Method for constructing circular RNA and vaccine against feline FIPV

Abstract

The present invention provides a method for constructing a circular RNA and a vaccine against feline FIPV. The method relates to a pharmaceutical formulation, the pharmaceutical formulation comprising a nucleic acid fragment, the nucleic acid fragment being a circular RNA comprising a first nucleic acid fragment and a second nucleic acid fragment, the first nucleic acid fragment encoding the M protein of feline infectious peritonitis virus, the second nucleic acid fragment encoding the N protein of feline infectious peritonitis virus, and the first nucleic acid fragment being ligated to or not ligated to the second nucleic acid fragment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to a method for constructing circular RNA and a vaccine against feline infectious peritonitis virus (FIPV) antigen, and more specifically to pharmaceutical preparations, methods for preparing pharmaceutical preparations, isolated nucleic acid molecules, expression vectors, recombinant viruses, liposomes, vaccines, recombinant cells, methods for constructing feline infectious peritonitis virus vaccines and their uses, or methods for preventing or treating feline infectious peritonitis virus infections.

Background Art

[0002] Feline coronavirus FCoV belongs to the coronavirus family. Coronaviridae viruses are all relatively large, round, and characterized by being forward-chain RNA viruses with an envelope. Their genomes are between 27 and 32 kb and encode replication polymerase, four structural proteins (S protein, M protein, N protein, and E protein), and several non-structural proteins. The S protein (spike protein) is one of the important structural proteins of coronaviruses. It is responsible for forming the surface protrusions of these viruses and is also an important element for the infection of these viruses. The S protein binds to receptors on host cells, thereby allowing the virus to enter the host cell and cause infection. In addition, the S protein is an important antigen in many coronavirus vaccines. The SII region of the S protein has few epitopes that mediate antibody-dependent enhanced immune responses (ADE). The M protein (membrane protein) is involved in the formation and positioning of the coronavirus particle morphology. It is a protein that penetrates the viral envelope and plays a role in forming the structure of the viral particle by interacting with other proteins. The N protein (Nucleocapsid protein) encloses the viral RNA genome and is involved in the replication and transcription of viral genes. At the same time, the N protein can induce the host immune system's response to the virus. The E protein (Envelope protein) is a protein on the envelope of the coronavirus and can interact with the M protein to form the structure of the viral particle, while also being involved in the infection and assembly processes of the virus.

[0003] Feline coronavirus (FCoV) is classified into feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV) based on biotype / pathogenicity. FECV has a very wide transmission range and infects intestinal epithelial cells, but is usually asymptomatic or causes only mild diarrhea. FIPV mainly infects feline monocytes and macrophages. Distinguishing between FECV and FIPV from genome sequences is difficult, and some studies have suggested that FIPV can be distinguished from FECV by spike protein amino mutations, although these mutations were later found to be more strongly associated with tissue preference.

[0004] Typical characteristics of FIPV include the development of pyogenic granulomatous lesions in various tissues and organs, including the lungs, liver, spleen, retina, and brain. Infection of macrophages and monocytes is considered key to the pathogenic mechanism. In the terminal stage of FIPV infection, a significant decrease in T cells in peripheral and lymphatic tissues and frequent hypergammaglobulinemia have been observed, suggesting the presence of severe immunodeficiency induced by the virus. Humoral immunity appears to be ineffective and may lead to "early death syndrome." When S antibodies are present at subneutralizing titers, they can enhance infection of target cells by binding to Fc receptors. Researchers have repeatedly attempted to develop FIPV vaccines based on humoral immunity, but many have failed. The main reason for failure is the existence of antibody-dependent enhancement (ADE) infection, where antibodies cannot exert an effective protective effect. Currently, researchers are attempting to control FIPV infection and elimination through cell-mediated immunity (CMI), but have not yet achieved good protective effects.

[0005] Therefore, research and development of vaccines against FIPV is urgently needed in this field. [Overview of the project] [Problems that the invention aims to solve]

[0006] This application is submitted by the inventor based on the following problems and facts: Current research and development of FIPV vaccines has failed to yield any significant breakthroughs over the long term, and FIPV infections have mostly resulted in cat deaths. [Means for solving the problem]

[0007] Therefore, in a first aspect of the present invention, the present invention provides a pharmaceutical formulation. According to an embodiment of the present invention, the pharmaceutical formulation comprises a nucleic acid fragment, the nucleic acid fragment being a circular RNA comprising a first nucleic acid fragment and a second nucleic acid fragment, the first nucleic acid fragment encoding the M protein of feline infectious peritonitis virus, the second nucleic acid fragment encoding the N protein of feline infectious peritonitis virus, and the first nucleic acid fragment being ligated to or not ligated to the second nucleic acid fragment. According to an embodiment of the present invention, a pharmaceutical formulation expressing either the M protein or the N protein of feline infectious peritonitis virus, or a combination thereof, can stimulate an animal somatic cell-mediated immune response.

[0008] Furthermore, in this invention, adaptive modification of the M or N protein of the wild-type FIPV virus can be performed as needed to reduce the toxicity of the FIPV virus, while simultaneously maintaining its immunogenicity without affecting its three-dimensional structure, thereby enabling the preparation of a novel FIPV virus vaccine. Based on the sequence comparison results (Tables 1 and 2), the M protein has an amino sequence with at least 89% homology to SEQ ID NO: 1, the nucleic acid fragment encoding the M protein has a nucleotide sequence with at least 67% homology to any sequence of SEQ ID NO: 4 to 7, the N protein has an amino sequence with at least 91% homology to SEQ ID NO: 2, and the nucleic acid fragment encoding the N protein has a nucleotide sequence with at least 70% homology to any sequence of SEQ ID NO: 8 to 11. The FIPV virus vaccine is not particularly limited as long as it can generate the receptor-binding region of the modified FIPV virus M or N protein in vivo and possesses immunogenicity that can induce an immune response in the animal body. Furthermore, the pharmaceutical formulation is used to stimulate the immune response of all animals, including but not limited to cats, that can be infected with feline infectious peritonitis virus.

[0009] According to embodiments of the present invention, the pharmaceutical formulation may include at least one of the following additional technical features.

[0010] According to an embodiment of the present invention, the first nucleic acid fragment is linked to the second nucleic acid fragment.

[0011] According to an embodiment of the present invention, the first nucleic acid fragment is not linked to the second nucleic acid fragment.

[0012] According to embodiments of the present invention, the pharmaceutical formulation further comprises a third nucleic acid fragment, the third nucleic acid fragment encoding the S, S_ec (S protein extracellular region), or SII protein of feline infectious peritonitis virus.

[0013] Based on the sequence comparison results (Tables 1 and 2), the S protein has an amino sequence with at least 45% homology to SEQ ID NO: 3, and the nucleic acid fragment encoding the S protein has a nucleotide sequence with at least 51% homology to any sequence from SEQ ID NO: 12 to 15. The S_ec protein has an amino sequence with at least 43% homology to the aminos at positions 1 to 1374 of SEQ ID NO: 3, and the nucleic acid fragment encoding the S_ec protein has a nucleotide sequence with at least 51% homology to any sequence from SEQ ID NO: 12 to 15 at positions 1 to 4122. The SII protein has an amino sequence with at least 62% homology to the aminos at positions 782 to 1433 of SEQ ID NO: 3, and the nucleic acid fragment encoding the SII protein has a nucleotide sequence with at least 57% homology to any sequence from SEQ ID NO: 12 to 15 at positions 2344 to 4299.

[0014] [Table 1]

[0015] [Table 2-1] [Table 2-2]

[0016] According to embodiments of the present invention, the first nucleic acid fragment and the second nucleic acid fragment are linked to or not linked to the third nucleic acid fragment.

[0017] According to an embodiment of the present invention, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:10. Optionally, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1 or 1:2 or 1:3 or 1:4 or 1:5 or 1:6 or 1:7 or 1:8 or 1:9 or 1:10 or 10:1 or 9:1 or 8:1 or 7:1 or 6:1 or 5:1 or 4:1 or 3:1 or 2:1. In some preferred examples of the present application, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1.

