Vaccine for preventing feline herpesvirus infection, and preparation method therefor and use thereof

Abstract

Provided are a vaccine for preventing feline herpesvirus infection, and a preparation method therefor and the use thereof. By means of antigen sequence design and screening, an antigen peptide and an mRNA nucleic acid molecule having excellent expression effects are obtained, and thus a vaccine prepared therefrom has a good immune effect and can induce the production of a highly-effective and durable level neutralizing antibody against a feline herpesvirus at a very low dose and dosing regimen; meanwhile, the mRNA vaccine has the characteristics of a good stability, a high delivery efficiency, safety, effectiveness and a controllable quality.

Description

A vaccine for preventing feline herpes virus infection and its preparation method and use Technical Field

[0001] The present invention relates to the technical field of vaccines, and in particular to a vaccine for preventing feline herpesvirus infection, a preparation method thereof, and application thereof in preventing feline herpesvirus disease. Background Art

[0002] Feline viral rhinotracheitis (FVRs), also known as feline infectious rhinotracheitis, is an acute, highly contagious disease caused by infection with the feline herpesvirus (FHV). Key clinical symptoms include fever, depression, loss of appetite, increased ocular and nasal discharge, facial or nasal dermatitis, conjunctivitis, and ulcerative keratitis. FHV is most susceptible to kittens aged 2 to 6 months, with a morbidity rate as high as 100%. While it does not cause mortality in adult cats, the mortality rate in kittens can reach 20% to 50%. FHV has a latent and latent infection mechanism and is one of the most serious known feline respiratory infections. The eight identified envelope glycoproteins of FHV include gB, gC, gD, gE, gG, gH, gI, and gL. The gB protein, a 949-amino acid envelope glycoprotein encoded by the UL27 gene, is a crucial component of the viral envelope and the primary immunogenic protein. The gD protein, a 374-amino acid protein encoded by the US6 gene, is highly conserved and antigenic, determining host cell selection, receptor recognition, and adsorption, inducing both cellular and humoral immunity. Therefore, gB and gD proteins could serve as candidate targets for vaccine development.

[0003] The development of vaccines to prevent feline herpesvirus infection, particularly mRNA vaccines, has significant implications for the prevention and control of major feline diseases. Compared to traditional vaccines, mRNA vaccines offer the following advantages: 1) safety, as mRNA does not integrate into the host genome and is naturally degraded by normal cells; 2) a simple production process, requiring no amplification in bacteria or cell culture and capable of in vitro transcription, offering the potential for rapid production; and 3) high efficacy, as mRNA vaccines can induce both humoral and cellular immunity and exhibit good immunogenicity.

[0004] Summary of the Invention

[0005] The present invention aims to provide a vaccine for preventing feline herpesvirus infection and the antigenic peptides and nucleic acid molecules required to form the vaccine. The antigenic peptides and nucleic acid molecules are derived from feline herpesvirus antigenic proteins gB, gC, gD, and / or gE. The present invention screens for sequences with excellent expression efficacy through antigen expression design and uses these sequences to prepare a vaccine. The resulting vaccine exhibits good safety, a simple production process, and excellent immunogenicity. This invention provides more options for vaccines for preventing feline herpesvirus infection.

[0006] The term "neutral lipid" herein refers to uncharged, non-phosphoglyceride lipid molecules.

[0007] The term "polyethylene glycol (PEG)-lipid" according to the present invention refers to a molecule comprising a lipid portion and a polyethylene glycol portion.

[0008] The term "lipid nanoparticle" according to the present invention refers to a particle having at least one dimension on the nanometer scale, which comprises at least one lipid.

[0009] The term "vaccine" in the present invention refers to a composition suitable for use in animals (including humans) that induces an immune response after administration, the strength of which is sufficient to minimally help prevent, improve or cure clinical diseases caused by microbial infection.

[0010] The term "N / P" in the present invention refers to the molar ratio of N in the cationic lipid to P in a single amino acid in the mRNA.

[0011] In one aspect of the present invention, an antigenic peptide comprising a feline herpesvirus antigen protein is provided.

