WO2025011593A1 - Vaccine for preventing feline panleukopenia, preparation method therefor and use thereof - Google Patents

Abstract

Provided are a vaccine for preventing feline panleukopenia, a preparation method therefor and a use thereof. An antigen peptide and an mRNA nucleic acid molecule that are obtained by means of antigen sequence design and screening are excellent in expression effect, so that a prepared vaccine has a good immune effect, and under a very low-dosage administration scheme, can induce production of a potent and lasting level neutralizing antibody for a feline panleukopenia virus. Moreover, an mRNA vaccine has the characteristics of being good in stability, high in delivery efficiency, safe, effective, and controllable in quality.

Description

A vaccine for preventing feline panleukopenia and its preparation method and use Technical Field

The present invention relates to the technical field of vaccines, and in particular to a vaccine for preventing feline panleukopenia, a preparation method thereof, and an application thereof in preventing feline panleukopenia.

Background Art

Feline panleukopenia (FP), also known as feline plague and feline infectious enteritis, is an acute, highly contagious disease of cats caused by feline parvovirus (FPV). Clinical manifestations are mainly characterized by sudden high fever, persistent vomiting, diarrhea, dehydration, circulatory disorders and a sharp decrease in white blood cells. Feline parvovirus (FPV) is listed as one of the three major viral infectious diseases of cats together with feline herpesvirus (FHV) and feline calicivirus (FCV). FPV is distributed in many countries and regions in the world and can infect cats, mice, raccoons, lynxes, leopards, tigers, lions and other animals, among which cats are the most susceptible animals. FPV mainly infects kittens under 1 year old. Under normal circumstances, the mortality rate for cats under 1 year old is 50% to 60%, and the mortality rate for kittens under 5 months old is as high as 80% to 90%. The VP2 protein encoded by FPV accounts for 90% of all structural proteins and contains multiple antigen recognition sites, which is the main antigenic protein. VP2 can induce the body to produce neutralizing antibodies, protecting the body from viral infection.

The development of vaccines to prevent feline panleukopenia, especially mRNA vaccines, has positive significance for the prevention and control of major cat diseases. Compared with traditional vaccines, mRNA vaccines have the following advantages: 1) Good safety, mRNA will not integrate into the host genome, and can be naturally degraded by normal cells; 2) Simple production process, no need to amplify in bacteria or cell culture, can be produced by in vitro transcription, and has the potential for rapid production; 3) High efficiency, mRNA vaccines can induce humoral and cellular dual immunity, and have good immunogenicity.

Summary of the invention

The purpose of the present invention is to provide a vaccine for preventing feline panleukopenia and the antigenic peptides and nucleic acid molecules required for the vaccine, wherein the antigenic peptides and nucleic acid molecules are derived from the VP2 protein of feline panleukopenia virus. The present invention selects sequence designs with excellent expression effects through antigen expression design, and prepares vaccines based on them. The prepared vaccine has good safety, simple production process and good immunogenicity. The present invention provides more options for vaccines for preventing feline panleukopenia.

The term "neutral lipid" in the present invention refers to uncharged, non-phosphoglyceride lipid molecules.

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

The term "lipid nanoparticle" according to the present invention refers to a particle having at least one dimension of the nanometer order, which comprises at least one lipid.

The term "vaccine" in the present invention refers to a composition suitable for application to 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 infection by microorganisms.

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

On the one hand, the present invention provides an antigenic peptide comprising a truncated form of the VP2 protein of feline panleukopenia virus (FPV), characterized in that the truncated form of the VP2 protein retains the protein region of the VP2 protein from position 33 to position 575 of the N-terminus to the C-terminus.

In some specific embodiments, the amino acid sequence encoding the truncated VP2 protein is as shown in SEQ ID NO:2, or has at least 75% homology with SEQ ID NO:2; preferably, it has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO:2.

Preferably, the antigenic peptide also includes a signal peptide.

In some specific embodiments, the components included in 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.

In another aspect, the present invention provides a nucleic acid molecule encoding any of the above-mentioned antigenic peptides comprising a truncated form of the feline panleukopenia virus VP2 protein.

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

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.

