A saRNA vaccine for the prevention of feline infectious peritonitis

Abstract

This invention provides a saRNA vaccine for the prevention of feline infectious peritonitis (FIP). The saRNA vaccine is a composition comprising saRNA A and saRNA B. The assembly elements of the saRNA include: a 5′ UTR, replicases nsp1-nsp4, a subgenomic promoter, a Kozak sequence, nucleic acid sequence A or nucleic acid sequence B, a 3′ UTR, and a polyadenylated tail (polyA). The saRNA vaccine provided by this invention is effective for the immunization prophylaxis of FIP types I and II. Furthermore, based on the development of this vaccine, by replacing the coding sequences of different antigens, it can be extended to the development of vaccines for other coronaviruses or other veterinary pathogens, demonstrating significant technological transfer value and industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of genetically engineered vaccines, and more specifically to a saRNA vaccine for the prevention of feline infectious peritonitis. Background Technology

[0002] Feline infectious peritonitis (FIP) is a highly lethal, immune-mediated systemic disease caused by the feline infectious peritonitis virus (FIPV), which mutates from feline enterovirus (FCoV) in cats. FIPV infects macrophages, causing systemic purulent granulomatous vasculitis. The disease primarily affects kittens, senior cats, and cats living in crowded multi-cat environments. Prevention and treatment of FIP face certain technical challenges; currently, there is no specific vaccine to prevent FIP, and standard feline triple vaccines do not provide protection against it.

[0003] In recent years, significant progress has been made in the research of vaccines based on self-amplifying RNA (saRNA). Compared with traditional mRNA vaccines, saRNA vaccines, with their unique self-replication mechanism, have shown great potential in reducing dosage requirements, prolonging antigen expression time, and enhancing immunogenicity. However, a mature solution for broad-spectrum saRNA vaccines targeting FIP has yet to be found. The transition from laboratory to clinical and market applications still faces several technical challenges, such as the instability of saRNA construction systems, the unstable delivery efficiency of delivery vectors, and the potential toxicity of non-structural proteins. Therefore, there is an urgent need to develop a broad-spectrum saRNA vaccine with stable immunogenicity and good safety profile. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problems of FIPV saRNA vaccines in the prior art, such as the poor stability of the saRNA construction system and the poor immunization effect.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A nucleotide sequence encoding a recombinant feline infectious peritonitis virus (FIPV) antigen, comprising nucleic acid sequences A and B, wherein nucleic acid sequence A is the sequence shown in SEQ ID NO:1 or a sequence having at least 90% identity with it and encoding the same functional antigen; and nucleic acid sequence B is the sequence shown in SEQ ID NO:2 or a sequence having at least 90% identity with it and encoding the same functional antigen.

[0006] A self-amplifying RNA (saRNA) composition comprising nucleic acid sequences A and B.

[0007] Furthermore, the nucleic acid sequence A is an antigen-coding sequence of N protein and M protein formed by the fusion sequence of the tandem FIPVI type S protein neutralizing epitope and T cell epitope to form self-assembled LS nanoparticles.

[0008] Furthermore, the nucleic acid sequence B is the coding sequence of an optimized fragment of a self-assembled LS nanoparticle tandemly connected to a FIPVII type S protein receptor-binding domain (RBD).

[0009] Furthermore, the assembly elements of the saRNA include: a 5′ cap structure, a 5′ UTR, a replicase coding region (nsp1-nsp4), a subgenomic promoter (SGP), a Kozak sequence, nucleic acid sequence A or nucleic acid sequence B, a 3′ UTR, and a polyadenylate tail polyA.

[0010] Furthermore, the saRNA is derived from any one of Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), or Western equine encephalitis virus (WEEV).

[0011] Furthermore, the saRNA is encapsulated in lipid nanoparticles (LNPs).

[0012] Furthermore, the lipid nanoparticles comprise: ionized lipids, MC3, cholesterol, phospholipids, and polyethylene glycol-modified lipids.

[0013] The use of the nucleotide sequence encoding the recombinant FIPV antigen in the preparation of feline infectious peritonitis vaccines and drugs.

[0014] Compared to existing technologies, the beneficial effects of this invention are: (1) Achieving broad-spectrum cross-protection against FIPVI and FIPVII serotypes. This invention employs a dual saRNA molecule co-design, one targeting the key S / N / M protein of FIPVI serotype and the other targeting the optimized RBD domain of FIPVII serotype. Codon optimization of the antigen sequence effectively enhances the in vitro expression, correct folding, and loading of the endogenous MHC pathway, thereby more effectively inducing highly efficient neutralizing antibodies and cross-cellular immune responses against both serotypes, overcoming the shortcomings of existing vaccines that have poor immunogenicity and difficulty in providing multi-serotype protection.

