Pharmaceutical composition for preventing and treating infectious peritonitis in animals

Abstract

Provided are a pharmaceutical composition for treating and preventing infectious peritonitis in animals. The pharmaceutical composition is mainly composed of M protein, N protein, and S protein which contain a FCoV virus strain, and is mainly used for preventing and treating feline infectious peritonitis. Experiments proved that the pharmaceutical composition can effectively play a role in protecting an animal body and has important significance in preventing FIP.

Description

A pharmaceutical composition for preventing and treating animal infectious peritonitis

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Chinese patent application No. 202310683619.6 filed on June 9, 2023, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a pharmaceutical composition for preventing and treating animal infectious peritonitis. Background Art

[0004] Infectious peritonitis often refers to tuberculous peritonitis and peritonitis caused by ascites due to hepatitis-induced cirrhosis. Both conditions are generally transmitted through the mouth and nose. Feline infectious peritonitis (FIP) is a systemic, fatal infectious disease caused by infection with the feline coronavirus (FCoV). Its main characteristics are peritonitis, the accumulation of large amounts of ascites, and a high mortality rate. Initial symptoms are subtle, primarily manifesting as loss of appetite, poor spirits, weight loss, and persistent fever. Later symptoms become more pronounced and can be divided into two forms: exudative (wet) and non-exudative (dry). The exudative form is characterized by the accumulation of fluid in body cavities (particularly the peritoneal cavity); the non-exudative form is characterized by the development of granulomatous lesions in various organs and associated clinical symptoms (such as ataxia and mild paralysis). FIP is more common in young cats aged 3-9 months, especially in cats raised in groups. Once the disease occurs, the mortality rate is almost 100%. Therefore, it is one of the most common diseases causing death in cats in recent years.

[0005] Feline coronavirus (FCoV) has two biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FIPV is a mutation of FECV and is the strain that causes FIP. FCoV is an enveloped, positive-strand RNA virus belonging to the family Coronaviridae, order Nidovirales, and genus Coronavirus. Its genome is approximately 29 kb and contains two nonstructural genes encoding replicases 1a and 1b, respectively; four structural genes encoding the S protein, envelope protein E, membrane protein M, and nucleocapsid protein N, respectively; and five accessory genes: 3a, 3b, 3c, 7a, and 7b. The full-length S protein consists of two parts, S1 and S2. S1 is primarily responsible for binding to ACE2 and consists of the N-terminal domain (NTD) and receptor binding domain (RBD). S2 is primarily responsible for fusion with the feline cell membrane and release of the genetic material. The M and E proteins are primarily involved in viral assembly and assembly. The N protein, abundant in coronaviruses, is a highly immunogenic protein involved in genome replication and regulation of cellular signaling pathways.

[0006] There is currently no effective treatment for FIP, and only medications can be used to prolong life and improve quality of life. Therefore, preventing the occurrence of FIP is particularly important. Currently, a commercial FCoV intranasal vaccine is available overseas, namely Pfizer's live attenuated vaccine Primucell FIP, which can be used for kittens over 16 weeks of age. However, it mainly targets the type II FCoV mutant DF2-FIPV. However, type I FCoV is more prevalent worldwide, so the protective effect of this vaccine is controversial. However, there is currently no effective vaccine available for type I FIPV.

[0007] mRNA vaccines are nucleic acid preparations that construct disease-specific antigen mRNA sequences. These are encapsulated in novel lipid nanoparticles (LNPs) and delivered to host cells. The host's ribosomes then translate the mRNA sequence into disease-specific antigen proteins, stimulating a specific immune response and ultimately providing immune protection. mRNA vaccines offer numerous advantages over traditional vaccines, including safety, effectiveness, rapid production speed, low cost, and the ability to combat viral mutations.

[0008] In summary, it is of great significance to study and develop mRNA vaccines for the prevention of FIP.

[0009] Summary of the Invention

[0010] definition

[0011] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0012] Definition of general terms

[0013] In the claims and / or description, when used in conjunction with the term "comprising", the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

[0014] As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0015] Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value. The term "etc." indicates that alternative features are reasonably expected within the recited range.

[0016] Although the disclosure supports a definition of the term "or" as only alternatives as well as "and / or," the term "or" in the claims means "and / or" unless explicitly stated as only alternatives or as mutually exclusive between alternatives.

[0017] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0018] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0019] The terms "identity" and "homology" as used in the present invention refer to the use of amino acid sequences or nucleotide sequences. Those skilled in the art can adjust the sequences according to actual work needs so that the used sequences have (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% similarity, and still have the same function as the original amino acid sequence or nucleotide sequence.

[0020] The term "variant" used with respect to a peptide or polypeptide refers to a peptide or polypeptide whose amino acid sequence differs due to insertion, deletion, or conservative substitution of amino acids but retains at least one biological activity. A variant may also refer to a protein having an amino acid sequence substantially identical to a reference protein, wherein the reference protein has an amino acid sequence that retains at least one biological activity. Conservative substitutions of amino acids, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are generally considered in the art to involve minor changes. As understood in the art, these minor changes can be identified, in part, by considering the hydropathic index of the amino acid (Kyte et al., 1982, J. Mol. Biol. 157: 105-132). The hydropathic index of an amino acid is based on considerations of its hydrophobicity and charge. It is known in the art that amino acids with similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids having a hydropathic index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that result in a protein retaining biological function. Considering the hydrophilicity of amino acids in the context of a peptide allows calculation of the maximum local average hydrophilicity of the peptide, a useful measurement that is reported to be closely related to antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety. Substituting amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity, as understood in the art. Amino acids with hydrophilicity values ​​that differ from each other by ±2 can be substituted. Both the hydrophobicity index and the hydrophilicity value of an amino acid are affected by the specific side chain of the amino acid. Consistent with this observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, especially those of their side chains, such as hydrophobicity, hydrophilicity, charge, size and other properties.

[0021] As used herein, the term "variant" with respect to nucleic acids refers to (i) a portion or fragment of a reference nucleotide sequence; (ii) a complementary sequence of a reference nucleotide sequence or a portion thereof; (iii) a nucleic acid substantially identical to a reference nucleic acid or its complementary sequence; (iv) a nucleic acid that hybridizes under stringent conditions to a reference nucleic acid, its complementary sequence, or a sequence substantially identical thereto. A variant can be a nucleic acid sequence that is substantially identical over the entire length of a gene sequence or a fragment thereof. A nucleic acid or protein sequence can be 66%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical over the entire length of a gene, protein sequence, or a fragment thereof.

[0022] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0023] As used herein, the term "fragment" refers to a target protein or polypeptide, as well as a target protein or polypeptide with N-terminal (N-terminus) or C-terminal (C-terminus) truncation, and / or internal deletion.

[0024] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) that serves as a template for the synthesis of other polymers and macromolecules in biological processes, which have a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a determined amino acid sequence and the resulting biological properties. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence, typically provided in a sequence listing) and the non-coding strand (which serves as a template for transcription of the gene or cDNA) can be said to encode the protein or other product of the gene or cDNA.