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

In another aspect, the present invention provides an mRNA composition for preventing feline panleukopenia, wherein the mRNA composition comprises any of the above-mentioned nucleic acid molecules.

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

In another aspect, the present invention provides any one of the following (a1)-(a4) biomaterials:

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

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

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

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

In another aspect, the present invention provides a kit for diagnosing or detecting feline panleukopenia virus, the kit comprising any of the above-mentioned antigenic peptides comprising a truncated form of the feline panleukopenia virus VP2 protein, any of the above-mentioned nucleic acid molecules, any of the above-mentioned mRNA compositions, or any of the above-mentioned biological materials.

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

In some specific embodiments, the vaccine is an mRNA vaccine, which is composed of the messenger RNA (mRNA) of the feline parvovirus protein or its protein homolog encapsulated by a delivery vehicle.

In some specific embodiments, 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 nanoparticles contain 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.

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

Preferably, the length of the mRNA sequence is 200-10000 amino acids. Further preferably, the length of the mRNA sequence is 500-8000 amino acids.

The mRNA vaccine can induce strong, long-lasting, high-level neutralizing antibodies against feline parvovirus after immunization.

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

Preferably, the cationic lipid has the following structure:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

^G1 and ^G2 are each independently an unsubstituted _C6 - _C10 alkylene group;

G ³ is an unsubstituted C ₁ -C ₁₂ alkylene group;

^R1 and ^R2 are each independently _C6 - _C24 alkyl or _C6 - _C24 alkenyl;

R ³ is OR ⁵ , CN, -C(=O)OR ⁴ , -OC(=O)R ⁴ or –NR ⁵ C(=O)R ⁴ ;

R ⁴ is a C ₁ -C ₁₂ hydrocarbon group; and

^R5 is H or a _C1 - _C6 hydrocarbon group.

Preferably, the cationic lipid compound has the following structure:

Preferably, the cationic lipid compound structure is:

DLin-MC3-DMA:

ALC-0315:

SM-102:

Preferably, the PEG-lipid is selected from: 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-dipalmitoyl, 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 of DMG-PEG2000, preferably DMG-PEG2000.

Preferably, the neutral lipid is selected from: 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), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) One or more combinations thereof, preferably DSPC.

Preferably, the steroidal lipids are selected from avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fucosterol, 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.

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

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 steroidal lipid in the lipid component is 25%-55%; and the molar percentage of the PEG-lipid in the lipid component is 0.1%-15%.

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

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

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.

Preferably, the prescription of the feline parvovirus mRNA vaccine further comprises other excipients, which are one or more combinations of sodium acetate, tromethamine, potassium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, and sucrose.

Preferably, in the feline parvovirus 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.

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.

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

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 is of formula (I):

wherein said R ₁ is selected from -OR ₄ , -NR ₄ R ₅ , -NR ₄ C(=O)R ₅ , -C(=O)OR ₅ , -OC(=O)R ₅ , -OC(=O)OR ₅ , -CN, a nitrogen-containing heterocyclic group or a guanidine group;

Said R ₄ and R ₅ 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;

The _G1 is selected from a chemical bond (-), a _C1-9 alkylene group, a _C2-9 alkenylene group, a _C3-9 alkynylene group, a _C3-8 cycloalkylene group or a _C3-8 cycloalkenylene group;

Preferably, the number of consecutive carbon atoms in the R ₁ -G ₁ - group does not exceed 10. For example, the number of consecutive carbon atoms may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Said G ₂ and G ₃ are independently selected from: a chemical bond (-), a C _1-12 alkylene group, a C _2-12 alkenylene group, a C _2-12 alkynylene group, a C _3-12 cycloalkylene group, a C _3-12 cycloalkenylene group, a C _3-12 cycloalkynylene group or a C _5-12 arylene group;

Said L ₁ and L ₂ are independently selected from: a chemical bond (-), -O-, -S-, -O(C=O)O-, -(C=O)NR _a -, -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 a combination of one or more of C _1-12 alkylene;

Wherein, x is selected from 0, 1 or 2;

Wherein, _Ra and _Rb are H, _C1-12 alkyl, _C1-12 alkenyl or _C1-12 alkyne;

The _R2 is selected from naturally occurring or non-naturally occurring steroids;

Said R ₃ 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 alkoxy.