[0015] (2) Highly efficient and sustained antigen expression, and potent immune response at low doses. This invention constructs a self-amplified RNA platform based on VEEV replicase (nsP1-nsP4). This system can continuously replicate RNA and perform subgenomic transcription within host cells, thereby achieving sustained antigen expression. Although this invention does not directly quantify the expression duration, based on the continuous amplification mechanism driven by the replicative subsystem, it can be reasonably inferred that it has a longer expression duration in vivo than traditional mRNA. Combining the known technical principles in this field, self-amplified RNA can maintain a longer expression time in vivo than traditional mRNA. The required immunization dose is significantly reduced. In Example 5 of this invention, under the same dosage conditions (50 μg), the immune response level and protective effect of the saRNA-LNP vaccine group were superior to those of the linear mRNA control group. At the same time, under lower dose conditions (10 μg and 5 μg), the saRNA-LNP vaccine can still maintain a high level of immune protection (the protection rate of the 10 μg group was 100%, and the protection rate of the 5 μg group was 83.3%), indicating that it has a significant advantage in reducing the immunization dose (showing a dose reduction trend of approximately 5–10 times). Simultaneously, through the multivalent display of LS nanoparticles and the synergy of N / M proteins, the strength of humoral and cellular immunity is significantly enhanced, achieving more comprehensive and longer-lasting immune protection.

[0016] (3) The LNP delivery system is highly efficient and stable. This invention uses ionizable lipid nanoparticles (ionized lipids, MC3, cholesterol, phospholipids and polyethylene glycol-modified lipids) with optimized ratios. Through microfluidic technology, it achieves uniform particle size (90-120nm), high encapsulation efficiency (>90%), protection of saRNA from degradation and promotion of its efficient entry into target cells, significantly improving the in vivo delivery efficiency and systemic immunization effect of the vaccine, and ensuring the systemic bioavailability of the vaccine.

[0017] (4) Possesses good safety and platform scalability: The preparation process is simple and can be rapidly scaled up. The saRNA vaccine of this invention is non-infectious and has no risk of genome integration. This invention involves in vitro operations throughout, including plasmid construction, in vitro transcription, and microfluidic LNP encapsulation, without the need for cell culture or viral amplification. It has a short production cycle, high batch-to-batch consistency, is easy to standardize and freeze-dry, and has the ability to rapidly respond to new variants and for large-scale manufacturing, making it suitable for the industrialization needs of various veterinary vaccines in the future.

[0018] (5) The self-amplified RNA (saRNA) platform based on the VEEV backbone constructed in this invention is highly modular and scalable. It is not only suitable for the development of a broad-spectrum vaccine for feline infectious peritonitis virus (FIPV), but can also quickly replace the antigen coding sequence and be extended to the development of vaccines for other coronaviruses or other veterinary pathogens.

[0019] In summary, the saRNA vaccine platform of this invention can be effectively used for the immunoprophylaxis of feline infectious peritonitis type I and II, and also has the potential to be extended to other animal infectious diseases (such as canine coronavirus, porcine epidemic diarrhea virus, etc.), veterinary tumor immunotherapy, and protein replacement therapy for rare genetic diseases, with significant technological transformation value and industrialization prospects. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the plasmid maps for saRNA A and saRNA B, where... Figure 1 A is the plasmid map of saRNA A. Figure 1 B is the plasmid map of saRNA B; Figure 2 This is a schematic diagram of saRNA molecules A and B. Figure 3 Linearization of saRNA A and saRNA B plasmids; Figure 4 The expression levels of saRNA and saRNA B in cells; Figure 5 The titers of anti-FIPV I RBD and FIPV II antibodies after immunization in the high, medium and low dose vaccine groups; Figure 6 Stimulation index for high, medium, and low dose vaccine groups; Figure 7 The survival status of the immunized animal group 4 weeks after challenge with the virus. Detailed Implementation

[0021] The following specific descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The following descriptions are merely preferred embodiments of the present invention and are not intended to limit the invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0022] Example 1. Design of FIPVI type antigen protein, design of FIPVII type antigen protein, and codon optimization This invention addresses the problem of significant antigenic differences and insufficient cross-protection between feline infectious peritonitis virus (FIPV) type I and type II. It designs a dual-antigen fusion self-assembled nanoparticle expression strategy to simultaneously induce humoral and cellular immune responses.