[0012] Specifically, the feline herpesvirus antigen protein is one or more of gB, gC, gD, or gE; wherein gB, gC, gD, and gE are connected by a linker, and preferably the linker is flexible, and its sequence has (GGGGS)n or (G)n, and preferably, the flexible linker is GSGS. Specifically, the gB, gC, gD, and gE may be full-length or truncated portions thereof, and the specific truncation method is to retain the extracellular region or the extracellular region + transmembrane region of gB, gC, gD, and gE; preferably, the amino acid sequence encoding the gB protein is SEQ ID NO: 1 or its truncated sequence SEQ ID NO: 2 or has at least 75% homology; preferably, it has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology; preferably, the gB protein is truncated by deleting the 3' end amino acid sequence 813-949; preferably, the amino acid sequence encoding the gC protein is SEQ ID NO: 3 or its truncated sequence SEQ ID NO:4 or has at least 75% homology; preferably, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology; preferably, the gC protein is truncated in such a way that the first 28 amino acids at the N-terminus and amino acids 488-534 at the C-terminus are removed; preferably, the amino acid sequence encoding the gD protein is SEQ ID NO:5 or a truncated sequence thereof, SEQ ID NO:6, or has at least 75% homology; preferably, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology, preferably, the gD protein is truncated in such a way that the 24 amino acids at the N-terminus and amino acids 289-374 at the C-terminus are removed; the amino acid sequence encoding the gE protein is SEQ ID NO:7 or a truncated sequence thereof, SEQ ID NO: 8 or has at least 75% homology; preferably, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology; preferably, the gE protein is truncated by removing the first 18 amino acids at the N-terminus and amino acids 421-534 at the C-terminus. Preferably, the feline herpesvirus antigen protein has at least 75% homology to one or more of SEQ ID NOs: 8-14; preferably, has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology.

[0013] In some embodiments, the feline herpes virus antigen protein contained in the antigenic peptide is selected from:

[0014] (A) The feline herpesvirus antigen protein is a fusion antigen protein of gD and gB proteins, wherein the gD and gB proteins are sequentially linked from the N-terminus to the C-terminus, and the gD protein is a gD protein truncation, wherein the gD protein truncation is performed by removing 24 amino acids at the N-terminus and amino acids 289-374 at the C-terminus;

[0015] (B) The feline herpesvirus antigen protein is a fusion protein of gD, gC and gE, wherein the gD, gC and gE antigen proteins are sequentially connected from the N-terminus to the C-terminus, and the gD, gC and gE antigen proteins are all truncated forms, the gD protein truncation form is truncated by removing the N-terminal 24 amino acids and the C-terminal amino acids 289-374, the gC protein truncation form is truncated by removing the first 28 amino acids of the N-terminus and the C-terminal amino acids 488-534, and the gE protein truncation form is truncated by removing the first 18 amino acids of the N-terminus and the C-terminal amino acids 421-534.

[0016] In some specific embodiments, the amino acid sequence encoding the gB protein is shown in SEQ ID NO: 1, the amino acid sequence encoding the gC protein truncation is shown in SEQ ID NO: 4, the amino acid sequence encoding the gD protein truncation is shown in SEQ ID NO: 6, and the amino acid sequence encoding the gE protein truncation is shown in SEQ ID NO: 8; or the amino acid sequences of the gB protein, gC protein truncation, gD protein truncation and gE protein truncation in the antigenic peptide have at least 75% homology to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively; preferably, the amino acid sequences of the gB protein, gC protein truncation, gD protein truncation and gE protein truncation in the antigenic peptide have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

[0017] Preferably, the antigenic peptide further includes a signal peptide.

[0018] In some specific embodiments, the components of the antigenic peptide are connected by a connecting peptide; preferably, the connecting peptide is a flexible connecting peptide; more preferably, the amino acid sequence of the connecting peptide is (GGGGS)n or (G)n; further preferably, the connecting peptide is GGGGS.

[0019] In another aspect, the present invention provides a nucleic acid molecule encoding any of the above-mentioned antigenic peptides comprising a feline herpesvirus antigen protein.

[0020] In some embodiments, the nucleic acid molecule is an mRNA nucleic acid molecule.

[0021] Specifically, the nucleic acid molecule is a natural or modified RNA, and the modified RNA includes modifying the RNA by partially or completely replacing natural uridine with modified uridine; preferably, the mRNA sequence is a modified RNA, and the modified RNA is modified by completely replacing natural uridine with 1-methyl-pseudouridine.

[0022] Specifically, the nucleic acid molecule further comprises a 5' cap structure, a 5' non-coding region, a 3' non-coding region and / or an mRNA sequence of a poly(A) tail.

[0023] In another aspect, the present invention provides an mRNA composition for preventing feline herpesvirus infection, wherein the mRNA composition comprises any of the nucleic acid molecules described above.

[0024] Preferably, the mRNA composition further comprises a pharmaceutically acceptable carrier.

[0025] In another aspect, the present invention provides any one of the following biomaterials (a1) to (a4):

[0026] (a1) an expression cassette containing any of the above nucleic acid molecules;

[0027] (a2) a recombinant vector containing any of the above nucleic acid molecules;

[0028] (a3) a recombinant bacterium containing any of the above nucleic acid molecules;

[0029] (a4) A transgenic cell line containing any of the above nucleic acid molecules.

[0030] In another aspect, the present invention provides a kit for diagnosing or detecting feline herpesvirus, the kit comprising any of the above-mentioned antigenic peptides comprising feline herpesvirus antigenic protein, any of the above-mentioned nucleic acid molecules, any of the above-mentioned mRNA compositions, or any of the above-mentioned biological materials.

[0031] In another aspect, the present invention provides a vaccine for preventing feline herpesvirus infection, wherein the vaccine comprises any one of the above-mentioned antigenic peptides or any one of the above-mentioned nucleic acid molecules.