[0018] According to an embodiment of the present invention, the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:10. Optionally, the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 1:1 or 1:2 or 1:3 or 1:4 or 1:5 or 1:6 or 1:7 or 1:8 or 1:9 or 1:10 or 10:1 or 9:1 or 8:1 or 7:1 or 6:1 or 5:1 or 4:1 or 3:1 or 2:1.

[0019] According to an embodiment of the present invention, the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment and the third nucleic acid fragment is 1:1:1. In some examples of the present application, when the mass ratio of the three unlinked nucleic acid fragments is 1:1:1, the prepared mRNA has a good immune effect against FIPV as a vaccine and can significantly improve each physiological index and survival rate of cats.

[0020] According to an embodiment of the present invention, the first nucleic acid fragment and the second nucleic acid fragment are not linked to the third nucleic acid fragment.

[0021] According to an embodiment of the present invention, the first nucleic acid fragment and the second nucleic acid fragment are linked to the third nucleic acid fragment.

[0022] According to an embodiment of the present invention, the first nucleic acid fragment is linked to the second nucleic acid fragment, the third nucleic acid fragment is not linked to the first nucleic acid fragment and the second nucleic acid fragment, or the first nucleic acid fragment is linked to the third nucleic acid fragment, the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment, or the second nucleic acid fragment is linked to the third nucleic acid fragment, and the first nucleic acid fragment is not linked to the second nucleic acid fragment and the third nucleic acid fragment.

[0023] According to an embodiment of the present invention, the 3'-end of the first nucleic acid fragment is linked to the 5'-end of the second nucleic acid fragment, the third nucleic acid fragment is not linked to the first nucleic acid fragment and the second nucleic acid fragment, or the 3'-end of the second nucleic acid fragment is linked to the 5'-end of the first nucleic acid fragment, the third nucleic acid fragment is not linked to the first nucleic acid fragment and the second nucleic acid fragment, or the 3'-end of the first nucleic acid fragment is linked to the 5'-end of the third nucleic acid fragment, the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment, or the 3'-end of the third nucleic acid fragment is linked to the 5'-end of the first nucleic acid fragment, the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment, or the 3'-end of the second nucleic acid fragment is linked to the 5'-end of the third nucleic acid fragment, the first nucleic acid fragment is not linked to the second nucleic acid fragment and the third nucleic acid fragment, or the 3'-end of the third nucleic acid fragment is linked to the 5'-end of the second nucleic acid fragment, the first nucleic acid fragment is not linked to the second nucleic acid fragment and the third nucleic acid fragment.

[0024] According to embodiments of the present invention, the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, the 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, or the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, or the 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment. The 3' end of the second nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment, the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, or the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, the 3' end of the first nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, or the 3' end of the third nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, and the 3' end of the second nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment.

[0025] Specifically, the linking scheme of the M, N, S, S_ec, or SII proteins of the feline infectious peritonitis virus (FIPV) can be any combination.

[0026] In some examples of the present invention, the M, N, and SII proteins of feline infectious peritonitis virus (FIPV) are preferably ligated to or unligated.

[0027] In some other examples of this application, the M, N, and SII proteins of feline infectious peritonitis virus (FIPV) are preferably not linked. The isolated single-antigen circular RNA vaccine can exhibit good immunoassay against FIPV, both as a pharmaceutical composition and as a multi-antigen circular RNA vaccine linked via different linking peptides. Furthermore, the isolated single-antigen circular RNA vaccine of this application can exhibit good immunoassay against different FIPV circulating strains, either as a pharmaceutical composition or as a multi-antigen circular RNA vaccine linked via different linking peptides.

[0028] According to an embodiment of the present invention, the M protein has an amino sequence that has at least 89% homology to the amino sequence shown in SEQ ID NO: 1.

[0029] According to an embodiment of the present invention, the M protein has an amino sequence that has at least 89% homology with SEQ ID NO: 1, and the amino at position 90 is Y, at position 102 is V, at position 120 is I, at position 144 is A, and at position 180 is L.

[0030] According to an embodiment of the present invention, the M protein has the amino sequence shown in SEQ ID NO: 1.

[0031] According to an embodiment of the present invention, the N protein has an amino sequence that has at least 91% homology to the amino sequence shown in SEQ ID NO: 2.

[0032] According to an embodiment of the present invention, the N protein has the amino sequence shown in SEQ ID NO: 2.

[0033] According to an embodiment of the present invention, the S protein has an amino sequence that has at least 45% homology to the amino sequence shown in SEQ ID NO: 3.

[0034] According to an embodiment of the present invention, the S protein has an amino acid sequence that has at least 45% homology to SEQ ID NO: 3, and the amino acid at position 515 is V, the amino acid at position 577 is Q, the amino acid at position 1385 is V, the amino acid at position 1386 is V, the amino acid at position 1397 is F, and the amino acid at position 1415 is I.

[0035] According to an embodiment of the present invention, the S protein has the amino sequence shown in SEQ ID NO: 3.

[0036] According to an embodiment of the present invention, the S_ec protein has an amino sequence that has at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO: 3.

[0037] According to an embodiment of the present invention, the S_ec protein has an amino sequence that has at least 43% homology with the aminos at positions 1 to 1374 compared to SEQ ID NO: 3, and the amino at position 515 is V and the amino at position 577 is Q.

[0038] According to an embodiment of the present invention, the S_ec protein has the amino sequence from position 1 to 1374 of SEQ ID NO: 3.

[0039] According to an embodiment of the present invention, the SII protein has an amino sequence that has at least 62% homology to the aminos at positions 782 to 1433 of the amino sequence shown in SEQ ID NO: 3.

[0040] According to an embodiment of the present invention, the SII protein has an amino sequence that has at least 62% homology with the aminos at positions 782 to 1433 compared to SEQ ID NO: 3, and the amino at position 1385 is V, the amino at position 1386 is V, the amino at position 1397 is F, and the amino at position 1415 is I.

[0041] According to an embodiment of the present invention, the SII protein has an amino sequence at positions 782-1433 of SEQ ID NO: 3.

[0042] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence that has at least 67% homology to any of the sequences SEQ ID NO: 4 to 7.

[0043] According to an embodiment of the present invention, the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 4-7.

[0044] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence that has at least 70% homology to any of the sequences with SEQ ID NO: 8 to 11.

[0045] According to an embodiment of the present invention, the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 8~11.

[0046] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that has at least 51% homology between the sequence of SEQ ID NO: 12-15 and the nucleotides at positions 1-4122.

[0047] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence from positions 1 to 4122 of SEQ ID NO: 12 to 15.

[0048] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence having at least 57% homology with the nucleotides at positions 2344 to 4299 of any sequence SEQ ID NO: 12 to 15.

[0049] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence from positions 2344 to 4299 of SEQ ID NO: 12 to 15.

[0050] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that has at least 51% homology to any of the sequences SEQ ID NO: 12 to 15.

[0051] According to an embodiment of the present invention, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 12-15.

[0052] According to embodiments of the present invention, the pharmaceutical formulation further comprises a fourth nucleic acid fragment, the fourth nucleic acid fragment encoding a signal peptide sequence of MHCI (major histocompatibility complex I) or a sequence having a similar function to the signal peptide of MHCI. According to embodiments of the present invention, by adding the MHCI signal peptide to the N-terminus of the antigen sequence, the ribosome attaches to the endoplasmic reticulum membrane and guides the transport of proteins within the cell.

[0053] Furthermore, in this application, sequences having a similar function to the signal peptide of MHCI can similarly attach ribosomes to the endoplasmic reticulum membrane and guide intracellular protein transport.

[0054] According to embodiments of the present invention, the signal peptide sequence of the MHCI does not include a transmembrane region.

[0055] According to an embodiment of the present invention, the signal peptide sequence of the MHCI has the amino sequence shown in SEQ ID NO: 16.

