Immunogenic fusion proteins against feline coronavirus

Abstract

The present invention relates to immunogenic fusion proteins against infectious animal diseases caused by feline coronavirus (FCoV). The present invention discloses a fusion protein comprising a CD40 binding domain; a FCoV antigen; 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. The present invention also discloses pharmaceutical compositions, expression vectors and the use of the fusion proteins of the present invention for eliciting an antigen-specific cell-mediated immune response, or for reducing, inhibiting, treating and / or ameliorating animal diseases caused by FCoV infection.

Description

Technical Field

[0001] [Reference Electronic Sequence List] The contents of the electronic sequence list (10040-005PCT_Sequence Listing_ST26_20250307.xml; size: 56KB; creation date: March 7, 2025) are incorporated herein by reference in their entirety.

[0002] This invention relates to fusion proteins, and more particularly to immunogenic fusion proteins for inducing antigen-specific cell-mediated immune responses against infectious animal diseases caused by feline coronavirus (FCoV). Background Technology

[0003] Feline coronavirus (FCoV) is a positive-sense RNA virus that infects cats globally. There are two biotypes of FCoV: feline enteric coronavirus (FECV), which infects the intestines, and feline infectious peritonitis virus (FIPV), which causes feline infectious peritonitis (FIP). FCoV is typically shed in the feces of healthy cats and is transmitted to other cats via the fecal-oral route. Transmission rates are much higher in multi-cat environments than in single-cat environments. The virus was initially not prominent until a mutation occurred that caused it to transform from FECV to FIPV. FIPV causes feline infectious peritonitis, and treatment is usually limited to symptomatic and palliative care. Currently, there are no effective preventative or control measures against FCoV. Therefore, the development of new vaccines to treat infections caused by FCoV remains necessary. Summary of the Invention

[0004] On one hand, the present invention relates to a fusion protein comprising: (a) a CD40 binding domain located at the N-terminus of the fusion protein; (b) an antigen of feline coronavirus (FCoV); and (c) a translocation domain comprising Pseudomonas aeruginosa (…). Pseudomonas (d) Exotoxin A (PE) translocation peptide, located between the CD40 binding domain and the antigen; and (d) furin and / or cathepsin L cleavage sites, located between the CD40 binding domain and the translocation domain.

[0005] On the other hand, the present invention relates to a DNA fragment encoding the fusion protein of the present invention or an expression vector containing the DNA fragment.

[0006] This invention also relates to pharmaceutical or vaccine compositions comprising the fusion protein of this invention, as well as pharmaceutically acceptable carriers and / or adjuvants.

[0007] On the other hand, the present invention relates to the use of the fusion protein, pharmaceutical composition or vaccine composition of the present invention in the manufacture of a medicament for inducing an antigen-specific cell-mediated immune response or for reducing, inhibiting, treating and / or improving infectious animal diseases caused by FCoV in animals in need.

[0008] In one embodiment, the animal is a feline. In another embodiment, the animal is selected from a group consisting of tigers, lions, and domestic cats.

[0009] This invention also relates to the fusion proteins, pharmaceutical compositions, or vaccine compositions of the present invention for inducing antigen-specific cell-mediated immune responses, or for reducing, inhibiting, treating, and / or improving infectious animal diseases caused by FCoV in animals in need.

[0010] Furthermore, the present invention relates to a method for inducing an antigen-specific cell-mediated immune response, or for reducing, suppressing, treating, and / or improving infectious animal diseases caused by FCoV in animals in need, the method comprising administering an effective amount of the fusion protein, pharmaceutical composition, or vaccine composition of the present invention to the animals in need. Attached Figure Description

[0011] Figure 1 and Figure 2 It is a vector map.

[0012] Figure 3 This describes the fusion protein CD40L-T of the present invention. PE A schematic diagram of -FCoV.

[0013] Figure 4 This is a graph illustrating the serum antigen-specific antibody levels in different animal groups. Placebo group: treated with PBS on days 0, 7, and 14; Group A: vaccinated on day 0; Group B: vaccinated on day 14. The x-axis represents time (number of days), and the y-axis represents the optical density reflecting serum antibody levels.

[0014] Figure 5 It is a display Figure 4 Image of IFN-γ+ immunospots in spleen cells of various animal groups.

[0015] Figures 6 to 8 These are explanations Figure 4 CD4 of each animal group + A diagram showing the levels of IFN-γ, IL-2, and TNF-α in memory T cells.

[0016] Figure 9 This is a quantitative fluorescence chromatogram, showing the shedding of FCoV in the feces of FIP cats treated with the fusion protein of this invention. The dashed line represents the detection limit of this detection method.