[0032] In some embodiments, the vaccine is an mRNA vaccine, which is composed of the messenger RNA (mRNA) of the feline herpesvirus protein or its protein homolog packaged in a delivery vehicle.

[0033] In some specific embodiments, the vaccine is an mRNA vaccine, and the nucleic acid molecules in the mRNA vaccine are encapsulated in a delivery vehicle; preferably, the delivery vehicle is a lipid nanoparticle (LNP), and the mRNA nucleic acid molecules are encapsulated in the lipid nanoparticle (LNP); more preferably, the lipid nanoparticles contain cationic lipids, neutral phospholipids, steroid lipids and polyethylene glycol (PEG)-lipids or the lipid nanoparticles contain steroid-cationic lipid compounds, neutral phospholipids, polyethylene glycol lipids.

[0034] Preferably, the lipid nanoparticles comprise cationic lipids, neutral phospholipids, steroidal lipids and polyethylene glycol (PEG)-lipids or steroid-cationic lipid compounds, neutral phospholipids, polyethylene glycol lipids.

[0035] Preferably, the length of the mRNA sequence is 200-10,000 amino acids. Further preferably, the length of the mRNA sequence is 500-8,000 amino acids.

[0036] The mRNA vaccine can induce potent, long-lasting, high-level neutralizing antibodies against feline herpes virus after immunization.

[0037] Preferably, the lipid nanoparticles comprise cationic lipids, neutral phospholipids, steroidal lipids and polyethylene glycol (PEG)-lipids or steroid-cationic lipid compounds, neutral phospholipids, polyethylene glycol lipids.

[0038] Preferably, the cationic lipid has the following structure:

[0039] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

[0040] G 1 and G 2 Each independently is an unsubstituted C6-C 10 alkylene;

[0041] G 3 For unsubstituted C1-C 12 alkylene;

[0042] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0043] R 3 OR 5 、CN、-C(=O)OR 4 、-OC(=O)R 4 or –NR 5 C(=O)R4 ;

[0044] R 4 C1-C 12 a hydrocarbon group; and

[0045] R 5 is H or a C1-C6 hydrocarbon group.

[0046] Preferably, the cationic lipid compound has the following structure:

[0047] Preferably, the cationic lipid compound structure:

[0048] DLin-MC3-DMA:

[0049] ALC-0315:

[0050] SM-102:

[0051] Preferably, the PEG-lipid is selected from the group consisting of: 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159), 1,2-dimyristoyl-sn-glyceromethoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disterylglycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglyceramide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) or one or more combinations thereof, preferably DMG-PEG2000.

[0052] Preferably, the neutral lipid is selected from the group consisting of: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), preferably DSPC.

[0053] Preferably, the steroidal lipid is selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol and polypeptide-modified cholesterol; one or more combinations of lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, preferably cholesterol.

[0054] Preferably, the polyethylene glycol (PEG)-lipid is DMG-PEG2000; the neutral phospholipid is DSPC; and the steroidal lipid is cholesterol.

[0055] Preferably, the lipid nanoparticles are characterized in that the molar percentage of the cationic lipid in the lipid component is 20-60%, the molar percentage of the neutral phospholipid in the lipid component is 5%-25%, the molar percentage of the steroid lipid in the lipid component is 25%-55%; and the molar percentage of the PEG-lipid in the lipid component is 0.1%-15%.

[0056] Preferably, the mass ratio (w / w) of total lipid to mRNA in lipid nanoparticles (LNP) is between 10-30:1.

[0057] Preferably, the lipid nanoparticles are characterized in that the molar ratio of the cationic lipid:neutral phospholipid:steroid lipid:PEG-lipid is 30-60:1-20:20-50:0.1-10.

[0058] Preferably, the molar ratio of the cationic lipid:neutral phospholipid:steroidal lipid:polyethylene glycol (PEG)-lipid conjugate is 40-60:10-20:30-50:1-5.

[0059] Preferably, the feline herpes virus mRNA vaccine further comprises other excipients in the prescription, and the excipients are one or more combinations of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.

[0060] Preferably, in the feline herpes virus mRNA vaccine, the average particle size of the nanoparticles is 50 to 200 nm, or the nanoparticles have a net neutral charge at neutral pH, or the nanoparticles have a polydispersity of less than 0.4.

[0061] More preferably, the molar ratio of cationic lipid:neutral phospholipid:steroidal lipid:polyethylene glycol (PEG)-lipid is 45:10:43:2 or 40:10:48:2.

[0062] Preferably, the lipid nanoparticles comprise cationic lipids, neutral phospholipids, steroidal lipids and polyethylene glycol (PEG)-lipids or steroid-cationic lipid compounds, neutral phospholipids, polyethylene glycol lipids.