[0056] According to an embodiment of the present invention, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 17-22.

[0057] According to an embodiment of the present invention, the fourth nucleic acid fragment is provided at the 5' end of the nucleic acid fragment or at the 5' end of the nucleic acid molecule.

[0058] The fourth nucleic acid fragment is provided upstream of the nucleic acid fragment or the nucleic acid molecule, and is selectively located at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

[0059] According to embodiments of the present invention, the pharmaceutical formulation further comprises a fifth nucleic acid fragment, the fifth nucleic acid fragment encoding an MITD (MHCI molecular transport signal or major histocompatibility complex I molecular transport signal) sequence or a sequence having a function similar to MITD. According to embodiments of the present invention, by adding the MITD sequence to the C-terminus of the nucleic acid molecule, CD4 + This can stimulate the proliferation of T cells and induce the production of more cellular factors.

[0060] In this application, the sequence having a function similar to the aforementioned MITD is similarly CD4. + It can stimulate the proliferation of T cells, induce the production of cellular factors, and trigger an immune response in animals.

[0061] According to an embodiment of the present invention, the MITD sequence has the amino sequence shown in SEQ ID NO: 23.

[0062] According to an embodiment of the present invention, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 24-32.

[0063] According to embodiments of the present invention, the fifth nucleic acid fragment is provided at the 3' end of the nucleic acid fragment or at the 3' end of the nucleic acid molecule.

[0064] The fifth nucleic acid fragment is provided downstream of the nucleic acid fragment or the nucleic acid molecule, and is selectively provided at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

[0065] According to embodiments of the present invention, the nucleic acid fragment or nucleic acid molecule has the nucleotide sequence shown in Table 4.

[0066] According to embodiments of the present invention, the pharmaceutical formulation further comprises a drug carrier, the drug carrier comprising at least one of liposomes, exosomes, polymer carriers, viral carriers, and nanoparticles.

[0067] The aforementioned drug carrier refers to a carrier that does not cause significant irritation to the test animal and does not remove the biological activity and properties of the pharmaceutical preparation.

[0068] In a second aspect of the present invention, the present invention provides a method for preparing the pharmaceutical formulation described in claim 1. According to an embodiment of the present invention, the method includes the step of mixing a first nucleic acid fragment and a second nucleic acid fragment in a predetermined ratio and processing them to obtain the pharmaceutical formulation. The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, the second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, and both the first and second nucleic acid fragments are circular RNAs.

[0069] According to embodiments of the present invention, this method can be used to prepare pharmaceutical formulations for the prevention or treatment of related diseases caused by feline infectious peritonitis virus.

[0070] According to embodiments of the present invention, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:10. Selectively, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0071] According to an embodiment of the present invention, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1.

[0072] According to embodiments of the present invention, the mixing treatment further comprises a third nucleic acid fragment encoding the S, S_ec, or SII protein of feline infectious peritonitis virus.

[0073] According to embodiments of the present invention, the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:10. Selectively, the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

[0074] According to an embodiment of the present invention, the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment is 1:1:1.

[0075] In a third aspect of the present invention, the present invention provides an isolated nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule comprises a first nucleic acid fragment encoding the M protein of feline infectious peritonitis virus and a second nucleic acid fragment encoding the N protein of feline infectious peritonitis virus, wherein the first nucleic acid fragment is ligated to the second nucleic acid fragment, and the nucleic acid molecule is a circular RNA.

[0076] According to embodiments of the present invention, nucleic acid molecules expressing the M and N proteins of feline infectious peritonitis virus can stimulate immune responses mediated in animal somatic cells.

[0077] Furthermore, in this invention, adaptive modification of the M or N protein of the wild-type FIPV virus can be performed as needed to reduce the toxicity of the FIPV virus, while simultaneously maintaining its immunogenicity without affecting its three-dimensional structure, thereby enabling the preparation of a novel FIPV virus vaccine. Based on the sequence comparison results (Tables 1 and 2), the M protein has an amino sequence with at least 89% homology to SEQ ID NO: 1, the nucleic acid fragment encoding the M protein has a nucleotide sequence with at least 67% homology to any sequence of SEQ ID NO: 4 to 7, the N protein has an amino sequence with at least 91% homology to SEQ ID NO: 2, and the nucleic acid fragment encoding the N protein has a nucleotide sequence with at least 70% homology to any sequence of SEQ ID NO: 8 to 11. The FIPV virus vaccine is not particularly limited as long as it can generate a binding region for the modified FIPV virus M or N protein in vivo and possesses immunogenicity that can induce an immune response in the animal body. Furthermore, the pharmaceutical formulation is used to stimulate the immune response of all animals, including but not limited to cats, that can be infected with feline infectious peritonitis virus.

[0078] According to embodiments of the present invention, the nucleic acid molecule may include at least one of the following additional technical features.

[0079] According to embodiments of the present invention, the nucleic acid molecule further comprises a third nucleic acid fragment, the third nucleic acid fragment encoding at least one of the S, S_ec, and SII proteins of feline infectious peritonitis virus.

[0080] Based on the sequence comparison results (Tables 1 and 2), the S protein has an amino sequence with at least 45% homology to SEQ ID NO: 3, and the nucleic acid fragment encoding the S protein has a nucleotide sequence with at least 51% homology to any sequence from SEQ ID NO: 12 to 15. The S_ec protein has an amino sequence with at least 43% homology to the aminos at positions 1 to 1374 of SEQ ID NO: 3, and the nucleic acid fragment encoding the S_ec protein has a nucleotide sequence with at least 51% homology to the nucleotides at positions 1 to 4122 of any sequence from SEQ ID NO: 12 to 15. The SII protein has an amino sequence with at least 62% homology to the aminos at positions 782 to 1433 of SEQ ID NO: 3, and the nucleic acid fragment encoding the SII protein has a nucleotide sequence with at least 57% homology to the nucleotides at positions 2344 to 4299 of any sequence from SEQ ID NO: 12 to 15. According to an embodiment of the present invention, the first nucleic acid fragment and the second nucleic acid fragment are linked to the third nucleic acid fragment.

[0081] According to embodiments of the present invention, the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, the 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, or the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, or the 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment. The 3' end of the second nucleic acid fragment is linked to the 5' end of the third nucleic acid fragment, the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, or the 3' end of the third nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment, the 3' end of the first nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, or the 3' end of the third nucleic acid fragment is linked to the 5' end of the second nucleic acid fragment, and the 3' end of the second nucleic acid fragment is linked to the 5' end of the first nucleic acid fragment.

[0082] Specifically, the linking scheme of the M, N, S, S_ec, or SII proteins of the feline infectious peritonitis virus (FIPV) can be any combination.

[0083] According to an embodiment of the present invention, the M protein has an amino sequence that has at least 89% homology to the amino sequence shown in SEQ ID NO: 1.

[0084] According to an embodiment of the present invention, the M protein has an amino sequence that has at least 89% homology with SEQ ID NO: 1, and the amino at position 90 is Y, at position 102 is V, at position 120 is I, at position 144 is A, and at position 180 is L.

[0085] According to an embodiment of the present invention, the M protein has the amino sequence shown in SEQ ID NO: 1.

[0086] According to an embodiment of the present invention, the N protein has an amino sequence that has at least 91% homology to the amino sequence shown in SEQ ID NO: 2.

[0087] According to an embodiment of the present invention, the N protein has the amino sequence shown in SEQ ID NO: 2.

[0088] According to an embodiment of the present invention, the S protein has an amino sequence that has at least 45% homology to the amino sequence shown in SEQ ID NO: 3.

[0089] According to an embodiment of the present invention, the S protein has an amino acid sequence that has at least 45% homology to SEQ ID NO: 3, and the amino acid at position 515 is V, the amino acid at position 577 is Q, the amino acid at position 1385 is V, the amino acid at position 1386 is V, the amino acid at position 1397 is F, and the amino acid at position 1415 is I.