[0025] mRNA vaccine terminology

[0026] As used herein, the term "immunogen" is intended to refer to a substance capable of inducing an adaptive immune response in an individual, wherein the adaptive immune response is capable of inducing an immune response that significantly targets a pathogen with which the immunogen shares immunological characteristics. An "immunogen" is any substance introduced into the body to produce an immune response. This substance can be a physical molecule, such as a protein, or it can be encoded by a vector, such as DNA, mRNA, or a virus.

[0027] The term "immune response", as used herein, refers to processes involving the activation and / or induction of effector functions, such as, by way of non-limiting example, T cells, B cells, natural killer (NK) cells, and / or antigen presenting cells (APCs). Thus, as will be appreciated by those skilled in the art, an immune response includes, but is not limited to, any detectable antigen-specific activation and / or induction of helper T cell or cytotoxic T cell activity or response, antibody production, antigen presenting cell activity or infiltration, macrophage activity or infiltration, neutrophil activity or infiltration, and the like.

[0028] The term "vaccine" refers to a composition that induces an immune response after inoculation into a subject. In some embodiments, the induced immune response provides protective immunity.

[0029] The term "antigen" or "Ag" is defined as a molecule that stimulates an adaptive immune response. This immune response may involve the production of antibodies, the activation of specific immunogenic cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. Those skilled in the art will appreciate that any DNA or RNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an adaptive immune response encodes an "antigen," as the term is used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be apparent that antigens can be produced synthetically or can be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0030] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0031] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked plasmids or contained in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0032] The mRNA is a monocistronic, bicistronic or polycistronic mRNA. The bicistronic or polycistronic mRNA is an mRNA containing two or more coding regions.

[0033] In one embodiment of the present invention, the coding regions of the bicistronic or polycistronic mRNA can be separated by at least one internal ribosome entry site (IRES) sequence. Useful IRES sequences include, but are not limited to, picornaviruses (e.g., FMDV), pestiviruses (e.g., CFFV), polioviruses (e.g., PV), encephalomyocarditis viruses (e.g., ECMV), foot-and-mouth disease viruses (e.g., FMDV), hepatitis C viruses (e.g., HCV), classical swine fever viruses (e.g., CSFV), mouse corneal leukemia viruses (e.g., MLV), simian immunodeficiency viruses (e.g., SIV), or cricket paralysis viruses (e.g., CrPV). The coding regions can also be separated by a "2A linker," which is a short viral peptide (18-25 amino acids), also known as a "self-cleaving" peptide, that enables a single transcript to produce multiple proteins, including, but not limited to, P2A / T2A / E2A / F2A.

[0034] Preferably, the mRNA is composed of several structural elements, that is, in addition to the above-mentioned coding region, the mRNA may also include a 5' cap structure, a promoter, a 5' non-coding region (5'UTR), a signal peptide, a 3' non-coding region (3'UTR) and / or an mRNA sequence of a polyadenylic acid tail (polyA), but is not limited to the above-mentioned elements. As long as they are necessary for the construction of mRNA, they are within the scope of protection of the present invention.

[0035] The M protein is a membrane protein of FCoV, which can interact with other structural proteins to promote the assembly of virus particles; the N protein is the nucleocapsid protein (N) of FCoV, which can bind to the FCoV genome, package the viral genome RNA into a ribonucleoprotein complex, and increase the efficiency of virus-like particle (VLP) assembly; the S protein is a spike protein (S), which mediates virus attachment and entry into host cells and determines the host range. The S1 region of the S protein is responsible for binding to receptors on host cells, and the S2 region is responsible for the fusion of the virus and the host cell membrane; the E protein is a small envelope protein (E) that is involved in virus assembly and budding.

[0036] Definitions of Therapy-Related Terms

[0037] As used herein, the term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases of mammals, particularly cats, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0038] The terms "subject," "individual," "patient," or "subject" include (optionally added if therapeutic use is present) mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0039] "Prevention" as used in the present invention refers to all actions to avoid symptoms or delay the onset of specific symptoms by administering the product of the present invention before or after the onset of the disease.

[0040] The "effective amount" of the present invention refers to the amount or dosage of the product of the present invention that provides the desired treatment or prevention after administration to a patient or organ in single or multiple doses.

[0041] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0042] The mRNA or composition of the present invention may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a carrier, a diluent, an adjuvant or a nucleotide sequence encoding an adjuvant, a solubilizer, an adhesive, a lubricant, a suspending agent, a transfection facilitator, or the like.

[0043] The transfection facilitating agent includes, but is not limited to, surfactants such as immunostimulating complexes, incomplete Freund's adjuvant, complete Freund's adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, latex adjuvant, liposome adjuvant, microbial adjuvant, LPS analogs (e.g., monophosphoryl ester A), muramyl peptides, benzoquinone analogs, squalene, hyaluronic acid, lipids, calcium ions, viral proteins, cations, polycations (e.g., poly-L-glutamic acid (LGS)) or nanoparticles or other known transfection facilitating agents.

[0044] The nucleotide sequence encoding the adjuvant is a nucleotide sequence encoding at least one of the following adjuvants: GM-CSF, IL-17, IFNγ, IL-15, IL-21, anti-PD1 / 2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, Lymphotoxin-α, hGH, MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac -1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,, CD40, CD40L, vascular growth factor, fibroblast growth factor, nerve growth factor, vascular endothelial growth factor, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

[0045] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0046] Delivery system

[0047] The terms "transformation," "transfection," "transduction," and "introduction" have the meanings generally understood by those skilled in the art, i.e., the process of introducing exogenous nucleic acid (DNA, RNA, etc.) into a host. The methods of transformation, transfection, and transduction include any method for introducing nucleic acid into a cell, including, but not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposomes, and lithium acetate-DMSO.

[0048] The composition of the present invention can be a lipid, a lipid complex, or a lipid nanoparticle. The lipid can be a lipid prepared in the following forms: 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) lipid, 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) lipid. The lipid complex or lipid nanoparticle can be formed from a lipid selected from the group consisting of DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, pegylated lipids, and amino alcohol lipids.

[0049] The "LNP" described in the present invention is lipid nanoparticles.

[0050] The effective amount of the mRNA, mRNA vaccine, protein or polypeptide vaccine, vector vaccine, conjugate or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0051] Dosage form

[0052] The mRNA, mRNA vaccine, protein or polypeptide vaccine, vector vaccine or pharmaceutical composition of the present invention can be incorporated into a drug suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms, such as liquid, semisolid and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders.

[0053] Route of administration

[0054] The term "administer" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, nasal or inhalation administration, but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intramuscular, intravenous or subcutaneous injection, particularly intramuscular administration.

[0055] The inhalation administration mode is selected from at least one of powder inhalation and atomization inhalation.

[0056] According to an embodiment of the present application, the number of administrations is 1 to 20 times, preferably 1 to 10 times, and more preferably 1, 2, 3, 4, 5 or 6 times.