[0063] In the composition, a hydrophobic chain of the cationic lipid in the steroid-cationic lipid is connected to the steroid to form a steroid-cationic lipid compound, and the steroid-cationic lipid compound has a structure of formula (I):

[0064] wherein said R1 is selected from -OR4, -NR4R5, -NR4C(=O)R5, -C(=O)OR5, -OC(=O)R5, -OC(=O)OR5, -CN, a nitrogen-containing heterocyclic group or a guanidine group;

[0065] Said R4 and R5 are independently selected from H, C 1-9 Alkyl, C 2-9 Alkenyl, C 2-9 Alkynyl, C 3-8 Cycloalkyl, C 3-8 Cycloalkenyl or C 3-8 cycloalkynyl;

[0066] The G1 is selected from chemical bonds (-), C 1-9 Alkylene, C 2-9 Alkenylene, C 3-9 Alkynylidene, C 3-8 Cycloalkylene or C 3-8 cycloalkenylene;

[0067] Preferably, the number of consecutive carbon atoms in R1-G1- does not exceed 10, for example, the consecutive carbon atoms may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0068] Said G2 and G3 are independently selected from: chemical bond (-), C 1-12 Alkylene, C 2-12 Alkenylene, C 2-12 Alkynylidene, C 3-12 Cycloalkylene, C 3-12 Cycloalkenylene, C 3-12 Cycloalkynylene or C 5-12 aromatic groups;

[0069] The L1 and L2 are independently selected from: chemical bond (-), -O-, -S-, -O(C=O)O-, -(C=O)NRa -、-NR a (C=O)-, -O(C=O)-, -(C=O)O-, -SS-, -S(O) x -、-OS(O) x O-, -C(=O)S-, -SC(=O)-, -NR a C(=O)NR b -、-OC(=O)NR a -、-NR a C(=O)O-, -OC(=O)S-, -SC(=O)O-, -P(O)(OR a )O-、-OP(O)(OR a )O- or C 1-12 a combination of one or more of alkylene;

[0070] Wherein, x is selected from 0, 1 or 2;

[0071] Among them, R a 、R b H, C 1-12 Alkyl, C 1-12 Alkenyl or C 1-12 of alkynes;

[0072] Said R2 is selected from naturally occurring or non-naturally occurring steroids;

[0073] Said R3 is independently selected from naturally occurring or non-naturally occurring steroids, C 6-24 Alkyl, C 6-24 Alkenyl, C 6-24 Alkyne or C 6-24 of alkoxy.

[0074] Furthermore, the R1 is selected from -OR4, -NR4R5, pyrazolyl, imidazolyl, piperazinyl, piperazinyl, piperidinyl, piperidinyl, guanidinyl, pyrrolyl or pyrrolidinyl; the R4 and R5 are independently selected from H, -C 1-9 Alkyl, -C 2-9 Alkenyl, -C 2-9 Alkynyl, -C 3-8 Cycloalkyl or -C 3-8 cycloalkenyl;

[0075] The G1 is a chemical bond (-), -C 1-9 Alkylene, -C 2-9 Alkenylene, -C 3-8 Cycloalkylene or -C 3-8 cycloalkenylene;

[0076] Preferably, the G1 is -C 1-6 Alkylene or -C2-6 alkenylene;

[0077] The number of consecutive carbon atoms in R1-G1- does not exceed 10. Specifically, the number of consecutive carbon atoms may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0078] Said G2 and G3 are independently selected from: chemical bond (-), -C 1-12 Alkylene, -C 2-12 Alkenylene or -C 2-12 Alkynylidene;

[0079] The L1 and L2 are independently selected from: -O(C=O)-, -(C=O)O-, -SS-, -O(C=O)O-, -NR a C(=O)O-, -(C=O)NR a -、-NR a (C=O)-, -C(=O)S-, -SC(=O)-, -OC(=O)NR a - or C 1-12 a combination of one or more of alkylene;

[0080] Among them, R a H, C 1-12 Alkyl, C 1-12 Alkenyl or C 1-12 of alkynes;

[0081] Further, the R1 is selected from -OR4, -NR4R5, imidazolyl, piperazinyl or guanidinyl;

[0082] Said R4 and R5 are independently selected from H, -C 1-5 Alkyl;

[0083] The G1 is -C 1-6 unsubstituted alkylene;

[0084] The number of consecutive carbon atoms in R1-G1- does not exceed 10. Specifically, the number of consecutive carbon atoms may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0085] Said G2 and G3 are independently selected from: chemical bond (-), -C 1-10 alkylene;

[0086] The L1 and L2 are independently selected from: -O-, -O(C=O)-, -(C=O)O-, -(C=O)S-, -O(C=O)O-, -NHC(=O)O-, -NHC(=O)-,

[0087] Further, the R2 is selected from: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fuccasterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid;

[0088] Preferably, the steroid is a sterol;

[0089] The sterol is a zoosterol, or an oxidized form or a reduced form thereof; and / or, the sterol is a phytosterol, or an oxidized form or a reduced form thereof;

[0090] and / or, the sterol is a synthetic sterol, or an oxidized or reduced form thereof;

[0091] Preferably, the sterol is selected from cholesterol, an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithocholate, stigmasterol, stigmasterol, campesterol, ergosterol or sitosterol;

[0092] More preferably, the sterol is an oxidized form of cholesterol, a reduced form of cholesterol, an alkyl lithocholate, stigmasterol, stigmasterol, campesterol, ergosterol or sitosterol;

[0093] More preferably, the structural formula of the sterol is as follows:

[0094] Where R is C 1-20 alkyl.