[0017] Figure 10A This is a graph showing the RT-PCR quantification of FCoV exfoliated in the feces of FCoV-infected (non-FIP) cats treated with the fusion protein of the present invention.

[0018] Figure 10B This is a graph showing the quantitative fluorescence of FCoV shedding in the feces of FCoV-infected (non-FIP) cats treated with the fusion protein of the present invention.

[0019] Figure 11 and Figure 12 The figures illustrate the body temperature and weight of cats pretreated with the fusion protein of the present invention and further exposed to FCoV.

[0020] Figure 13 and Figure 14 The figures illustrate the ALT and AST levels in cats pretreated with the fusion protein of the present invention and further exposed to FCoV.

[0021] Figure 15 This is a graph illustrating the survival rate of cats pretreated with the fusion protein of the present invention and then further exposed to FCoV.

[0022] Figure 16 This is a graph illustrating the fold increase in Th1-based immune induction for each adjuvant group. The symbol # indicates a significant increase in the fold increase in cell-mediated immunity across all five adjuvant groups compared to the placebo group.

[0023] Figures 17 to 19 These are graphs illustrating the induced levels of IFN-γ, IL-2, and TNF-α in CD4+ memory T cells of each adjuvant group. Detailed Implementation

[0024] definition Professional APCs and non-professional APCs both present endogenous peptides on the cell membrane using MHC class I molecules. These peptides originate from the cell itself, unlike the exogenous antigens presented by professional APCs using MHC class II molecules. Cytotoxic CD8 + T cells can interact with antigens presented by MHC class I molecules.

[0025] CD40 is a co-stimulatory protein expressed on antigen-presenting cells, such as dendritic cells, macrophages, and B cells. Binding of CD40L to CD40 activates antigen-presenting cells and triggers a variety of downstream effects. CD40 is a drug target for cancer immunotherapy.

[0026] The term "CD40 binding domain" refers to a protein that can recognize and bind to CD40. The CD40 binding domain can be selected from either: "CD40 ligand (CD40L) or a functional fragment thereof", or "anti-CD40 antibody or a functional fragment thereof".

[0027] The terms "CD40L," "CD40 ligand," and "CD154" are used interchangeably. CD40L binds to CD40 (a protein) on antigen-presenting cells (APCs) and produces a variety of effects depending on the target cell type. CD40L plays an important role in the co-stimulation and regulation of immune responses by initiating and activating CD40-expressing immune cells via T cells. US 5,962,406 discloses the nucleotide and amino acid sequences of CD40L. CD40L can be in monomeric, dimeric, or trimeric form. In some embodiments, the CD40L used in this invention is a monomer.

[0028] The terms “anti-CD40 antibody,” “CD40-specific antibody,” and “antibody specifically targeting CD40” are used interchangeably.

[0029] When the term "consistently made up of..." is used to describe the amino acid sequence of a polypeptide, it means that the polypeptide may or may not have the N-terminal starting amino acid "M" (derived from the start codon AUG) as part of the polypeptide, depending on the requirements of protein translation. For example, when a second antigen fuses with a first antigen, the starting amino acid "M" of the second antigen may be omitted or retained.

[0030] As used in this article, a "translocation domain" is a polypeptide with the biological activity of transporting fused or linked antigens across the endosomal membrane into the cytoplasm of the cell. Translocation domains guide or facilitate antigen presentation toward the major histocompatibility complex class I (MHC-1) pathway (i.e., the cytotoxic T cell pathway).

[0031] The term "pseudomonas exotoxin A (PE) translocation peptide (T)" PE ")" refers to PE domain II peptides or their functional fragments that have translocation biological activity.

[0032] The terms “furin protease and / or cathepsin L” or “furin protease / cathepsin L” are used interchangeably.

[0033] 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. The cleavage site is a protease-sensitive site of furin and / or cathepsin L. It can be a peptide linker containing the cleavage site introduced into the fusion protein. Furthermore, the furin and / or cathepsin L cleavage site can be a PE intrinsic protease cleavage site located within the translocation domain of the fusion protein or a PE intrinsic protease cleavage site adjacent to the translocation domain of the fusion protein.

[0034] The terms "antigen" and "immunogen" are used interchangeably. An antigen refers to an antigenic protein or polypeptide derived from feline coronavirus (FCoV). The antigen contains at least one epitope for inducing a desired immune response. In some embodiments, the antigen is a polypeptide derived from FCoV with a length of at least 8 amino acids. In some embodiments, the antigen comprises at least one antigenic polypeptide selected from the 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 independent antigenic polypeptides selected from the S, M, or N proteins of FCoV. In some embodiments, the antigen is selected from the group consisting of FCoV S1, S2, M, N proteins and any antigenic fragments thereof.