Further, the R ₁ is selected from -OR ₄ , -NR ₄ R ₅ , pyrazolyl, imidazolyl, piperazinyl, piperazinyl, piperidinyl, piperidinyl, guanidinyl, pyrrolyl or pyrrolidinyl; the R ₄ and R ₅ 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} ;

The _G1 is a chemical bond (-), _-C1-9 alkylene, _-C2-9 alkenylene, _-C3-8 cycloalkylene or _-C3-8 cycloalkenylene;

Preferably, G ₁ is -C _1-6 alkylene or -C _2-6 alkenylene;

The number of consecutive carbon atoms in the R ₁ -G ₁ - group does not exceed 10. Specifically, the number of consecutive carbon atoms may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

The G ₂ and G ₃ are independently selected from: a chemical bond (-), -C _1-12 alkylene, -C _2-12 alkenylene or -C _2-12 alkynylene;

L ₁ and L ₂ 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 a combination of one or more of C _1-12 alkylene;

wherein _Ra is H, a _C1-12 alkyl group, a _C1-12 alkenyl group or a _C1-12 alkyne group;

Further, the R ₁ is selected from -OR ₄ , -NR ₄ R ₅ , imidazolyl, piperazinyl or guanidinyl;

Said R ₄ and R ₅ are independently selected from H, -C _1-5 alkyl;

The _G1 is _-C1-6 unsubstituted alkylene;

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

The G ₂ and G ₃ are independently selected from: a chemical bond (-), a -C _1-10 alkylene group;

The L ₁ and L ₂ are independently selected from: -O-, -O(C=O)-, -(C=O)O-, -(C=O)S-, -O(C=O)O-, -NHC(=O)O-, -NHC(=O)-,

Further, the R ₂ is selected from: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, cholic acid, glycocholic acid, taurocholic acid, deoxycholic acid or lithocholic acid;

Preferably, the steroid is a sterol;

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;

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

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 and sitosterol;

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

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

wherein R is a C _1-20 alkyl group.

Further, the _R3 is selected from the following structures:

Preferably, the R ₃ is selected from the following structures:

Furthermore, the structure of the compound is:

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.

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

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

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

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

In another aspect of the present invention, a method for preparing the mRNA vaccine is provided, wherein the vaccine is prepared by encapsulating any of the above-mentioned mRNA nucleic acid molecules in lipid nanoparticles.

In some specific embodiments, the method for preparing the mRNA vaccine comprises dissolving cationic lipids, neutral phospholipids, steroid lipids, polyethylene glycol (PEG)-lipids into a solvent or dissolving steroid-cationic lipid compounds, neutral phospholipids, polyethylene glycol lipids into a solvent and then mixing them with any of the above-mentioned mRNA nucleic acid molecules to obtain the vaccine.

Preferably, the method for preparing the feline parvovirus 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.

Preferably, the method for preparing the feline parvovirus 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 them; 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 them; preferably, the ultrafiltration method is tangential flow filtration; more preferably, the mixing method can be turbulent mixing, laminar mixing or microfluidic mixing.

The preparation method comprises the steps of preparing biological materials, including: (a) a recombinant vector, including any of the above-mentioned nucleic acid molecule constructs and plasmid vectors; (b) a transformant, including any 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, Marco 145, PK 15, Hela, 293T or insect cells, and more preferably 293T cells.

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 promoter sequence, a 5'UTR sequence, a Kozak sequence, a signal peptide coding sequence, a 3'UTR sequence of an mRNA molecule, 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.

Preferably, in the method for preparing the feline parvovirus mRNA vaccine, the diluent can be acetate buffer, citrate buffer, phosphate buffer or tris buffer.

Preferably, in the method for preparing the feline parvovirus mRNA vaccine, the buffer solution has a pH of 3 to 6 and a concentration of 6.25 to 200 mM.

Preferably, in the method for preparing the feline parvovirus mRNA vaccine, the N/P when the mRNA is lipid-encapsulated is 2-10, and the preferred N/P is 3-9.

