Immunogenic fusion proteins against feline coronaviruse

Abstract

Immunogenic fusion proteins against infectious animal diseases caused by feline coronavirus (FCoV). A fusion protein is disclosed, which comprises a CD40-binding domain; an antigen of FCoV; a translocation domain located between the CD40-binding domain and the antigen, and a furin and / or cathepsin L cleavage site located between the CD40-binding domain and the translocation domain. Also disclosed are pharmaceutical compositions, expression vectors and use of the fusion proteins of the invention for eliciting an antigen-specific cell-mediated immune response, or for reducing, inhibiting, treating and / or ameliorating an animal disease caused by FCoV infection.

Description

[0001] IMMUNOGENIC FUSION PROTEINS AGAINST FELINE CORONAVIRUSE

[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (10040-005PCT_Sequence

[0004] ListingL_ST26_20250307.xml; size 56 KB; creation date March 7, 2025) are herein incorporated by reference in their entireties.

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to fusion proteins, and more specifically to immunogenic fusion proteins for eliciting antigen-specific cell-mediated immune responses against infectious animal diseases caused by feline coronavirus (FCoV).

[0007] BACKGROUND OF THE INVENTION

[0008] Feline coronavirus (FCoV) is a positive-stranded RNA virus that infects cats worldwide.

[0009] FCoV has two biotypes: feline enteric coronavirus (FECV), which infects the intestines, and feline infectious peritonitis virus (FIPV), which causes the disease feline infectious peritonitis (FTP). Feline coronavirus is typically shed in feces by healthy cats and transmitted by the fecal-oral route to other cats. In environments with multiple cats, the transmission rate is much higher compared to single-cat environments. The virus is insignificant until mutations cause it to be transformed from FECV to FIPV. FIPV causes feline infectious peritonitis, for which treatment is generally symptomatic and palliative only. There is no effective prevention or control measures against FCoV. There remains, therefore, a need for developing new vaccines for treating the infection caused by FCoV.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect, the invention relates to a fusion protein comprising: (a) a CD40-binding domain, located at the N-terminal of the fusion protein; (b) an antigen of feline coronavirus

[0012] (FCoV); (c) a translocation domain, which comprises a Pseudomonas Exotoxin A (PE) translocation peptide and is located between the CD40-binding domain and the antigen; and (d) a furin and / or cathepsin L cleavage site, located between the CD40-binding domain and the translocation domain.

[0013] In another aspect, the invention relates to a DNA fragment, or an expressing vector comprising a DNA fragment encoding a fusion protein of the invention.

[0014] The invention further relates to a pharmaceutical or a vaccine composition comprising a fusion protein of the invention and a pharmaceutical acceptable carrier and / or an adjuvant.

[0015] Yet in another aspect, the invention relates to use of a fusion protein, a pharmaceutical composition, or a vaccine composition of the invention in the manufacture of a medicament for eliciting an antigen-specific cell-mediated immune response, or for reducing, inhibiting, treating, and / or ameliorating an infectious animal disease caused by FCoV in an animal in need thereof.

[0016] In one embodiment, the animal is a felid species. In another embodiment, the animal is selected from the group consisting of tigers, lions, and domestic cats.

[0017] The invention also relates to a fusion protein, a pharmaceutical composition, or a vaccine composition of the invention for use in eliciting an antigen-specific cell-mediated immune response, or for use in reducing, inhibiting, treating, and / or ameliorating an infectious animal disease caused by FCoV in an animal in need thereof.

[0018] Alternatively, the invention relates to a method for eliciting an antigen-specific cell- mediated immune response, or for reducing, inhibiting, treating, and / or ameliorating an infectious animal disease caused by FCoV in an animal in need thereof, said method comprising administering an effective amount of a fusion protein, a pharmaceutical composition or a vaccine composition of the invention to the animal in need thereof.

[0019] BRIEF DESCRIPTION OF DRAWINGS

[0020] FIGs. 1- 2 are vector maps.

[0021] FIG. 3 is a schematic drawing depicting the fusion protein CD40L-TPE-FCoV of the invention. FIG. 4 is a graph illustrating the serum antigen-specific antibody levels in different animal groups. Placebo group: PBS treated on Days 0, 7, 14; group A: vaccinated on Day 0; group B: vaccinated on Day 14. The x- axis is time in days, y-axis is optical density which reflects serum antibody levels.

[0022] FIG. 5 is a graph showing IFN-γ+immunospots in the splenocytes from each animal group of FIG. 4.

[0023] FIGs. 6-8 are graphs illustrating the levels of IFN-γ, IL-2, and TNF-α, respectively, in the

[0024] CD4+memory T cells of each animal group of FIG. 4.

[0025] FIG. 9 is a graph of fluorescence quantification, indicating FCoV shedding in the feces of

[0026] FIP cats treated with a fusion protein of the invention. The dot line is the detection limits of the assay.

[0027] FIG. 10A is a graph showing the RT-PCR quantification of FCoV shedding in the feces of

[0028] FCoV-infected (non-FIP) cats treated with the fusion protein of the invention.

[0029] FIG. 10B is a graph showing the fluorescence quantification of FCoV shedding in feces of a FCoV-infected (non-FIP) cat treated with the fusion protein of the invention.

[0030] FIG. 11 and 12 are graphs respectively illustrating the body temperature and body weight of the cats pretreated with the fusion protein of the invention and further exposed to FCoV.

[0031] FIG. 13 and 14 are graphs respectively illustrating ALT and AST levels of the cats pretreated with the fusion protein of the invention and further exposed to FCoV.

[0032] FIG. 15 is a graph illustrating the survival rate of the cats pretreated with the fusion protein of the invention and further exposed to FCoV.

[0033] FIG. 16 is a graph illustrating the fold-induction of Thl-based immunity of each adjuvant group. The symbol # indicates all five adjuvant groups significantly increased fold changes in cell-mediated immunity as compared with the placebo group. FIG. 17-19 are graphs illustrating the induction levels of IFN-γ, IL-2, and TNF-α, respectively, in CD4 memory T cells of each adjuvant group.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] Definitions

[0036] Professional APCs and non-professional APCs use an MHC class I molecule to display endogenous peptides on the cell membrane. These peptides originate within the cell itself, in contrast to the exogenous antigen displayed by professional APCs using MHC class II molecules. Cytotoxic CD8* T cells can interact with antigens presented by the MHC class I molecule.

