A secreted self-assembling nanoparticle feline infectious peritonitis mRNA vaccine and uses thereof

Abstract

This invention belongs to the field of biomedicine, specifically relating to a secretory self-assembling nanoparticle mRNA vaccine for feline infectious peritonitis and its application. First, this invention constructs a Ferritin fusion antigen by fusing the RBD or S1 fragment of the FIPV S protein with ferritin. Second, this invention constructs an mRNA encoding the aforementioned Ferritin fusion antigen. The mRNA is delivered into host cells via lipid nanoparticles (LNPs), where it is translated and expressed in the cytoplasm. The Ferritin fusion antigen then forms a nanoparticle structure within the cell using its natural self-assembly properties, eliminating the need for in vitro assembly steps. Compared to traditional nanoparticle vaccines, this method avoids steps such as in vitro induction expression, purification, and adjuvant addition, simplifying the production process, improving immunogenicity, reducing the risk of adverse drug reactions (ADE), and enhancing safety. Furthermore, it significantly improves humoral and cellular immunity.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to a secretory self-assembling nanoparticle feline infectious peritonitis mRNA vaccine and its application. Background Technology

[0002] Feline infectious peritonitis (FIP) is a fatal disease caused by mutations in feline coronavirus (FCoV), with an extremely high mortality rate. Currently, there is a lack of safe and effective preventive vaccines in clinical practice. FIP is mainly divided into serotypes I and II, with serotype I being the dominant strain circulating globally. However, its isolation and culture in vitro are difficult, severely hindering vaccine development.

[0003] The spike protein (S) of coronaviruses is a key structural protein mediating viral adsorption, membrane fusion, and invasion of host cells. It consists of two functional subunits, S1 and S2, with the receptor-binding domain (RBD) in the S1 subunit being the core antigenic region that induces neutralizing antibodies. Previous studies have shown that immune responses against the S protein or RBD play a decisive role in viral neutralization.

[0004] However, the development of FIPV vaccines has long been constrained by the risk of antibody-dependent enhancement (ADE). Some vaccines based on the full-length S protein or viral vectors have induced non-neutralizing antibodies in animal experiments, which has exacerbated the disease progression. Therefore, how to induce highly effective neutralizing antibodies while avoiding ADE is a core technical challenge in FIPV vaccine design.

[0005] In existing research, some approaches have attempted to use ferritin nanoparticles as an antigen display platform, fusing the RBD or S1 fragment of FIPV S protein with ferritin, expressing and purifying the fusion protein in vitro, and utilizing the self-assembly properties of ferritin to form multivalent antigen nanoparticles, thereby enhancing immunogenicity. Such approaches typically require: (1) expressing the fusion protein in vitro in a prokaryotic or eukaryotic expression system; (2) obtaining nanoparticles through multi-step purification and in vitro assembly; and (3) relying on chemical adjuvants to enhance the immune response during immunization. Although this strategy improves the immunogenicity of antigens to some extent, it still has the following limitations: (1) the in vitro protein expression and purification process is complex, the process is difficult to scale up, and it is not conducive to rapid large-scale production; (2) the conformation of the antigen assembled in vitro is difficult to completely simulate the natural translation and folding environment in vivo, which may affect the presentation of key neutralizing epitopes; (3) it relies on exogenous adjuvants, making the immunization procedure complex; and (4) it cannot achieve in situ expression and continuous antigen presentation of antigens in host cells.

[0006] In recent years, the successful application of mRNA vaccine technology in novel coronavirus vaccines, combining mRNA technology with polymerized antigen design, holds promise for overcoming the shortcomings of traditional protein subunit vaccines in terms of conformation, delivery, and duration of immunity. Therefore, developing a FIPV antigen vaccine based on an mRNA platform that can be expressed in situ in vivo and self-assembled into nanoparticles is an inevitable trend in current technological development. However, due to differences in virus types and the complexity of vaccine preparation, this strategy has not yet been successfully applied to FIPV, and there is currently no technical solution for expressing nanoparticles using mRNA vaccines. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention first optimizes the antigen sequence encoding FIPV by fusing the RBD or S1 fragment of the FIPV S protein with ferritin to construct a Ferritin fusion antigen. Secondly, this invention constructs an mRNA encoding the aforementioned Ferritin fusion antigen. This mRNA is delivered into host cells via lipid nanoparticles (LNPs), where it is translated and expressed in the cytoplasm, and then forms a nanoparticle structure within the cell using its natural self-assembly properties. Specifically, it includes the following:

[0008] In a first aspect, the present invention provides a recombinant antigen for feline infectious peritonitis, wherein the recombinant antigen is any one of the Ferritin fusion antigens obtained through the following two methods:

[0009] (1) The fusion antigen S1-FT obtained by linking the S1 subunit of the S protein of type I or type II feline infectious peritonitis to ferritin via a flexible linker;

[0010] (2) The fusion antigen RBD-FT is obtained by linking the RBD of type I or type II feline infectious peritonitis S protein with ferritin through a flexible linker.

