days 10, 17, 23, 26, and 27 post-infection, confirming the ADE effect. The average death time of groups 2 was faster than that of group 1. Figure 6 Groups 2 (1:2346) and 3 (1:4069) both induced high levels of neutralizing antibodies. Figure 7 The IFN-γ expression level in group 3 (610.342 pg / mL) was significantly higher than that in group 2 (371.933 pg / mL) and group 1 (80.650 pg / mL) (p<0.001). Figure 8 ); 3 groups showed no specific positive signal for FIPV; 1 group showed diffuse brownish-yellow staining at the villus tip, indicating a moderate positive signal; 2 groups showed dark brown positive signal at both the villus tip and basal region, indicating a strong positive signal. Figure 9This demonstrates that the mutant mRNA vaccine of this application successfully overcomes the ADE effect of the S protein, while simultaneously maintaining high immunogenicity. The vaccine provides stable protection for cats, with 100% survival rate in cats vaccinated with the mutant mRNA group. Humoral and cellular immunity levels are simultaneously higher than those of the wild-type S protein, breaking through the barriers of high immunogenicity and high ADE risk of FIPV; achieving higher immunogenicity and lower ADE risk.

[0060] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

[0061] Sequence List: Wild-type S protein amino acid sequence (SEQ ID NO.1): The 5' UTR sequence (SEQ ID NO.2) in the pIVT-FIPV-S-mut recombinant vector is: ACTCTTCTGGTCCCCACAGACTCAGAGAGAACCC The 3' UTR sequence (SEQ ID NO.3) in the pIVT-FIPV-S-mut recombinant vector is as follows: CCCAACGGGCCCTCCTCCCC The Poly(A) tail (SEQ ID NO.4) in the pIVT-FIPV-S-mut recombinant vector is: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA A1 site - Protein WT amino acid sequence (SEQ ID NO.5): LTANLNNGFYPVASSEVGFVNKSVVLLPSFFTYTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDVYCIRSNQFSVYVHSTCKSSLWDNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVAARTRTNEQVVRSLYVIYEEGDNIVGVPSDNSGLHDLSV A1 site - Protein A1-1 amino acid sequence (SEQ ID NO.6): LTANLNNGFYPVASSEVGFVNKSVVLLPSFFTYTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDVTCIRSNQASVTVHSTCKSSAWNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVAARTRTNEQVVRSLYVIYEEGDNIVGVPSDNSGLHDLSV A1 site - Protein A1-2 amino acid sequence (SEQ ID NO.7): LTANLNNGFYPVASSEVGFVNKSVVLLPSFFTYTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDSSCIRSNQASVTTHSTCKSSLWNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVAARTRTNEQVVRSLYVIYEEGDNIVGVPSDNSGLHDLSV Amino acid sequence of Site A1 - Protein A1-3 (SEQ ID NO. 8): LTANLNNGFYPVASSEVGFVNKSVVLLPSFFTYTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDVACIRSNQASVSVHSTCKSSSWNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVAARTRTNEQVVRSLYVIYEEGDNIVGVPSDNSGLHDLSV Amino acid sequence of Site A2 - Protein A2-1 (SEQ ID NO. 9): LTANLNNGFYPVASSEVGFVNKSVVLLPSFFTYTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDVYCIRSNQFSVYVHSTCKSSLWDNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVSAKTKTNEQAVRSLYVIYEEGDNIVGVPSDNSGLHDLSV Amino acid sequence of Site A2 - Protein A2-2 (SEQ ID NO. 10): LTANLNNGFYPVASSEVGFVNKSVVLLPSFFTYTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDVYCIRSNQFSVYVHSTCKSSLWDNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDTAAKTRTNQQVVRSLYVIYEEGDNIVGVPSDNSGLHDLSV Amino acid sequence of Site A2 - Protein A2-3 (SEQ ID NO. 11): LTANLNNGFYPVASSEVGFVNKSVVLLPSFFTYTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDVYCIRSNQFSVYVHSTCKSSLWDNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVAARTKTNQQVVRSLYVIYEEGDNIVGVPSDNSGLHDLSV Amino acid sequence of ProteinA1-2 / 2-1 (SEQ ID NO. 12): LTANLNNGFYPVASSEVGFVNKSVVLLPSFFTYTAVNITIDLGMKLSGYGQPIASTLSNITLPMQDNNTDSSCIRSNQASVTTHSTCKSSLWNIFNQDCTDVLEATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVSAKTKTNEQAVRSLYVIYEEGDNIVGVPSDNSGLHDLSV Amino acid sequence of S-mut (SEQ ID NO. 13): S-mut mRNA sequence (SEQ ID NO.14):

Claims

1. A feline infectious peritonitis S protein mutant mRNA vaccine, characterized in that, Including S protein mutant mRNA, the mutant sequence is shown in SEQ ID NO:

14.

2. The feline infectious peritonitis S protein mutant mRNA vaccine according to claim 1, characterized in that, This also includes pharmaceutically acceptable excipients or delivery systems.

3. The feline infectious peritonitis S protein mutant mRNA vaccine according to claim 2, characterized in that, The delivery system is a lipid nanoparticle, which contains ionizable lipids, auxiliary lipids, cholesterol, or polyethylene glycol-modified lipids.

4. The feline infectious peritonitis S protein mutant mRNA vaccine according to claim 3, characterized in that, The auxiliary lipid is a PEG-modified lipid or an auxiliary structural phospholipid.

5. The feline infectious peritonitis S protein mutant mRNA vaccine according to claim 4, characterized in that, The excipients include at least one of buffer salts, osmotic pressure regulators, stabilizers, and preservatives; the buffer salt is a phosphate buffer or a Tris-HCl buffer, and the osmotic pressure regulator is sucrose, trehalose, or mannitol.

6. The feline infectious peritonitis S protein mutant mRNA vaccine according to claim 5, characterized in that, The vaccine can be administered via intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, or nebulized inhalation.

7. A method for preparing a feline infectious peritonitis S protein mutant mRNA vaccine according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Synthesize a DNA template encoding the mRNA of the S protein mutant as shown in SEQ ID NO:14; (2) Using the DNA template as raw material, prepare an amplification plasmid vector, and generate a recombinant DNA sequence through in vivo amplification by microorganisms; (3) Synthesize modified recombinant mRNA molecules through in vitro transcription reaction; (4) Purify the obtained recombinant mRNA molecules to remove residual DNA template, free nucleotides and extraneous proteins; (5) The purified recombinant mRNA molecules are combined with a pharmaceutical carrier, and pharmaceutical excipients are added to prepare a mutant mRNA vaccine formulation. The formulation is then sterilely filtered, dispensed, and stored.

8. The method for preparing a feline infectious peritonitis S protein mutant mRNA vaccine according to claim 7, characterized in that, The amplification plasmid vector in step (2) is selected from: pIVT-D1-Kan-BsaI, pUC19, pUC18, pGEM-3Z, pGEM-4Z or pET-28a plasmid vector.

9. The method for preparing a feline infectious peritonitis S protein mutant mRNA vaccine according to claim 7, characterized in that, Step (3) also includes the following sub-steps: Step (3.1): Plasmid extraction; Step (3.2): Plasmid linearization; Step (3.3): Linear plasmid purification; Step (3.4): In vitro transcription.

Images