[0035] In some embodiments, FCoV is feline infectious peritonitis virus (FIPV) or feline enteric coronavirus (FECV). In the working embodiment of the present invention, the FCoV is a fatal biotype, i.e., feline infectious peritonitis virus (FIPV).

[0036] CD28 (differentiation cluster 28) is one of the proteins expressed on T cells, providing the co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation via CD28 provides a strong signal for the production of various interleukins, especially IL-6. CD28 is the receptor for both CD80 (B7.1) and CD86 (B7.2) proteins. CD80 expression is upregulated in antigen-presenting cells (APCs) upon activation by Toll-like receptor ligands. CD28 is the only B7 receptor constitutively expressed on naive T cells. The absence of CD28:B7 interaction between the TCR and the MHC:antigen complex on naive T cells leads to a non-responsive state.

[0037] The term "effective dose" refers to the amount of active fusion protein required to produce a therapeutic effect in a treated subject. As those skilled in the art will appreciate, the effective dose will vary depending on the route of administration, the use of excipients, and the possibility of co-administration with other treatments.

[0038] The term "treatment" or "treatment" refers to the administration of an effective amount of a fusion protein to a subject in need of cancer or infection, or symptoms or predisposition to such diseases, with the aim of curing, alleviating, mitigating, remedying, improving, reducing, or suppressing the disease, its symptoms, or predisposition to it. Healthcare professionals can identify such subjects based on the results of any appropriate diagnostic method.

[0039] "0 to 12 repetitions" or "2 to 6 repetitions" refers to all integer unit quantities within the range of "0 to 12" or "2 to 6" that are specifically disclosed as part of this invention. Therefore, unit quantities of "0, 1, 2, 3, 4...10, 11 and 12" or "2, 3, 4, 5 and 6" are included as embodiments of this invention.

[0040] When mentioning "fusion protein CD40L-T" PE When "-FCoV" is used, the term "FCoV" stands for feline coronavirus (FCoV) antigen.

[0041] Abbreviations: MCS, multiple replication site; Rap1, Ras-proximate-1 or Ras-associated protein 1; CD40, differentiation cluster 40; CDR, complementarity-determining region; sc, subcutaneous; aa, amino acid; FCoV, feline coronavirus; FCoV-N, N protein of FCoV; S1, receptor-binding domain (RBD); S2, fusion domain (FP); M, membrane protein; N, nucleocapsid protein of FCoV.

[0042] Fusion protein The present invention relates to a fusion protein comprising: (a) a CD40 binding domain located at the N-terminus of the fusion protein; (b) a feline coronavirus (FCoV) antigen; (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 protease and / or cathepsin L cleavage site located between the CD40 binding domain and the translocation domain.

[0043] The fusion proteins of this invention can trigger antigen-specific T-cell immune responses via the MHC class I antigen presentation pathway. They share a common mechanism of action. (The text then mentions CD40L-T...) PE Taking -Ag (where Ag represents an appropriate antigen) as an example, its mechanism of action is as follows: (1) CD40L-T PE -Ag binds to cells expressing CD40 (such as dendritic cells or macrophages) and is internalized through CD40-mediated endocytosis; (2) CD40L-T PE-Ag is cleaved in endosomes by furin and / or cathepsin L to release the CD40L fragment from T PE -Removed from Ag fragment; (3) T PE -Ag fragments cross the endosomal membrane and enter the cytoplasm; (4) T PE -Ag fragments are digested by cytoplasmic proteasomes to generate small antigens containing epitopes; (5) Small antigens are delivered via the MHC class I pathway for antigen presentation; and (6) Inducing or enhancing CD8 by recognizing these presented antigens by T cells. + T-cell specific immune response.

[0044] In the fusion protein of this invention, there are no furin and / or cathepsin L cleavage sites between the antigen and the translocation domain. The presence and location of furin and / or cathepsin L cleavage sites in the fusion protein allow for the removal of the CD40 binding domain from the fusion protein after furin and / or cathepsin L cleavage.

[0045] In one embodiment, the furin and / or cathepsin L cleavage site comprises or is composed of 4 to 20 amino acids, preferably 4 to 10 amino acids, more preferably 4 to 6 amino acids. In another embodiment, the furin and / or cathepsin L cleavage site comprises, is composed of, or is selected from the following amino acid sequence: (1) RX 1 X 2 R, where X 1 and X 2 It is any amino acid residue, or (2) RX 1 RX 2 X 3 R, where X 1 and X 2 It is any amino acid residue, and X 3 It is K, F, or R.