[0011] Preferably, the amino acid sequence of the fusion antigen S1-FT is as shown in SEQ ID NO.1 or SEQ ID NO.2; and the amino acid sequence of the fusion antigen RBD-FT is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0012] In a second aspect, the present invention provides a feline infectious peritonitis mRNA, wherein the feline infectious peritonitis mRNA encodes the feline infectious peritonitis recombinant antigen described in the first aspect above.

[0013] Preferably, the coding sequence of the feline infectious peritonitis recombinant antigen described in the first aspect above is as shown in any one of SEQ ID NO. 5-8.

[0014] Preferably, non-coding region sequences 5'UTR and 3'UTR are added to the 5' and 3' ends of the encoded sequence.

[0015] Preferably, the 5' end of the 5'UTR is modified with a Cap1-type cap; and the 3' end of the 3'UTR is modified with polyadenine.

[0016] Preferably, the mRNA sequence is as shown in any one of SEQ ID NO. 9-12.

[0017] Thirdly, the present invention provides the application of the feline infectious peritonitis mRNA described in the second aspect above in the preparation of a feline infectious peritonitis mRNA vaccine.

[0018] Fourthly, the present invention provides an mRNA vaccine for feline infectious peritonitis, the mRNA vaccine comprising the mRNA described in the second aspect above, and a vaccine vector for encapsulating the mRNA.

[0019] Preferably, the vaccine carrier is a lipid nanoparticle that can be utilized by mRNA.

[0020] Preferably, the liposome nanoparticles comprise: 40-60 parts by weight of ionizable cationic lipids, such as 40, 45, 50, 55, or 60 parts by weight; 8-16 parts by weight of phospholipids, such as 8, 10, 12, 14, or 16 parts by weight; 30-45 parts by weight of cholesterol lipids, such as 30, 35, 40, or 45 parts by weight; and 1-5 parts by weight of PEG-modified lipids.

[0021] Preferably, the ionizable cationic lipid is selected from at least one of the following: amino cationic lipid SM102, 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)-2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)aminodiyl)di(dodecane-2-ol) (C12-200), (6Z,9Z,28Z,31Z)-heptadecene-6,9, 28,31-Tetraen-19-yl 4-(dimethylamino)butyrate (MC3), N,N-dimethyl-2,3-bis((9Z,12Z)-octadec-9,12-dien-1-oxy)propylamine (DLinDMA), 2-(2,2-di((9Z,12Z)-octadec-9,12-dien-1-yl)-1,3-dioxane-4-yl)-N,N-dimethylethylamine (DLinKC2DMA) [XTC2]), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12), 10,13-dimethyl-17-(6-methylheptyl-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopentanphenanthrene-3-yl-3-(1H-imidazol-5-yl)propionate (ICE), (15Z,18Z)-N,N-dimethyl-6 -((9Z,12Z)-octadec-9,12-dien-1-yl)tetradec-15,18-dien-1-amine (HGT5000), (4Z,15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadec-9,12-dien-1-yl)tetradec-4,15,18-trien-1-amine (HGT5001), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N- Dimethylammonium bromide (DDAB), 1,2-dimyristoxypropyl-3-dimethylhydroxyethylammonium chloride (DMRIE), dioleoyloxy-N-[2-arginine formamide)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), octadecanoamide arginine (DOGS), 1,2-dioleoyl-3-dimethylaminopropane (DODAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), and N,N-dimethyl-(2,3-dioleoyloxy)propylamine -Dimyristyl(oxy)propylamine (DMDMA), 1,2-dilininyloxy-N,N-dimethylaminopropane (DLenDMA), (2S)-2-(4-((10,13-dimethyl-17-(6-methylheptyl-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopentan[a]phenanthrene-3-yl)oxy)butoxy)-N,N-dimethyl-3-((9Z,12Z)-octadec-9,12-Dien-1-oxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3[β]-oxy)-3'-oxopentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylamino-3-dimethylaminopropane (DOcarbDAP), (9Z,9'Z,12Z,12'Z)- 3-(dimethylamino)propane-1,2-diylbis(octadec-9,12-dienoate) (DLinDAP), 1,2-dilininylamino-3-dimethylaminopropane (DLinCDAP), 2,2-dilininyl-4-dimethylaminomethyl-[1,3]-dioxane (DLin-K-DMA), 2-((2,3-bis((9Z,12Z)-octadec-9,12-dien-1-oxy)propyl)dithio)-N,N-dimethylethylamine (HGT4003).

[0022] Preferably, the cholesterol lipids are selected from cholesterol or PEGylated cholesterol.

[0023] The beneficial effects of this invention are:

[0024] This invention first constructs a Ferritin fusion antigen by fusing the RBD or S1 fragment of the FIPV S protein with ferritin; and then constructs an mRNA that encodes the above-mentioned Ferritin fusion antigen. The mRNA is delivered into the host cell via lipid nanoparticles (LNPs), where it is translated and expressed in the cytoplasm. The mRNA also forms a nanoparticle structure within the cell by utilizing its natural self-assembly properties. This avoids the difference between the antigen conformation and the natural viral state, and solves the problem that fusion proteins expressed in vitro cannot completely mimic the natural translation, folding, and modification environment within the host cell, which may affect the presentation of key neutralizing epitopes.