[0057] The first aspect of the present invention provides a DNA element

[0058] A DNA element is obtained by connecting nucleotides of at least one of the three elements of the M protein, N protein, and S protein of the FCoV virus strain;

[0059] Furthermore, the elements may be connected by or without a connecting peptide. When the number of connecting peptides is greater than one, the multiple connecting peptides may be the same or different, but are preferably the same.

[0060] Furthermore, the number of the elements is preferably 1-3 or 2-3, and can be 1 or 2 or 3;

[0061] Furthermore, the structure of the recombinant DNA molecule can be any one of the following combinations: M protein, N protein, and S protein single elements; a double-element nucleotide sequence of M+N, N+M, M+S, S+M, N+S, or S+N connected in sequence; or a triple-element nucleotide sequence of N+M+S, N+S+M, S+N+M, M+N+S, M+S+N, or S+M+N connected in sequence; the "sequence" refers to the order from the 5' end to the 3' end.

[0062] For example only, the preferred sequential connection combinations may include M+N, N+M, N+M+S, N+S+M, S+N+M, M+N+S, M+S+N, and S+M+N; and the more preferred sequential connection combinations may include N+M, N+M+S, and N+S+M.

[0063] Furthermore, the elements of the recombinant DNA molecule can be connected by a connecting peptide; the connecting peptide can be an internal ribosome entry site (IRES) or a self-cleaving 2A peptide (2A), and the 2A connecting peptide includes a P2A connecting peptide, a T2A connecting peptide, an E2A connecting peptide and an F2A connecting peptide; preferably a P2A connecting peptide or a T2A connecting peptide.

[0064] The S protein can be any one of the full-length S, S1 protein, and S2 protein, but the full-length S and S2 proteins are preferred;

[0065] The DNA element is preferably at least one of M+N(T2A), N+M(T2A), M+N(P2A), N+M(P2A), N+M+S(T2A), N+S+M(T2A), N+M+S2(T2A), N+S2+M(T2A), N+M+S(P2A), N+S+M(P2A), N+M+S2(P2A), and N+S2+M(P2A).

[0066] More preferably, the nucleotide and protein sequences may be as shown in Table 1, as well as sequences with 66% or more, 75% or more, 85% or more, or 95% or more identity that can achieve the same function.

[0067] For example, the DNA sequence of the element is shown in SEQ ID NO: 7-23, and the amino acid sequence is shown in SEQ ID NO: 60-76; the DNA sequence of the connecting peptide is shown in SEQ ID NO: 4-5, and the amino acid sequence is shown in SEQ ID NO: 58-59; and sequences that can achieve the same functional identity of more than 66%, more than 75%, more than 85%, or more than 95%.

[0068] The second aspect of the present invention provides a recombinant nucleic acid molecule

[0069] A recombinant nucleic acid molecule, comprising a 5' untranslated region, a Kozak sequence region, at least one antigen encoding region, a 3' untranslated region, and polyA; the sequence is not particularly limited, but is preferably connected sequentially from 5' to 3';

[0070] The antigen coding region is a recombinant DNA molecule composed of the above-mentioned multiple elements; the nucleic acid molecule can be a DNA or RNA molecule, and also includes various specific subtypes.

[0071] The poly A is formed by connecting different numbers of A, including 1-200 A connections, specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 ,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113 3,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160, 161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200 A's are connected, preferably 50-150 A's are connected, more preferably 100 A's are connected.

[0072] Furthermore, a promoter may be further included at the 5' end of the 5' untranslated region; the promoter of the template DNA may be a promoter capable of initiating translation, including but not limited to T7, CMV or SP6 promoter;

[0073] Furthermore, the 3' end of the 5' untranslated region may further include a signal peptide coding region, and the signal peptide may be selected from TPA, IgE, self-signal peptide, etc. However, it is worth noting that the DNA molecule may or may not include a signal peptide. In fact, not including a signal peptide does not affect the translation expression of the molecule;

[0074] The amino acid sequence of the linker is at least one of a 2A connecting peptide (P2A, T2A, E2A and F2A) and an IRES. Preferably, the DNA sequence of the connecting peptide is shown in SEQ ID NOs: 4-5, and the amino acid sequence is shown in SEQ ID NOs: 58-59;

[0075] The nucleotide and protein sequences are shown in Table 1 and Table 2, as well as sequences with 66% or more, 75% or more, 85% or more, or 95% or more identity that can achieve the same function.

[0076] For example, the recombinant DNA molecule, the 5' untranslated region sequence is shown as SEQ ID NO: 1; the Kozak sequence is shown as SEQ ID NO: 2; the 3' untranslated region is shown as SEQ ID NO: 3; the polyA sequence is shown as SEQ ID NO: 6; the recombinant DNA molecule, the DNA sequence is SEQ ID NO: 24-40; and the sequence identity that can achieve the same function is 66% or more, 75% or more, 85% or more, or 95% or more.

[0077] The mRNA molecules transcribed from the aforementioned DNA molecules may preferably be the mRNAs shown in SEQ ID NOs: 41-57.

[0078] The third aspect of the present invention relates to a structure comprising the aforementioned DNA element, recombinant nucleic acid molecule

[0079] A recombinant vector comprising the aforementioned recombinant DNA element or recombinant DNA molecule; the vector may be a vector known in the prior art, including but not limited to pUC57, pTZ19R, PMD18T, PQE30, pUC18, and pUC19;

[0080] An mRNA transcribed from the aforementioned recombinant DNA molecule;

[0081] A protein, a protein translated from the aforementioned recombinant DNA molecule; and various DNA molecules capable of expressing the protein, which means that the scope of protection of the present invention includes all DNA molecules that can be deduced through codon expression rules, not just the DNA sequences shown in the examples.

[0082] A recombinant cell, into which the aforementioned recombinant vector is introduced; the cell includes but is not limited to human or animal cell lines, such as CRFK feline kidney cells, PG-4 (S+L-) feline brain cells, F81 feline kidney cells, FCA-S1 feline skin cells, CATK1 feline kidney cells, MDCK (NBL-2) canine kidney cells, SCBN canine duodenal cells, DH82 canine renal malignant histiocytosis cells, HEK293T, HEK293, BHK, CHO, 3T3, NS0, Hela, HT-1080, PERC6, CAP, HKB-11, Huh-7, etc.;

[0083] The introduction method includes but is not limited to liposome transfection, microvesicle transfection, exosome transfection, electroporation, nanocarrier transfection, and chemical transfection using a transfection reagent; the nanocarrier includes lipids, polymers, or lipid-polymer hybrids.

[0084] A delivery system for delivering the above-mentioned recombinant DNA molecules, recombinant nucleic acid vectors, and mRNA.

[0085] The mRNA may have moderate modifications, including but not limited to pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), 5-methoxy-uridine (5moU), N6-methyl-adenosine (m6A), 5-methyl-cytidine (5m5C), etc.