[0037] CD40 is a costimulatory protein expressed on antigen-presenting cells (e.g., dendritic cells. macrophages, and B cells). The binding of CD40L to CD40 activates antigen-presenting cells and induces a variety of downstream effects. CD40 is a drug target for cancer immunotherapy.

[0038] The term “a CD40-binding domain" refers to a protein that can recognize and binds to

[0039] CD40. A CD40-binding domain may be selected from one of the following: “CD40 ligand

[0040] (CD40L) or a functional fragment thereof*, “an anti-CD40 antibody or a functional fragment thereof.”

[0041] The terms “CD40L”, “CD40 ligand” and “CD 154” are interchangeable. CD40L binds to

[0042] CD40 (protein) on antigen-presenting cells (APC), which leads to many effects depending on the target cell type. CD40L plays a significant role in co-stimulation and regulation of the immune response via T cell priming and activation of CD40-expressing immune cells. US 5,962,406 discloses the nucleotide and amino acid sequence of CD40L. The CD40L could be in the form of monomer, dimer or trimer. In some embodiments, the CD40L used in the invention is a monomer.

[0043] The terms “anti-CD40 antibody”, “CD40-specific antibody”, and “an antibody specifically against CD40” are interchangeable. When the term “consist substantially of is used in describing an amino acid sequence of a polypeptide, it means that the polypeptide may or may not have a starting amino acid “M”

[0044] (translated from a start codon AUG) at N-terminal as a part of the polypeptide, depending on protein translation requirements. For example, when the second antigen fused to the first antigen, the starting amino acid “M” of the second antigen could be omitted or kept.

[0045] As used herein, “a translocation domain” is a polypeptide having biological activity in translocating a fused or linked antigen across an endosomal membrane into cytosol of a cell. The translocation domain guides or facilitates the antigen toward class I major histocompatibility complex (MHC-1) pathway (i.e., a cytotoxic T cell pathway) for antigen presentation.

[0046] The term “a Pseudomonas Exotoxin A (PE) translocation peptide (TPE)” refers to a PE domain n peptide or a functional fragment thereof that has the biological activity in translocation.

[0047] The terms “furin and / or cathepsin L” or “furm / cathepsin L” are interchangeable.

[0048] The term “furin and / or cathepsin L cleavage site” refers to a short peptide sequence having at least four amino acids that can be cleaved by furin or cathepsin L, or by both furin and cathepsin L. Said cleavage site is a furin and / or cathepsin L protease sensitive site. It may be a peptide linker comprising said cleavage site that is introduced into the fusion protein. In addition, the furin and / or cathepsin L cleavage site may be a PE intrinsic protease cleavage site present in or adjacent to the translocation domain of the fusion protein.

[0049] The terms “antigen” and “immunogen” are interchangeable. An antigen refers to an antigenic protein or polypeptide derived from feline coronavirus (FCoV). Said antigen comprises at least one epitope for inducing desirable immune response. In some embodiments, the antigen is a polypeptide of at least 8 amino acids in length derived from FCoV. In some embodiments, the antigen comprises at least one antigenic polypeptide selected from spike (S) protein, membrane (M) protein or nucleocapsid (N) protein of FCoV. In some embodiments, the antigen is a fusion antigen comprising at least two antigenic polypeptides independently selected from S protein, M protein or N protein of FCoV. In some embodiments, the antigen is selected from the group consisting of FCoV SI, S2, M, N proteins, and any antigenic fragment thereof.

[0050] In some embodiments, the FCoV is feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV). In the working examples of the invention, said FCoV is a fatal biotype, i.e., feline infectious peritonitis virus (FIPV).

[0051] CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. T cell stimulation through CD28 in addition to the T-cell receptor (TCR) can provide a potent signal for the production of various interleukins (IL-6 in particular). CD28 is the receptor for CD80 (B7.1) and

[0052] CD86 (B7.2) proteins. When activated by Toll-like receptor ligands, the CD80 expression is upregulated in antigen-presenting cells (APCs). CD28 is the only B7 receptor constitutively expressed on naive T cells. Association of the TCR of a naive T cell with MHC:antigen complex without CD28:B7 interaction results in a T cell that is anergic.

[0053] The term “an effective amount" refers to the amount of an active fusion protein that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on rout of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.

[0054] The term “treating”, or “treatment” refers to administration of an effective amount of the fusion protein to a subject in need thereof, who has cancer or infection, or a symptom or predisposition toward such a disease, with the purpose of cure, alleviate, relieve, remedy, ameliorate, or reduce, inhibit the disease, the symptoms of it, or the predisposition towards it.

[0055] Such a subject can be identified by a health care professional based on results from any suitable diagnostic method. By ”0 to 12 repeats" or “2 to 6 repeats”, it means that all integer unit amounts within the range “0 to 12” or “2 to 6" are specifically disclosed as part of the invention. Thus, 0, 1 , 2, 3,

[0056] 4, . . . 10, 11 and 12" or “2, 3, 4, 5 and 6" unit amounts are included as embodiments of this invention.

[0057] When the phrase “the fusion protein CD40L-TPE-FCoV” is referred, the term “FCoV” therein stands for an antigen of feline coronavirus (FCoV).

[0058] Abbreviations: MCS, multiple cloning sites; Rapl, Ras-proximate-1 or Ras-related protein

[0059] 1; CD40, Cluster of differentiation 40; CDR, Complementarity-determining region; s.c.. subcutaneously; a.a., amino acid; FCoV, feline coronavirus; FCoV-N, N protein of FCoV; SI, receptor binding domain (RBD); S2, fusion domain (FP); M, membrane protein; N, nucleocapsid protein of FCoV.

[0060] Fusion proteins

[0061] The invention relates to a fusion protein comprising: (a) a CD40-binding domain, located at the N-terminal of the fusion protein; (b) an antigen of feline coronavirus (FCoV); (c) a translocation domain, which comprises a Pseudomonas Exotoxin A (PE) translocation peptide and is located between the CD40-binding domain and the antigen; and (d) a fiirin and / or cathepsin L cleavage site, located between the CD40-binding domain and the translocation domain.