[0025] By using mRNA technology and ferritin self-assembly technology, the level of immune response is significantly improved, solving the technical problems that traditional FIPV inactivated or attenuated vaccines cannot induce sufficient neutralizing antibodies and cannot effectively activate cellular immunity.

[0026] By precisely screening the S1 and RBD regions of type I (UU4) and type II (DF2), the interference of non-neutralizing epitopes is reduced, thus mitigating the risk of ADE (antibody-dependent enhancement) effects. Furthermore, the vaccine design can take into account different FIPV serotypes, providing broad-spectrum protection against prevalent type I and type II FIPV.

[0027] By utilizing mRNA for in situ expression, secretion, and self-assembly within the host, complex purification processes and adjuvant additions are eliminated, thus solving the problems of complex and costly production processes of existing ferritin nanoparticle vaccines. Attached Figure Description

[0028] Figure 1 Schematic diagram of mRNA structure.

[0029] Figure 2 Recombinant plasmid map of FIPV-I S mRNA vaccine.

[0030] Figure 3 Recombinant plasmid map of FIPV-I S1-FT mRNA vaccine.

[0031] Figure 4 Recombinant plasmid map of FIPV-I RBD-FT mRNA vaccine.

[0032] Figure 5 Recombinant plasmid map of FIPV-II S mRNA vaccine.

[0033] Figure 6 Recombinant plasmid map of FIPV-II S1-FT mRNA vaccine.

[0034] Figure 7 Recombinant plasmid map of FIPV-II RBD-FT mRNA vaccine.

[0035] Figure 8 Western blotting was used to verify the antigen expression results.

[0036] Figure 9 TEM verifies the formation results of nanoparticles. From left to right, they are S1-FT, RBD-FT, FIPV-I (top), and FIPV-II (bottom).

[0037] Figure 10 Results of specific IgG titer assay in mouse serum after immunization with FIPV-I mRNA vaccine (Day 14 was the second week after the first immunization, and Day 28 was the second week after the second immunization).

[0038] Figure 11 Two weeks after the second dose of FIPV-II mRNA vaccine immunization, the results of the specific IgG titer determination in mouse serum (Day 14 was the second week after the first immunization, and Day 28 was the second week after the second immunization).

[0039] Figure 12After immunization with FIPV-II mRNA vaccine, the results of neutralizing antibody titer determination in mouse serum were verified using a pseudovirus system.

[0040] Figure 13 ELISPOT results of mouse spleen cells three weeks after the second dose of FIPV-II mRNA vaccine immunization. Detailed Implementation

[0041] The following are various exemplary embodiments of the present invention, but they should not be considered as limitations on the present invention, but rather as a more detailed description of certain aspects, features and implementations of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] Example 1: Preparation of mRNA vaccine

[0046] 1. mRNA construction and preparation

[0047] 1.1 Antigen Design

[0048] (1) FIPV type I (FIPV-I) mRNA vaccine

[0049] S antigen: The C-terminus of the type I FIPV spike protein is truncated by 20 amino acids, and the specific amino acid sequence is shown in SEQ ID NO. 13.

[0050] S1-Ferritin (S1-FT) fusion antigen: The S1 domain of the type I FIPV spike protein is extracted and coupled to the ferritin self-assembly element Ferritin through a 16-GS amino acid repeat sequence as a flexible linker. The amino acid sequence is shown in SEQ ID NO.1.

[0051] RBD-Ferritin (RBD-FT) fusion antigen: The receptor-binding domain (RBD) of the type I FIPV spike protein is used as a flexible linker to Ferritin via a 16-GS amino acid repeat sequence, as shown in SEQ ID NO.2.

[0052] (2) FIPV II mRNA vaccine

[0053] S antigen: The C-terminus of the type II FIPV spike protein is truncated by 20 amino acids, and the specific amino acid sequence is shown in SEQ ID NO.14.

[0054] S1-Ferritin (S1-FT) fusion antigen: The S1 domain of the type II FIPV spike protein is extracted and coupled to Ferritin, a ferritin self-assembly component, through a 16-GS amino acid repeat sequence as a flexible linker. The amino acid sequence is shown in SEQ ID NO.3.

[0055] RBD-Ferritin (RBD-FT) fusion antigen: The receptor-binding domain (RBD) of the type II FIPV spike protein is used as a flexible linker to Ferritin via a 16-GS amino acid repeat sequence, as shown in SEQ ID NO.4.

[0056] To facilitate subsequent detection of antigen expression, a flag tag fragment was added to the N-terminus of all antigens. At the same time, to avoid conflict with the signal peptide carried on the expression vector, the original signal peptide of the spike protein was identified and removed by the protein signal peptide prediction website Protter. Finally, codon optimization was performed on all sequences.

[0057] 1.2 mRNA framework

[0058] Untranslated regions (UTRs) are added to the 5' and 3' ends of the antigen sequence to stabilize and regulate the mRNA. Cap1-type capping is performed at the 5' end of the 5' UTR to improve mRNA structural stability, enhance translation efficiency, and reduce the immunogenicity of the mRNA in cells. Polyadenine modification is performed at the 3' end of the 3' UTR to further improve mRNA structural stability and enhance translation efficiency. A schematic diagram of the specific structure is shown below. Figure 1 As shown.