[0086] The fourth aspect of the present invention provides a pharmaceutical composition

[0087] The composition comprises the aforementioned DNA elements, recombinant DNA molecules, mRNA, protein, recombinant cells, recombinant vectors, and delivery systems.

[0088] Furthermore, the composition may be a pharmaceutical composition, for example, a vaccine composition, a therapeutic pharmaceutical composition, more preferably a DNA vaccine, an mRNA vaccine, a protein vaccine, a therapeutic protein, a therapeutic nucleic acid composition, etc.;

[0089] Ingredients:

[0090] The DNA elements, recombinant nucleic acid molecules (DNA, RNA), mRNA, proteins, etc. provided in the present invention can be separate components or mixed in different proportions; the components used in the mixture can be the same or different;

[0091] For example, an N protein mRNA vaccine and an M protein mRNA vaccine can be mixed; an N protein mRNA vaccine, an M protein mRNA vaccine, and an S2 protein mRNA vaccine can be mixed; or mRNA vaccines expressed by recombinant vectors combining multiple components can be mixed in a certain proportion.

[0092] When there are two components, the mixing ratio can be various ratios such as 1:1, 2:1, 3:1, 1:2, 1:3, 1:4, 4:1, etc., but preferably an equal ratio of 1:1 is used; when there are three components, the mixing ratio can be 1:1:1, 1:1:2, 2:1:1, 1:2:1, etc., but preferably an equal ratio of 1:1:1 is used.

[0093] Excipients:

[0094] In addition to the main ingredients, the pharmaceutical composition may optionally further comprise one or more pharmaceutically acceptable excipients, carriers, adjuvants, and vehicles. Examples of excipients, carriers, adjuvants, and vehicles include, but are not limited to, anti-adherents, adhesives, coatings, compression aids, disintegrants, dyes, lubricants, emulsifiers, fillers (diluents), film formers or coatings, flavorings, fragrances, glidants (flow enhancers), lubricants, adsorbents, suspending or dispersing agents, or sweeteners. Exemplary excipients, carriers, adjuvants, and vehicles include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (monohydrogen), calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, cross-linked povidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0095] The pharmaceutical composition can be prepared in the form of an injectable preparation or an oral preparation. The injectable preparations are classified by physical state into liquid injections, powders for injection, and tablets for injection; and by injection site into intradermal injections, subcutaneous injections, intramuscular injections, intravenous injections, and spinal injections. The solvent for the injectable preparations is preferably water for injection or physiological saline.

[0096] Formulations for oral use include tablets containing the active ingredient mixed with nontoxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starch including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrants (e.g., cellulose derivatives including microcrystalline cellulose, starch including potato starch, croscarmellose sodium, alginates, or alginic acid); binders (e.g., sucrose, glucose, sorbitol, gum arabic, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinyl pyrrolidone, or polyethylene glycol); and lubricants, glidants, and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oil, or talc). Formulations for oral use may also be in the form of chewable tablets, or in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin or olive oil). Powders, granules and pills can be prepared using the ingredients mentioned above under tablets or capsules in a conventional manner using, for example, a mixer, fluidized bed equipment or spray drying equipment.

[0097] Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to, colorants, flavorings, plasticizers, humectants, and buffers. Preparations for oral use may also be in the form of chewable tablets, or in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate, or kaolin), or in the form of soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil). Powders, granules, and pills can be prepared in a conventional manner using, for example, a mixer, fluidized bed apparatus, or spray drying apparatus using the ingredients mentioned above under tablets or capsules.

[0098] Application method

[0099] In some embodiments, administration comprises administering a composition described herein intramuscularly, intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, transperitoneally, subcutaneously, subconjunctivally, intracapsularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally (e.g., as a tablet, capsule, caplet, gel-tablet, or syrup), topically (e.g., as a cream, gel, lotion, or ointment), topically, by inhalation, by injection, or by infusion (e.g., continuous infusion as a cream or lipid composition, local perfusion directly bathing target cells, catheterization, lavage).

[0100] Liposomal delivery

[0101] The composition may further include a cationic or polycationic compound. The cationic or polycationic compound may be free or bound to the mRNA. The form in which the cationic or polycationic compound is bound to the mRNA may be selected to make the composition more stable.

[0102] Preferably, the composition further comprises lipids. Preferably, the lipids include, but are not limited to, lipids that promote self-assembly to form virus-sized particles (~100 nm), lipids that facilitate the release of mRNA from endosomes into cells, lipids that support phospholipid bilayer structures, or lipids that serve as stabilizers.

[0103] More preferably, in order to increase the half-life of LNP, the lipid may further comprise PEGylated lipid.

[0104] In a specific embodiment of the present invention, the lipid comprises a cationic lipid, a PEGylated lipid, cholesterol and / or a phospholipid.

[0105] The liposome structure can be obtained by mixing phospholipid MC3, distearoylphosphatidylcholine (DSPC), cholesterol and lipid-polyethylene glycol (such as DMG-PEG2000) with ethanol in a certain ratio, for example, mixing with ethanol in a molar ratio of 50:10:38.5:1.5.

[0106] The fifth aspect of the present invention provides a method for preparing a vaccine

[0107] A method for preparing an mRNA vaccine comprises the following steps:

[0108] (1) DNA template synthesis

[0109] (2) mRNA transcription

[0110] In vitro transcription of mRNA

[0111] (3) mRNA capping step

[0112] The steps include mRNA dissolution and dilution, denaturation step, and preparation of a capping reaction system.

[0113] In the dissolution and dilution step, the recovered mRNA is appropriately diluted with RNase-free water;

[0114] The denaturation step can be performed by heating followed by cooling, for example, heating at 65°C for 5 minutes and then quickly placing on ice for 5 minutes;

[0115] The capping reaction involves incubating denatured RNA with a capping enzyme.

[0116] (4) mRNA purification step

[0117] The purification methods include but are not limited to oligo(dT)-cellulose column chromatography, oligo(dT)-cellulose liquid centrifugation, and oligo(dT)-magnetic bead method; electrophoresis includes but is not limited to denaturing gel electrophoresis such as denaturing agarose gel electrophoresis, and capillary electrophoresis.

[0118] The sixth aspect of the present invention provides a method for the prevention and treatment of

[0119] The use of the aforementioned DNA elements, recombinant DNA molecules, mRNA, recombinant vectors, recombinant cells, and pharmaceutical compositions in treating and / or preventing animal coronavirus infections, as well as their use in preparing drugs for treating and / or preventing diseases caused by animal coronavirus infections.

[0120] Furthermore, the animal coronavirus infection disease is a disease caused by enteric coronavirus infection or a disease caused by infectious peritonitis virus infection;

[0121] A method for treating and / or preventing animal coronavirus infection, comprising administering the aforementioned DNA element, recombinant nucleic acid molecule, mRNA, recombinant cell, or pharmaceutical composition to a subject in need of treatment or prevention;

[0122] The administration method is single or multiple administrations, and the interval between two administrations can be 18-26 days, such as 18, 19, 20, 21, 22, 23, 24, 25, 26 days, preferably 20, 21, 22, 23, 24 days, and more preferably 22 days.