[0095] Furthermore, the R3 is selected from the following structures:

[0096] Preferably, the R3 is selected from the following structures:

[0097] Furthermore, the structure of the compound is:

[0098] Furthermore, the nitrogen to phosphorus ratio of the nucleic acid-lipid particle is (1-15):1, including but not limited to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or 15:1.

[0099] Preferably, the nucleic acid-lipid particle has a diameter of 15 nm to 300 nm;

[0100] Preferably, the nucleic acid-lipid particle has a diameter of 60 nm to 102 nm;

[0101] More preferably, the nucleic acid-lipid particle has a diameter of 80 nm to 90 nm.

[0102] Furthermore, the dosage form of the nucleic acid delivery vector of the composition is a liquid preparation or a lyophilized powder.

[0103] On the other hand, the present invention provides a method for preparing the mRNA vaccine, wherein the vaccine is prepared by encapsulating any of the above-mentioned mRNA nucleic acid molecules in lipid nanoparticles.

[0104] In some specific embodiments, the method for preparing the mRNA vaccine is to dissolve cationic lipids, neutral phospholipids, steroid lipids, polyethylene glycol (PEG)-lipids into a solvent or dissolve steroid-cationic lipid compounds, neutral phospholipids, polyethylene glycol lipids into a solvent and then mix them with any of the above-mentioned mRNA nucleic acid molecules to prepare the vaccine.

[0105] Preferably, the preparation method of the feline herpes virus mRNA vaccine is to dissolve cationic lipids, neutral phospholipids, steroid lipids, PEG-lipids or steroid-cationic lipids, compound neutral phospholipids and polyethylene glycol lipids in a solvent and then mix them with nucleic acids to obtain the vaccine.

[0106] Preferably, the preparation method of the feline herpes virus mRNA vaccine is to dissolve cationic lipids, neutral phospholipids, steroid lipids, PEG-lipids or steroid-cationic lipids, compound neutral phospholipids and polyethylene glycol lipids in ethanol, mix them with the diluted mRNA diluent, and then ultrafilter, dilute, and filter to obtain the vaccine; preferably, cationic lipids, neutral phospholipids, steroid lipids, PEG-lipids or steroid-cationic lipids, compound neutral phospholipids and polyethylene glycol lipids in ethanol, mix them with the diluted mRNA diluent at a certain flow rate ratio, and then ultrafilter, dilute, and filter to obtain the vaccine; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent mixing, laminar mixing or microfluidic mixing.

[0107] The preparation method comprises the steps of preparing a biological material, including: (a) a recombinant vector, including any one of the above-mentioned nucleic acid molecule constructs and plasmid vectors; (b) a transformant, including any one of the above-mentioned nucleic acid molecule constructs, or (a) a host cell of the recombinant vector; preferably, the plasmid vector includes pcDNA3.1, CET 1019HS puro, pIRES or pRL SV40, and more preferably pcDNA3.1; preferably, the host cell includes a prokaryotic cell or a eukaryotic cell; further preferably, the prokaryotic cell includes DH5α, JM109 or BL21, and more preferably DH5α; further preferably, the eukaryotic cell includes yeast cells, Marc-145, PK 15, Hela, 293T or insect cells, and more preferably 293T cells.

[0108] The preparation method of the construct comprises: (1) synthesizing a gene fragment of an expression construct, wherein the amino acid sequence of the construct comprises, in order from the 5' end to the 3' end, a 5'UTR sequence, a Kozak sequence, a signal peptide coding sequence, a 3'UTR sequence, and a poly(A) sequence; (2) inserting the gene fragment obtained in step (1) into a plasmid vector to obtain a recombinant vector, and then transforming the recombinant vector into a host cell to obtain a transformant, extracting the recombinant vector after the transformant proliferates, and obtaining the construct after in vitro transcription.

[0109] Preferably, in the preparation method of the feline herpesvirus mRNA vaccine, the diluent can be acetate buffer, citrate buffer, phosphate buffer or tris buffer.

[0110] Preferably, the method for preparing the feline herpesvirus mRNA vaccine is characterized in that the buffer solution has a pH of 3 to 6 and a concentration of 6.25 to 200 mM.

[0111] Preferably, in the preparation method of the feline herpes virus mRNA vaccine, the N / P ratio when lipid-encapsulated mRNA is 2-10, preferably 3-9.