[0062] The fusion proteins of the invention can elicit an antigen-specific T cell immune response via MHC class I antigen presentation pathway. They share a common mechanism of action.

[0063] Using CD40L-TPE-Ag (Ag stands for a suitable antigen) as an example, the mechanism of action is as follows:

[0064] (1) CD40L-TPE-Ag binds to a CD40-expressing cell (e.g., dendritic cell or macrophage) and is internalized via a CD40-mediated endocytosis; (2) CD40L-TPE-Ag is cleaved by furin protease and / or cathepsin L protease within an endosome to remove the CD40L fragment away from the TPE-Ag fragment;

[0065] (3) the TPE-Ag fragment is translocated across the endosomal membrane and enter the cytosol;

[0066] (4) the TPE-Ag fragment is digested by cytosol proteasomes to generate small antigens comprising epitopes;

[0067] (5) the small antigens are delivered via MHC class I pathway for antigen presentation; and

[0068] (6) a CDS* T cell specific immune response is induced or enhanced by T-cell recognizing these presented antigens.

[0069] No furin and / or cathepsin L cleavage site is present between the antigen and the translocation domain in the fusion protein of the invention. The presence of a furin and / or cathepsin L cleavage site and its location in the fusion protein permits removal of CD40-binding domain from the fusion protein after the furin and / or cathepsin L cleavage.

[0070] In one embodiment, the furin and / or cathepsin L cleavage site comprises or consists of 4-20 amino acids, preferred 4-10 amino acids, and more preferred 4-6 amino acids. In another embodiment, a furin and / or cathepsin L cleavage site comprises, consist of, or is an amino acid sequence selected from ( 1) RX’X2R, wherein X1and X2are any amino acid residue, or (2)

[0071] RXlRX2X3R, wherein X1 and X2are any amino acid residue, and X3is K, F or R.

[0072] In another embodiment, the fusion protein of the invention further comprises a peptide linker between the CD40-binding domain and the translocation domain, wherein the furin and / or cathepsin L cleavage site is present in said peptide linker. The peptide linker may comprise (a) a rigid linker (EAAAAK)n or (SEQ ID NO: 38)n; and (b) a cleavable linker comprising a furin and / or cathepsin L cleavage site, wherein n is an integer from 0-12, preferably from 2-6, more preferably from 3-4, and said furin and / or cathepsin L cleavage site comprises an amino acid sequence selected from (1) RX'X2R, wherein X1and X2are any amino acid residue, or (2) RX1RX2X3R, wherein X* and X2are any amino acid residue, and X3is K, F or R. In one embodiment, the peptide linker comprises (EAAAAK)3 and RX*RX2X3R (wherein X1is A, X2is

[0073] Y, X3is K). In another embodiment, the peptide linker comprises RXxX*R (wherein X1is V, X2is A) and (EAAAAKh.

[0074] In one embodiment, a fusion protein of the invention sequentially (from N- to C-tenninal) comprises: (a) a CD40-binding domain; (b) a furin and / or cathepsin L cleavage site; (c) a translocation domain comprising a PE translocation peptide (Tre); and (d) an antigen of FCoV.

[0075] In another embodiment, the fusion protein of the invention sequentially (from N- to C- terminal) comprises: (a) a CD40-binding domain; (b) a peptide linker comprising a furin and / or cathepsin L cleavage site; (c) a translocation domain comprising a PE translocation peptide

[0076] (TPE); and (d) an antigen of FCoV.

[0077] The TPEis a functional moiety having a biological activity in translocation. The furin and / or cathepsin L cleavage site is an amino acid sequence selected from (1) RX'X2R, wherein

[0078] X1and X2are any amino acid residue, or (2) RX1RX2X3R, wherein X1and X2are any amino acid residue, and X3is K, F or R or (3) an intrinsic fiirin cleavage site within or derived from PE.

[0079] In one embodiment, the PE translocation peptide (TPE) is domain II (a.a. residues 253-364;

[0080] SEQ ID NO: 9) of Pseudomonas Exotoxin A protein (full-length PE, SEQ ID NO: 4) or a functional moiety thereof.

[0081] In another embodiment, the PE translocation peptide (TPE) consists of 26-112 a.a. residues in length. The PE translocation peptide (Tre) comprises a minimal functional fragment of

[0082] GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO: 5).

[0083] In one embodiment, a PE translocation peptide (TPE) comprises an amino acid sequence that is at least 85%, 88%, 90%, 95%, 97% or 99% identical to SEQ ID NO: 5, 6, 7, 8 or 9. In another embodiment, a TPEcomprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 8 and 9. In another embodiment, a TPEis PE280-305 (SEQ ID NO: 5), PE28M13 (SEQ ID NO: NO: 6), PE268-313 (SEQ ID NO: NO: 7), PE253-313 (SEQ ID NO. 8), or

[0084] PE253-364 (SEQ ID NO: 9; full-length PE domain II).

[0085] A CD40-binding domain permits the fusion protein of the invention to bind to a CD40 receptor on a CD40-expressing cell (e.g., dendritic cell or macrophage). A CD40-binding domain may be one selected from the group consisting of (i) a CD40 ligand (CD40L) or a functional fragment thereof; and (ii) a CD40-specific antibody or a functional fragment thereof. In one embodiment, a functional fragment of CD40L is a truncated CD40L substantially lacking transmembrane and cytoplasmic regions of the full-length CD40L.

[0086] In another embodiment, a CD40L or a functional fragment thereof consists of 153-261 a.a. residues in length. In another embodiment, a CD40L comprises a minimal functional fragment of

[0087] SEQ ID NO: 16 or 19. Further in another embodiment, a CD40L or a functional fragment thereof consists of 153-261 a.a. residues in length and said CD40L comprises a minimal functional fragment of SEQ ID NO: 16 or 19.

[0088] In one embodiment, a CD40L comprises an amino acid sequence that is at least 85%, 88%,

[0089] 90%, 95%, 97% or 99% identical to SEQ ID NO: 14, 15, 16, 17, 18 or 19. In another embodiment, the CD40L is selected from the group consisting of SEQ ID NO: 14, 15, 16, 17, 18 or 19.