[0123] The medication administered each time can be the same or different;

[0124] The administration dose is 1-50 μg / kg, preferably 1-30 μg / kg, more preferably 1-20 μg / kg;

[0125] Furthermore, the infected subjects are preferably mammals, including but not limited to humans, cats, dogs, mice, horses, cattle, sheep, pigs, monkeys, etc.; the inventors used the cell lines of the above-mentioned mammals to establish a verification model and conducted in vivo animal challenge verification experiments to prove the universality and effectiveness of prevention and treatment in mammals.

[0126] Furthermore, it can be preferably used to prevent and / or treat feline infectious peritonitis (FIP) caused by feline coronavirus (FcoV);

[0127] According to an embodiment of the present application, the number of administrations is 1 to 20 times, preferably 1 to 10 times, and more preferably 1, 2, 3, 4, 5 or 6 times.

[0128] A seventh aspect of the present invention provides variant verification

[0129] In order to further verify the technical effect, the inventors further verified the M protein, N protein, S full length, S1 protein, S2 protein and the recombinant DNA molecules and protein structures of different element combinations recorded in the present invention on the basis of the sequence disclosure of the present invention, and verified the results of at least 66%, 67%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 ...0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, 90%, On the basis of 9.5% and 99.9% identity, for example, verification of multiple different types and sources of introns, verification of spacers of different lengths, etc., by truncation, mutation, segmentation, etc. to ensure more than 66% homology, by adopting the verification method in the following invention summary and specific embodiments, first synthesize the recombinant nucleic acid molecule, prepare it into mRNA vaccine in vitro, and verify it by the same method in the embodiment at various cell levels and animal levels. The results show that the same technical effect can be achieved, and therefore the protection scope of more than 66% identity can be supported. More preferably, it can be a protection scope of more than 75% identity, more preferably, it can be a protection scope of more than 85% identity, and most preferably, it can be a protection scope of more than 95% identity.

[0130] The variants can be verified by comparing the new sequence with the query sequence to determine the percentage of sequence identity. Standard methods for comparing the similarity and amino acid position of two nucleic acids or polypeptides that are commonly used can be used to perform such comparisons. Using a computer program such as BLAST or FASTA, for example, two polypeptides can be aligned to perform optimal alignment of their respective amino acids (either along the full length of one or both sequences, or along a predetermined portion of one or both sequences). Such programs provide "defective" open penalties and "defective" gap penalties. A scoring matrix such as PAM250 (a standard scoring matrix; see Dayhoff et al., Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978)) or the BLOSUM scoring matrix can be used in conjunction with the computer program. The percentage of identity is then calculated. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] FIG1 is an electrophoretic diagram of 1% formaldehyde denaturing agarose gel electrophoresis to identify the integrity of mRNA;

[0132] FIG2 is a diagram illustrating the expression level of mRNA in cells;

[0133] FIG3 is a diagram illustrating the expression level of mRNA in cells;

[0134] FIG4 is an ELISA assay for detecting binding and neutralizing antibody levels;

[0135] FIG5 is the antibody titer determination experimental results;

[0136] Figure 6A is a graph showing the FCoV detection results; Figure 6B is a graph showing the body temperature detection results; Figure 6C is a graph showing the weight measurement results; Figure 6D is a graph showing the N and S2 protein binding antibody detection; Figure 6E is a graph showing the neutralizing antibody detection; and Figure 6F is a graph showing the grouping information;

[0137] Figure 7A shows the response observation after immunization, and Figures 7B and C show the body temperature and body weight after immunization, respectively;

[0138] Figures 8A and 8B are binding antibody assays; Figure 8C is a neutralizing antibody assay; and Figure 8D is a lymphocyte proliferation assay.

[0139] Figure 9A shows the body temperature after the challenge, and Figure 9B shows the body weight after the challenge;

[0140] FIG10A shows the statistical results of survival rate; FIG10B shows the results of the detection of the viral titer of feline infectious peritonitis virus in the main organs of the animals in each group. DETAILED DESCRIPTION

[0141] Example 1 Synthesis of mRNA sequences

[0142] 1) Obtaining target genes:

[0143] Element gene source: The DNA sequences of the M protein (MW030108.1: 26093-26881), N protein (MW030108.1: 26894-28024) and S protein (MW030109.1: 20437-24795) of the FCoV virus strain rQS-79 were searched in the GenBank database; 5'-UTR sequence (derived from cat DNAH2, cat hemoglobin or cat albumin gene); 3'UTR sequence (derived from cat hemoglobin or cat albumin gene), where the S protein includes S full-length protein, S1 protein, and S2 protein; the specific sequences are shown in Table 1.

[0144] Recombinant gene combinations: The element sequences (as shown in Table 1) were combined. The sequence structure from the 5' to 3' end of each combination was: 5'-UTR (SEQ ID NO: 1) + Kozak (SEQ ID NO: 2) + target DNA sequence (SEQ ID NO: 7-23) + 3'-UTR (SEQ ID NO: 3) + 100 polyA (SEQ ID NO: 6). DNA templates were synthesized by a gene synthesis company and ligated into the pUC57 vector (GenScript, SD1176) to obtain recombinant plasmids. The different target DNA sequences in each combination were connected by a linker. Two different 2A peptide linkers were selected, with the amino acid sequences being T2A peptide: GSGEGRGSLLTCGDVEENPGP (SEQ ID NO. 58) and P2A peptide: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO. 59).

[0145] Table 1

[0146] The recombinant plasmids represented by the 5'-UTR + Kozak + target DNA sequence + 3'-UTR + 100 polyA structure (Table 2, SEQ ID NOs: 24-40) were transformed into competent DH5α cells. Large numbers of cells were obtained through cloning, screening, and amplification. A large amount of recombinant plasmid was obtained using the endotoxin-free plasmid extraction kit (Tian Gen, DP117). The recombinant plasmids were linearized using restriction endonucleases and recovered using a recovery kit to obtain the template for mRNA synthesis.

[0147] Table 2

[0148] 2) mRNA transcription:

[0149] mRNA in vitro transcription kit (NEB, E2050) was used for in vitro synthesis of mRNA. The reaction system is shown in Table 3 below:

[0150] Table 3

[0151] Incubate at 37°C for 2 hours and recover mRNA using an mRNA recovery kit.

[0152] 3) mRNA capping:

[0153] Use the mRNA capping kit (NEB, M2080, M0366) for mRNA capping reaction. Cap1 structure can be added. The reaction system is as follows:

[0154] a) Dilute 10 g of the recovered mRNA to 13.5 L with RNase-free water;

[0155] b) Heat at 65°C for 5 minutes and immediately place on ice for 5 minutes;

[0156] c) Prepare the reaction system according to Table 4:

[0157] Table 4

[0158] d) Incubate at 37°C for 1 hour;

[0159] e) Purification of the capped mRNA.

