[go: up one dir, main page]

WO2025189435A1 - Procédé d'utilisation d'un antigène multi-épitope pour construire un vaccin à arn pour virus pif - Google Patents

Procédé d'utilisation d'un antigène multi-épitope pour construire un vaccin à arn pour virus pif

Info

Publication number
WO2025189435A1
WO2025189435A1 PCT/CN2024/081756 CN2024081756W WO2025189435A1 WO 2025189435 A1 WO2025189435 A1 WO 2025189435A1 CN 2024081756 W CN2024081756 W CN 2024081756W WO 2025189435 A1 WO2025189435 A1 WO 2025189435A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
seq
optionally
protein
acid fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/081756
Other languages
English (en)
Chinese (zh)
Inventor
刘强
李翠翠
廖微曦
黄慧雅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Syngentech Co Ltd
Original Assignee
Beijing Syngentech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Syngentech Co Ltd filed Critical Beijing Syngentech Co Ltd
Priority to PCT/CN2024/081756 priority Critical patent/WO2025189435A1/fr
Publication of WO2025189435A1 publication Critical patent/WO2025189435A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biotechnology, and specifically, to a method for constructing an RNA vaccine against the multi-epitope antigens of feline FIPV. More specifically, it relates to an isolated nucleic acid molecule, an expression vector, a recombinant virus, a liposome, a vaccine, a recombinant cell, and a method and use of constructing a feline infectious peritonitis virus vaccine.
  • Feline coronavirus belongs to the coronavirus family.
  • Viruses in the Coronaviridae family are characterized by relatively large, round, enveloped, positive-strand RNA viruses with genomes ranging from 27 to 32 kb. They encode replication polymerases, four structural proteins (S, M, N, and E), and several nonstructural proteins.
  • S protein spike protein
  • the S protein is a key structural protein of coronaviruses. It forms the surface protrusions of these viruses and is a key factor in their infection.
  • the S protein binds to receptors on host cells, allowing them to enter and infect.
  • the S protein is a key antigen in many coronavirus vaccines.
  • the M protein (membrane protein) is involved in the formation and localization of coronavirus particles. It spans the viral envelope and interacts with other proteins to form the structure of the virion.
  • the N protein (nucleocapsid protein) encapsulates the viral RNA genome and is involved in viral gene replication and transcription. It also stimulates the host immune system to respond to the virus.
  • E protein (Envelope protein) is a protein on the coronavirus envelope that can interact with M protein to form the structure of virus particles. It is also involved in the infection and assembly process of the virus.
  • the N-terminus of the N protein has a conserved amino acid site that binds to nucleic acids, which is relatively conserved among coronaviruses and has multiple T cell or B cell epitopes; the C-terminus of the coronavirus's N protein can form a special secondary structure; and studies have reported that the area where T cell or B cell epitopes are concentrated mainly exists in the NSP12 non-structural protein; the two B cell epitopes HR2_4 and HR2_11 are derived from the SII region of FIPV.
  • Feline coronaviruses are categorized by biotype and pathogenicity as feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV).
  • FECV is highly transmissible, infecting intestinal epithelial cells and causing little to no symptoms or only mild diarrhea.
  • FIPV primarily infects feline monocytes and macrophages. Distinguishing FECV from FIPV based on genomic sequence is difficult. Although some studies have suggested that FIPV can be distinguished from FECV by amino acid mutations in the spike protein, these mutations have subsequently been found to be more correlated with tissue tropism.
  • FIPV FIPV-induced granulomatous lesions in various tissues and organs, including the lungs, liver, spleen, omentum and brain. Infection of macrophages and monocytes is considered to be the key to the pathogenic mechanism. At the end of FIPV infection, a large decrease in T cells in peripheral and lymphoid tissues can be observed, and hypergammaglobulinemia is often present, indicating the presence of severe virus-induced immune disorders. Humoral immunity does not seem to have a protective effect and may lead to "early death syndrome". When S antibodies are present in sub-neutralizing titers, they can enhance the infection of target cells by binding to Fc receptors.
  • the present invention provides an isolated nucleic acid molecule.
  • the isolated nucleic acid molecule comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, a third nucleic acid fragment, a fourth nucleic acid fragment, and a fifth nucleic acid fragment; wherein the first nucleic acid fragment is derived from the N-terminus (NTD) of the N protein of the QS strain of feline infectious peritonitis virus; the second nucleic acid fragment is derived from the C-terminus (CTD) of the N protein of the QS strain of feline infectious peritonitis virus; the third nucleic acid fragment encodes the NSP12 protein of the QS strain of feline infectious peritonitis virus; the fourth nucleic acid fragment is derived from the epitope HR2_4 of the S protein of the 79-1146 strain of feline infectious peritonitis virus; and the fifth nucleic acid fragment is derived