[0112] Preferably, the vaccine is in the form of an oral preparation, an intramuscular injection preparation, an intravenous injection preparation, an inhalation preparation, a liquid preparation, a lyophilized powder, an aerosol inhalation preparation or a dry powder inhalation preparation.

[0113] The vaccine of the present invention may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a carrier, a diluent, an adjuvant or an amino acid sequence encoding an adjuvant, a solubilizing agent, a binder, a lubricant, a suspending agent, a transfection facilitator, etc. The transfection facilitator includes, but is not limited to, surfactants such as immunostimulating complexes, Freunds incomplete adjuvant, LPS analogs (e.g., monophosphoryl ester A), muramyl peptides, benzoquinone analogs, squalene, hyaluronic acid, lipids, lipids, calcium ions, viral proteins, cations, polycations (e.g., poly-L-glutamic acid (LGS)), or nanoparticles or other known transfection facilitators. The amino acid sequence encoding the adjuvant is an amino acid sequence encoding at least one of the following adjuvants: GM-CSF, IL-17, IFNg, IL-15, IL-21, anti-PD1 / 2, lactoferrin, protamine, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INF-α, INF-γ, Lymphotoxin-α, hGH, MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac -1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,, CD40, CD40L, vascular growth factor, fibroblast growth factor, nerve growth factor, vascular endothelial growth factor, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAP K, SAP-1, JNK, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof.

[0114] Another aspect of the present invention provides a use of any of the above-mentioned antigenic peptides, any of the above-mentioned nucleic acid molecules, any of the above-mentioned mRNA compositions, or any of the above-mentioned biological materials in the preparation of a vaccine or a drug for preventing or treating feline herpesvirus infection.

[0115] The vaccine of the present invention can deliver the biologically active substance by oral administration, inhalation or injection.

[0116] The beneficial effects of the present invention compared to the prior art are:

[0117] First, the present invention designs and screens antigen sequences to obtain antigen peptides and mRNA nucleic acid molecules with better expression effects;

[0118] Second, the vaccine prepared by the present invention has a good immune effect. At a very low dose and administration schedule, it can induce the production of strong and long-lasting neutralizing antibodies against feline herpes virus, which is better than the effect of existing vaccines on the market.

[0119] 3. The mRNA vaccine preparation prepared by the present invention has good stability. The expression of mRNA in vivo can avoid contamination from exogenous factors such as viruses and proteins, and has better antiviral effects and fewer side effects.

[0120] 4. The vaccine prepared by the present invention is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] Figures 1A and 1B show the fluorescence expression of different FHV antigen mRNA stock solutions;

[0122] Figure 2 is a schematic diagram of the immunization procedure. DETAILED DESCRIPTION

[0123] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0124] Example 1: mRNA sequence screening

[0125] The plasmid DNA sequence was designed according to the protein sequence shown in Table 1.

[0126] Table 1 Summary of sequence screening information

[0127] Artificially synthesize a plasmid DNA sequence containing elements related to RNA transcription and adding an EGFP fluorescent marker to facilitate screening of the expression of different antigen designs. The plasmid is transformed into Escherichia coli for amplification. The plasmid after fermentation and purification is linearized with the restriction endonuclease BspQ1. Transcription is performed using a T7 in vitro transcription kit to obtain uncapped mRNA. The transcription template is digested with DNaseI, and the mRNA is purified by precipitation. The mRNA is capped using a Cap1 capping kit, and the capped mRNA is purified using an mRNA purification kit. The purified mRNA is dissolved in acidic sodium citrate buffer, and its concentration is detected using a nanodrop and set aside.

[0128] In vitro expression detection of mRNA stock solution:

[0129] a. Plate: CRFK cells (10 6 cell / well), and the cell plate was placed in a CO2 incubator and cultured at 37°C for 24 h;

[0130] b. Transfection: Remove the culture plate and replace the culture medium with serum-free medium. Mix 2.5 μg of mRNA stock solution with Lipo3000 to prepare a premix, incubate at room temperature for 5 minutes, and slowly add the premix dropwise to the culture plate.

[0131] c. Incubation: Place the culture plate in a CO2 incubator at 37°C for 48 hours.

[0132] d. Fluorescence photography: Take photos every 12 hours to track the expression.

[0133] The results are shown in Figures 1A and 1B: The antigen designs of gD truncation + gB or gD truncation + gC truncation + gE truncation in FHV expressed better, so the designs with better expression were used as antigen designs for vaccine development for further research.

[0134] Example 2: Lipid Nanoparticle Encapsulation of mRNA Antigen

[0135] The mRNA solution in Example 1 was encapsulated with a formulation containing cationic lipids ALC-0315, SM-102, DLin-MC3-DMA, or steroid-cationic lipid compound 5, compound 11, or compound 17. A molar ratio of cationic lipid: neutral phospholipid: steroid lipid: polyethylene glycol (PEG)-lipid of 45:10:43:2 or a molar ratio of steroid-cationic lipid: neutral phospholipid: polyethylene glycol (PEG)-lipid of 49.25:49.25:1.5 was dissolved and mixed in ethanol. The total flow rate of the nanomedicine manufacturing equipment was set at 12 ml / min. The mRNA solution and the lipid mixture were encapsulated at a flow rate ratio of 3:1. After encapsulation, the sample was collected by ultrafiltration through a tangential flow filtration system, and a stabilizer solution (e.g., sucrose) was added to obtain the mRNA vaccine (mRNA-LNP). Sampling was measured for encapsulation efficiency, average particle size, PDI, and zeta potential.