[0090] In another embodiment, a CD40-binding domain is a CD40-specific antibody (or anti-

[0091] CD40 antibody). A CD40-specific antibody is an antibody specifically recognizing and binding to CD 40 protein. A CD40-specific antibody can bind to CD40 protein on a CD40-expressing cell.

[0092] In one embodiment, the CD40-specific antibody comprises a heavy chain variable domain

[0093] (VH) and a light chain variable domain (VL), wherein the VH comprises the amino acid sequence of SEQ ID NO: 22; and the VL comprises the amino acid sequence of SEQ ID NO: 23. In another embodiment, the CD40-specific antibody is selected from the group consisting of a single chain variable fragment (scFv), a diabody (dscFv), a triabody, a tetrabody, a bispecific- scFv, a scFv-Fc, a scFc-CH3, a single chain antigen-binding fragment (scFab), an antigen- binding fragment (Fab), Faba, a minibody and a fully antibody.

[0094] In another embodiment, the CD40-binding domain is a CD40-specific scFv (anti-CD40 scFv) comprising a heavy chain variable domain (VH), a light chain variable domain (VL) and a flexible linker (L) connecting the VH and the VL. In one embodiment, the CD40-specific scFv comprises SEQ ID NO: 20 or 21.

[0095] In another embodiment, the CD40-binding domain according to the invention is (i) a CD40- specific antibody or a binding fragment thereof, or (ii) a CD40-specific single chain variable fragment (scFv) or a binding fragment thereof; said CD40-specific antibody or said CD40- specific scFv comprising a VH and a VL, wherein: (a) the VH comprises SEQ ID NO: 22; and (b) the VL comprises SEQ ID NO: 23.

[0096] In another embodiment, the CD40-specific antibody or CD40-specific scFv comprises a VH and a VL, the VH comprising VH CDR1 , VH CDR2 and VH CDR3; and the VL comprising VL

[0097] CDR1, VL CDR2 and VL CDR3, wherein: (i) the VH CDR1, VH CDR2 and VH CDR3 comprises

[0098] SEQ ID NO: 24, 25 and 26, respectively; and (ii) the VL CDR1 , VL CDR2 and VL CDR3 comprises SEQ ID NO: 27, 28 and 29, respectively.

[0099] In another embodiment, the CD40-binding domain is a CD40-specific scFv comprising a

[0100] VH and a VL, wherein: (a) the VH comprises SEQ ID NO: 22; and (b) the VL comprises SEQ ID

[0101] NO: 23.

[0102] In another embodiment, the fusion protein of the invention further comprises an endoplasmic reticulum (ER) retention sequence located at the C-terminal of the antigen, with the proviso that the translocation domain comprises a PE translocation peptide (TPE). The ER retention sequence may comprise SEQ ID NO: 30, 31, 32, 33 or 34. In one embodiment, the ER retention sequence is SEQ ID NO: 30.

[0103] In another embodiment, the fusion protein of the invention further comprises a CD28- activating peptide located between the CD40-binding domain and the fiirin and / or cathepsin L cleavage site.

[0104] In one embodiment, the CD28-activating peptide consists of 28-53 a.a. residues in length. In another embodiment, the CD28-activating peptide comprises a minimal functional fragment of

[0105] SEQ ID NO: 35. In another embodiment, the CD28-activating peptide consists of 28-53 a.a. residues in length and said CD28-activating peptide comprises a minimal functional fragment of

[0106] SEQ ID NO: 35.

[0107] In another embodiment, the CD28-activating peptide comprises an amino acid sequence that is at least 85%, 88%, 90%, 95%, 97% or 99% identical to SEQ ID NO: 35, 36 or 37. In another embodiment, the CD28-activating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36 and 37. In another embodiment, the CD28- activating peptide is SEQ ID NO: 35, 36 or 37.

[0108] In one embodiment, an antigen comprises an ammo acid sequence that is at least 80%, 85%,

[0109] 90%, 95% or 99% identical to SEQ ID No: 39, 40, 41 or 42. In another embodiment, an antigen is a peptide that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID No: 39, 40, 41 or

[0110] 42. In another embodiment, an antigen is a peptide with an amino acid sequence of SEQ ID No:

[0111] 39, 40, 41 or 42.

[0112] In one embodiment, said antigen comprises at least one epitope for inducing a desired immune response, preferably containing 1 to 50 epitopes, more preferably containing 1 to 20 epitopes. The antigen may be a single antigen or an antigenic fragment thereof, or a fusion antigen comprising at least two antigenic polypeptides fused together with or without a linker between the two antigenic polypeptides.

[0113] A fusion antigen may have a rigid linker, (EAAAAK)n, connecting two different antigenic polypeptides, wherein n is an integer from 0-12, preferably from 2-6, more preferably from 3-4.

[0114] In other words, the rigid linker comprises 0 to 12 repeats, 2 to 6 repeats or 3 to 4 repeats of the sequence EAAAAK (SEQ ID NO. 38).

[0115] The fusion protein of the invention may further comprise a rigid linker between the CD40- binding domain and the furin and / or cathepsin L cleavage site. The rigid linker comprises 0 to 12 repeats of the amino acid sequence EAAAAK (SEQ ID NO: 38). The rigid linker may be

[0116] (EAAAAK)n, or (SEQ ID NO: 38)n, wherein n is an integer from 0-12, preferably from 2-6, more preferably from 3-4. In one embodiment, the rigid linker comprises 2 to 6 repeats or 3 to 4 repeats of SEQ ID NO: 38.

[0117] In another embodiment, the fusion protein of the invention comprises, or consists substantially of, an amino acid sequence that is at least 85%, 88%, 90%, 95% or 99% identical to

[0118] SEQ ID NO: 43. Further in another embodiment, the fusion protein of the invention comprises, or consists substantially of, an amino acid sequence selected from the group consisting of SEQ

[0119] ID NO: 43. In one embodiment, the fusion protein of the invention has 300-1500 amino acids in length.

[0120] The invention further relates to a fusion protein in the manufacture of a medicament for eliciting an antigen-specific cell-mediated immune response and / or an antigen-specific humoral immune response, or for reducing, inhibiting, treating, and / or ameliorating an infectious animal disease caused by FCoV in an animal in need thereof. In one embodiment, the animal are domestic and nondomestic felid species. The domestic felid species are domestic cats. The nondomestic felid species include, but not limited to, Cheetahs (Acinonyx jubatns), European wildcats (Felis Silvestris'), a tiger (Panthera tigris), a mountain lion (Puma concolor), and lion

[0121] (Panthera leo).