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.

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, an adhesive, a lubricant, a suspending agent, a transfection facilitator, etc. The transfection facilitator includes, but is not limited to, surfactants such as immunostimulatory complexes, Freunds incomplete adjuvant, LPS analogs (e.g., monophosphoryl ester A), cell wall 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, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, CD40, CD40L, vascular endothelial 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 NI K, 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 their functional fragments.

Another aspect of the present invention provides 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 panleukopenia.

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

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention designs and screens antigen sequences to obtain antigen peptides and mRNA nucleic acid molecules with better expression effects;

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 panleukopenia virus, which is better than the existing vaccines on the market.

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;

4. The vaccine prepared by the present invention is suitable for large-scale production.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a fluorescence expression diagram of different FPV antigen mRNA stock solutions;

Figure 2 is a schematic diagram of the immunization procedure.

DETAILED DESCRIPTION

The technical solutions in the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1: mRNA sequence screening

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

Table 1 Summary of sequence screening information

Artificially synthesize plasmid DNA sequence, the DNA sequence contains elements related to RNA transcription, and at the same time increase the signal peptide sequence and add EGFP fluorescent tag to facilitate the screening of in vitro expression of different antigen designs. The plasmid is transformed into Escherichia coli for amplification. The plasmid purified by fermentation is linearized with restriction endonuclease BspQ1. Transcribe with T7 in vitro transcription kit to obtain uncapped mRNA. Digest the transcription template with DNaseI respectively, and purify the mRNA by precipitation method. Cap the mRNA with Cap1 capping kit, and purify the capped mRNA with mRNA purification kit respectively. Dissolve the purified mRNA in acidic sodium citrate buffer, detect its concentration with nanodrop, and set aside.

In vitro expression detection of mRNA stock solution:

a. Plate laying: CRFK cells (10 ⁶ cells/well) were cultured in a 24-well cell culture plate, and the cell plate was placed in a CO ₂ incubator at 37°C for 24 h;

b. Transfection: Take out the culture plate and replace the culture medium with serum-free medium; mix 2.5ug mRNA stock solution and lipo3000 to make a premix, incubate at room temperature for 5 minutes; slowly add the premix to the culture plate;

c. Culture: Place the culture plate in a _CO2 incubator and culture at 37°C for 48 hours;

d. Fluorescence photography: Take photos every 12 hours to track the expression status.

The results are shown in Figure 1: The antigen design with truncated N-terminus and C-terminus of FPV VP2 protein expressed better in vitro, better than the other three designs. Therefore, this design with better expression was used as a candidate antigen design for vaccine development for further study.

Example 2: Lipid Nanoparticles Encapsulating mRNA Antigens

The mRNA solution in Example 1 is encapsulated with a formula containing cationic lipid ALC-0315, SM-102, DLin-MC3-DMA or steroid-cationic lipid compound 5, compound 11, or compound 17; the cationic lipid: neutral phospholipid: steroid lipid: polyethylene glycol (PEG)-lipid is dissolved and mixed in ethanol at a molar ratio of 45:10:43:2 or steroid-cationic lipid: neutral phospholipid: polyethylene glycol (PEG)-lipid at a molar ratio of 49.25:49.25:1.5. The total flow rate of the nano drug manufacturing equipment is set to 12ml/min. The mRNA solution and the lipid mixed solution are encapsulated at a flow rate ratio of 3:1. After the encapsulation is completed, the tangential flow filtration system is ultrafiltered to exchange the liquid and collect the sample, and a stabilizer solution (such as sucrose) is added to obtain an mRNA vaccine (mRNA-LNP). Sampling is measured to obtain the encapsulation rate, average particle size, PDI and Zeta potential.

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

Example 3: Cat Immunization and Detection

Immunoassay:

Twenty cats aged 2-3 months were divided into groups and the immunization experiment was performed as shown in Table 3: after immunization, the vital signs and clinical manifestations (body temperature, mental state, food intake, feces, and drinking water) of the experimental cats were observed and recorded every day, body weight was measured every 7 days, venous blood was collected every 14 days, serum was separated, and the neutralizing antibody titer against FPV was detected.