[0059] Specifically, the mRNA sequence of FIPV-I S1-FT is shown in SEQ ID NO.9; the mRNA sequence of FIPV-I RBD-FT is shown in SEQ ID NO.10; the mRNA sequence of FIPV-II S1-FT is shown in SEQ ID NO.11; the mRNA sequence of FIPV-II RBD-FT is shown in SEQ ID NO.12; the mRNA sequence of FIPV-I S is shown in SEQ ID NO.15; and the mRNA sequence of FIPV-II S is shown in SEQ ID NO.16.

[0060] 2. Preparation of mRNA vaccines

[0061] The lipid ethanol phase (organic phase A) consists of: 50 parts by weight of ionizable cationic lipids (SM-102), 12 parts by weight of phospholipids (DSPC), 35 parts by weight of cholesterol lipids (cholesterol), and 3 parts by weight of PEG-modified lipids (DMG-PEG2000). The above lipid mixture was dissolved in 200 ml of proof ethanol to make the total lipid concentration 16 mg / mL. The mixture was stirred at 30°C in the dark until completely dissolved, and then sterilized by a 0.22 µm PTFE filter to obtain organic phase A.

[0062] Aqueous phase of mRNA (Aqueous Phase B): The mRNA (approximately 4 kb) encoding the above antigen was dissolved in 25 mM sodium citrate, 125 mM NaCl, and pH 4.0 buffer to prepare a solution of 0.25 mg / mL; the solution was sterilized using a 0.22 µm PES filter to obtain aqueous phase B.

[0063] Organic phase A and aqueous phase B are mixed to prepare an mRNA vaccine.

[0064] In this embodiment, the KNAUER IJM NanoScaler benchtop impingement jet mixing (IJM) system was used to collide and self-assemble the lipid ethanol phase and the mRNA aqueous phase, which are composed of the above weight parts, within milliseconds to prepare an mRNA-LNP vaccine with a particle size of 80±10 nm, PDI≤0.20, and encapsulation efficiency≥90%.

[0065] Example 2 Antigen purification and nanoparticle assembly

[0066] 1. Validation of antigen expression and secretion

[0067] To facilitate in vitro testing of the expression and secretion function of the mRNA sequence, a recombinant plasmid capable of transcribing the mRNA sequence described in Example 1 was constructed, as shown in the diagram. Figure 2-7 As shown. This plasmid was used to verify the expression and secretion function of the mRNA sequence in eukaryotic cells in this embodiment. The recombinant plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd. After obtaining the recombinant plasmid, the following methods were used for verification:

[0068] Plasmid transfection: HEK-293T cells were transfected using recombinant plasmids. Calcium phosphate transfection was performed when cell confluence reached approximately 70%. For a 12-well plate, the total DNA mass transfected per well was 1 μg. The specific transfection method was as follows: the transfection system was 10% of the cell culture medium, with 1 mL of cell culture medium per well in a 12-well plate, resulting in a total transfection system of 100 μL. One 1.5 mL EP tube was filled with 50 μL of 2×HBS. Another 1.5 mL EP tube was filled with 5 μL of 2.5 M calcium chloride solution and the plasmid, and ddH2O was added to bring the total to 50 μL. The components in the tubes were then mixed thoroughly and evenly added to the 2×HBS solution. Bubbles were created in the liquid using a pipette to produce small, uniform calcium phosphate particles. This mixture was then evenly added to the cell plate and shaken well. The cells were then returned to the incubator and cultured for another 24 h.

[0069] Supernatant sample preparation: Use a pipette to transfer the cell culture medium into a new 1.5 mL ep tube, centrifuge at 3000 rpm for 5 min to remove cell pellet, take 50 μL of the centrifuged cell culture medium and add it to a new 1.5 mL ep tube, add 5×SDS loading buffer solution to prepare the sample.

[0070] Preparation of cell pellet samples: Remove the supernatant of cell culture medium, add 150 μL of 1×SDS loading buffer solution to the cell plate, and after complete lysis, pipette the sample into a 1.5 mL ep tube.

[0071] Sample preparation: Place the 1.5 mL ep tube containing the sample into a metal bath and heat at 95°C for 10 min. Remove and centrifuge briefly, then vortex to mix. Place the tube back into the metal bath and heat at 95°C for 10 min. Remove and centrifuge briefly again, then vortex to mix. Finally, centrifuge briefly again to complete the preparation of Western Blot samples.

[0072] Gel Preparation: Gel preparation was performed using the Omni-Easy One-Step PAGE Gel Rapid Preparation Kit (10%). Taking a 1.5 mm thick gel as an example, first clean the glass plate and allow it to dry. Set up the gel rack and check for leaks with ultrapure water. After ensuring there are no leaks, drain the liquid and allow it to dry completely. Take a clean 50 mL centrifuge tube and add 4 mL of the lower gel solution A and B, respectively, followed by 80 μL of coagulant. Cap and invert to mix thoroughly to prepare the lower gel. Then, take another clean 50 mL centrifuge tube and add 1 mL of the lower gel solution A and B, respectively, followed by 20 μL of coagulant. Cap and invert to mix thoroughly to prepare the upper gel. First, add the upper gel to the glass plate interlayer, leaving a 2 cm gap, then pour in the remaining upper gel. Finally, insert the comb and wait 15 minutes. After the gel solidifies, gently and slowly remove the comb under running water.