[0023] FIPVI-type S (Spike) protein, N (Ncleocapsid) protein, and M (Membrane) protein. To achieve the co-expression of multiple antigens within the same host cell, this invention connects the coding sequences of FIPVI-type S, N, and M proteins sequentially via a self-cleaving peptide P2A, constructing a multi-antigen expression cassette within the same open reading frame. The structure is: S protein coding sequence - P2A - N protein coding sequence - P2A - M protein coding sequence. To achieve high-density repetitive display of antigens, the aforementioned multi-antigen expression cassette is fused to a self-assembling LS nanoparticle backbone protein at its C-terminus, allowing the expression product to self-assemble into a regular nanoparticle structure within the cell, thereby improving antigen presentation efficiency and immunogenicity.

[0024] The FIPV type II antigen selects the receptor-binding domain (RBD) in its S protein as the type II specific antigen. The RBD contains a key neutralizing antibody binding region and is a core target for inducing humoral immune responses. The RBD is fused to the LS nanoparticle backbone protein at its C-terminus and expressed. After in vivo expression, it can self-assemble into a highly repetitive nanoparticle antigen structure, thereby enhancing immunogenicity.

[0025] Both the FIPV type I multi-antigen expression cassette and the FIPV type II RBD fusion expression cassette described above were constructed using a self-amplified RNA (saRNA) vector system. In vivo expression via the self-amplified RNA platform resulted in highly repetitive display of nanoparticle antigen structures, enhancing immunogenicity.

[0026] To improve the expression efficiency of the antigen protein in cat host cells, codon optimization was performed on the antigen coding sequence. This involved adopting feline-preferred codon frequencies, increasing the GC content to 50%–60%, removing potential splicing sites, removing unstable internal sequences, avoiding strong secondary structures, removing consecutive homobasic repeats, and deleting potential termination signal-like sequences. After optimization, the codon fit index (CAI) was ≥0.85, and the 5′ secondary structure free energy was optimized, reducing the absolute value of ΔG near the start codon and thus enhancing ribosome binding efficiency. An optimized 3′ untranslated region (UTR) was designed downstream of the coding sequence to enhance mRNA stability. The nucleic acid sequence A of the FIPVI type S-P2A-N-P2A-M-LS fusion protein is shown in SEQ ID NO:1, and the nucleic acid sequence B of the self-assembled nanoparticle antigen composed of the FIPVII type RBD-LS fusion protein is shown in SEQ ID NO:2.

[0027] Example 2: Construction of saRNA molecule A and saRNA molecule B as transcription template plasmids The saRNA composition contains saRNA molecule A and saRNA molecule B, both designed and constructed using a transcription template based on the Venezuelan equine encephalitis virus (VEEV) TC-83 attenuated strain. Their core structures, from the 5' to the 3' end, include: 5' UTR, nsP1-4 (encoding the VEEV replicase complex), subgenomic promoter (SGP), FIPV antigen coding sequences (i.e., nucleic acid sequences A and B designed in Example 1), 3' UTR, and a ploy(A) tail. Schematic diagrams of the structures of saRNA molecule A and saRNA molecule B are shown below. Figure 1 As shown in A and 1B, nsP1-4 encode the viral replicase complex and are responsible for RNA replication and subgenomic transcription. The subgenomic promoter (SGP), located downstream of nsP4, drives the efficient expression of downstream FIPV antigens. The 3'UTR and polyA sequences enhance mRNA stability.

[0028] The designed sequences were commissioned to a professional gene synthesis company for whole-genome synthesis and cloned downstream of the T7 promoter of the pEAVrMLVB plasmid vector, yielding recombinant plasmid A (containing nucleic acid sequence A) and recombinant plasmid B (containing nucleic acid sequence B). The synthesized plasmids were transformed into *E. coli* Stbl3 competent cells (this strain is suitable for plasmids containing stable repetitive sequences or large inserts), plated on LB agar plates containing ampicillin, and incubated overnight at 37°C. The following day, single colonies were picked for preliminary identification by colony PCR and sent for Sanger sequencing to verify the correctness of the inserted sequences. Positive clones with completely correct sequencing were used as linearization templates for subsequent in vitro transcription. The mRNA sequence of saRNA molecule A is shown in SEQ ID NO. 3, and the mRNA sequence of saRNA molecule B is shown in SEQ ID NO. 4.