[0160] The integrity and size of mRNA were identified by 1% formaldehyde denaturing agarose gel electrophoresis. The electrophoresis diagram is shown in FIG1 : Lanes 1-6 in FIG1A are mRNAs of the target mRNA sequences M, N, M+N(T2A), N+M(T2A), M+N(P2A), and N+M(P2A) (i.e., SEQ ID NOs: 41, 42, 46-49) shown in Table 2, and their corresponding target DNA sequences are shown in SEQ ID NOs: 24, 25, 29-32; Lanes 7-15 in FIG1B are mRNAs of the target mRNA sequences S2, N+M+S(T2A), N+S+M(T2A), N+M+S2(T2A), N+S2+M(T2A), N+M+S(P2A), N+S+M(P2A), N+M+S2(P2A), N+S2+M(P2A) (i.e., SEQ ID The mRNAs synthesized in vitro were characterized by the presence of mRNAs having the correct molecular weight and no degradation.

[0161] Example 2 Cell-level expressibility

[0162] To detect the intracellular expression levels of some mRNA combinations listed in Table 2, including M, N, M+N(T2A), N+M(T2A), M+N(P2A), and N+M(P2A), experiments were performed using cat kidney cells (CRFK). Following the sequence combinations listed in Table 2, an HA tag was fused to the C-terminus of the M protein, and an EGFP tag was fused to the C-terminus of the N protein. CRFK cells were seeded in 24-well plates and cultured for 18 hours. One hour before transfection, the medium was replaced with serum-free Opti-MEM medium. M, N, M+N(T2A), N+M(T2A), M+N(P2A), and N+M(P2A) mRNA combinations were transfected using lipofectamine 2000. After 6 hours of culture, the medium was replaced with fresh medium and cultured for an additional 42 hours. The cell supernatant was removed, the cells were digested, and resuspended in PBS containing 1% FBS. Anti-HA antibody conjugated to APC fluorescent protein was added. The cells were incubated at 4°C for 1 hour, washed twice with cold PBS containing 1% FBS, and resuspended in 200 mL of PBS containing 1% FBS. FACS analysis was performed using a flow cytometer (Beckman Coulter, CytoFLEX).

[0163] The results are shown in Figure 2: Figure 2A shows the expression levels of the M protein; Figure 2B shows the expression levels of the N protein. The expression levels of the P2A peptide-linked group were significantly higher than those of the T2A peptide-linked group. Furthermore, among the P2A peptide-linked combinations, the expression levels of both proteins in the N+M tandem combination were significantly higher than those in the M+N tandem combination, and were comparable to the expression levels of the individual M and N proteins.

[0164] From the above results, it can be seen that the N protein is more conducive to the expression of proteins upstream and downstream of the connecting peptide upstream of the 2A peptide. Therefore, we designed the target RNA sequences shown in Table 2 as S2, N+M+S(T2A), N+S+M(T2A), N+M+S2(T2A), N+S2+M(T2A), N+M+S(P2A), N+S+M(P2A), N+M+S2(P2A), N+S2+M(P2A) (i.e., SEQ ID NO: 45, 50-57), wherein the C-terminus of the S2 protein is fused with a MYC tag. The expression level was detected using the above-mentioned flow cytometry experimental method, and the S2 protein was detected using anti-MYC labeled with PE-Cyanine7 fluorescent protein. The detection results are shown in Figure 3.

[0165] Similar to the results in Figure 2, the expression levels of the P2A peptide-linked group were significantly higher than those of the T2A peptide-linked group; and among the P2A peptide-linked combinations, there was no significant difference in the expression levels of M, N, and S proteins between different tandem combinations, and all were comparable to the expression levels of individual proteins.

[0166] Example 3 Preparation process of mRNA vaccine

[0167] Based on the expression levels of mRNA combinations at the cellular level, we selected P2A peptide-linked N+M, N+M+S, N+S+M, N+M+S2, and N+S2+M groups for the preparation of lipid nanoparticles. Separate M, N, and S2 mRNA lipid nanoparticles were also prepared. The preparation process was as follows:

[0168] 1) Loading mRNA: FCoV-M-mRNA, FCoV-N-mRNA, FCoV-S2-mRNA, FCoV-N+M-mRNA, FCoV-N+M+S-mRNA, FCoV-N+S+M-mRNA, FCoV-N+M+S2-mRNA and FCoV-N+S2+M-mRNA were mixed with citric acid buffer at a ratio of 1:1, and incubated at a certain temperature and time to allow sufficient cross-linking to form a suitable complex.

[0169] 2) Preparation of liposomes: Phospholipid MC3, distearoylphosphatidylcholine (DSPC), cholesterol and lipid-polyethylene glycol (DMG-PEG2000) (Aiweituo Pharmaceutical Technology Co., Ltd.) were mixed with ethanol at a molar ratio of 50:10:38.5:1.5, and the raw materials were uniformly dissolved using ultrasonication or other methods.

[0170] 3) Mixing liposomes and mRNA: Mix the buffer solution in step 1 and the liposome solution in step 2, and fully mix the two substances by shearing action such as ultrasound or glass beads.

[0171] 4) Purification: The mixed reagents are purified to remove excess liposomes, junk RNA, unloaded mRNA, or impurities.

[0172] 5) Determination of particle size and concentration: The particle size and concentration of liposome particles were determined using a dynamic light scattering instrument.

[0173] It should be noted that contamination by RNA hydrolases and other degrading enzymes should be avoided during the preparation process to ensure the integrity and stability of mRNA.

[0174] The test results showed that the diameter of the lipid nanoparticles after packaging was between 70-100nM and had good uniformity.

[0175] Example 4 Evaluation of Antibody Titer of Feline Infectious Peritonitis mRNA Vaccine in Mice

[0176] Six- to eight-week-old male BALB / c mice were randomly divided into eight groups (five mice per group): FCoV-M-mRNA + FCoV-N-mRNA, FCoV-M-mRNA + FCoV-N-mRNA + FCoV-S2-mRNA, FCoV-N + M-mRNA, FCoV-N + M + S-mRNA, FCoV-N + S + M-mRNA, FCoV-N + M + S2-mRNA, and FCoV-N + S2 + M-mRNA vaccine groups, and a PBS blank control group. Immunization was performed by intramuscular injection, with a primary immunization on day 1 and a booster immunization on day 28. Each group received a 10g dose. Tail bleeding was performed 14 days after the second immunization, and serum was collected for binding and neutralizing antibody levels using ELISA.

[0177] The results are shown in Figure 4. Compared to the control group, all vaccine groups achieved very high binding antibody levels, and the binding antibody levels of the tandem combination vaccine were comparable to those of the mixed immunizations with different vaccines. (In the figure: N / M represents a 1:1 mixture of FCoV-N-mRNA vaccine and FCoV-N-mRNA vaccine; N / M / S2 represents a 1:1:1 mixture of FCoV-N-mRNA vaccine, FCoV-M-mRNA vaccine, and FCoV-S2-mRNA vaccine; the plus sign indicates that two or three proteins were co-expressed in tandem and packaged into a single vaccine.)