  • the NTD, CTD, NSP12, HR2_4 or HR2_11 protein sequences of the wild-type FIPV virus can also be adaptively modified as needed to improve antigen expression and reduce the toxicity of the FIPV virus without affecting its three-dimensional structure and retaining its immunogenicity, so as to prepare a new type of FIPV virus vaccine.
  • the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO: 1, and the NTD protein nucleic acid fragment has a nucleotide sequence that is at least 71% identical to SEQ ID NO: 16-19;
  • the CTD protein has an amino acid fragment that is at least 92% homologous to SEQ ID NO: 2
  • the CTD protein nucleic acid fragment has a nucleotide sequence that is at least 72% identical to SEQ ID NO: 20-23;
  • the NSP12 protein has an amino acid fragment that is at least 97% homologous to SEQ ID NO: 3
  • the CTD protein nucleic acid fragment has a nucleotide sequence that is at least 72% identical to SEQ ID NO: 20-23;
  • the NSP12 protein has an amino acid fragment that is at least 97% homologous to SEQ ID NO: 3.
  • the NSP12 protein nucleic acid fragment has a nucleotide sequence that is at least 71% identical to SEQ ID NOs: 24-27;
  • the HR2_4 protein has an amino acid segment that is at least 50% homologous to SEQ ID NO: 4, and the HR2_4 protein nucleic acid segment has a nucleotide sequence that is at least 50% identical to SEQ ID NOs: 28-31;
  • the HR2_11 protein has an amino acid segment that is at least 95% homologous to SEQ ID NO: 5, and the HR2_11 protein nucleic acid segment has a nucleotide sequence that is at least 73% identical to SEQ ID NOs: 32-35.
  • FIPV virus vaccines are not particularly limited, as long as they can produce the modified FIPV virus NTD, CTD, NSP12, HR2_4, and/or HR2_11 protein receptor binding region in an organism, are immunogenic, and can stimulate the organism to produce a corresponding immune response.
  • the isolated nucleic acid molecule can be used to stimulate an immune response in all animals that can be infected with feline infectious peritonitis virus, including but not limited to cats.
  • linker is a flexible or rigid amino acid chain that acts as a link between two fusion proteins, such as 3 ⁇ flag, EAAAK, GGGS, AAY, GPGPG, (GGGGS)n, etc.
  • linker sequences in experiments can be applied to the embodiments of this application.
  • the NTD, CTD, NSP12, HR2_4, and HR2_11 are connected via a linker, but the connection method is not particularly limited.
  • the nucleic acid molecules can be connected in the form of NTD-linker-CTD-linker-NSP12-linker-HR2_4-linker-HR2_11, or NTD-linker-NSP12-linker-CTD-linker-HR2_4-linker-HR2_11, etc.
  • the isolated nucleic acid molecule may further include at least one of the following technical features:
  • the nucleic acid fragments are connected or not connected.
  • the nucleic acid fragments are connected by a linker.
  • the first to fifth nucleic acid fragments are connected by a linker as a nucleic acid molecule, which can stimulate an immune response mediated by animal somatic cells.
  • the nucleic acid fragments are not connected.
  • any of the first to fifth nucleic acid fragments can stimulate an animal's somatic cell-mediated immune response. For example, immunizing a test animal with the first nucleic acid fragment alone can stimulate an animal's somatic cell-mediated immune response.
  • the first to fifth nucleic acid fragments can be freely combined to stimulate an immune response mediated by animal somatic cells.
  • the first nucleic acid fragment and the second nucleic acid fragment are connected to immunize the test animal, which can also stimulate an immune response mediated by animal somatic cells.
  • the combination method can be set based on actual experimental needs. In some examples of the present application, any combination method can achieve the effect of stimulating an immune response mediated by animal somatic cells.
  • any one of the nucleic acid fragments comprises at least 15 amino acids.
  • the inventors have found through extensive experimental verification that 15 amino acids from any nucleic acid fragment can stimulate an animal's somatic cell-mediated immune response.
  • the 15 amino acids in any nucleic acid fragment can also be connected or not connected.
  • Experimental verification shows that the effect of stimulating the immune response mediated by animal somatic cells can be achieved under the conditions of connection or not.
  • the NTD protein has an amino acid sequence that is at least 91% homologous to SEQ ID NO:1.
  • the NTD protein has the amino acid sequence shown in SEQ ID NO:1.
  • the CTD protein has an amino acid sequence that is at least 92% homologous to SEQ ID NO:2.
  • the CTD protein has the amino acid sequence shown in SEQ ID NO:2.
  • the NSP12 protein has an amino acid sequence that is at least 97% homologous to SEQ ID NO:3.
  • the NSP12 protein has the amino acid sequence shown in SEQ ID NO:3.