[0122] Pharmaceutical composition / Vaccine of the invention

[0123] The invention further relates to a pharmaceutical composition or a vaccine, which comprises:

[0124] (a) the fusion protein of the invention; and

[0125] (b) a pharmaceutical acceptable carrier and / or an adjuvant.

[0126] The vaccine of the invention is a prophylactic and / or therapeutic vaccine. In one embodiment, the fusion protein is for use in eliciting an antigen-specific cell-mediated immune response and antigen-specific humoral response against FCoV in an uninfected or infected animal.

[0127] The term “carrier" or “pharmaceutically acceptable carrier” includes any and all solvents. dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art

[0128] (see, for example, Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company,

[0129] 1990, pp. 1289-1329).

[0130] Suitable adjuvants include, but not limited to, (1) oil-in-water (O / W) adjuvant (e.g.,

[0131] MONTANIDE™ ISA 15A VG, MONTANIDE™ ISA 35 VG, MONTANIDE™ ISA28R VG, and SUMMIT™ S350), (2) water-in-oil (W / O) adjuvant (e.g., MONTANIDE™ ISA 61 VG,

[0132] MONTANIDE™ ISA 71 VG, MONTANIDE™ ISA 71R VG, MONTANIDE™ ISA 761 VG,

[0133] MONTANIDE™ ISA 78 VG, MONTANIDE™ ISA 763B VG, MONTANIDE™ ISA 660 VG, and Freund’s incomplete adjuvant), (3) water-in-oil-in-water (W / O / W) adjuvant (e.g.,

[0134] MONTANIDE™ ISA 201 VG, MONTANIDE™ ISA 206 VG, and MONTANIDE™ ISA 207 VG); (4) aluminum salts adjuvant (e.g., aluminum hydroxide and aluminum phosphate); (5) saponin-based adjuvant (e.g., GPI-0100, Quil A or QS-21); (6) Toll-like receptor (TLR) agonist adjuvant (e.g., Poly I:C, monophosphoryl lipid A and CpG oligonucleotide); (7) chitosan-based adjuvant (e.g. ViscoGel and ZenoParticle CH-100) and (8) a combination of above mentioned two or more adjuvants.

[0135] The CpG oligonucleotide adjuvant includes, but not limited to, class A CpG (i.e., CpG 1585,

[0136] CpG2216 or CpG2336), class B CpG (i.e„ CpG1668, CpG1826, CpG2006, CpG2007, CpG

[0137] BW006 or CpG D-SL01) and class C CpG (i.e„ CpG2395, CpG M362 or CpG D-SL03).

[0138] Another suitable CpG adjuvant is CpG1018. In one embodiment, the adjuvant is a CpG oligonucleotide (ODN).

[0139] Chitosan was researched and developed to enhance the effectiveness of different vaccines by optimizing formulations. Chitosan-based adjuvants are known for their ability to enhance the antigenicity of vaccines, promoting innate and adaptive immune responses (Aishwarya Rathore et al. (2022), Biomed J Sci A Tech Res, 45(I)-2022. BJSTR. MS.ID.007140.). A chitosan adjuvant could be prepared by a known method, such as the method disclosed in patent

[0140] CN109833474B. In one embodiment, the adjuvant is a chitosan-based adjuvant.

[0141] In one embodiment, the fusion protein of the invention could be formulated with at least one adjuvant selected from a water-in-oil (W / O) adjuvant, a Toll-like receptor (TLR) agonist adjuvant or a chitosan-based adjuvant. In one embodiment, the fusion protein of the invention could be formulated with a water-in-oil (W / O) adjuvant. In one embodiment, the fusion protein of the invention could be formulated with a Toll-like receptor (TLR) agonist adjuvant. In one embodiment, the fusion protein of the invention could be formulated with a CpG oligonucleotide

[0142] (ODN) adjuvant. In one embodiment, the fusion protein of the invention could be formulated with a chitosan-based adjuvant. The formulated pharmaceutical compositions or vaccines are expected to exhibit potent immune response against FCoV and prevent and / or treat FIP. The pharmaceutical composition / vaccine may be an enteral or a parenteral dosage form, suitable for transdennal, transmucosal, nasopharyngeal, pulmonary, or direct injection, or for systemic (e.g., parenteral) or local (e.g., intratumor or intralesional injection) administration.

[0143] Parenteral injection may be via intravenous (z.v.), intraperitoneal (z.p.), intramuscular (z.zn.), subcutaneous (s.c.) or intradermal (z.<Z.) routes. The pharmaceutical composition may also be administered orally, e.g., in the form of tablets, coated tablets, dragdes, hard and soft gelatine capsules.

[0144] The dosage of the fusion protein may vary, depending on the disease to be controlled, the age and the individual condition of the animal and the mode of administration. The dosage may be fitted to individual requirements in each case to obtain a therapeutically effective amount of the fusion protein of the invention to achieve a desired therapeutic response.

[0145] For infected animals, a single dosage of about 0.01 to 10 mg, especially about 0.01 to 5 mg, comes into consideration. Depending on severity of the disease and the precise pharmacokinetic profile, the fusion protein may be administered with one dosage unit per week, bi-week, or month, and totally give 1 to 6 dosage units per cycle to satisfy such treatment.

[0146] In one embodiment, the invention provides a kit or a packaged pharmaceutical composition comprising a fusion protein of the invention and instructions for using thereof to treat one or more symptoms of an infectious disease caused by FCoV in an animal in need thereof.

[0147] EXAMPLES

[0148] Methods and Materials

[0149] Table 1 shows SEQ ID numbers of and the corresponding polypeptides / fusion proteins.

[0150] Table 1

[0151] Flow cytometry. Splenocytes were treated with a stimulator at 37°C for 2 hrs, incubated with Brefeldin A and Monensin for 2 hours, and harvested for immunostaining simultaneously with antibodies APC / Cy7-conjugated anti-CD3, PerCP / Cy5.5-conjugated anti-CD4, FITC- conjugated anti-CD8, PE-conjugated anti-CD44 and APC-conjugated anti-CD62L. The splenocytes were washed, permeabilized, fixed and intracellularly stained simultaneously with antibodies PE-conjugated anti-IFN-γ and PE / Cy7-conjugated anti-IL-2 and eFluor450- conjugated anti-TNF-α. Intracellular cytokines (IFN-γ, IL-2 or TNF-α) induced in the CD8+ and / or CD4+ memory T cells were analyzed by Gallios Flow Cytometer and Kaluza software.