[0178] The neutralizing antibody titer table is shown in Table 5 below: Compared with the control group, the neutralizing antibody titer of each vaccine group was significantly increased; among the tandem combination vaccines, the top three vaccines with the highest neutralizing antibody titer were FCoV-M+N+S2-mRNA vaccine group, FCoV-N+M-mRNA vaccine group and FCoV-N+M+S-mRNA vaccine group, among which the neutralizing antibody titer of the FCoV-M+N+S2-mRNA vaccine group was equivalent to that of the FCoV-M-mRNA vaccine + FCoV-N-mRNA vaccine + FCoV-S2-mRNA vaccine mixed group, and the FCoV-N+M-mRNA vaccine group was equivalent to that of the FCoV-M-mRNA vaccine + FCoV-N-mRNA vaccine mixed group.

[0179] Table 5

[0180] The results of the antibody titer determination experiment are shown in Figure 5: the antibody titers of the FCoV-N+M-mRNA, FCoV-N+M+S-mRNA and FCoV-N+M+S2-mRNA vaccine groups were all higher than 2 6The antibody titer of the FCoV-M+N+S2-mRNA vaccine group was comparable to that of the FCoV-M-mRNA vaccine + FCoV-N-mRNA vaccine + FCoV-S2-mRNA vaccine mixed group, and the antibody titer of the FCoV-N+M-mRNA vaccine group was comparable to that of the FCoV-M-mRNA vaccine + FCoV-N-mRNA vaccine mixed group.

[0181] From the above results, it can be seen that the FCoV-N+M-mRNA, FCoV-N+M+S-mRNA and FCoV-N+M+S2-mRNA vaccines prepared by the present invention can play a good protective effect, and the protective effect is comparable to that of mixed immunization with vaccines expressing different proteins alone.

[0182] Example 5 Evaluation of the effectiveness of feline infectious peritonitis mRNA vaccine in target animals

[0183] In order to test the effectiveness of feline infectious peritonitis mRNA vaccine in animals, the feline infectious peritonitis target animal model was used to test the protective effects of FCoV-N+M-mRNA, FCoV-N+M+S-mRNA and FCoV-N+M+S2-mRNA vaccines against feline infectious peritonitis virus disease.

[0184] The specific experimental process and results are as follows:

[0185] 1) Phase 1 - Pre-immunization

[0186] One-year-old breed cats with strong bodies, no external injuries and good mental state were selected for laboratory examination, and finally 12 test animals that met the experimental standards were screened out.

[0187] The screening results are shown in Figure 6: Figure 6A is FCoV detection, all results are negative; Figure 6B is body temperature detection, all are within the normal range (37.8℃~39.5℃); Figure 6C is weight measurement, all are within the normal range; Figure 6D is N and S protein binding antibody detection, no antibodies were detected; Figure 6E is neutralizing antibody detection, no neutralizing antibodies were detected; Figure F is grouping information.

[0188] 2) Second stage - immunization and post-immunization response observation

[0189] Animals were observed for 7 days before immunization. The first subcutaneous immunization was administered on day 8, and the second subcutaneous immunization was administered on day 29. The vaccine dose for each group was 15 g per animal. Following each immunization, the animal's temperature, weight, food intake, subcutaneous swelling and pain, and other abnormalities were recorded daily.

[0190] The recorded results are shown in Figure 7: Figure 7A shows the reaction observation after immunization, and Figures 7B and C show the body temperature and body weight after immunization, respectively. The FCoV-N+M+S-mRNA group showed a one-time fever symptom after the first and second immunizations, while the other groups were normal.

[0191] 3) Phase III - Post-immunization testing

[0192] Serum was collected before immunization, 14 days after the first immunization, 14 days after the second immunization, and 28 days after the second immunization for detection of binding antibodies and neutralizing antibodies to the N and S proteins. Whole blood was also collected on the 14th day after the first immunization, 14 days after the second immunization, and 28 days after the second immunization for isolation of PBMCs and lymphocyte proliferation assays.

[0193] The experimental results are shown in Figure 8: Figures 8A and 8B are binding antibody detection, binding antibodies were detected in all groups, and there was no significant difference in antibody levels; Figure 8C is neutralizing antibody detection, and the groups with neutralizing antibody levels from high to low are N+M+S2 group, N+M group, and N+M+S group; Figure 8D is a lymphocyte proliferation experiment, and the groups with lymphocyte proliferation promotion levels from high to low are N+M+S2 group, N+M group, and N+M+S group, which is consistent with the neutralizing antibody detection results.

[0194] 4) The fourth stage - attacking the virus

[0195] The animals were observed for 28 days after the second immunization and challenged on the 29th day. 5 TCID50 (50% tissue culture infective dose) was administered orally to the test animals, followed by a 28-day observation period. Daily temperature, weight, water intake, food consumption, and other abnormalities were recorded, and supportive care was provided as needed. If an animal died during the study, a necropsy was performed promptly, and the titer of FIPV in major organs was determined.

[0196] The body temperature and weight tests after the infection are shown in Figures 9A and B: all cats in the N+M+S2 group were normal; cat No. 11 in the N+M group continued to have a fever for several days after the infection, and the other two cats were normal; cats No. 3 and 12 in the N+M+S group continued to have a fever for several days after the infection, and cat No. 3 died 18 days after the infection; all three cats in the PBS group showed fever symptoms after the infection, and cats No. 1 and No. 6 died 20 days after the infection.

[0197] Twenty-eight days after challenge, survival rates were calculated on day 29. All surviving animals were euthanized and necropsied, and viral titers of FIPV in major organs were measured. The survival rate statistics are shown in Figure 10A: The survival rates of the N+M+S2 and N+M groups were both 100%; the survival rate of the N+M+S group was second highest; and the survival rate of the PBS group was the lowest. The viral titers of FIPV in major organs of each group are shown in Figure 10B: The organs tested included the heart, liver, spleen, lung, kidney, mesenteric lymph nodes (MLN), and ascites. The viral titer in the N+M+S group was comparable to that in the control group, followed by the N+M group, and the lowest in the N+M+S2 group.

[0198] The above results indicate that the selected N+M+S2 tandem mRNA vaccine offers excellent protection against feline infectious peritonitis, with no significant adverse reactions in cats following immunization, demonstrating safety and effectiveness. The N+M tandem mRNA vaccine offers less protection, with some cats experiencing transient fevers after immunization. The N+M+S tandem mRNA vaccine offers the least protection. This has important implications for the clinical prevention of feline infectious peritonitis.