  • the HR2_4 protein has an amino acid sequence that is at least 50% homologous to SEQ ID NO:4.
  • the HR2_4 protein has the amino acid sequence shown in SEQ ID NO:4.
  • the HR2_11 protein has an amino acid sequence that is at least 95% homologous to SEQ ID NO:5.
  • the HR2_11 protein has the amino acid sequence shown in SEQ ID NO:5.
  • the first nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NOs: 16 to 19.
  • the first nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 16 to 19.
  • the second nucleic acid fragment has a nucleotide sequence that is at least 72% identical to any one of SEQ ID NOs: 20 to 23.
  • the second nucleic acid fragment has a nucleotide sequence shown in SEQ ID NOs: 20 to 23.
  • the third nucleic acid fragment has a nucleotide sequence that is at least 71% identical to any one of SEQ ID NO: 24 to 27.
  • the third nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 24 to 27.
  • the fourth nucleic acid fragment has a nucleotide sequence that is at least 50% identical to any one of SEQ ID NO: 28 to 31.
  • the fourth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 28 to 31.
  • the fifth nucleic acid fragment has a nucleotide sequence that is at least 73% identical to any one of SEQ ID NO: 32 to 35.
  • the fifth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 32 to 35.
  • the homology of the amino acid sequence refers to the similarity between two amino acid sequences; the identity of the nucleotide sequence refers to the similarity between two nucleotide sequences.
  • the nucleic acid molecule further includes a sixth nucleic acid segment encoding a signal peptide sequence (MHC-I sp) of MHC-I (major histocompatibility complex I).
  • MHC-I sp signal peptide sequence of MHC-I (major histocompatibility complex I).
  • the purpose of adding the MHC-I signal peptide to the N-terminus of the antigen sequence is to enable ribosomes to attach to the endoplasmic reticulum membrane and guide protein transport within the cell.
  • the signal peptide sequence of MHC-I does not contain a transmembrane region.
  • the signal peptide sequence of MHC-I has an amino acid sequence as shown in SEQ ID NO:6.
  • the sixth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 36 to 38.
  • the sixth nucleic acid fragment is arranged at the 5' end of the nucleic acid molecule.
  • the nucleic acid further comprises a seventh nucleic acid segment encoding a MITD (major histocompatibility complex class I molecule transport signal) sequence.
  • adding the MITD sequence to the C-terminus of the nucleic acid molecule can stimulate CD4 + T cell proliferation and induce the production of more cytokines.
  • the MITD sequence comprises a transmembrane region.
  • the MITD sequence has the amino acid sequence shown in SEQ ID NO:7.
  • the seventh nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 39 ⁇ 41.
  • the seventh nucleic acid fragment is arranged at the 3' end of the nucleic acid molecule.
  • the eighth nucleic acid fragment encodes an HBHA (heparin-binding hemagglutinin protein of Mycobacterium tuberculosis) adjuvant sequence.
  • HBHA heparin-binding hemagglutinin protein of Mycobacterium tuberculosis
  • HBHA has a strong immunostimulatory effect, can induce the maturation of DC cells, further planning CD4 + and CD8 + T cells, secreting IFN- ⁇ , and inducing T cell-mediated cytotoxicity.
  • the HBHA sequence has the amino acid sequence shown in SEQ ID NO:8.
  • the eighth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 42 to 44.
  • the ninth nucleic acid fragment encodes a PADRE (Pan HLA-DR reactive epitope) sequence; according to an embodiment of the present invention, PADRE belongs to a "universal" 13-amino acid pan-HLA DR peptide epitope used to activate CD4+T cells.
  • PADRE Pan HLA-DR reactive epitope
  • the PADRE sequence has the amino acid sequence shown in SEQ ID NO:9.
  • the ninth nucleic acid fragment has a nucleotide sequence shown in SEQ ID NO: 45 to 47.
  • the nucleic acid molecule further comprises a rigid or flexible linker sequence.
  • the connecting sequence has an amino acid sequence shown in SEQ ID NO: 11 to 15.
  • the connecting sequence has a nucleotide sequence shown in SEQ ID NO: 51 to 70.
  • the nucleic acid molecule is linear.
  • the present invention provides an expression vector.
  • the expression vector carries the nucleic acid molecule described in the first aspect of the present invention.
  • the expression vector can be expressed in cells, bacteria, yeast, or feline organisms.
  • the above-mentioned expression vector further includes at least one of the following technical features:
  • the expression vector is a non-pathogenic viral vector.
  • the non-pathogenic virus is selected from at least one of a retrovirus, a lentivirus, an adenovirus and an adeno-associated virus.
  • the present invention provides a recombinant virus.
  • the recombinant virus carries the nucleic acid molecule described in the first aspect of the present invention.
  • the recombinant virus containing the nucleic acid molecule described in the first aspect can be stably propagated in large quantities.
  • the present invention provides a liposome.