[0152] Enzyme-linked immunospot (ELISpot) assay. Splenocytes, 2x105cells / well, were seeded in triplicate in a murine IFN-γ 96-well CTL IMMUNOSPOT® plate. The cells were incubated for 24 hours at 37°C in the presence or absence of a stimulator and discarded.

[0153] Captured IFN-γ was detected by biotin-conjugated anti-murine IFN-γ antibody. IFN-γ- immunospots were developed according to the manufacturer’s instructions. IFN-γ-immunospots were scanned and counted with a CTL IMMUNOSPOT® S5 Micro analyzer. The results were presented as IFN-γ+" immunospots per million splenocytes.

[0154] Indirect enzyme-linked immunosorbent assay (ELISA). To capture antigen-specific antibodies of interest, corresponding antigenic protein or peptide was synthesized and coated on 96-well plates at 1 pg / well overnight at 4°C. After washing, the plates were blocked with BSA in

[0155] PBS. Serum samples were serially diluted and 100 pl of the dilutions were incubated on the antigen-coated plates for 2 hours at 37°C. Captured antigen-specific antibodies were detected by horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for 30 minutes at 37°C, color developed with TMB (tetramethylbenzidine), and the relative titers of antigen-specific antibodies in the serum samples were determined.

[0156] Reverse transcription polymerase chain reaction (RT-PCR). Feline fecese were collected for RNA analysis. RNA was extracted from 200 μl of a 10% fecal suspension in PBS using a nucleic acid detection kit (BioGuard, Taiwan) according to the manufacturer's instructions. After RNA extraction, the extracted RNA was amplified with a Bioguard Real-time

[0157] PCR analyzer. The results were presented as Ct value, which reflects the quantity of RNA in the sample.

[0158] Feline Coronavirus (FCoV) Quantitative Test. The pathogen FCoV was detected with a commercial fluorescence immunochromatographic rapid test (Dawnsail Biotech, China).

[0159] Swabed fecal samples were dissolved in preservation buffer and an 80 pl aliquot was added to the test paper in the Dawnsail fluorescent reader and analyzed. The results were collected after

[0160] 10 minutes and represented as IU values, which reflects the levels of virus.

[0161] ALT and AST activity determination. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities were determined by substrate assays. Both were measured by conversion of NADH to NAD+. The decrease in NADH was measured as a reduction in light absorbance at 340 nm. The results were calibrated to IFCC reference materials and expressed as units per liter (U / L) where one unit represents the conversion of 1 pmol of substrate per minute.

[0162] Statistical analysis. Using t-test, results considered significant when p<0.05. The symbol * means statistical significance, p<0.05. Example 1

[0163] Construction of Expression Vector

[0164] Vector CD40L-TPE-FCoV for expression of a fusion protein with a FCoV-N antigen was generated (FIG. 1). Plasmid pTAC-MAT-Tag-2 (Catalog No. E5405, Sigma-Aldrich) was digested with HtnSXUSalL A DNA fragment encodingm^nCD40L-Linker-PEAco1-xw*Sail, which has CD40L108-261, a cleavable linker and a PE translocation peptide (PE280-305), was PCR synthesized, digested with HmdlU / San. and inserted into the plasmid pTAC-MAT-Tag-2 to generate an intermediate vector P07-His-pNC (FIG. 2). A DNA fragment encoding FCoV-N protein (SEQ ID NO: 42) carrying a His tag was inserted into the intermediate vector P07-His- pNC to obtain a final vector CD40L-Tre-FCoV for expression of a fusion protein with a FCoV N antigen (FIG. 1).

[0165] Other FCoV antigens of interest, e.g., spike protein S 1 , membrane protein M, or antigenic peptides of FCoV antigens may be inserted into the intermediate vector P07-His-pNC (FIG. 2) for expression of alternate fusion protein antigens (FIGs. 1 and 3).

[0166] Vector CD40L-TPE-FCoV. A vector CD40L-TPE-FCoV (FIG. 1) was constructed to exp-ess the fusion protein CD40L-TPE-FCoV (SEQ ID NO: 43; FIG. 3), which comprises: (a) a truncated CD40 ligand CD4OLios-26i (SEQ ID NO: 19); (b) a cleavable peptide linker, comprising (EAAAAK); (SEQ ID NO: 3) and RX’RX2X3R (wherein X1is A, X2is Y, X3is K);

[0167] (c) a PE translocation peptide; and (d) an antigenic FCoV N protein (SEQ ID NO: 42).

[0168] The cleavable linker allows furin and / or cathepsin L protease to cut the fusion protein of the invention for releasing the TPE-FCoV fragment from the fusion protein.

[0169] Using a similar method described above, any other antigen(s) of interest (e.g. spike protein, membrane protein or desired antigenic peptides) from FCoV may replace the antigen FCoV N protein and be inserted into the plasmid of FIG. 2 to generate an expression vector like FIG. 1 for expressing a fusion protein comprising tiie antigen(s) of interest. Example 2

[0170] Protein Expression

[0171] E. coli BL21 cells harboring the expression vector CD40L-TPE-FCoV were grown and fusion protein expression induced by IPTG. The fusion protein was purified and refolded from guanidine hydrochloride solubilized inclusion bodies.

[0172] Example 3

[0173] Both Humoral and Cell-mediated Immune Responses were Promoted

[0174] After Vaccination with the Fusion Protein

[0175] The fusion protein CD40L-TPE-FCoV as constructed above was used for studies described below. It was dissolved in 5 mM citrate buffer, pH 4.0. Unless otherwise stated, 0.5% Chitosan was used as the adjuvant. The final vaccine formulation contained 0.2 mg / mL of the fusion protein.