[0073] Electrophoresis: After securing the gel plate to the gel holder, correctly install it in the electrophoresis tank and pour in an appropriate amount of 1×Tris-glycine-SDS (TGS) running buffer. Carefully add the prepared sample and protein marker to the wells using a pipette. For wells without sample, add the same volume of 1×loading buffer. Close the electrophoresis tank lid, plug in the power, and set the electrophoresis conditions: constant voltage, initially 70 V for 30 min; then increase to 150 V for 1 h.

[0074] Transfer: After electrophoresis, open the lid, open the electrophoresis tank, and remove the glass clamps. Prepare a stainless steel tray and pour in an appropriate amount of transfer buffer. Remove the gel from the glass clamps, remove the top layer of gel, and place the gel in the tray to moisten it with transfer buffer. Mark the non-protein side of the NC membrane with a ballpoint pen, and place the NC membrane, filter paper, and sponge sheet in the tray to moisten them with transfer buffer. Assemble the gel, filter paper, and NC membrane into a sandwich structure in the following order: negative electrode - filter paper - gel - NC membrane - filter paper - positive electrode, ensuring there are no air bubbles in between. Use constant voltage conditions, 100 V for 90 min, for transfer.

[0075] Blocking: Prepare blocking solution (using TBST to prepare 5% skim milk), place the NC membrane after transfer into the blocking box, add an appropriate amount of blocking solution, and block at room temperature for 30-60 min.

[0076] Primary antibody incubation: Prepare a 1% skim milk solution using TBST, and add the primary antibody according to the recommended dilution ratio in the antibody instructions. After blocking, discard the blocking solution, add the primary antibody, and incubate at room temperature with shaking at 60 rpm for 1 hour or at 4°C overnight on a shaker.

[0077] Secondary antibody incubation: After primary antibody incubation, recover the primary antibody. Add an appropriate amount of TBST and wash at room temperature with a shaker at 90 rpm for 10 min, repeating three times. Prepare secondary antibody for the corresponding species: Dilute the secondary antibody with TBST according to the recommended dilution ratio in the antibody instructions. Add the prepared secondary antibody and incubate at room temperature with a shaker at 60 rpm for 1 h.

[0078] Chemiluminescent development: After incubation with the secondary antibody, discard the secondary antibody. Add an appropriate amount of TBST and wash at room temperature on a shaker at 90 rpm for 10 min. Repeat three times. Then prepare the chemiluminescent developing solution according to the instructions. Lay a smooth sheet of plastic wrap on the table, evenly drip the prepared developing solution onto it, gently lift the membrane with tweezers, drain the excess water on absorbent paper, ensure the protein side of the membrane is in full contact with the developing solution, and then place it in the electronic developing solution to begin development.

[0079] Test results are as follows Figure 8 As shown, the protein expression of the recombinant plasmid was identified by Western blotting. The results showed that the recombinant plasmid described in this application can be transcribed and translated normally into a protein of the expected size in cells. The ferritin-conjugated antigen protein can be detected in the cell culture supernatant, indicating that the protein can be secreted normally.

[0080] 2. Antigen assembly verification

[0081] To facilitate in vitro testing of the antigen expressed by the mRNA sequence, a recombinant plasmid capable of transcribing the mRNA sequence described in Example 1 was constructed, as shown in the diagram. Figure 2-7 As shown. This plasmid was used to verify the antigen assembly of the mRNA sequence expressed in eukaryotic cells in this embodiment. The recombinant plasmid was synthesized by Sangon Biotech (Shanghai) Co., Ltd. After obtaining the recombinant plasmid, it was verified using the following methods:

[0082] Plasmid transfection: HEK 293T cells were transfected using PEI transfection reagent. For example, 10 15 cm circular cell culture dishes were used, with each dish transfected with 15 μg of recombinant plasmid. Sixteen hours after transfection, the culture medium was replaced with protein purification medium, and samples were collected 72 hours later.

[0083] Sample preparation: Transfer the cell culture medium to centrifuge tubes and centrifuge at 300 g for 5 min at 4°C, discarding cell debris. Using 10 15 cm circular cell culture dishes as an example, concentrate all cell culture supernatant to 50 mL using a Merck 30 kDa Amicon® Ultra centrifuge tube. The supernatant sample is taken.

[0084] Resin equilibration: Purification was performed using Anti-DYKDDDDK G1 AffinityResin (catalog number: L00432) from Genscript Biotech Inc. An appropriate amount of resin was taken according to the manufacturer's instructions, centrifuged at 3000 rpm for 1 min at 4°C, the supernatant was discarded, and the resin was resuspended in three column volumes of TBS buffer. This process was repeated three times.

[0085] Protein adsorption: The equilibrated resin was added to the concentrated supernatant and placed in a rotary shaker at 4°C for adsorption by rotation for 3 hours.