  • the liposome comprises a liposome carrier and a nucleic acid fragment, wherein the nucleic acid fragment is as defined in the first aspect of the present invention.
  • the liposome containing the liposome carrier and the nucleic acid fragment plays an important role in improving nucleic acid stability, cellular uptake, reducing toxic side effects, and improving delivery efficiency.
  • the present invention provides a vaccine.
  • the vaccine comprises the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, or the liposome described in the fourth aspect.
  • the aforementioned vaccines can efficiently activate cell-mediated immune responses in animals.
  • the vaccine contains only proteins capable of activating cellular immune responses, thus avoiding toxic side effects and providing enhanced safety.
  • the above vaccine may further include at least one of the following additional technical features:
  • the vaccine includes at least one selected from RNA vaccine, DNA vaccine, protein recombinant vaccine, inactivated vaccine, attenuated vaccine, and viral vector vaccine.
  • the vaccine is an RNA vaccine.
  • the vaccine further comprises an adjuvant.
  • the adjuvant includes at least one of a TLR agonist and Mn 2+ .
  • the TLR agonist includes at least one of HBHA, CpG, R837, MPLA and derivatives thereof.
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect of the present invention, or the recombinant virus described in the third aspect of the present invention.
  • the recombinant cell is used to package a virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine to stimulate an immune response in the body.
  • the present invention provides a method for constructing a feline infectious peritonitis virus vaccine.
  • the method comprises introducing the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect, or the recombinant virus described in the third aspect into a recipient cell.
  • the method according to embodiments of the present invention can package a virus carrying the nucleic acid molecule for use in preparing a nucleic acid vaccine. This method for constructing an infectious peritonitis virus vaccine is safe, simple, and highly effective.
  • the above method further includes at least one of the following technical features:
  • the method prior to introduction into the recipient cells, the method further includes encapsulating the nucleic acid, expression vector, or recombinant virus with an encapsulation vector.
  • encapsulating the nucleic acid, expression vector, or recombinant virus with an encapsulation vector can protect the vaccine components from external environmental interference that affects their potency.
  • the encapsulation vector can also reduce the contact of the vaccine components with the external environment, thereby reducing the risk of contamination of the vaccine components and improving the safety of the vaccine.
  • some encapsulation vectors also have the effect of enhancing the infectivity of the vaccine components, thereby enabling them to more effectively stimulate an immune response.
  • the encapsulation carrier is selected from at least one of liposomes, polymer carriers, viral carriers, and nanoparticles.
  • the carrier is a nanoparticle.
  • the recipient cell is a CRFK cell, HEK293FT, HEK293T or BHK cell.
  • the recipient cells are CRFK cells.
  • the present invention provides a use of the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, or the recombinant cell described in the sixth aspect in the preparation of a drug or vaccine.
  • the drug or vaccine is used to prevent or treat diseases associated with feline infectious peritonitis virus infection.
  • the drug or vaccine prepared based on the aforementioned nucleic acid molecule, expression vector, recombinant virus, or recombinant cell has high safety and can activate an animal cell-mediated immune response in a short period of time.
  • the present invention proposes a method for preventing or treating feline infectious peritonitis virus infection.
  • the method comprises: administering the nucleic acid molecule described in the first aspect of the present invention, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, the vaccine described in the fifth aspect, or the recombinant cell described in the sixth aspect to the test animal.
  • administering an effective dose of a pharmaceutical preparation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine or recombinant cell to a test animal infected with FIPV can significantly improve the various physiological indicators and survival rate of the test animal.
  • the aforementioned treatment methods have good immune effects on various epidemic strains of FIPV.
  • the term “effective dose” refers to an amount that can produce a function or activity on a subject animal and can be accepted by the subject animal.
  • the effective amount of the nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine or recombinant cell of the present invention may vary depending on the mode of administration and the severity of FIPV infection in the test animal.
  • 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).