[0090] According to an embodiment of the present invention, the S protein has the amino sequence shown in SEQ ID NO: 3.

[0091] According to an embodiment of the present invention, the S_ec protein has an amino sequence that has at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO: 3.

[0092] According to an embodiment of the present invention, the S_ec protein has an amino sequence that has at least 43% homology with the aminos at positions 1 to 1374 compared to SEQ ID NO: 3, and the amino at position 515 is V and the amino at position 577 is Q.

[0093] According to an embodiment of the present invention, the S_ec protein has the amino sequence from position 1 to 1374 of SEQ ID NO: 3.

[0094] According to an embodiment of the present invention, the SII protein has an amino sequence that has at least 62% homology to the aminos at positions 782 to 1433 of the amino sequence shown in SEQ ID NO: 3.

[0095] According to an embodiment of the present invention, the SII protein has an amino sequence that has at least 62% homology with the aminos at positions 782 to 1433 compared to SEQ ID NO: 3, and the amino at position 1385 is V, the amino at position 1386 is V, the amino at position 1397 is F, and the amino at position 1415 is I.

[0096] According to an embodiment of the present invention, the SII protein has an amino sequence at positions 782-1433 of SEQ ID NO: 3.

[0097] According to an embodiment of the present invention, the first nucleic acid fragment has a nucleotide sequence that has at least 67% homology to any of the sequences SEQ ID NO: 4 to 7.

[0098] According to an embodiment of the present invention, the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 4-7.

[0099] According to an embodiment of the present invention, the second nucleic acid fragment has a nucleotide sequence that has at least 70% homology to any of the sequences with SEQ ID NO: 8 to 11.

[0100] According to an embodiment of the present invention, the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 8~11.

[0101] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that has at least 51% homology to the nucleotides at positions 1 to 4122 of any sequence SEQ ID NO: 12 to 15.

[0102] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence from position 1 to 4122 of SEQ ID NO: 12 to 15.

[0103] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence having at least 57% homology to the nucleotides at positions 2344 to 4299 of any sequence SEQ ID NO: 12 to 15.

[0104] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence from positions 2344 to 4299 of SEQ ID NO: 12 to 15.

[0105] According to an embodiment of the present invention, the third nucleic acid fragment has a nucleotide sequence that has at least 51% homology to any of the sequences SEQ ID NO: 12 to 15.

[0106] According to an embodiment of the present invention, the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 12-15.

[0107] According to embodiments of the present invention, the pharmaceutical formulation further comprises a fourth nucleic acid fragment, the fourth nucleic acid fragment encoding a signal peptide sequence of MHCI (major histocompatibility complex I) or a sequence having a similar function to the signal peptide of MHCI. According to embodiments of the present invention, by adding the MHCI signal peptide to the N-terminus of the antigen sequence, ribosomes can be attached to the endoplasmic reticulum membrane, guiding the transport of proteins within the cell.

[0108] Furthermore, in this application, sequences having a similar function to the signal peptide of MHCI similarly attach ribosomes to the endoplasmic reticulum membrane and guide the transport of proteins within the cell.

[0109] According to embodiments of the present invention, the signal peptide sequence of the MHCI does not include a transmembrane region.

[0110] According to an embodiment of the present invention, the signal peptide sequence of the MHCI has the amino sequence shown in SEQ ID NO: 16.

[0111] According to an embodiment of the present invention, the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 17-22.

[0112] According to embodiments of the present invention, the fourth nucleic acid fragment is provided at the 5' end of the nucleic acid fragment or at the 5' end of the nucleic acid molecule.

[0113] The fourth nucleic acid fragment is provided upstream of the nucleic acid fragment or the nucleic acid molecule and is selectively located at the 5' end of the nucleic acid fragment or the 5' end of the nucleic acid molecule.

[0114] According to embodiments of the present invention, the pharmaceutical formulation further comprises a fifth nucleic acid fragment, the fifth nucleic acid fragment encoding an MITD (MHCI molecular transport signal or major histocompatibility complex I molecular transport signal) sequence or a sequence having a function similar to MITD. According to embodiments of the present invention, by adding the MITD sequence to the C-terminus of the nucleic acid molecule, CD4 + This can stimulate the proliferation of T cells and induce the production of more cellular factors.

[0115] In this application, the sequence having a function similar to the aforementioned MITD is similarly CD4. + It can stimulate the proliferation of T cells, induce the production of more cellular factors, and trigger an immune response in the animal body.

[0116] According to an embodiment of the present invention, the MITD sequence has the amino sequence shown in SEQ ID NO: 23.

[0117] According to an embodiment of the present invention, the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 24-32.

[0118] According to embodiments of the present invention, the fifth nucleic acid fragment is provided at the 3' end of the nucleic acid fragment or at the 3' end of the nucleic acid molecule.

[0119] The fifth nucleic acid fragment is provided downstream of the nucleic acid fragment or the nucleic acid molecule, and is preferably provided at the 3' end of the nucleic acid fragment or the 3' end of the nucleic acid molecule.

[0120] According to embodiments of the present invention, the nucleic acid fragment or nucleic acid molecule has the nucleotide sequence shown in Table 4.

[0121] In a fourth aspect of the present invention, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector supports a nucleic acid molecule described in a third aspect of the present invention. According to an embodiment of the present invention, the expression vector can be expressed in vivo in cells, bacteria, yeast, or felines.

[0122] According to embodiments of the present invention, the expression vector may include at least one of the following additional technical features.

[0123] According to embodiments of the present invention, the expression vector is a nonviral carrier.

[0124] In a fifth aspect of the present invention, the present invention provides a recombinant virus. According to an embodiment of the present invention, the recombinant virus carries a nucleic acid molecule described in the third aspect of the present invention. The recombinant virus containing the nucleic acid molecule described in the third aspect can be mass-produced, and the recombinant virus plays an important role in vaccine research and development.

[0125] In a sixth aspect of the present invention, the present invention provides liposomes. According to embodiments of the present invention, the liposome comprises a liposome carrier and a nucleic acid fragment, the nucleic acid fragment being limited by the first and third aspects of the present invention. The liposome comprising the liposome carrier and the nucleic acid fragment plays an important role in improving nucleic acid stability, improving cell uptake rate, reducing toxic side effects, and improving delivery efficiency.

[0126] In a seventh aspect of the present invention, the present invention provides a vaccine. According to an embodiment of the present invention, the vaccine comprises a pharmaceutical formulation according to the first aspect of the present invention, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant virus according to the fifth aspect, or a liposome according to the sixth aspect of the present invention. According to an embodiment of the present invention, by employing the aforementioned vaccine, the cell-mediated immune response of animals can be efficiently activated. Furthermore, the vaccine contains only proteins that activate the immune response of cells, thus avoiding the occurrence of toxic side effects and providing higher safety.

[0127] According to embodiments of the present invention, the vaccine may include at least one of the following additional technical features.

[0128] According to embodiments of the present invention, the vaccine further comprises an adjuvant.

[0129] According to embodiments of the present invention, the adjuvant is a TLR agonist, Mn 2+ It includes at least one of the following.

[0130] According to embodiments of the present invention, the TLR agonist comprises at least one of CpG, R837, MPLA, and its derivatives.

[0131] In an eighth aspect of the present invention, the present invention provides recombinant cells. According to an example of the present invention, the recombinant cells carry a nucleic acid fragment, a nucleic acid molecule as described in the third aspect of the present invention, an expression vector as described in the fourth aspect of the present invention, or a recombinant virus as described in the fifth aspect of the present invention. The nucleic acid fragment comprises a first nucleic acid fragment encoding the M protein of feline infectious peritonitis virus, a second nucleic acid fragment encoding the N protein of feline infectious peritonitis virus, and a third nucleic acid fragment encoding the S, S_ec, or SII protein of feline infectious peritonitis virus, wherein the first and second nucleic acid fragments are ligated to or unligated to the third nucleic acid fragment, and the nucleic acid molecule is circular RNA. According to embodiments of the present invention, the recombinant cells are used to package a virus carrying the nucleic acid molecule, to prepare a nucleic acid vaccine to induce a stronger immune response in an animal body, or to express the M, N, and S, S_ec, or SII proteins of the FIPV virus.