[0086] Gravity column packing: The supernatant after adsorption is added to the gravity column, and gravity is used to make the liquid flow out, trapping the packing material, collecting the outflow liquid, and taking a sample for flow through sampling.

[0087] Impurity washing: Wash the packing material with 10 to 20 column volumes of TBS buffer to remove any non-specific bound impurities. When the liquid level is tangent to the resin plane for the last time, proceed with the elution process.

[0088] Protein elution: Prepare a competitive elution buffer solution, FLAG® peptide solution, at a concentration of 500 μg / mL. Load 2–3 column volumes of the competitive elution buffer solution into the column using gravity flow. When approximately one column volume of elution buffer solution remains above the packing material, seal the liquid outlet at the bottom of the column. Incubate at room temperature for 30–60 minutes. Open the end of the gravity column and collect the eluent; this is the purified protein sample.

[0089] The purified antigen protein from Flag was diluted to 0.1 mg / mL, and after sample preparation, it was imaged using a 200KV cryo-transmission electron microscope at the Electron Microscopy Center of Lanzhou University. Results Figure 9 As shown, the antigens (S1-Ferritin and RBD-Ferritin) designed in this application were successfully formed into nanoparticle structures in vitro. The FIPV-I S1-Ferritin particles are approximately 50 nm in size, and the FIPV-II RBD-Ferritin particles are approximately 12 nm in size. Similarly, the FIPV-II S1-Ferritin particles are approximately 50 nm in size, and the FIPV-II RBD-Ferritin particles are approximately 12 nm in size. However, the S antigen cannot be secreted and cannot autonomously assemble into nanoparticles in vitro.

[0090] Example 3: Immunological evaluation of FIPV mRNA vaccine in mice

[0091] 1. Route of administration and dosage

[0092] The vaccine was administered via intramuscular injection into the thigh muscle of the mouse. Each mouse received a total of 5 μg of mRNA. The packaged mRNA vaccine was diluted to the same volume with sterile PBS, and each mouse received the same volume. The control group received the same volume of PBS. The injection was administered to half of each thigh.

[0093] 2. Immunization program

[0094] Female BALB / c mice aged 6-8 weeks were selected for the experiment, with five mice in each group. The control group and the experimental group were randomly assigned to either group. The mice were immunized twice. Blood samples were collected two weeks after the first immunization and on the same day as the second immunization. Blood samples were collected again two weeks after the second immunization and three weeks after the second immunization. The mice were then dissected.

[0095] 3. Animal and challenge models

[0096] Female BALB / c mice aged 6-8 weeks were selected for the experiment and were not challenged with the virus.

[0097] 4. Determination of specific IgG titer in mouse serum

[0098] Coating-specific antigen: The S1 domain of the laboratory-purified viral S protein was used as the antigen. Diluted to 2 μg / ml with coating buffer, 100 μL was added to each well of a 96-well plate. The plate was capped and incubated overnight at 4 °C. The next day, the liquid in the wells was discarded. Each well was filled with washing buffer three times. After the first wash, the plate was allowed to stand for 2 min before discarding. The subsequent two washes were allowed to stand for 1 min each. The reaction plate was then inverted onto filter paper to remove any remaining liquid.

[0099] Sealing: Fill each well with sealing solution (about 300 μL), cover or seal the plate with sealing film, incubate at 37°C for 1 h, pour off the liquid in the well, and wash 3 times.

[0100] Serum incubation: Dilute the serum to be tested with diluent according to the serial dilution method (1:100, 1:200, 1:400, etc.), add 100 μL of the antibody to be tested and blank control at different dilutions to the corresponding wells, cover or seal the plate, and incubate at room temperature for 1 h. After incubation, pour off the liquid in the wells and wash repeatedly 3 times.

[0101] Secondary antibody incubation: Add HRP-labeled secondary antibody, 100 μL per well, incubate at room temperature for 1 h, wash at least 5 times as described above, and blot dry on filter paper.

[0102] Color development: After washing, remove the washing solution and add 100 μL of TMB color development solution. Incubate at room temperature in the dark for 3-30 min until the color reaches the expected depth.

[0103] To terminate the reaction, add 100 μL of 2 mol / L H2SO4 to each well. After stabilizing for 3-5 minutes, the sample can be measured colorimetrically.

[0104] Detection: The light absorption of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) instrument.

[0105] Calculation: Calculate the absorbance ratio of the antibody to be tested to the blank control (Positive / negative, P / N). When P / N is greater than 2, it is positive; when P / N is less than 2, it is negative. The positive result with the highest dilution factor is taken as the measured antibody titer.

[0106] The results of the specific IgG titer test in mouse serum after immunization with FIPV-I mRNA vaccine are as follows: Figure 10 As shown; the results of the additional assay of specific IgG titer in mouse serum after immunization with FIPV-II mRNA vaccine are as follows. Figure 11 As shown in the figure. The results indicate that, compared to the S antigen mRNA vaccine group, the mRNA vaccine described in this invention produced high-titer specific IgG in mouse serum after only one immunization, and the specific IgG titer in mouse serum continued to increase after two immunizations.