  • Various factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of FIPV infection in the test animal, the weight of the test animal, the immune status of the test animal, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.
  • the above method may further include at least one of the following technical features:
  • the test animal is selected from cats.
  • the present invention proposes a use of the nucleic acid molecule described in the first aspect, the expression vector described in the second aspect, the recombinant virus described in the third aspect, the liposome described in the fourth aspect, the vaccine described in the fifth aspect or the recombinant cell described in the sixth aspect in preventing or treating feline infectious peritonitis virus infection.
  • administering an effective dose of nucleic acid molecules, expression vectors, recombinant viruses, liposomes, vaccines or recombinant cells to a test animal infected with FIPV can significantly improve the various physiological indicators and survival rates of the test animals.
  • the aforementioned treatment methods have good immune effects on various epidemic strains of FIPV.
  • FIG1 is a result of detecting the expression of target mRNA encapsulated by LNP according to Example 1 of the present invention.
  • FIG2 shows the change in survival rate after immunization with the target mRNA packaged in LNP according to Example 2 of the present invention.
  • FIG3 is a median statistical result of ELISPOT detection stimulated by a multi-epitope antigen polypeptide library after immunization with the target mRNA packaged by LNP according to Example 3 of the present invention.
  • FIG4 is a statistical result of ELISPOT detection of multi-epitope antigen polypeptide stimulation after immunization with LNP-encapsulated target mRNA according to Example 3 of the present invention.
  • FIG5 shows the change in survival rate after immunization with the target mRNA packaged in LNP according to Example 4 of the present invention.
  • first and second are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to.
  • a feature defined as “first” or “second” may explicitly or implicitly include at least one such feature.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • non-pathogenic viral vector refers to a class of viruses that can express target antigens and are non-pathogenic, and are usually used to prepare vaccines. These vectors are genetically modified and the gene sequence of the target antigen is added, which is then expressed and replicated by the viral vector.
  • non-pathogenic viral vectors as vaccine vectors are that they can induce the immune system to produce a strong immune response, thereby stimulating the body's immune response to the target antigen; non-pathogenic viral vectors are usually designed to be unable to replicate, so they will not reproduce in the body and will not cause disease; compared with other traditional vaccine preparation methods, vaccines prepared with non-pathogenic viral vectors are more convenient and safer in production and storage, and have better stability and purity.
  • the RNA vaccine for preventing feline infectious peritonitis described in the present invention is prepared by constructing a vector encoding multiple epitope antigens of the FIPV virus, and then preparing an RNA vaccine for preventing the FIPV virus through lipid nanoparticles (LNP).
  • the vaccine can produce a strong immune response after immunizing cats.
  • the vaccine described in the present invention uses the N-terminus, C-terminus, NSP12 protein sequence of the N protein of the FIPV virus and the HR2-4 and HR2-11 sequences of the S protein as the main components of the constructed nucleic acid molecule.
  • the RNA vaccine expressed by the nucleic acid molecule has the advantages of simple preparation process, high safety, no toxic side effects, and industrial production; sufficient protection effect can be achieved using a very small dose, and it is superior to existing treatment methods in terms of safety and effectiveness.
  • sequences of the products corresponding to the names in Table 5 or Table 6 are formed by connecting the corresponding sequences in "SEQ ID NO:" in the 5' to 3' direction.
  • sequence of MHC-I sp-HBHA-L-PADRE-L-NTD-L-CTD-L-NSP12-L-HR2_4-L-HR2_11-L-MITD is composed of the MHC-I sp sequence shown in SEQ ID NO:38, the HBHA sequence shown in SEQ ID NO:44, the L (linker) sequence shown in SEQ ID NO:53, the PADRE sequence shown in SEQ ID NO:47, the L (linker) sequence shown in SEQ ID NO:56, the NTD sequence shown in SEQ ID NO:19, the L (linker) sequence shown in SEQ ID NO:59, the CTD sequence shown in SEQ ID NO:23, the L (linker) sequence shown in SEQ ID NO:62, the N
  • the 3’ end of the sequence shown in SEQ ID NO:38 is linked to the 5’ end of the sequence shown in SEQ ID NO:44
  • the 3’ end of the gene sequence shown in SEQ ID NO:44 is linked to the 5’ end of the sequence shown in SEQ ID NO:53
  • the 3’ end of the gene sequence shown in SEQ ID NO:53 is linked to the 5’ end of the sequence shown in SEQ ID NO:47
  • the 3’ end of the gene sequence shown in SEQ ID NO:47 is linked to the 5’ end of the sequence shown in SEQ ID NO:56