[0029] Implementation Case 3: Preparation Method of Feline Infectious Peritonitis (FIP) saRNA Vaccine 1. Preparation of plasmid templates Correct positive clones containing the full-length saRNA expression framework were inoculated into kanamycin-containing liquid medium and cultured at 37°C and 200 rpm for 12–16 h. The cultured bacterial suspension was collected. The bacterial cells were subjected to lysis, neutralization, clarification, supernatant separation, and DNA purification to extract and purify the plasmid. The concentration and purity of the obtained plasmid DNA were determined, with the A260 / A280 ratio controlled between 1.8 and 2.0. The integrity of the plasmid DNA was verified by agarose gel electrophoresis. After confirming the correctness of the saRNA replicon region, target antigen coding region, and untranslated region by sequencing, the plasmid was linearized using the restriction endonuclease MluI at 37°C. The results are shown below. Figure 3 As shown. After the enzyme digestion reaction, the enzyme was inactivated in an 80℃ metal bath. 1 / 10 volume of sodium acetate solution (3 mol / L, pH 5.2) was added to the linearized enzyme digestion reaction system and mixed thoroughly. Then, 2 volumes of pre-cooled anhydrous ethanol were added, and the mixture was incubated at -80℃ for at least 30 minutes. The mixture was then centrifuged at 4℃ and 10,000–14,000 rpm for 30 minutes, and the supernatant was discarded. The resulting precipitate was washed three times with pre-cooled 70%–75% ethanol, centrifuged for 10 minutes each time at 2–8℃ and 10,000–14,000 rpm, and the supernatant was discarded. The precipitate was dried at room temperature or under vacuum until no obvious liquid residue remained. An appropriate amount of nuclease-free water was added to the dried precipitate to dissolve it, and the concentration of linearized plasmid DNA was determined to obtain the purified linearized template, which was stored at -20℃ or lower for later use.

[0030] 2. Preparation and purification of saRNA by in vitro transcription The in vitro transcription reaction was carried out using a co-transcriptional capping method, that is, a cap structure analog was added to the transcription reaction system. The reaction system is shown in Table 1. This method allows the obtained saRNA to form a cap structure at the 5' end during transcription, thereby improving the stability of saRNA and the in vivo translation efficiency.

[0031] The full-length saRNA is approximately 10 kb to 15 kb, containing the complete self-amplifying RNA replicase coding region and the antigen expression region regulated by the subgene promoter. Purification steps employed a combination of column chromatography and membrane filtration with buffer replacement to obtain high-purity saRNA. The integrity of the obtained saRNA was detected by capillary electrophoresis (5200 Fragment Analyzer System) or equivalent methods, showing that it was predominantly full-length RNA, with residual nucleic acids and proteins within acceptable limits.

[0032] The purified saRNA was stored in a low-ionic-strength, RNase-free buffer system and preserved at low temperature to maintain its structural stability and biological activity.

[0033] Table 1. Reaction system for in vitro transcription to prepare mRNA

[0034] 3. Preparation of saRNA-LNP A microfluidic chip mixing technique was used to encapsulate saRNA in lipid nanoparticles (LNPs) to obtain a saRNA-LNP vaccine formulation with uniform particle size distribution and high encapsulation efficiency. The specific steps are as follows.

[0035] (1) Preparation of aqueous and oil phases Using 50 mM citrate buffer (pH 4.0) as the aqueous buffer system, the saRNA A and saRNA B solutions transcribed and purified in vitro were diluted to a final concentration of 0.12 mg / mL. The saRNA A and saRNA B were mixed at a 1:1 ratio and set aside for later use.

[0036] The LNP oil phase consists of cationic lipids, MC3, cholesterol (CHO), structural lipid DSPC, and modified lipid DMG-PEG2000. The lipids are mixed in a predetermined molar ratio, preferably 45:25:10:19:1, and optimized according to the target particle size (approximately 100 nm) and nucleic acid encapsulation requirements, with an N / P ratio controlled at 6. After converting each lipid to its corresponding mass based on its molecular weight, it is dissolved in anhydrous ethanol to prepare a lipid stock solution with a total lipid concentration of 10 mg / mL.

[0037] (2) Microfluidic encapsulation preparation of saRNA-LNP Microfluidic chip cleaning: Before formal fabrication, anhydrous ethanol was injected into both channels of the chip for rinsing. The total flow rate was set at 12 mL / min, the ratio of aqueous phase to oil phase flow rate was 1:1, and the volume of each rinse was 4 mL. After draining the residual liquid, the rinsing was repeated 3 times to ensure the cleanliness of the chip channels.