[0107] 5. Determination of neutralizing antibody titer in mouse serum

[0108] According to the literature, the receptor for FIPV type II virus is aminopeptidase N (APN). This protein is present on the surface of cat kidney cells (CRFK), so CRFK cells were used as target cells in this experiment to test the neutralizing antibody titer of FIPV-II mRNA vaccine.

[0109] 5.1 Materials and Reagents

[0110] Cells: CRFK cells (confirmed to be free of mycoplasma contamination).

[0111] Virus: A recombinant pseudovirus expressing the S protein of the DF2 strain.

[0112] Serum sample: Post-immunization mouse serum (heat inactivated: 56℃ for 30 min).

[0113] Positive control: only virus solution was added.

[0114] Negative control: only diluted serum was added.

[0115] Virus control: virus only + culture medium.

[0116] Cell control: Cells + culture medium only.

[0117] Culture medium: DMEM containing 10% FBS (with antibiotics).

[0118] Test reagents: Luciferase test kits (such as Promega Steady-Glo®).

[0119] 5.2 Equipment

[0120] Biosafety cabinet, CO2 incubator, multi-functional microplate reader, centrifuge, pipette.

[0121] 5.3 Experimental Procedure

[0122] (1) Serum pretreatment

[0123] 1) Heat inactivation: 56℃ water bath for 30 minutes to inactivate complement (avoid complement-mediated pseudo-neutralization).

[0124] 2) Dilution: Use a 96-well plate for dilution. Starting concentration: 1:10 dilution. Serial dilution: Perform 10-fold serial dilutions (e.g., 1:10 to 1:100) with culture medium in a 96-well plate, bringing the volume to 75 μL in each well.

[0125] Objective: To determine the effective dilution range of neutralizing antibodies.

[0126] (2) Virus-serum incubation

[0127] Virus dosage: Add 75 μL of virus.

[0128] Incubation conditions: 37°C, 1 hour (simulating the in vivo neutralization process).

[0129] Mixing ratio: 75 μL serum diluent + 75 μL virus solution per well (final volume 150 μL).

[0130] Objective: To allow antibodies to bind to viral surface antigens and block viral infection.

[0131] (3) Cell infection

[0132] 1) Cell preparation: CRFK cells were seeded 24 hours in advance (96-well plates, 2×10⁶ cells / wells). 4 (cells / pores).

[0133] 2) Infection procedure: Aspirate cell culture medium, add 100 μL of virus-serum mixture, and continue culturing for 24 hours. Objective: To allow the virus to enter the cells and express Luciferase.

[0134] (4) Luciferase activity assay

[0135] 1) Remove the virus-serum mixture.

[0136] 2) Lysis of cells: Add 100 μL of Steady-Glo® reagent to each well and shake at room temperature in the dark for 10 minutes.

[0137] 3) Detection: The luminescence value (RLU) is read using an ELISA reader. Purpose: To quantify the residual viral infectivity (RLU is positively correlated with viral activity).

[0138] (5) Neutralization valence calculation

[0139] 1) Method: The dose-response curve was fitted using GraphPad Prism nonlinear regression, and the ID was calculated. 50 (Serious dilution that inhibits 50% viral activity).

[0140] Example: If ID 50 =1:1000 means that serum diluted 1000 times can still neutralize 50% of the virus.

[0141] Blood was collected from the orbital rim of mice three weeks after the second immunization, and serum was obtained. Neutralizing antibody titers were then detected using pseudoviruses. The results of the neutralizing antibody titer assay in the serum of mice immunized with FIPV-II mRNA vaccine are as follows: Figure 12 As shown in the figure. These results indicate that, compared to the S antigen mRNA vaccine group, the mRNA vaccine described in this application can stimulate mice to produce higher levels of neutralizing antibodies. Specifically, the antigen RBD-Ferritin can more efficiently stimulate mice to produce neutralizing antibodies, and this component produces the highest neutralizing antibody titer. The FIPV-I mRNA vaccine described in this application also exhibits similar activity.

[0142] 6. Evaluation of mouse cellular immune levels

[0143] Mouse spleen cells were tested using the Mouse IFN-γ Precoated ELISPOT Kit (strips) (catalog number: 2210005) from Shenzhen Dako Biotechnology Co., Ltd. The ELISPOT results of IFN-γ can reflect the strength of Th1-type cellular immunity. The specific method is as follows:

[0144] Reagent preparation: The kit used was the Mouse IFN-γ Precoated ELISPOT Kit (strips) from Shenzhen Dakwei Biotechnology Co., Ltd., catalog number: 2210005. The reagents were prepared according to the kit instructions.

[0145] Antigen protein: The S protein S1 Domain of the DF2 strain was expressed in eukaryotic cells using the pFUSE-hIgG1-Fc vector as the antigen. This vector carries an Fc tag, and the antigen protein can be purified using Beyotime's Protein A+G Agarose.

[0146] Activation of pre-coated plates: Add 200 μL of serum-free medium or RPMI-1640 medium to each well, let stand at room temperature for 5-10 min, and then remove the plates.

[0147] Add cell suspension: Add the adjusted cell suspension to each well, 100 μL / well.