  • the 3’ end of the sequence shown in SEQ ID NO:56 is linked to the 5’ end of the sequence shown in SEQ ID NO:19
  • the 3’ end of the gene sequence shown in SEQ ID NO:19 is linked to the 5’ end of the sequence shown in SEQ ID NO:59
  • the 3’ end of the gene sequence shown in SEQ ID NO:59 is linked to the 5’ end of the sequence shown in SEQ ID NO:23
  • the 3’ end of the gene sequence shown in SEQ ID NO: 31 is connected to the 5’ end of the sequence shown in SEQ ID NO: 67
  • the 3’ end of the sequence shown in SEQ ID NO: 67 is connected to the 5’ end of the sequence shown in SEQ ID NO: 35
  • the 3’ end of the gene sequence shown in SEQ ID NO: 35 is connected to the 5’ end of the sequence shown in SEQ ID NO: 70
  • the 3’ end of the gene sequence shown in SEQ ID NO: 70 is connected to the 5’ end of the sequence shown in SEQ ID NO: 41.
  • the linker is not particularly limited, and the above-mentioned linker can be selected, or other flexible peptide or rigid peptide amino acid sequences can be selected for connection according to experimental requirements.
  • the mRNA sequence is synthesized in vitro and then encapsulated using lipid nanoparticles (LNP) and expressed in CRFK (cat kidney) cells and cat DC (dendritic cells).
  • LNP lipid nanoparticles
  • CRFK cat kidney cells
  • cat DC dendritic cells
  • RNA sequences were synthesized in vitro and expressed in CRFK (cat kidney) cells, and the sequences with the highest expression levels were selected as the final target sequences for vaccine preparation.
  • the specific steps are as follows:
  • Codon optimization of multi-epitope antigen sequences was performed using the cat universal codon library and the cat mesenteric lymph node codon library, with three optimizations for each antigen;
  • lipid nanoparticles are prepared. The specific steps are as follows:
  • lipid solution The average molecular weight of the liposome system is approximately 620.62. To prepare a 12 mM lipid solution, weigh 42.61 mg of SM-102, 4.52 mg of PEG-DMG, 9.48 mg of DSPC, and 17.86 mg of Chol, dissolve in 10 mL of anhydrous ethanol, and filter through a 0.22 ⁇ m filter membrane.
  • the effectiveness of the vaccine was evaluated by assessing changes in physiological indicators such as body temperature, body weight, and survival rate of the test animals after mRNA vaccine immunization.
  • the LNP preparation method was the same as in Example 1.
  • D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP.
  • SEQ ID NO: 71 cat-derived 5'HBB-UTR
  • SEQ ID NO: 72 3'HBA-UTR
  • the sequence description and composition are shown in Tables 4 and 5.
  • test animals meeting the test criteria are screened through physical examination and laboratory tests.
  • Physical examination items include: body temperature and weight; screening items include: PCR detection, N and S binding antibodies, and neutralizing antibody testing.
  • the specific experimental steps are as follows:
  • the screened test animals were immunized with the target mRNA encapsulated in LNPs.
  • the immunization procedure is as follows:
  • the first vaccination was performed on D0
  • the second vaccination was performed on D21
  • the virus was challenged on D28 after the second vaccination;
  • the challenge virus strain was QS-1146; 5 kittens/group.
  • LNP-encapsulated target mRNA was transfected into CRFK cells, harvested 24 hours later, and immunoblotting was performed to detect protein expression. The results, as shown in Figure 2, demonstrated normal expression. Following challenge with LNP-encapsulated target mRNA in each group, all five kittens in the PBS group developed fever and weight loss, and autopsies revealed typical feline infectious peritonitis. Physiological indicators of the kittens in the group immunized with multi-epitope antigen mRNA significantly improved, with a significant increase in survival rate, as shown in Figure 2. Only one kitten died after 21 days, and the survival rate remained at 80% by day 30.
  • each antigen epitope was evaluated by detecting the stimulation response of the PBMC of the test animals to each epitope after immunization with the multi-epitope antigen mRNA vaccine.
  • the LNP preparation method and the test animal screening method were the same as those in Examples 1 and 2.
  • D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP.
  • SEQ ID NO: 71 cat-derived 5'HBB-UTR
  • SEQ ID NO: 72 3'HBA-UTR
  • the sequence description and composition are shown in Tables 4 and 5.
  • the antigen epitope polypeptide library is synthesized, and the specific implementation steps are as follows:
  • Each epitope peptide sequence was window-cut with a window size of 15 amino acids and a step size of 7 amino acids, and each window peptide and the full-length peptide were synthesized (Sino Biological).
  • PBMC of the test animal is extracted after immunization, stimulated with each epitope, and the response is detected.
  • the specific implementation steps are as follows:
  • PBMCs were extracted from the serum of the test animals and added to activated IFN- ⁇ coated plates. Negative stimulators PBS and positive stimulators PMA were added and incubated with each epitope peptide for 16-24 hours. The cells were counted and statistically analyzed using an ELISPOT detection kit (Dakoway).