[0038] Sample loading: Use a syringe to draw up the aqueous saRNA solution and the oil lipid solution respectively, remove air bubbles, and install them into the corresponding inlet of the microfluidic device.

[0039] Microfluidic mixing and encapsulation: The total flow rate of the microfluidic system was set to 12 mL / min, and the volumetric flow rate ratio of the aqueous phase to the oil phase was 3:1. During operation, the generated saRNA-LNP was collected within 3–13 seconds by controlling the switching time between the waste liquid and the sample collection tube, thus obtaining the preliminary encapsulation product.

[0040] Preliminary sample processing: The collected saRNA-LNP was immediately diluted 20-fold with sterile PBS buffer and temporarily stored at 2–8°C to prevent nanoparticle aggregation or nucleic acid degradation.

[0041] (3) Ultrafiltration concentration and buffer replacement of saRNA-LNP The diluted saRNA-LNP suspension was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa and concentrated by centrifugation at 4°C and 3000 g.

[0042] After discarding the filtrate, the sample was buffer-replaced with sterile PBS and then sterilized by passing it through a 0.22 μm filter membrane. Ultrafiltration was then performed again to concentrate the LNP to the target volume and concentration. The resulting saRNA-LNP can be used for in vitro cell transfection expression experiments or animal immunization experiments. The prepared formulation can be stored for short periods at 4°C or for long periods at –80°C.

[0043] (4) Encapsulation efficiency test The encapsulation efficiency of saRNA was determined using the Ribo Green fluorescence method. Prepared saRNA-LNP samples were divided into two groups: an unlysed group (for detecting free RNA) and a lysed group (for detecting total RNA content after complete disruption of the lipid structure with a nonionic surfactant). In the unlysed group, fluorescent dye was added directly to detect free RNA content. In the lysed group, an appropriate amount of nonionic surfactant was added to lyse the LNPs before adding the fluorescent dye to detect total RNA content. Fluorescence intensity was measured using a fluorescence microplate reader, and RNA concentration was calculated based on a standard curve.

[0044] Encapsulation efficiency was calculated using the following formula: Encapsulation efficiency (%) = (Total RNA content - Free RNA content) / Total RNA content × 100%. Results showed: The encapsulation efficiency of three independently prepared batches of samples was 90%–97%, with an inter-batch variation (RSD) of less than 5%; the encapsulation efficiency of different dosage forms (5 μg, 10 μg, and 50 μg) remained above 90%. This indicates that the formulation of the present invention has good encapsulation efficiency and process stability.

[0045] (5) Particle size and polydispersity index (PDI) detection Particle size and particle size distribution (PDI) were determined using dynamic light scattering (DLS). An appropriate amount of saRNA-LNP sample was diluted with sterile PBS and analyzed using a particle size analyzer at 25°C. Results showed that the average hydrated particle size was 60–120 nm, and the PDI was ≤0.25. This indicates that the formulation has a uniform particle size distribution, good stability, and potential for in vivo delivery.

[0046] (6) The Zeta potential was determined by electrophoretic light scattering method.

[0047] After appropriate dilution, the samples were tested at 25°C. The results showed that the zeta potential ranged from -5 mV to +15 mV; the preferred range was -5 mV to +5 mV. This potential range is beneficial for maintaining colloidal stability under physiological conditions and reducing non-specific protein adsorption. The results are shown in Table 2.

[0048] Table 2. Average values ​​of three replicates of the physicochemical properties of the mRNA-LNP vaccine

[0049] Example 4. In vitro cell transfection and expression of saRNA-LNP This embodiment uses cat kidney cells (CRFK) as an in vitro model to verify the transfection efficiency of the prepared saRNA-LNP in cat-derived cells and the expression of the target antigen protein.

[0050] Cell transfection: CRFK cells were seeded into 6-well cell culture plates, with 2 mL of cell suspension added to each well, at a cell density of 4 × 10⁶ cells / well. 5 Cells were cultured at 37°C and 5% CO2 for 24 h at a concentration of / mL to allow for cell adhesion and optimal confluence. Different doses of saRNA-LNP vaccine were added to different wells, resulting in saRNA concentrations of 10 μg, 2 μg, and 1 μg in each well, with three replicates per sample. After gentle mixing, cells were incubated at 37°C and 5% CO2 for another 24 h. CRFK cells without saRNA-LNP were used as a negative control for background signal and nonspecific response assessment.