[0136] Table 2 Characterization of parameters of LNP-mRNA compositions formed by four-component cationic lipids and three-component sterol-cationic lipid compounds

[0137] Example 3: Cat Immunization and Detection

[0138] Immunity experiments:

[0139] Thirty-eight cats aged 2-3 months were divided into 9 groups and grouped as shown in Table 3 for immunization experiments: After immunization, the clinical manifestations of the experimental cats (body temperature, mental state, food intake, feces, and drinking water) were observed and recorded every day, body weight was measured every 7 days, and venous blood was collected every 14 days to separate serum and detect the neutralizing antibody titer against FHV-1.

[0140] Table 3: Design of cat immunization experiment

[0141] 1. Evaluation of humoral immunity against FHV

[0142] The collected serum was separated and neutralization experiments were performed. The test results showed that the mRNA vaccine designed and prepared with two antigens, gD truncation + gB or gD truncation + gC truncation + gE truncation, stimulated the body to produce high levels of neutralizing antibodies against FHV-1. The neutralizing antibody titer could reach 1:1024 two weeks after the second immunization. Compared with the positive control group of Miao Sanduo, it was not only able to produce neutralizing antibodies more quickly, but also the neutralizing antibody level was significantly higher.

[0143] Table 4: Feline herpesvirus neutralizing antibody titer (1:n)

[0144] 2. FHV immune challenge evaluation

[0145] The challenge evaluation was conducted on the 42nd day after the first vaccination: Groups 6, 8, and 10 were treated with FHV / AS strain (10 7.5 Cats were challenged with intranasal drops of 100 mg / mL (PFU / ml / cat) and observed for 28 consecutive days for clinical symptoms, morbidity, and mortality. The results showed that 100% of the experimental cats were protected after immunization with either the gD truncated + gB or gD truncated + gC truncated + gE truncated mRNA vaccines. Although some symptoms appeared, they recovered well within 2-3 days. Compared with the positive control vaccine of Myo-Tri-Dox, the protection rate was higher and the clinical symptoms were milder. Therefore, the mRNA vaccine provided in this protocol can provide good protection for cats, as shown in Table 5:

[0146] Table 5: Protection effect of FHV-1 challenge

[0147] In summary, the FHV-1 mRNA vaccine described in the present invention can provide better protection for cats.

[0148] Among them, the amino acid sequence of SEQ ID NO: 1 encoding FHV-1gB protein is:

[0149] SEQ ID NO: 2 encodes the amino acid sequence of the FHV-1 truncated gB protein:

[0150] SEQ ID NO: 3 encodes the amino acid sequence of FHV-1gC protein:

[0151] The amino acid sequence of SEQ ID NO:4 encoding the FHV-1 truncated gC protein is:

[0152] SEQ ID NO:5 encodes the amino acid sequence of FHV-1gD protein:

[0153] SEQ ID NO:6 encodes the amino acid sequence of the FHV-1 truncated gD protein:

[0154] SEQ ID NO:7 encodes the amino acid sequence of FHV-1gE protein:

[0155] SEQ ID NO:8 encodes the amino acid sequence of the FHV-1gE truncated protein:

[0156] SEQ ID NO:9 encodes the amino acid sequence of gB+gD protein:

[0157] The amino acid sequence of SEQ ID NO: 10 encoding the FHV-1gB+gD+gC fusion protein is:

[0158] SEQ ID NO:11 encodes the amino acid sequence of gD truncated + gC truncated + gE truncated:

[0159] SEQ ID NO: 12 encodes the amino acid sequence of the FHV-1 truncated gD protein + gB protein:

[0160] The amino acid sequence of SEQ ID NO: 13 encoding the FHV-1 gD protein + gC protein + gE truncated protein is:

Claims

1. An antigen peptide comprising a feline herpes virus antigen protein, characterized in that: The feline herpes virus antigen protein contained in the antigen peptide is selected from: (A) the feline herpesvirus antigen protein is a fusion antigen protein of gD and gB proteins, wherein the gD and gB proteins are sequentially connected from the N-terminus to the C-terminus, and the gD protein is a gD protein truncate, wherein the gD protein truncate is truncated by removing 24 amino acids at the N-terminus and amino acids 289-374 at the C-terminus; (B) The feline herpesvirus antigen protein is a fusion protein of gD, gC and gE, wherein the gD, gC and gE antigen proteins are sequentially connected from the N-terminus to the C-terminus, and the gD, gC and gE antigen proteins are all truncated forms, the gD protein truncation form is truncated by removing the N-terminal 24 amino acids and the C-terminal amino acids 289-374, the gC protein truncation form is truncated by removing the first 28 amino acids at the N-terminus and the C-terminal amino acids 488-534, and the gE protein truncation form is truncated by removing the first 18 amino acids at the N-terminus and the C-terminal amino acids 421-534.