  • PBMCs were extracted from the test animals and stimulated with each epitope peptide library.
  • the stimulation response to the entire epitope peptide library was tested, and the results are shown in Figure 3.
  • HR2-4 and HR2-11 are shorter in length and have a correspondingly smaller peptide library size. However, they still produced detectable responses after stimulation with the PBMCs of the immunized test animals.
  • the PBMCs of the test animals immunized with the multi-epitope antigen mRNA vaccine showed significant responses to stimulation with each epitope peptide library compared to the PBS immunization control group.
  • Using the entire peptide library to stimulate the PBMCs of the immunized test animals can obtain a response that is even stronger than when stimulated with each epitope peptide library.
  • PBMCs of the test animals were extracted and stimulated with a single peptide from each antigen epitope peptide library.
  • the stimulation response to each antigen epitope peptide was detected, and the overall distribution of the stimulation response of the peptides in each antigen epitope peptide library was statistically analyzed. The results are shown in Figure 4.
  • the overall distribution of the stimulation response of the peptides in each antigen epitope peptide library showed a lower distribution of the stimulation response of the peptides in the HR2-4 and HR2-11 peptide libraries, while the distribution of the stimulation response of the peptides in the NTD, CTD, and NSP12 peptide libraries was higher. However, they all had stronger responses than the PBS immunized control group. Stimulating the PBMCs of the immunized test animals with the full-length peptides can obtain a significantly stronger response than that of the peptides in each antigen peptide library.
  • Example 4 Validation of the potency of multi-epitope antigen mRNA vaccines against different epidemic strains
  • This example evaluated the effectiveness of a multi-epitope antigen mRNA vaccine against different circulating FIPV strains.
  • the LNP preparation method, test animal screening method, immunization schedule, and evaluation method were the same as in Examples 1 and 2.
  • the challenge strains were HF1902 and SH2211.
  • D1 was constructed between the cat-derived 5'HBB-UTR (SEQ ID NO: 71) and 3'HBA-UTR (SEQ ID NO: 72), transcribed into mRNA in vitro, and packaged with LNP.
  • SEQ ID NO: 71 cat-derived 5'HBB-UTR
  • SEQ ID NO: 72 3'HBA-UTR
  • the sequence description and composition are shown in Tables 4 and 5.
  • the multi-epitope antigen mRNA vaccine can effectively improve the physiological indicators and survival rate of the test animals after challenge with different FIPV epidemic strains.
  • the survival rate is shown in Figure 5.
  • the survival rate of the control group dropped below 50% around 20 days after challenge with the two epidemic strains, while the survival rate of the experimental group remained at 100% until 30 days after challenge.
  • the reference terms “one embodiment”, “some embodiments”, “example”, “specific example”, or “some examples” mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention.
  • the schematic representations of the above terms do not necessarily refer to the same embodiment or example.
  • the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
  • those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Communicable Diseases (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Pulmonology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne un procédé d'utilisation d'un antigène multi-épitope pour construire un vaccin à ARN pour le virus de la péritonite infectieuse féline (PIF). Le procédé concerne une molécule d'acide nucléique isolée. La molécule d'acide nucléique comprend : au moins un d'un premier fragment d'acide nucléique, d'un deuxième fragment d'acide nucléique, d'un troisième fragment d'acide nucléique, d'un quatrième fragment d'acide nucléique et d'un cinquième fragment d'acide nucléique ; le premier fragment d'acide nucléique étant dérivé du domaine N-terminal (NTD) de la protéine N de la souche QS du virus PIF ; le deuxième fragment d'acide nucléique étant dérivé du domaine C-terminal (CTD) de la protéine N de la souche QS du virus PIF ; le troisième fragment d'acide nucléique codant pour la protéine NSP12 de la souche QS du virus PIF ; le quatrième fragment d'acide nucléique étant dérivé de l'épitope HR2_4 de la protéine S de la souche 79-1146 du virus PIF, le cinquième fragment d'acide nucléique étant dérivé de l'épitope HR2_11 de la protéine S de la souche 79-1146 du virus PIF et la molécule d'acide nucléique étant de l'ARN.
PCT/CN2024/081756 2024-03-14 2024-03-14 Procédé d'utilisation d'un antigène multi-épitope pour construire un vaccin à arn pour virus pif Pending WO2025189435A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2024/081756 WO2025189435A1 (fr) 2024-03-14 2024-03-14 Procédé d'utilisation d'un antigène multi-épitope pour construire un vaccin à arn pour virus pif