[0051] Cell lysis and sample preparation: 24 h after transfection, the culture medium was discarded, and the cells were washed twice with PBS buffer. 0.4 mL of trypsin was added to each well to digest the cells. After complete cell detachment, 1 mL of LDM medium was added to stop the digestion. The cell suspension was collected in centrifuge tubes and centrifuged at 3500 rpm for 1 min at 4 °C, and the supernatant was discarded. 150 μL of cell lysis buffer was added to the cell pellet, and lysis was performed on ice for 30 min, gently shaking every 10 min. After lysis, the cells were centrifuged at 13000 rpm for 10 min at 4 °C, and the supernatant was collected as the sample for subsequent analysis.

[0052] ELISA detection of target protein expression: Anti-FIPV-IRBD / N / M protein antibodies and anti-FIPV-IIRBD protein antibodies were diluted to 1 μg / mL using coating buffer, and 100 μL was added to each well. The mixture was then incubated overnight at 4°C. The cells were subsequently washed three times with PBST for 3 min each time.

[0053] Add 200 μL of blocking buffer to each well and block at 37 °C for 1 h, followed by washing three times with PBST. Then add 100 μL of the test sample or standard (FIPV-IRBD / N / M protein or FIPV-IIRBD protein) to each well and incubate at 37 °C for 1 h, followed by washing five times with PBST. Add 100 μL of HRP-labeled anti-FIPV-IRBD / N / M protein antibody or anti-FIPV-IIRBD protein antibody (1:5000 dilution) to each well and incubate at 37 °C for 30 min, followed by washing five times with PBST. Then add 100 μL of TMB substrate solution and incubate in the dark for 15 min. Stop the reaction by adding 50 μL of stop solution and detect the OD value of each well using a microplate reader at 450 nm.

[0054] The transfection efficiency of saRNA-LNP in CRFK cells and its ability to drive the expression of FIPV-I and FIPV-II related antigen proteins were evaluated by comparing the OD values ​​of the transfected group and the negative control group. Results are as follows: Figure 4 As shown, different doses of saRNA-LNP efficiently express FIPV-IMEV / N / M and FIPV-IIRBD proteins in CRFK cells.

[0055] Example 5. Safety and efficacy evaluation of saRNA-LNP vaccine in a cat model. This embodiment systematically evaluates the safety of the saRNA-LNP vaccine and its immunoprotective effect against FIPV I and FIPV II viruses in actual target animals by conducting in vivo immunization and viral challenge experiments in SPF-grade healthy kittens.

[0056] Experimental animals and grouping: Forty-two healthy SPF kittens aged 4 weeks were selected and randomly divided into 7 groups of 6 kittens each. The specific grouping is as follows: 1) High-dose vaccine group (vaccine group 1): The saRNA-LNP vaccine of this invention was injected intramuscularly into the leg, with a volume of 0.5 mL / animal, containing 50 μg of saRNA. A booster immunization was performed 2 weeks after the first immunization, and challenged with FIPV type I and FIPV type VII virus 4 weeks after the first immunization. The animals were observed for 28 days after the challenge.

[0057] 2) Medium-dose vaccine group (vaccine group 2): The saRNA-LNP vaccine of this invention was injected intramuscularly into the leg, with a volume of 0.5 mL / animal, of which the saRNA content was 10 μg; booster immunization was performed 2 weeks after the first immunization; challenge was performed with FIPV type I and FIPVII virus 4 weeks after the first immunization, and the animals were observed for 28 days after the challenge.

[0058] 3) Low-dose vaccine group (vaccine group 3): The saRNA-LNP vaccine of this invention was injected intramuscularly into the leg, with a volume of 0.5 mL / animal, containing 5 μg of saRNA; a booster immunization was performed 2 weeks after the initial immunization. Four weeks after the initial immunization, the animals were challenged with FIPV type I and FIPV type VII viruses, and were observed for 28 days after the challenge.

[0059] 4) Linear mRNA control group (control group 1): Linear mRNA-LNP vaccine was injected intramuscularly into the leg. The mRNA encoding antigen sequence is consistent with that of the saRNA vaccine but does not contain the replicase coding region. The administration volume was 0.5 mL / animal, and the mRNA content was 50 μg. The immunization procedure was the same as that of the vaccine group. Four weeks after the first immunization, the animals were challenged with FIPV type I and FIPV type II viruses, respectively. The animals were observed for 28 days after the challenge.

[0060] 5) Challenge control group 1 (control group 2): No immunization treatment was performed. Four weeks after the first immunization, the virus was challenged with FIPV type I virus and observed for 28 days after the challenge.