2. The antigen peptide according to claim 1, characterized in that The amino acid sequence encoding the gB protein is shown in SEQ ID NO: 1, the amino acid sequence encoding the gC protein truncation is shown in SEQ ID NO: 4, the amino acid sequence encoding the gD protein truncation is shown in SEQ ID NO: 6, and the amino acid sequence encoding the gE protein truncation is shown in SEQ ID NO: 8; or the amino acid sequences of the gB protein, gC protein truncation, gD protein truncation and gE protein truncation in the antigenic peptide have at least 75% homology with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively; preferably, the amino acid sequences of the gB protein, gC protein truncation, gD protein truncation and gE protein truncation in the antigenic peptide have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

3. The antigen peptide according to claim 1 or 2, characterized in that A signal peptide is inserted into the N-terminus of the antigenic peptide.

4. The antigen peptide according to claim 3, characterized in that The components included in the antigen peptide are connected by a connecting peptide; preferably, the connecting peptide is a flexible connecting peptide; more preferably, the amino acid sequence of the connecting peptide is (GGGGS)n or (G)n; further preferably, the connecting peptide is GGGGS.

5. A nucleic acid molecule encoding the antigenic peptide according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule is an mRNA nucleic acid molecule.

7. The nucleic acid molecule according to claim 6, characterized in that The nucleic acid molecule is a natural or modified RNA, wherein the modified RNA comprises modifying the RNA by partially or completely replacing natural uridine with modified uridine; preferably, the mRNA sequence is a modified RNA, wherein the modified RNA is modified by completely replacing natural uridine with 1-methyl-pseudouridine.

8. The nucleic acid molecule according to claim 7, characterized in that The nucleic acid molecule further comprises a 5' cap structure, a 5' non-coding region, a 3' non-coding region and / or an mRNA sequence of a polyadenylic acid tail.

9. An mRNA composition for preventing feline herpes virus infection, characterized in that: The mRNA composition comprises the nucleic acid molecule according to any one of claims 6 to 8.

10. The mRNA composition according to claim 9, characterized in that The mRNA composition also includes a pharmaceutically acceptable carrier.

11. Any one of the following biological materials (a1) to (a4): (a1) an expression cassette containing the nucleic acid molecule of claim 5; (a2) a recombinant vector containing the nucleic acid molecule of claim 5; (a3) a recombinant bacterium containing the nucleic acid molecule of claim 5; (a4) A transgenic cell line containing the nucleic acid molecule of claims 5-8.

12. A kit for diagnosing or detecting feline herpes virus, characterized in that: The kit comprises the antigen peptide according to any one of claims 1 to 4, the nucleic acid molecule according to any one of claims 4 to 8, the mRNA composition according to any one of claims 9 to 10, or the biological material according to claim 11.

13. A vaccine for preventing feline herpes virus infection, characterized in that: The vaccine comprises the antigenic peptide according to any one of claims 1 to 4 or comprises the nucleic acid molecule according to any one of claims 4 to 8.

14. The vaccine according to claim 13, characterized in that The vaccine is an mRNA vaccine, and the nucleic acid molecules in the mRNA vaccine are encapsulated in a delivery vector; preferably, the delivery vector is a lipid nanoparticle (LNP), and the mRNA nucleic acid molecules are encapsulated in the lipid nanoparticle (LNP); more preferably, the lipid nanoparticle contains cationic lipids, neutral phospholipids, steroidal lipids and polyethylene glycol (PEG)-lipids or the lipid nanoparticles contain steroid-cationic lipid compounds, neutral phospholipids, polyethylene glycol lipids.

15. A method for preparing an mRNA vaccine for preventing feline herpes virus infection, characterized in that: The vaccine is prepared by encapsulating the mRNA nucleic acid molecule according to any one of claims 4 to 8 in lipid nanoparticles.

16. The method for preparing the mRNA vaccine according to claim 15, characterized in that: The vaccine is prepared by dissolving cationic lipids, neutral phospholipids, steroid lipids, polyethylene glycol (PEG)-lipids in a solvent or dissolving steroid-cationic lipid compounds, neutral phospholipids, polyethylene glycol lipids in a solvent and then mixing them with the mRNA nucleic acid molecule according to any one of claims 4 to 8.

17. Use of the antigenic peptide according to any one of claims 1 to 3, the nucleic acid molecule according to any one of claims 4 to 8, the mRNA composition according to any one of claims 9 to 10, or the biomaterial according to claim 11 in the preparation of a vaccine or a drug for preventing or treating feline herpes virus infection.

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