[0132] In a ninth aspect of the present invention, the present invention provides a method for constructing a feline infectious peritonitis virus vaccine. According to an example of the present invention, the method comprises the step of introducing a nucleic acid fragment, a nucleic acid molecule according to a third aspect of the present invention, an expression vector according to a fourth aspect of the present invention, or a recombinant virus according to a fifth aspect of the present invention into a receptor cell. The nucleic acid fragment comprises a first nucleic acid fragment encoding the M protein of feline infectious peritonitis virus, a second nucleic acid fragment encoding the N protein of feline infectious peritonitis virus, and a third nucleic acid fragment encoding the S, S_ec, or SII protein of feline infectious peritonitis virus, wherein the first and second nucleic acid fragments are ligated to or not ligated to the third nucleic acid fragment, and the nucleic acid molecule is circular RNA.

[0133] The method according to the embodiments of the present invention can be used to package a virus carrying the nucleic acid molecule and to prepare a nucleic acid vaccine. The method for constructing an infectious peritonitis virus vaccine is safe, convenient, and efficient.

[0134] According to embodiments of the present invention, the method for constructing the above-mentioned feline infectious peritonitis virus vaccine may include at least one of the following additional technical features.

[0135] According to embodiments of the present invention, the method further comprises the step of coating the nucleic acid, expression vector, or recombinant virus using a coating carrier before introducing receptor cells.

[0136] According to embodiments of the present invention, the coated carrier is selected from at least one of liposomes, exosomes, polymer carriers, viral carriers, and nanoparticles.

[0137] According to embodiments of the present invention, the coating carrier is a nanoparticle. By selectively coating RNA with nanoparticles, the RNA can be protected from degradation and, by binding to the cell membrane, the delivery of RNA into the cell can be promoted.

[0138] According to embodiments of the present invention, the receptor cells are CRFK cells, HEK293FT, HEK293T, BHK cells, or insect cells.

[0139] According to the embodiment of the present invention, the receptor cell is a CRFK cell. According to the embodiment of the present invention, the CRFK cell did not show a rejection reaction in the body of the test animal.

[0140] In a tenth aspect of the present invention, the present invention provides the use of the pharmaceutical formulation described in the first aspect of the present invention, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant virus described in the fifth aspect, and the liposome described in the sixth aspect in the preparation of a pharmaceutical or vaccine. According to an example of the present invention, the pharmaceutical or vaccine is used to prevent or treat a disease associated with feline infectious peritonitis virus infection. According to an example of the present invention, the pharmaceutical 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 time.

[0141] In an eleventh aspect of the present invention, the present invention provides a method for preventing or treating feline infectious peritonitis virus infection. According to an embodiment of the present invention, the method includes administering to a test animal a pharmaceutical formulation according to the first aspect of the present invention, a nucleic acid molecule according to the third aspect, an expression vector according to the fourth aspect, a recombinant virus according to the fifth aspect, a liposome according to the sixth aspect, a vaccine according to the seventh aspect, or a recombinant cell according to the eighth aspect. According to an embodiment of the present invention, administering an effective dose of a pharmaceutical formulation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine, or recombinant cell to a test animal infected with FIPV can significantly improve various physiological indicators and survival rates of the test animal. The above-described treatment method shows good immunological effects against various circulating strains of FIPV.

[0142] In this specification, the term "effective dose" means the amount that can produce function or activity in a test animal and is acceptable to the test animal.

[0143] The effective dose of the pharmaceutical formulation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine, or recombinant cell described in the present invention varies depending on the method of administration, the severity of FIPV infection in the test animal, and other factors. The selection of a preferred effective dose can be determined by those skilled in the art based on various factors (e.g., clinical trials). These factors include, but are not limited to, pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life, the severity of FIPV infection in the test animal, the body weight of the test animal, the immune status of the test animal, and the route of administration. For example, depending on the urgency of the treatment situation, the dose may be administered in multiple divided doses per day, or the dose may be reduced proportionally.

[0144] According to embodiments of the present invention, the above method may include at least one of the following additional technical features.

[0145] According to an embodiment of the present invention, the test animal is selected from cats.

[0146] In a twelfth aspect of the present invention, the present invention proposes the use of the pharmaceutical formulation described in the first aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant virus described in the fifth aspect, the liposome described in the sixth aspect, the vaccine described in the seventh aspect, or the recombinant cell described in the eighth aspect, in the prevention or treatment of feline infectious peritonitis virus infection. According to examples of the present invention, administering an effective dose of the pharmaceutical formulation, nucleic acid molecule, expression vector, recombinant virus, liposome, vaccine, or recombinant cell to a test animal infected with FIPV can significantly improve various physiological indicators and survival rates of the test animal. Furthermore, the above-described treatment method shows good immunological effects against various circulating strains of FIPV.

[0147] Additional aspects and advantages of the present invention are shown in part in the following description, some of which become apparent from the following description or are understood through the practice of the present invention. [Brief explanation of the drawing]

[0148] The above and / or additional aspects and advantages of the present invention will become apparent and readily apparent from the description of the embodiments with reference to the following drawings. [Figure 1] This is the expression detection result of the target circular RNA coated with LNP according to Example 2 of the present invention. [Figure 2] This shows the change in viability after immunization of target circular RNA coated with LNP according to Example 2 of the present invention, followed by a viral attack. [Figure 3] This is the expression detection result of the target circular RNA coated with LNP according to Example 3 of the present invention. [Figure 4] This shows the change in viability after immunization of target circular RNA coated with LNP according to Example 3 of the present invention, followed by a viral attack. [Figure 5] This shows the change in viability after immunization of target circular RNA coated with LNP according to Example 4 of the present invention, followed by a viral attack. [Figure 6] This shows the change in viability after immunization of target circular RNA coated with LNP according to Example 5 of the present invention, followed by a viral attack. [Modes for carrying out the invention]

[0149] Embodiments of the present invention shown in the drawings will be described in detail below. The embodiments described hereafter with reference to the drawings are illustrative and used solely to illustrate the present invention and should not be understood as limiting the present invention.

[0150] Furthermore, the terms “first” and “second” are used solely for descriptive purposes and should not be understood as indicating the number of technical features of relative importance, whether explicitly or implicitly. Therefore, features limited by “first” and “second” may explicitly or implicitly include at least one such feature. In this description, “multiple” means at least two, for example, two, three, etc., unless otherwise specified.

[0151] Beneficial effects The RNA vaccine for preventing feline infectious peritonitis described in the present invention involves constructing a carrier encoding at least one of the M, N, S, S_ec, or SII proteins of the FIPV virus, and then preparing the RNA vaccine for preventing the FIPV virus using lipid nanoparticles (LNPs). The antibodies stimulated and produced by the vaccine inhibit the recognition of FCoV serotype II virus and host cells, avoiding the induction of antibody-dependent enhancement (ADE) in the body. After immunizing cats, the vaccine can produce a relatively strong immune response and neutralizing antibodies with protective efficacy. The vaccine described in the present invention has advantages such as a simple preparation process, high safety, no toxic side effects, and the ability to be industrially produced, with RNA containing the M, N, S, S_ec, or SII amino sequences of the FIPV virus as the main component. Using extremely small doses can achieve sufficient protective effects, and it is superior to existing therapeutic methods in terms of safety and efficacy.

[0152] The arrangement relating to this application is shown in Table 3.