[0061] 6) Challenge control group 2: No immunization was performed. Four weeks after the first immunization, the virus was challenged with FIPV type II virus and observed for 28 days after the challenge.

[0062] 7) Placebo group: 0.5 mL of nuclease-free PBS was injected intramuscularly into the leg. No viral challenge was performed. The group was observed for 28 consecutive days.

[0063] 2. Safety Evaluation: From immunization to the end of the experiment, the mental state, activity level, diet, and injection site reactions of the kittens in each group were observed daily, and weight changes were measured and recorded weekly. Results showed that no significant adverse reactions were observed in the vaccine group kittens within two weeks after immunization; their mental state was good; their diet was normal; there was no redness or inflammation at the injection site; and their weight gain trend was not significantly different from the blank control group, indicating that the saRNA-LNP vaccine has good in vivo safety at the tested dose.

[0064] 3. Effectiveness evaluation: (1) Detection of specific antibody response Serum samples were collected from kittens in each group on day 14 after secondary immunization, and the level of specific IgG antibodies against FIPV was detected using an indirect ELISA method. Data were analyzed statistically using one-way ANOVA.

[0065] The results showed that, compared with the challenge control group, all saRNA-LNP vaccine immunization groups (high-dose, medium-dose, and low-dose groups) induced significantly increased specific IgG antibody titers. Among them, the antibody levels in the high-dose group (50 μg / animal) and the medium-dose group (10 μg / animal) were significantly higher than those in the challenge control group (P < 0.001), and the low-dose group (5 μg / animal) also showed a significant increasing trend (P < 0.01).

[0066] Further comparisons showed that, under the same dosage (50 μg), the antibody titer in the saRNA-LNP vaccine group was significantly higher than that in the linear mRNA control group (P < 0.01), indicating that the self-amplified RNA platform can significantly improve the efficiency of antigen expression in vivo, thereby enhancing the level of humoral immune response. In addition, no significant antibody response was detected in the blank control group.

[0067] The above results indicate that the saRNA-LNP vaccine of the present invention can induce high-titer FIPV-specific antibodies under low-dose conditions and has good immunogenicity.

[0068] (2) Detection of cellular immune response On day 14 after the second immunization, the spleens of kittens in each group (including the high-dose vaccine group, the medium-dose vaccine group, the low-dose vaccine group, the linear mRNA control group, the challenge control group, and the blank control group) were taken. Splenic lymphocytes were isolated and single-cell suspensions were prepared. After adjusting the cell concentration, the cells were seeded into 96-well cell culture plates, with 100 μL of cell suspension added to each well.

[0069] FIPV type I specific antigenic peptide or FIPV N and M proteins were added to each experimental well as stimulating antigens, with a final concentration of 10 μg / mL; phytohemagglutinin (ConA) was added to the positive control wells, with a final concentration of 5 μg / mL; and an equal volume of culture medium was added to the negative control wells. Each treatment group had three replicates.

[0070] The culture plates were incubated at 37℃ and 5% CO2 for 72 h. Four h before the end of the incubation period, 10 μL CCK-8 reagent was added to each well, and incubation continued for another 4 h. The absorbance (OD) of each well was then measured at 450 nm. The Stimulation Index (SI) was calculated based on the data from each group using the formula: SI = OD value of antigen-stimulated well / OD value of negative control well. An SI ≥ 2.0 was considered indicative of positive lymphocyte proliferation.

[0071] The results are as follows Figure 6As shown, compared with the challenge control group and the blank control group, all saRNA-LNP vaccine immunization groups showed significant lymphocyte proliferation response. Among them, the SI values ​​of the high-dose group (50 μg / animal) and the medium-dose group (10 μg / animal) were significantly higher than those of the control group (P<0.01), and the low-dose group (5 μg / animal) also met the positive criteria (SI≥2.0), indicating that the vaccine can effectively induce FIPV-specific cellular immune response.

[0072] Further comparisons showed that, under the same or lower dosage conditions, the cellular immune response induced by the saRNA-LNP vaccine group was generally better than that of the linear mRNA-LNP control group, suggesting that the self-amplified RNA platform has certain advantages in inducing cellular immunity.

[0073] 4. Virus challenge protection test After viral challenge, kittens in each group were continuously observed, and their mental state, changes in appetite, weight changes, and clinical symptoms such as pleural or peritoneal effusion were recorded. Morbidity and mortality rates were also calculated, and Kaplan-Meier survival curves were plotted.