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2024/081756 WO2025189435A1 (fr) 2024-03-14 2024-03-14 Procédé d'utilisation d'un antigène multi-épitope pour construire un vaccin à arn pour virus pif

Publications (1)

Publication Number Publication Date
WO2025189435A1 true WO2025189435A1 (fr) 2025-09-18

Family

ID=97062581

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/081756 Pending WO2025189435A1 (fr) 2024-03-14 2024-03-14 Procédé d'utilisation d'un antigène multi-épitope pour construire un vaccin à arn pour virus pif

Country Status (1)

Country Link
WO (1) WO2025189435A1 (fr)

Similar Documents

Publication Publication Date Title
CN115551545A (zh) SARS-COV-2 mRNA结构域疫苗
WO2021185310A1 (fr) Vecteur de virus mvsv, vaccin à vecteur viral associé et nouveau vaccin contre la pneumonie à coronavirus basé sur la médiation par mvsv
US9801927B2 (en) Pharmaceutical compositions comprising a polypeptide comprising at least one CXXC motif and heterologous antigens and uses thereof
CN113151184B (zh) 基于细胞膜展示冠状病毒免疫原以诱导中和抗体的方法
WO2020063370A2 (fr) Composition immunitaire, son procédé de préparation et application associée
CN103747797A (zh) 脂质体制剂
AU2024205755B2 (en) Treatment of covid-19 and methods therefor
WO2022110099A1 (fr) Vaccins à coronavirus et leurs utilisations
CN113666990A (zh) 一种诱导广谱抗冠状病毒的t细胞疫苗免疫原及其应用
CN114213548B (zh) 同时诱导抗多种病毒的免疫应答的方法
US20250000968A1 (en) Sars-cov-2 spike fused to a hepatitis b surface antigen
WO2023051701A1 (fr) Arnm, protéine et vaccin contre l'infection par sars-cov-2
EP4205762A1 (fr) Vaccin à adn amélioré pour sars-cov-2
WO2023023940A1 (fr) Immunogène destiné à induire un vaccin à lymphocytes t anti-coronavirus à large spectre et son utilisation
WO2021254270A1 (fr) Procédé pour induire un anticorps neutralisant sur la base d'une membrane cellulaire pour afficher un immunogène de coronavirus
WO2021214703A1 (fr) Vaccin contre sras-cov-2 et sa préparation
WO2024055273A1 (fr) Vaccin contre l'arnm de la rage et sa préparation et son utilisation
WO2025189435A1 (fr) Procédé d'utilisation d'un antigène multi-épitope pour construire un vaccin à arn pour virus pif
WO2025189436A1 (fr) Procédé de construction d'un vaccin polytope à arn circulaire pour le virus de la péritonite infectieuse féline (pif)
WO2024234754A1 (fr) Procédé de construction d'arn circulaire et vaccin contre le fipv
WO2024234755A1 (fr) Développement d'un vaccin à arnm à antigènes multiples contre le vpif félin
WO2024234756A1 (fr) Procédé de construction d'arnm et vaccin ciblant m et n antigènes de fipv féline
CN118389541A (zh) 针对猫fipv的多表位抗原构建rna疫苗的方法
CN118185961A (zh) 针对猫fipv的多表位抗原构建环状rna疫苗的方法
He et al. A novel nanoparticle vaccine, based on S1-CTD, elicits robust protective immune responses against porcine deltacoronavirus

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24928907

Country of ref document: EP

Kind code of ref document: A1