Table 3: Experimental design for FPV immunogenicity assessment

Evaluation of humoral immunity of FPV

The serum collected above was separated and tested for FPV, and a neutralization titer experiment was performed. The results showed that the neutralizing antibody titer of the virus was qualified, as shown in Table 4. The test results showed that the mRNA vaccine prepared by the present invention stimulated the body to produce a higher level of neutralizing antibodies against FPV, especially design 2, the antibody level could reach 1:1024 two weeks after the second immunization. Compared with the positive control group of Miao Sanduo, it can not only produce neutralizing antibodies more quickly, but also the level of neutralizing antibodies is significantly higher.

Table 4: Feline parvovirus neutralizing antibody titer (1:n)

In the present invention, SEQ ID NO: 1 is the amino acid sequence encoding the FPV VP2 protein:

SEQ ID NO: 2 is an amino acid truncated sequence encoding FPV VP2 protein:

SEQ ID NO: 3 is the N-terminal truncated sequence of VP2 protein:

SEQ ID NO: 4 is the C-terminal truncated sequence of VP2 protein

Claims (17)

An antigenic peptide comprising a truncated form of the VP2 protein of feline panleukopenia virus (FPV), characterized in that the truncated form of the VP2 protein retains the protein region of the VP2 protein from position 33 to position 575 from the N-terminus to the C-terminus. The antigenic peptide according to claim 1 is characterized in that the amino acid sequence encoding the truncated form of the VP2 protein is as shown in SEQ ID NO: 2, or has at least 75% homology with SEQ ID NO: 2; preferably, it has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with SEQ ID NO: 2. The antigen peptide according to claim 1 or 2, characterized in that a signal peptide is inserted into the N-terminus of the antigen peptide. The antigen peptide according to claim 3 is 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. A nucleic acid molecule encoding an antigenic peptide comprising a truncated form of the feline panleukopenia virus VP2 protein as described in any one of claims 1 to 4. The nucleic acid molecule according to claim 5 is characterized in that the nucleic acid molecule is an mRNA nucleic acid molecule. The nucleic acid molecule according to claim 6 is characterized in that 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 by completely replacing natural uridine with 1-methyl-pseudouridine. The nucleic acid molecule according to claim 7 is characterized in that the nucleic acid molecule also includes a 5' cap structure, a 5' non-coding region, a 3' non-coding region and/or an mRNA sequence of a poly(A) tail. An mRNA composition for preventing feline panleukopenia, characterized in that the mRNA composition comprises the nucleic acid molecule according to any one of claims 6 to 8. The mRNA composition according to claim 9, characterized in that the mRNA composition also includes a pharmaceutically acceptable carrier. 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. A kit for diagnosing or detecting feline panleukopenia virus, characterized in that the kit comprises an antigenic peptide comprising a truncated form of the feline panleukopenia virus VP2 protein as described in any one of claims 1 to 4, a nucleic acid molecule as described in any one of claims 5 to 8, an mRNA composition as described in any one of claims 9 to 10, or a biomaterial as described in claim 11. A vaccine for preventing feline panleukopenia, characterized in that the vaccine comprises the antigenic peptide according to any one of claims 1 to 4 or the nucleic acid molecule according to any one of claims 5 to 8. The vaccine according to claim 13 is 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 nanoparticles contain 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. A method for preparing an mRNA vaccine for preventing feline panleukopenia, characterized in that the vaccine is prepared by encapsulating the mRNA nucleic acid molecules described in any one of claims 5 to 8 in lipid nanoparticles. The method for preparing an mRNA vaccine according to claim 15 is characterized in that the vaccine is prepared by dissolving cationic lipids, neutral phospholipids, steroid lipids, and polyethylene glycol (PEG)-lipids in a solvent or dissolving steroid-cationic lipid compounds, neutral phospholipids, and polyethylene glycol lipids in a solvent and then mixing them with the mRNA nucleic acid molecule according to any one of claims 5 to 8. Use of the antigen peptide according to any one of claims 1 to 4, the nucleic acid molecule according to any one of claims 5 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 panleukopenia.