[0199] Example 6 Evaluation of the therapeutic efficacy of feline infectious peritonitis mRNA vaccine in target animals infected with feline infectious peritonitis virus

[0200] To further evaluate the therapeutic effect of feline infectious peritonitis mRNA vaccine on animals infected with feline infectious peritonitis virus, we selected 12 one-year-old cats with strong bodies, no external injuries and good mental state. 5 The animals were orally administered TCID50 (TCID50) and 6 hours later, divided into four groups of three animals each. They were vaccinated three times (day 0, day 7, and day 21) with FCoV-N+M-mRNA, FCoV-N+M+S-mRNA, and FCoV-N+M+S2-mRNA vaccines using a P2A linker peptide, respectively, at a dose of 20 g per animal. Clinical observation was performed, and the number of surviving cats was recorded and the survival rate was calculated. The results showed that the survival rate of the N+M+S2 and N+M groups was 100%.

[0201] The safety of the feline infectious peritonitis mRNA vaccine was further verified to obtain the maximum tolerable dose for the target animal cats. Preliminary results showed that the vaccine was safe.

[0202] Sequence Listing (Disclaimer: Sequence 2 is too short and sequences 41-57 are RNA containing U, which makes them unrecognizable by WIPO Sequence. Therefore, they are intentionally skipped in the software, but this does not affect the search. The above sequences are listed below for reference. The original sequences shall prevail as the original public sequence)

Claims

1. A DNA element, comprising the nucleotides of at least one of the three elements of the M protein, N protein and S protein of the FCoV virus strain connected.

2. The DNA element according to claim 1, wherein the DNA elements can be connected by a connecting peptide; preferably, when there is more than one connecting peptide, the multiple connecting peptides can be the same or different, but are preferably the same; preferably, the connecting peptide can be a 2A linker or an IRES sequence; preferably a 2A linker (P2A / T2A / E2A / F2A).

3. The DNA element according to claim 1, wherein the number of elements of the DNA element is preferably 2-3.

4. The DNA element according to claim 1, the structure of the recombinant DNA molecule can be any one of the combinations of the nucleotide sequences of M protein, N protein, S protein, M+N, N+M, M+S, S+M, N+S, S+N, N+M+S, N+S+M, S+N+M, M+N+S, M+S+N, and S+M+N; preferably M+N, N+M, N+M+S, N+S+M, S+N+M, M+N+S, M+S+N, and S+M+N; more preferably N+M, N+M+S, and N+S+M.

5. The DNA element according to any one of claims 1 to 4, wherein the S protein can be any one of the full-length S protein, the S1 protein, and the S2 protein.

6. The DNA element according to claim 4, wherein the DNA sequence of the element is shown in SEQ ID NO: 7-23, and the amino acid sequence is shown in SEQ ID NO: 60-76; the DNA sequence of the connecting peptide is shown in SEQ ID NO: 4-5, and the amino acid sequence is shown in SEQ ID NO: 58-59; and sequences that can achieve the same functional identity of 66%, 75%, 85% or 95% or more.

7. A recombinant nucleic acid molecule, which comprises a 5' untranslated region, a Kozak sequence region, at least one antigen coding region, a 3' untranslated region and polyA, wherein the antigen coding region is the DNA element according to claims 1-6; and the nucleic acid molecule can be a DNA molecule or an RNA molecule.

8. The recombinant nucleic acid molecule according to claim 7, wherein the 5' untranslated region sequence is derived from DNAH2, cat hemoglobin or cat albumin gene; the 3' untranslated region sequence is derived from cat hemoglobin or cat albumin gene; and the polyA element is a sequence formed by connecting 1-200 A's.

9. The recombinant nucleic acid molecule according to any one of claims 7-8, wherein the 5' end of the 5' untranslated region may further include a promoter; and the 3' end of the 5' untranslated region may further include a signal peptide coding region.

10. The recombinant nucleic acid molecule according to claim 9, wherein the 5' untranslated region sequence is shown in SEQ ID NO: 1; the Kozak sequence is shown in SEQ ID NO: 2; the 3' untranslated region is shown in SEQ ID NO: 3; the polyA sequence is shown in SEQ ID NO: 6; the recombinant nucleic acid molecule, the DNA sequence is SEQ ID NO: 24-40; and sequences that can achieve the same functional identity of 66%, 75%, 85% or 95% or more.

11. A recombinant vector comprising the DNA element according to claims 1-6 or the recombinant nucleic acid molecule according to claims 7-10.

12. An mRNA obtained by transcription of the recombinant nucleic acid molecule according to claims 7-10; preferably, it can be shown in SEQ ID NO: 41-57.

13. A protein obtained by translation of the DNA element according to claims 1-6 or the recombinant nucleic acid molecule according to claims 7-10.

14. A nucleotide sequence that expresses the protein of claim 13.

15. A recombinant cell obtained by introducing the DNA element according to claims 1 to 6, the recombinant nucleic acid molecule according to claims 7 to 10, the recombinant vector according to claim 11, or the mRNA according to claim 12 into the cell.

16. A pharmaceutical composition comprising the DNA element of claims 1-6 or the recombinant nucleic acid molecule of claims 7-10 or the recombinant vector of claim 11 or the mRNA of claim 12; the pharmaceutical composition may be a therapeutic composition or a vaccine composition.

17. The pharmaceutical composition according to claim 16, which can be in the form of a single component or a mixture of multiple components, and the multiple components can be mixed in different proportions; the components used in the mixture can be the same or different.

18. The pharmaceutical composition according to claim 16, further comprising pharmaceutically acceptable excipients, auxiliary materials, vehicles, lipids.

19. The pharmaceutical composition according to claim 18, wherein the lipid comprises a cationic lipid, a PEGylated lipid, cholesterol and / or a phospholipid.

20. A method for preparing an mRNA vaccine, comprising the following steps: (1) DNA template synthesis; (2) mRNA transcription; (3) mRNA capping; (4) mRNA purification, wherein the DNA template is the DNA element described in claims 1-6 or the recombinant DNA molecule described in claims 7-10.

21. Use of the DNA element of claims 1-6, the recombinant DNA molecule of claims 7-10, the recombinant vector of claim 11, the mRNA of claim 12, the protein of claims 13-14, the recombinant cell of claim 15, and the pharmaceutical composition of claims 16-19 in treating and / or preventing animal coronavirus infection diseases, or in the preparation of drugs for treating and / or preventing diseases caused by animal coronavirus infection.

22. The use according to claim 21, wherein the animal coronavirus infection disease is a disease caused by enteric coronavirus infection or a disease caused by infectious peritonitis virus infection.

23. A method for treating and / or preventing an animal coronavirus infection disease, the method comprising administering to an administration subject the DNA element of claims 1-6, the recombinant DNA molecule of claims 7-10, the recombinant vector of claim 11, the mRNA of claim 12, the protein of claims 13-14, the recombinant cell of claim 15, or the pharmaceutical composition of claims 16-19.

24. The use and method of claims 21-23, wherein the animal is a mammal, including but not limited to humans, cats, dogs, mice, horses, cows, sheep, pigs, monkeys, etc.

25. The use according to claim 24, further comprising the use for treating and / or preventing feline infectious peritonitis (FIP) caused by feline coronavirus (FcoV).

26. The method according to claim 25, wherein the administration method is single or multiple, and the administration interval is 18-26 days; when the administration method is multiple, the drugs administered twice can be the same or different.

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