[0153] Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6 Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 3-17 Table 3-18 Table 3-19 Table 3-20 Table 3-21 Table 3-22 Table 3-23 Table 3-24 Table 3-25 Table 3-26 Table 3-27 Table 3-28

[0154] Table 4-1 Table 4-2

[0155] Table 5

[0156] In Tables 4 and 5, the product sequences corresponding to the names are formed by linking the sequences corresponding to "SEQ ID NO:" in the 5' to 3' directions. In Table 4, the sp-Flag-SII6-MITD sequence is formed by linking the signal peptide sequence shown in SEQ ID NO:22, the gene sequence shown in SEQ ID NO:15, and the MITD sequence shown in SEQ ID NO:32. The 3' end of the signal peptide sequence shown in SEQ ID NO:22 is linked to the 5' end of the gene sequence shown in SEQ ID NO:15, and the 3' end of the gene sequence shown in SEQ ID NO:15 is linked to the 5' end of the MITD sequence shown in SEQ ID NO:32.

[0157] The present invention will be described below with reference to specific examples, which are illustrative and do not limit the invention in any way. Where specific techniques or conditions are not shown in the examples, the invention must be carried out in accordance with the techniques or conditions described in the literature in the art or in accordance with the product specifications. Where the manufacturer of the reagents or equipment used is not indicated, they are conventional products available commercially.

[0158] Example 1: Vaccine titer verification

[0159] An embodiment of the present invention evaluates the effectiveness of a vaccine by evaluating changes in physiological indicators such as body temperature and body weight, as well as changes in survival rate, after immunization with a circular RNA vaccine in test animals and subsequent viral attack.

[0160] According to the embodiments of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed downstream of CVB3 IRES (SEQ ID NO: 44), transcribed in vitro, and constructed as circular RNAs, the sequences of which are shown in Table 4. According to the embodiments of the present invention, lipid nanoparticles (LNPs) are prepared. The specific steps are shown below. 1) Preparation of lipid solution: The average molecular weight of the liposome system is approximately 620.62. A 12 mM lipid solution was prepared, and SM-102: 42.61 mg, PEG-DMG: 4.52 mg, DSPC: 9.48 mg, and Chol: 17.86 mg were added. After dissolving in 10 mL of anhydrous ethanol, the solution was filtered through a 0.22 μm filter membrane. 2) The target circular RNA was diluted with citrate buffer (pH 4), mixed uniformly, and then prepared using a rapid nanopharmaceutical preparation system (Meitai) with the flow rate precondition set to 1:3 (organic phase X volume (containing cationic lipids): aqueous phase Y volume (containing nucleic acids) = 1:3). The circular RNA vaccine liposome solution was then placed in 30 times the volume of PBS, concentrated in a 15 ml ultrafiltration tube with a cutoff volume of 100 kDa, and centrifuged at 3000 rpm for 20 mins. Finally, it was diluted by equivolute in a 600 mM sucrose solution (PBS prepared, filtered through a 0.22 μm filter membrane). The sample was stored at -20°C and prepared for use. According to embodiments of the present invention, test animals meeting the test criteria were screened by physical examination and laboratory examination. Physical examination items included body temperature and weight, and screening items included PCR detection, N and S bound antibody testing, and neutralizing antibody testing. The specific experimental steps are as follows. 1) Physical examination: For 7 days prior to immunization, the kittens' body temperature and weight were measured daily. The normal body temperature was approximately 38.5°C, and the weight of a 1-year-old pet cat was approximately 3 kg. 2) Screening of FIPV-negative cats: The FIPV 7ab gene was detected by PCR, and the N and S proteins were used as antigens by ELISA to detect bound antibodies in cat serum. Neutralizing antibodies against FIPV were detected using pseudovirus neutralization experiments.

[0161] According to an embodiment of the present invention, test animals screened with target circular RNA coated with LNPs were immunized. The immunization program was as follows: The D0 virus was first-stage, the D21 virus was second-stage, and after the second-stage virus was second-stage, the D28 virus attack was carried out. The toxic strain used in the virus attack was QS-1146, and it involved 5 kittens per group.

[0162] CRFK cells were transfected with LNP-coated target circular RNAs, collected after 24 hours, and immunoblotting was performed to detect protein expression. The results are shown in Figure 1, and all cells were expressed normally. After immunoviral attack on the LNP-coated target circular RNAs in each group, all five kittens in the PBS group successively developed symptoms of fever and weight loss. Autopsy revealed that these were typical feline transmissible abdominal lesions. The physiological indicators of the kittens were clearly improved in the groups immunized with circular RNAs expressing M, N, and SII antigens and their combinations. All kittens in the M+N+SII group were normal. The survival rates are shown in Figure 2. Circular RNAs expressing M, N, and SII antigens all improved survival rates after viral attack following immunization. Multi-antigen combinations further improved survival rates after viral attack, with the M+N+SII group achieving a 100% survival rate after viral attack.

[0163] The results above indicate that circular RNAs expressing the M, N, and SII antigens, as well as combinations thereof, exhibit good immune effects against FIPV as vaccines, significantly improving various physiological indicators and survival rates, with the M+N+SII combination being the most effective.

[0164] Example 2: Validation of titer of multi-antigen circular RNA vaccines using different ligation methods

[0165] Examples of the present invention evaluate the efficacy of multi-antigen cyclic RNA vaccines with different linkage schemes. The LNP preparation and expression verification methods, test animal screening methods, immunization programs, and evaluation methods are the same as in Example 1.

[0166] According to the embodiments of the present invention, sp-HA-M6-MITD, sp-His-N2, sp-HA-M-MITD-2A-His-N, and sp-HA-M-GS-N-MITD were constructed downstream of CVB3 IRES (SEQ ID NO: 44), and circular RNAs were constructed by in vitro transcription. The sequences are shown in Tables 4 and 5. The expression verification results after LNP coating the target circular RNA are shown in Figure 3, and in all cases, normal expression was achieved. After immunoviral attack, all multi-antigen circular RNA vaccines with different ligation methods effectively improved the physiological indicators and survival rates of the test animals after viral attack. The survival rates are shown in Figure 4, and there were no significant differences between the different ligation methods.

[0167] These results demonstrate that both isolated single-antigen circular RNA vaccines and multi-antigen circular RNA vaccines linked via different linking peptides can exhibit favorable immune effects against FIPV.

[0168] Example 3: Verification of titers of multi-antigen cyclic RNA vaccines with different proportions.

[0169] Examples of the present invention evaluate the efficacy of multi-antigen cyclic RNA vaccines in different proportions. The LNP preparation method, test animal screening method, immunization program, and evaluation method are the same as in Example 1.

[0170] According to the embodiments of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed downstream of CVB3 IRES (SEQ ID NO: 44), and circular RNAs were constructed by in vitro transcription. The sequences are shown in Table 4. After immune viral attack, all multi-antigen cyclic RNA vaccines with different proportions effectively improved the physiological indicators and survival rates of the test animals after viral attack, as shown in Figure 5.

[0171] These results demonstrate that multi-antigen cyclic RNA vaccines with different proportions can all exhibit good immune efficacy against FIPV.

[0172] Example 4: Verification of multi-antigen cyclic RNA vaccine titers for different circulating strains.

[0173] Examples of the present invention evaluated the efficacy of a multi-antigen circular RNA vaccine against different FIPV epidemic strains. The LNP preparation method, test animal screening method, immunization program, and evaluation method were the same as in Example 1. The virulent strains used for viral attack were HF1902 and SH2211.

[0174] According to the embodiments of the present invention, sp-HA-M6-MITD, sp-His-N2, and sp-SII2-MITD were constructed downstream of CVB3 IRES (SEQ ID NO: 44), and circular RNAs were constructed by in vitro transcription. The sequences are shown in Table 4. After immune viral attack, the multi-antigen circular RNA vaccine effectively improved physiological indicators and survival rates in test animals against different FIPV epidemic strains, as shown in Figure 6.

[0175] These results demonstrate that multi-antigen cyclic RNA vaccines can exhibit favorable immune responses against a wide range of circulating FIPV strains.

[0176] In this specification, any description referring to terms such as “one example,” “several examples,” “example,” “specific example,” or “several examples” means that a particular feature, structure, material, or property described with reference to that example is included in at least one example of this disclosure. In this specification, the general expressions of the above terms do not necessarily apply to the same example. In addition, any particular feature, structure, material, or property described may be incorporated in an appropriate manner in any one or more examples. Furthermore, those skilled in the art can combine and combine the various examples and features relating to the various examples described herein without contradiction.