[0176] Female C57BL / 6NCrlBltw mice of 5 to 6-weeks-old were randomly divided into placebo and vaccinated groups, A and B (n=5 per group). The placebo group received PBS s.c. on days 0,

[0177] 7 and 14; the group A received the vaccine containing 100 μg fusion protein, s.c. on day 0, and the group B received the vaccine containing 100 pg, s.c. on day 14 according to the dosing schedule in Table 2. Sera were collected on days 0, 7, 14 and 21 for determination of antigen- specific humoral immune responses. Mice were sacrificed on day 21.

[0178] The levels of antigen-specific antibody in sera were analyzed by using ELISA.

[0179] Thioredoxin-FCoV nucleocapsid (N) fusion protein was used to coat the ELISA plates as a capture antigen.

[0180] Splenocytes harvested from day 21 mice were cultured and stimulated with an antigenic stimulator, which is a short peptide pool covering the sequence of the antigen FCoV N protein, for determination of antigen-specific cell-mediated immune responses by ELISpot assays of

[0181] IFN-γ-secretion. The splenocytes were also used to analyze intracellular cytokine induction (IFN-γ, IL-2 and TNF-α) in memory T cells by using flow cytometry. The frequency of IFN-γ- secreting splenocytes was analyzed by using Enzyme-linked immunospot (ELISpot) assay.

[0182] Table 2 shows the dosing schedule. “V”: vaccinated; not vaccinated.

[0183] Table 2

[0184] The level of antigen-specific antibody in the vaccinated group A sera was significantly higher than the placebo and group B by days 14 and 21. The antibody level in the group B sera was not different from the placebo group. The data indicates that 7 days are insufficient to elicit a significant antibody titer (group B) but 14 days are sufficient to elicit a significant antibody titer that lasts at least two weeks (group A, FIG. 4).

[0185] Both groups A and B showed a significant increase in the frequency of IFN-γ-secreting splenocytes. The data in group B (vaccinated on day 14) indicates that the vaccine of the invention can trigger a robust increase in the cellular immune response by day 7 after immunization. The data in group A (vaccinated on day 0) indicates that the robust increase in the cellular immune response triggered by the vaccine of the invention can sustain for at least 21 days after immunization (FIG. 5).

[0186] The levels of IFN-γ, IL-2 and TNF-α induction in the CD4+memory T cells from group A

[0187] (vaccinated on day 0) and group B (vaccinated on day 14) were found all increased as compared to the placebo group. The results indicates that the fusion protein vaccine of the invention can elicit a specific cell-mediated immune response (FIGs. 6-8). Therefore, the fusion protein of the invention can promote both humoral and cell-mediated immune responses. The prompt cellular immune response occurs early and can be beneficial for treating cats with FIP in the initial stages of the disease.

[0188] Example 4

[0189] Virus Levels Are Decreased After Vaccination of FIP Cats with the Fusion Protein

[0190] Two cats (Cl and C2) with FIP were subcutaneously administrated on days 0, 7, 14 and 21 with the fusion protein CD40L-TPE-FCoV formulated as mentioned in Example 3. Cat fecal samples were collected and the FCoV levels determined by Feline Coronavirus (FCoV)

[0191] Quantitative Test on days 7, 14, 21, 28 and 35. The results of fluorescence quantification were shown in FIG. 9. The results from days 7 and 14 were pooled and compared to the pool of days

[0192] 21, 28, 35 by unpaired T-test The two pools were statistically significant from each other at P <

[0193] 0.05. This indicates a statistically significant decrease in the virus levels after vaccination with the fusion protein CD40L-TPE-FCoV.

[0194] Cats C3, C4, C5, C6 diagnosed with FCoV infection but without symptoms of FIP were used to assess the effect of the fusion protein CD40L-TPE-FCoV on clearing the virus. The fusion protein CD40L-TPE-FCoV formulated as described in Example 3 was administered subcutaneously. Cat C3 received two injections on Days 0 and 14; cat C4 received one injection on day 0. Cats C5 and C6 as a control group were not vaccinated. Fecal samples were collected for analysis of the viral levels by reverse transcription polymerase chain reaction (RT-PCR) on

[0195] Day 0, 14, 21 and 35 (FIG.10A).

[0196] Cats C3, C4, C5, C6 all had detectable FCoV RNA levels by RT-PCR on Day 0. By day 21, cats C3 and C4, both vaccinated, were alive and had FCoV RNA levels decreased. In contrast, the control cats C5 and C6 had died, therefore no FCoV viral RNA measurements were possible on day 21. In another experiment, cat C7 diagnosed with FCoV infection but without symptoms of FIP was injected with the same fusion protein formulated as above on days 0 and 14, and fecal viral levels were measured by fluorescence immunoassay. On Day 0, cat 7 had detectable viral levels.

[0197] By Day 14, the fecal viral levels had dropped below the detection limits of the assay (FIG.10B).

[0198] Both the RT-PCR and fluorescence immunoassay methods show that cats with the FCoV infection vaccinated with the fusion protein CD40L-TPE-FCoV had their fecal viral load reduced to below the detection level and survived. In contrast, the control animals, which had received no vaccination, died before the end of the experiment. This indicates that the fusion protein CD40L-

[0199] TPE-FCOV vaccination was effective in keeping the animals alive.

[0200] Example 5

[0201] Vaccinalion of Cats with the Fusion Protein Prevents Fever, Weight Loss, Liver Damage and

[0202] Mortality caused by FCoV

[0203] Cats (4-10 months) were randomly divided into placebo and vaccinated groups (n=3 per group). The placebo group was treated s.c. with PBS. The vaccinated group was treated s.c. with the fusion protein as previously described. Twenty-one and forty-two days after the PBS or the fusion protein treatments, the cats were challenged i.p with the virus type II strain FIPV 79-1146

[0204] (102TCID50). Body temperature and weight were measured routinely, and sera were collected for determination of ALT and AST activities.

[0205] After the virus challenge, the vaccinated group showed a relatively stable body temperature and weight up to 35 days, while the placebo group showed persistently elevated body temperature with spikes over 39°C and weight loss (FIGs. 11-12). The serum liver enzyme levels of ALT and AST in the vaccinated group were stable without changes for at least 20 days post viral challenge. The placebo group showed elevated ALT and AST levels beginning at day 7 post viral challenge (FIGs. 13-14). All the animals in the vaccinated group survived during the entire observation period of 35 days. All the placebo group cats died by day 21 post viral challenge

[0206] (FIG. 15).