[0074] As shown in Table 3, kittens in both the challenge control group 1 (FIPV type I) and the challenge control group 2 (FIPV type II) developed typical FIP clinical symptoms within 7–14 days after challenge, including lethargy, decreased appetite, weight loss, and pleural and peritoneal effusions, ultimately all of which resulted in death. In contrast, all saRNA-LNP vaccine immunization groups showed significant protective effects. In the high-dose group (50 μg / kit) and the medium-dose group (10 μg / kit), all animals survived after challenge with FIPV type I and FIPV type II viruses, achieving a protection rate of 100%; in the low-dose group (5 μg / kit), only 5 / 6 kittens survived after challenge, achieving a protection rate of 83.3%. Figure 7 As shown.

[0075] In the linear mRNA control group, 5 / 6 birds survived under the same conditions, with a protection rate of 83.3%. Further comparisons showed that, under the same dosage conditions, the saRNA-LNP vaccine group exhibited superior protective efficacy compared to the linear mRNA control group in both the high-dose (50 μg / bird) and medium-dose (10 μg / bird) groups, demonstrating higher survival rates and milder clinical symptoms. This suggests that the self-amplified RNA platform possesses higher antigen expression efficiency and immunoprotective capacity in vivo. In the low-dose group (5 μg / bird), the protection rates of the saRNA-LNP vaccine group and the linear mRNA control group were comparable, both demonstrating some immunoprotective effect, indicating that both platforms can provide basic immunoprotection at lower dosages.

[0076] The kittens in the blank control group remained healthy throughout the observation period and showed no abnormal clinical manifestations.

[0077] The above results demonstrate that the saRNA-LNP vaccine described in this invention provides significant immune protection against FIPV types I and II under different dosage conditions, with the medium- and high-dose groups achieving complete protection (100% survival). At the same dosage, the saRNA-LNP vaccine exhibits superior protective efficacy compared to the linear mRNA control group, demonstrating higher survival rates and milder clinical symptoms; however, at low doses, the protective effects of both platforms are comparable. These results further demonstrate that vaccines based on a self-amplified RNA platform possess higher antigen expression efficiency and immune activation capacity in vivo, maintaining or even enhancing immune protection while reducing dosage, thus demonstrating significant application advantages.

[0078] Table 3. Results of challenge protection test of feline infectious peritonitis saRNA vaccine

Claims

1. A nucleotide sequence encoding a recombinant feline infectious peritonitis virus (FIPV) antigen, characterized in that, It comprises nucleic acid sequences A and B, wherein nucleic acid sequence A is the sequence shown in SEQ ID NO:1 or a sequence having at least 90% identity with it and encoding the same functional antigen; and nucleic acid sequence B is the sequence shown in SEQ ID NO:2 or a sequence having at least 90% identity with it and encoding the same functional antigen.

2. A self-amplifying RNA (saRNA) composition, characterized in that, The saRNA composition comprises the nucleic acid sequences A and B as described in claim 1.

3. The nucleotide sequence encoding the FIPV antigen according to claim 1, characterized in that, The nucleic acid sequence A is an antigen-coding sequence of N protein and M protein formed by the fusion sequence of the neutralizing epitope of the FIPVI type S protein and the epitope of the T cell.

4. The nucleotide sequence encoding the FIPV antigen according to claim 1, characterized in that, The nucleic acid sequence B is The coding sequence for an optimized fragment of a self-assembled LS nanoparticle tandemly connected to the FIPVII type S protein receptor-binding domain (RBD).

5. The saRNA composition according to claim 2, characterized in that, The assembly elements of the saRNA include: a 5′UTR, replicases nsp1-nsp4, a subgenomic promoter, a Kozak sequence, nucleic acid sequence A or nucleic acid sequence B, a 3′UTR, and a polyadenylate tail polyA.

6. The saRNA composition according to claim 2, characterized in that, The saRNA is derived from any one of Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), or Western equine encephalitis virus (WEEV).

7. The saRNA composition according to claim 2, characterized in that, The saRNA was encapsulated in lipid nanoparticles (LNPs).

8. The saRNA composition according to claim 7, characterized in that, The lipid nanoparticles comprise: ionized lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipids.

9. The use of a nucleotide sequence encoding a recombinant FIPV antigen as described in claim 1 in the preparation of a feline infectious peritonitis vaccine and medicament.

10. The use of the saRNA composition as described in claim 2 in the preparation of feline infectious peritonitis vaccines and medicaments.

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