[0177] Although embodiments of the present invention have been presented and described, these embodiments are illustrative and should not be understood as limiting the disclosure. Those skilled in the art will understand that various changes, modifications, substitutions, and variations are possible with respect to the embodiments within the scope of the disclosure.

Claims

1. It is a pharmaceutical preparation, It is a circular RNA and contains a nucleic acid fragment that includes a first nucleic acid fragment and a second nucleic acid fragment. The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus. The second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus. A pharmaceutical formulation characterized in that the first nucleic acid fragment is linked to or not linked to the second nucleic acid fragment.

2. The pharmaceutical formulation according to claim 1, wherein the nucleic acid fragment further comprises a third nucleic acid fragment, the third nucleic acid fragment encoding the S, S_ec, or SII protein of feline infectious peritonitis virus.

3. The pharmaceutical formulation according to claim 2, characterized in that the first nucleic acid fragment and the second nucleic acid fragment are linked to or not linked to the third nucleic acid fragment.

4. The pharmaceutical formulation according to claim 3, characterized in that the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:10, preferably 1:

1.

5. The pharmaceutical formulation according to claim 3, characterized in that the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:

10.

6. The pharmaceutical formulation according to claims 3 to 5, characterized in that the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment is 1:1:

1.

7. The pharmaceutical formulation according to claim 3, characterized in that the first nucleic acid fragment and the second nucleic acid fragment are linked to the third nucleic acid fragment.

8. A method for preparing a pharmaceutical preparation according to any one of claims 1 to 7, The process includes the step of mixing a first nucleic acid fragment and a second nucleic acid fragment in a predetermined ratio and processing them to obtain the pharmaceutical preparation, The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus. The second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus. A method characterized in that the first nucleic acid fragment and the second nucleic acid fragment are circular RNA.

9. The method according to the 8th method, characterized in that the mass ratio of the first nucleic acid fragment to the second nucleic acid fragment is 10:1 to 1:

10.

10. The method according to 9, characterized in that the mass ratio is 1:

1.

11. The method according to 9, wherein the mixing treatment further comprises a third nucleic acid fragment, the third nucleic acid fragment encoding the S, S_ec, or SII protein of feline infectious peritonitis virus.

12. The method according to 11, characterized in that the mass ratio of the second nucleic acid fragment to the third nucleic acid fragment is 10:1 to 1:

10.

13. The method according to 12, characterized in that the mass ratio of the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment is 1:1:

1.

14. Isolated nucleic acid molecules, The first nucleic acid fragment encoding the M protein of feline infectious peritonitis virus, It comprises a second nucleic acid fragment encoding the N protein of feline infectious peritonitis virus, The first nucleic acid fragment is linked to the second nucleic acid fragment, The isolated nucleic acid molecule is characterized by being a circular RNA.

15. The nucleic acid molecule according to claim 14, further comprising a third nucleic acid fragment, the third nucleic acid fragment encoding the S, S_ec, or SII protein of feline infectious peritonitis virus, and the first nucleic acid fragment and the second nucleic acid fragment being linked to the third nucleic acid fragment.

16. The first nucleic acid fragment is linked to the second nucleic acid fragment, and the third nucleic acid fragment is not linked to the first and second nucleic acid fragments, or The first nucleic acid fragment is linked to the third nucleic acid fragment, and the second nucleic acid fragment is not linked to the first nucleic acid fragment and the third nucleic acid fragment, or The second nucleic acid fragment is linked to the third nucleic acid fragment, and the first nucleic acid fragment is not linked to the second and third nucleic acid fragments, or The 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the third nucleic acid fragment is not connected to the first nucleic acid fragment and the second nucleic acid fragment, or The 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the third nucleic acid fragment is not connected to the first nucleic acid fragment and the second nucleic acid fragment, or The 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the second nucleic acid fragment is not connected to the first nucleic acid fragment and the third nucleic acid fragment, or The 3' end of the third nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the second nucleic acid fragment is not connected to the first nucleic acid fragment and the third nucleic acid fragment, or The 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the first nucleic acid fragment is not connected to the second nucleic acid fragment and the third nucleic acid fragment, or The 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the first nucleic acid fragment is not connected to the second nucleic acid fragment and the third nucleic acid fragment, or The 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, or The 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, or The 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the 3' end of the first nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, or The 3' end of the second nucleic acid fragment is connected to the 5' end of the third nucleic acid fragment, and the 3' end of the third nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, or The 3' end of the third nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment, and the 3' end of the first nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, or The pharmaceutical formulation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that the 3' end of the third nucleic acid fragment is connected to the 5' end of the second nucleic acid fragment, and the 3' end of the second nucleic acid fragment is connected to the 5' end of the first nucleic acid fragment.

17. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that the M protein has an amino sequence having at least 89% homology with the amino sequence shown in SEQ ID NO:

1.

18. The pharmaceutical preparation or nucleic acid molecule according to claim 17, characterized in that the M protein has an amino sequence having at least 89% homology with SEQ ID NO: 1, and the amino at position 90 is Y, at position 102 is V, at position 120 is I, at position 144 is A, and at position 180 is L.

19. The pharmaceutical preparation or nucleic acid molecule according to claim 18, characterized in that the M protein has the amino sequence shown in SEQ ID NO:

1.

20. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that the N protein has an amino sequence having at least 91% homology with the amino sequence shown in SEQ ID NO:

2.

21. The pharmaceutical preparation or nucleic acid molecule according to claim 20, characterized in that the N protein has the amino sequence shown in SEQ ID NO:

2.

22. The pharmaceutical formulation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that the S protein has an amino sequence having at least 45% homology with the amino sequence shown in SEQ ID NO:

3.

23. The pharmaceutical preparation or nucleic acid molecule according to claim 22, characterized in that the S protein has an amino sequence having at least 45% homology with SEQ ID NO: 3, and the amino at position 515 is V, the amino at position 577 is Q, the amino at position 1385 is V, the amino at position 1386 is V, the amino at position 1397 is F, and the amino at position 1415 is I.

24. The pharmaceutical preparation or nucleic acid molecule according to claim 23, characterized in that the S protein has the amino sequence shown in SEQ ID NO:

3.

25. The pharmaceutical formulation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that the S_ec protein has an amino sequence having at least 43% homology to the aminos at positions 1 to 1374 of the amino sequence shown in SEQ ID NO:

3.

26. The pharmaceutical preparation or nucleic acid molecule according to claim 25, characterized in that the S_ec protein has an amino sequence having at least 43% homology with the aminos at positions 1 to 1374 compared to SEQ ID NO: 3, and the amino at position 515 is V and the amino at position 577 is Q.

27. The pharmaceutical preparation or nucleic acid molecule according to claim 26, characterized in that the S_ec protein has an amino sequence at positions 1 to 1374 of SEQ ID NO:

3.

28. The pharmaceutical formulation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that the SII protein has an amino sequence having at least 59% homology to the aminos at positions 661 to 1433 of the amino sequence shown in SEQ ID NO:

3.

29. The pharmaceutical preparation or nucleic acid molecule according to claim 28, characterized in that the SII protein has an amino sequence having at least 59% homology with the aminos at positions 661 to 1433 compared to SEQ ID NO: 3, and the amino at position 1385 is V, the amino at position 1386 is V, the amino at position 1397 is F, and the amino at position 1415 is I.

30. The pharmaceutical preparation or nucleic acid molecule according to claim 29, characterized in that the SII protein has an amino sequence at positions 661 to 1433 of SEQ ID NO:

3.

31. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that the first nucleic acid fragment has a nucleotide sequence having at least 67% homology with any of the sequences of SEQ ID NO: 4 to 7.

32. The pharmaceutical preparation or nucleic acid molecule according to claim 31, characterized in that the first nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 4 to 7.