[0207] The results indicate that vaccination with the fusion protein of the invention can prevent fever, weight loss, liver damage and mortality caused by the virus FIPV 79-1146.

[0208] Example 6

[0209] Adjuvants Enhance Cell-mediated Immune Response of the Fusion Protein

[0210] Multiple adjuvants were evaluated for enhancement of the efficacy of the fusion protein vaccine in providing active acquired immunity to the infectious disease caused by FCoV. Five groups of adjuvants were tested, including: (1) chitosan: a chitosan adjuvant (0.5%); (2) ODN:

[0211] CpG1826 (200 ug / dose); (3) oil in water (O / W): SUMMIT™ S35O (20%); (4) water in oil

[0212] (W / O): ISA 660 VG (50%); and (5) mineral salts: aluminum phosphate (10%).

[0213] The fusion protein CD40L-TPE-FCOV as constructed above was dissolved in 5 mM citrate buffer (pH 4.0) and formulated in suspension with an adjuvant. Female C57BL / 6NCrlBltw mice of 5 to 6-weeks-old were randomly divided into placebo and adjuvant groups (n=5 per group).

[0214] On Day 0, the placebo group had no treatment, the adjuvant group received the fusion protein vaccine with an adjuvant. On day 7, mice were sacrificed, splenocytes harvested and cultured for determination of antigen-specific cell-mediated immune response.

[0215] Enhancement of Th 1 -based immunity by the adjuvants was evaluated by IFN-γTELISpot assay and expressed as fold changes. All five adjuvant groups significantly increased fold changes in cell-mediated immunity as compared with the placebo group (FIG. 16 - #).

[0216] The chitosan, ODN and W / O adjuvant groups showed significantly more enhancement of the effect of the fusion protein on induction of IFN-γ+splenocytes than the O / W and mineral salt group, each of which showed 10-fold of placebo (FIG. 16 - *). The ODN adjuvant group was the most efficacious, showing nearly 200-fold of placebo. The chitosan and W / O adjuvant groups each had nearly 100-fold increase in Th 1 -based immunity (FIG. 16). It was unexpected that the adjuvants chitosan, ODN and W / O groups exhibited more potent enhancement of the FCoV fusion protein vaccine effect than the O / W and mineral salts groups. It was also unexpected that the ODN group was the most potent adjuvant in enhancing the immunity of the fusion protein vaccine of the invention. These findings indicate that the enhancement efficacy of adjuvants are not predictable.

[0217] The intracellular cytokines IFN-γ, IL-2 and TNF-α in CD4+memory T cells were measured by flow cytometry. All adjuvant groups showed enhancement in induction of the cytokines IFN- y, IL-2 and TNF-α as compared with the placebo group (FIGs. 17-19).

[0218] The currently available vaccine for FCoV is inadequate. For example, VANGUARD®

[0219] Feline FIP Intranasal non-adjuvanted vaccine containing attenuated live virus is licensed for preventing FIP caused by feline infectious peritonitis virus (FIPV). However, according to the

[0220] American Animal Hospital Association (AAHA), it is not generally recommended at this time because its uncertain ability to uniformly prevent disease in North American cat populations does not justify its routine use. In addition, the benefits and risks of vaccination remain unclear.

[0221] We provide evidence showing that vaccination of cats with the fusion protein of the invention can prevent fever, weight loss, liver damage and mortality caused by FCoV. In cats afflicted with FIP, vaccination with the fusion protein of the invention can decrease viral load and prevent mortality.

[0222] All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

Claims

CLAIMSWhat is claimed is:

1. A fusion protein comprising:(a) a CD40-binding domain, located at the N-terminal of the fusion protein;(b) an antigen of feline coronavirus (FCoV);(c) a translocation domain, comprising a Pseudomonas Exotoxin A (PE) translocation peptide and located between the CD40-binding domain and the antigen; and(d) a furin and / or cathepsin L cleavage site, located between the CD40L and the translocation domain.

2. The fusion protein of claim 1, wherein the PE translocation peptide comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 5, 6, 7, 8, or 9, with 26-112 amino acids in length.

3. The fusion protein of claim 1, wherein the furin and / or cathepsin L cleavage site comprises an amino acid sequence of RX*X2R or RX1RX2X3R, wherein X* and X2are any amino acids, and X3is K, F or R.

4. The fusion protein of claim 1, wherein the CD40-binding domain is a CD40 ligand(CD40L) or a functional fragment thereof, or is an antibody specifically against CD40 or a functional fragment thereof.

5. The fusion protein of claim 4, wherein the CD40L comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 14, 15, 16, 17, 18 or 19, with 153-261 amino acids in length.

6. The fusion protein of claim 1 , wherein the antigen is selected from the group consisting ofFCoV SI, S2, M, N proteins, and any antigenic fragment thereof.

7. The fusion protein of claim 1, wherein the FCoV is feline infectious peritonitis virus(FIPV).

8. The fusion protein of claim 2, further comprising an endoplasmic reticulum (ER) retention sequence located at the C-terminus of the antigen.

9. A pharmaceutical composition, comprising:(i) the fusion protein of claim 1; and(ii) a pharmaceutical acceptable carrier and / or an adjuvant.

10. The pharmaceutical composition of claim 10, wherein the adjuvant is selected from the group consisting of a water-in-oil (W / O) adjuvant, a Toll-like receptor (TLR) agonist adjuvant and a chitosan-based adjuvant.

11. The fusion protein of claim 1 , wherein the antigen is FCoV nucleocapsid protein.

12. A pharmaceutical composition, comprising:(i) the fusion protein of claim 11 ; and(ii) an adjuvant, selected from chitosan-based adjuvants, water-in-oil (W / O) adjuvants, orToll-like receptor (TLR) agonist adjuvants.

13. Use of a fusion protein as claimed in any one of claims 1-8, or a pharmaceutical composition of claim 9, in the manufacture of a medicament for preventing fever, weightLoss, liver damage and mortality caused by FCoV in an animal in need thereof, or for decreasing viral load and preventing mortality in an animal afflicted with feline infectious peritonitis.

14. The use of claim 13, wherein the animal is a felid species.

15. The use of claim 13, wherein the animal is selected from the group consisting of tigers, lions, and domestic cats.

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