[0148] Positive control wells: Cell concentration can be 5 × 10⁻⁶ 5 cells / well;

[0149] Negative control wells: Cell concentration can be 5 × 10⁻⁶. 5 cells / well;

[0150] Experimental wells: Cell concentration can be 5 × 10⁻⁶ 5 cells / well;

[0151] Add irritant: 10 μL / well, as follows:

[0152] Positive control well: Add positive stimulant working solution;

[0153] Negative control wells: The culture medium used to resuspend the cells was added;

[0154] Experimental wells: Add antigen protein (prepared to a final concentration of 10× using serum-free medium or RPMI 1640).

[0155] Incubation: After adding all samples and stimulants, cover the plate. Incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0156] Cell lysis: Pour out the cells and culture medium from the wells. Add ice-cold deionized water, 200 μL / well, and incubate at 4°C for 10 min to lyse the cells in a hypotonic manner.

[0157] Washing: Shake out the liquid in the well, add 1× Washing buffer, 260 μL / well, let stand for 1 min, then discard the liquid in the well. Repeat six times, and each time invert the plate onto absorbent paper to dry.

[0158] Antibody incubation test: Add diluted biotinylated antibody working solution to each well, 100 μL / well. Incubate at 37℃ for 1 h.

[0159] Washing: Shake out the liquid in the well, add 1× Washing buffer, 260 μL / well, let stand for 1 min, then discard the liquid in the well. Repeat six times, and each time invert the plate onto absorbent paper to dry.

[0160] Enzyme-linked avidin incubation: Add diluted enzyme-labeled avidin (Streptavidin-HRP) working solution to each well, 100 μL / well. Incubate at 37℃ for 1 h.

[0161] Washing the plate: Shake out the liquid in the wells, add 1× Washing buffer, 260 μL / well, let stand for 1 min, then discard the liquid in the wells. Repeat five times, pat dry on absorbent paper each time. Then remove the plate base and wash the bottom of the membrane and the base with deionized water / tap water. Carefully pat dry the base and the bottom of the membrane with absorbent paper. Close the base, add 1× Washing buffer, 260 μL / well, let stand for 1 min, then discard the liquid in the wells. Invert the plate on absorbent paper to air dry.

[0162] Developing color: Add freshly prepared AEC developing solution to each well, 100 μL / well. Incubate at room temperature in the dark for 5-30 min, stopping the development time according to the spot formation. Check every 5-10 min.

[0163] To stop color development: Pour out the liquid from the hole, remove the base of the plate, and wash both sides and the base with tap water 3-5 times to stop color development. Place the plate in a cool, shady place at room temperature and allow it to air dry naturally before reattaching the base.

[0164] Photo counting: Photo counting was performed using a Mabtech ELISA spot analyzer (model: ASTOR 2).

[0165] The measurement results are as follows Figure 13 As shown, compared to the S antigen mRNA vaccine group, the mRNA vaccine described in this application can significantly enhance the level of cellular immunity. The FIPV-I mRNA vaccine described in this application also has similar activity.

[0166] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant antigen for feline infectious peritonitis, characterized in that, The recombinant antigen is any one of the Ferritin fusion antigens obtained through the following two methods: (1) The fusion antigen S1-FT obtained by linking the S1 subunit of the S protein of type I or type II feline infectious peritonitis virus to ferritin via a flexible linker; (2) The fusion antigen RBD-FT is obtained by linking the RBD of type I or type II feline infectious peritonitis virus S protein with ferritin through a flexible linker.

2. The recombinant antigen for feline infectious peritonitis as described in claim 1, characterized in that, The amino acid sequence of the fusion antigen S1-FT is shown in SEQ ID NO.1 or SEQ ID NO.2; the amino acid sequence of the fusion antigen RBD-FT is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. A feline infectious peritonitis mRNA, characterized in that, The feline infectious peritonitis mRNA encodes the feline infectious peritonitis recombinant antigen as described in claim 1 or 2.

4. The feline infectious peritonitis mRNA as described in claim 3, characterized in that, The sequence encoding the recombinant antigen of feline infectious peritonitis as described in claim 1 or 2 is shown in any one of SEQ ID NO. 5-8.

5. The feline infectious peritonitis mRNA as described in claim 4, characterized in that, Uncoding regions 5'UTR and 3'UTR are added to the 5' and 3' ends of the sequence encoding the recombinant feline infectious peritonitis antigen as described in claim 1 or 2.

6. The feline infectious peritonitis mRNA as described in claim 5, characterized in that, The 5' end of the 5'UTR is modified with Cap1 type capping; the 3' end of the 3'UTR is modified with polyadenine.

7. The feline infectious peritonitis mRNA as described in claim 6, characterized in that, The mRNA sequence is shown in any of SEQ ID NO. 9-12.

8. The use of feline infectious peritonitis mRNA as described in any one of claims 3-7 in the preparation of feline infectious peritonitis mRNA vaccine.

9. An mRNA vaccine for feline infectious peritonitis, characterized in that, The mRNA vaccine comprises the mRNA as described in any one of claims 3-8, and a vaccine vector for encapsulating the mRNA.

10. The mRNA vaccine as described in claim 9, characterized in that, The vaccine carrier is a lipid nanoparticle that can be utilized by mRNA.

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