33. The pharmaceutical formulation according to claim 1 or the nucleic acid molecule according to claim 14, characterized in that the second nucleic acid fragment has a nucleotide sequence having at least 70% homology with any of the sequences of SEQ ID NO: 8 to 11.

34. The pharmaceutical preparation or nucleic acid molecule according to claim 33, characterized in that the second nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 8 to 11.

35. The pharmaceutical formulation according to claim 2 or the nucleic acid molecule according to claim 15, characterized in that the third nucleic acid fragment has a nucleotide sequence having at least 51% homology with the nucleotides at positions 1 to 4122 of any sequence SEQ ID NO: 12 to 15.

36. The pharmaceutical preparation or nucleic acid molecule according to claim 35, characterized in that the third nucleic acid fragment has a nucleotide sequence from position 1 to 4122 of SEQ ID NO: 12 to 15.

37. The pharmaceutical preparation or nucleic acid molecule according to claim 36, characterized in that the third nucleic acid fragment has a nucleotide sequence having at least 56% homology with the nucleotides at positions 1981 to 4299 of any of the sequences of SEQ ID NO: 12 to 15.

38. The pharmaceutical preparation or nucleic acid molecule according to claim 37, characterized in that the third nucleic acid fragment optionally has a nucleotide sequence from positions 1981 to 4299 of SEQ ID NO: 12 to 15.

39. The pharmaceutical formulation or nucleic acid molecule according to claim 38, characterized in that the third nucleic acid fragment has a nucleotide sequence having at least 51% homology with any of the sequences of SEQ ID NO: 12 to 15.

40. The pharmaceutical preparation or nucleic acid molecule according to claim 39, characterized in that the third nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 12 to 15.

41. A pharmaceutical formulation according to claim 1 or 2, or a nucleic acid molecule according to claim 14 or 15, further comprising a fourth nucleic acid fragment, wherein the fourth nucleic acid fragment encodes an MHCI signal peptide sequence or a sequence having a similar function to an MHCI signal peptide.

42. The pharmaceutical formulation or nucleic acid molecule according to claim 41, characterized in that the signal peptide sequence of the MHCI does not contain a transmembrane region.

43. The pharmaceutical preparation or nucleic acid molecule according to claim 41, characterized in that the signal peptide sequence of the MHCI has the amino sequence shown in SEQ ID NO:

16.

44. The pharmaceutical formulation or nucleic acid molecule according to claim 41, characterized in that the fourth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 17 to 22.

45. The pharmaceutical formulation or nucleic acid molecule according to claim 41, characterized in that the fourth nucleic acid fragment is provided at the 5' end of the nucleic acid fragment or at the 5' end of the nucleic acid molecule.

46. A pharmaceutical formulation according to claim 1 or 2, or a nucleic acid molecule according to claim 14 or 15, further comprising a fifth nucleic acid fragment, wherein the fifth nucleic acid fragment encodes an MITD sequence or a sequence having a similar function to an MITD sequence.

47. The pharmaceutical preparation or nucleic acid molecule according to claim 46, characterized in that the MITD sequence has the amino sequence shown in SEQ ID NO:

23.

48. The pharmaceutical preparation or nucleic acid molecule according to claim 46, characterized in that the fifth nucleic acid fragment has the nucleotide sequence shown in SEQ ID NO: 24 to 32.

49. The pharmaceutical formulation or nucleic acid molecule according to claim 46, characterized in that the fifth nucleic acid fragment is provided at the 3' end of the nucleic acid fragment or at the 3' end of the nucleic acid molecule.

50. The nucleic acid fragment or nucleic acid molecule is characterized by having the nucleotide sequence shown in Table 4, as described in claim 1 or the nucleic acid molecule described in claim 14.

51. The pharmaceutical formulation according to claim 1, further comprising a drug carrier, wherein the drug carrier comprises at least one of liposomes, exosomes, polymer carriers, viral carriers, and nanoparticles.

52. An expression vector characterized by supporting a nucleic acid molecule according to any one of claims 14 to 50.

53. The expression vector according to claim 52, characterized in that the expression vector is a nonviral carrier.

54. A recombinant virus characterized by carrying a nucleic acid molecule as described in any one of claims 14 to 50.

55. A liposome comprising a liposome carrier and a nucleic acid fragment, wherein the nucleic acid fragment is limited by any one of claims 1 to 7 or 15 to 50.

56. A vaccine comprising a pharmaceutical preparation according to any one of claims 1 to 7 or 15 to 51, a nucleic acid molecule according to any one of claims 14 to 50, an expression vector according to any one of claims 52 to 53, a recombinant virus according to claim 54, or a liposome according to claim 55.

57. The vaccine according to claim 56, further comprising an adjuvant.

58. The aforementioned adjuvant is a TLR agonist, Mn ２＋ The vaccine according to claim 57, characterized by comprising at least one of the following.

59. The vaccine according to claim 58, characterized in that the TLR agonist comprises at least one of CpG, R837, MPLA, and its derivatives.

60. Recombinant cells carrying a nucleic acid fragment, a nucleic acid molecule according to any one of claims 14 to 50, an expression vector according to any one of claims 52 to 53, or a recombinant virus according to claim 54, The nucleic acid fragment is a circular RNA and comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment. The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus. The second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus. The third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus. Recombinant cells characterized in that the first nucleic acid fragment and the second nucleic acid fragment are linked to or not linked to the third nucleic acid fragment.

61. A method for constructing a feline infectious peritonitis virus vaccine, The process includes the step of introducing a nucleic acid fragment, a nucleic acid molecule according to any one of claims 14 to 50, an expression vector according to any one of claims 52 to 53, and a recombinant virus according to claim 54 into a receptor cell. The nucleic acid fragment is a circular RNA and comprises at least one of a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment. The first nucleic acid fragment encodes the M protein of feline infectious peritonitis virus, The second nucleic acid fragment encodes the N protein of feline infectious peritonitis virus, The third nucleic acid fragment encodes the S, S_ec, or SII protein of feline infectious peritonitis virus. A method characterized in that the first nucleic acid fragment and the second nucleic acid fragment are linked to or not linked to the third nucleic acid fragment.

62. The method according to 61, further comprising the step of coating the nucleic acid, expression vector, or recombinant virus using a coating carrier before introducing receptor cells.

63. The method according to 62, characterized in that the coated carrier is selected from at least one of liposomes, exosomes, polymer carriers, viral carriers, and nanoparticles.

64. The method according to 63, characterized in that the coating carrier is a nanoparticle.

65. The method according to 61, characterized in that the receptor cells are CRFK cells, HEK293FT, HEK293T, BHK cells, or insect cells.

66. The method according to 65, characterized in that the receptor cells are CRFK cells.

67. Use of a pharmaceutical formulation according to any one of claims 1 to 7, 15 to 51, a nucleic acid molecule according to any one of claims 14 to 50, an expression vector according to any one of claims 52 to 53, a recombinant virus according to claim 54, or a liposome according to claim 55 in the preparation of a pharmaceutical or vaccine for the prevention or treatment of a disease associated with feline infectious peritonitis virus infection.

68. A method for preventing or treating feline infectious peritonitis virus infection, comprising the step of administering to a test animal a pharmaceutical preparation according to any one of claims 1 to 7 or 16 to 51, a nucleic acid molecule according to any one of claims 14 to 50, an expression vector according to any one of claims 52 to 53, a recombinant virus according to claim 54, a liposome according to claim 55, a vaccine according to any one of claims 56 to 59, or a recombinant cell according to claim 60.

69. The method according to 68, characterized in that the animal is selected from cats.

70. Use of a pharmaceutical preparation according to any one of claims 1 to 7 or 16 to 51, a nucleic acid molecule according to any one of claims 14 to 50, an expression vector according to any one of claims 52 to 53, a recombinant virus according to claim 54, a liposome according to claim 55, a vaccine according to any one of claims 56 to 59, or a recombinant cell according to claim 60, in the prevention or treatment of feline infectious peritonitis virus infection.

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