[0046] In another embodiment, the fusion protein of the present invention further includes a peptide linker between a CD40 binding domain and a translocation domain, wherein furin and / or cathepsin L cleavage sites are present in the peptide linker. The peptide linker may include (a) a rigid linker (EAAAAK). n Or (SEQ ID NO: 38) n ; and (b) a cleavable linker comprising furin and / or cathepsin L cleavage sites, wherein n is 0 to 12, preferably 2 to 6, more preferably an integer of 3 to 4, and said furin and / or cathepsin L cleavage sites comprise an amino acid sequence selected from: (1) RX1 X 2 R, where X 1 and X 2 For any amino acid residue; or (2) RX 1 RX 2 X 3 R, where X 1 and X 2 X can be any amino acid residue. 3 The peptide linker is K, F, or R. In one embodiment, the peptide linker comprises (EAAAAK)3 and RX. 1 RX 2 X 3 R (where X) 1 Let A and X be the two numbers. 2 Let Y and X be the numbers. 3 (K). In another embodiment, the peptide linker includes RX. 1 X 2 R (where X) 1 Let V, X 2 For A) and (EAAAAK)3.

[0047] In one embodiment, the fusion protein of the present invention sequentially (from N-terminus to C-terminus) comprises: (a) a CD40 binding domain; (b) a furin and / or cathepsin L cleavage site; and (c) a PE translocation peptide (T... PE ) translocation domain; and (d) FCoV antigen.

[0048] In another embodiment, the fusion protein of the present invention sequentially (from N-terminus to C-terminus) comprises: (a) a CD40 binding domain; (b) a peptide linker comprising furin and / or cathepsin L cleavage sites; and (c) a PE translocation peptide (T... PE ) translocation domain; and (d) FCoV antigen.

[0049] T PE It is a functional part with translocation biological activity. The cleavage site of furin and / or cathepsin L is selected from the following amino acid sequences: (1) RX 1 X 2 R, where X 1 and X 2 For any amino acid residue; or (2) RX 1 RX 2 X 3 R, where X 1 and X 2 X can be any amino acid residue. 3 For K, F or R; or (3) an intrinsic furin cleavage site within or derived from PE.

[0050] In one implementation, PE translocation peptide (T) PE ) is domain II (aa residues 253 to 364; SEQ ID NO: 9) or a functional portion thereof of the Pseudomonas exotoxin A protein (full-length PE, SEQ ID NO: 4).

[0051] In another embodiment, PE translocation peptide (T PE It consists of 26 to 112 amino acid residues in length. PE translocation peptide (T...) PE The smallest functional fragment containing GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO: 5).

[0052] In one implementation, PE translocation peptide (T) PE It contains at least 85%, 88%, 90%, 95%, 97%, or 99% of the same amino acid sequence as SEQ ID NO: 5, 6, 7, 8, or 9. In another embodiment, T PE It comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, 6, 7, 8, and 9. In another embodiment, T PE For PE 280-305 (SEQ ID NO: 5), PE 280-313 (SEQ ID NO: 6), PE 268-313 (SEQ ID NO: 7), PE 253-313 (SEQ ID NO: 8) or PE 253-364 (SEQ ID NO: 9; full-length PE domain II).

[0053] The CD40 binding domain enables the fusion protein of the present invention to bind to the CD40 receptor on cells expressing CD40 (e.g., dendritic cells or macrophages). The CD40 binding domain may be selected from one of the following groups: (i) a CD40 ligand (CD40L) or a functional fragment thereof; and (ii) a CD40-specific antibody or a functional fragment thereof. In one embodiment, the functional fragment of CD40L is a truncated CD40L, substantially lacking the transmembrane and cytoplasmic regions of the full-length CD40L.

[0054] In another embodiment, CD40L or a functional fragment thereof consists of 153 to 261 aa residues in length. In another embodiment, CD40L comprises the minimum functional fragment of SEQ ID NO: 16 or 19. Additionally, in yet another embodiment, CD40L or a functional fragment thereof consists of 153 to 261 aa residues in length, and said CD40L comprises the minimum functional fragment of SEQ ID NO: 16 or 19.

[0055] In one embodiment, CD40L comprises at least 85%, 88%, 90%, 95%, 97%, or 99% of the same amino acid sequence as SEQ ID NO: 14, 15, 16, 17, 18, or 19. In another embodiment, CD40L is selected from the group consisting of SEQ ID NO: 14, 15, 16, 17, 18, or 19.

[0056] In another embodiment, the CD40 binding domain is a CD40-specific antibody (or anti-CD40 antibody). A CD40-specific antibody is an antibody that specifically recognizes and binds to the CD40 protein. A CD40-specific antibody can bind to the CD40 protein on cells expressing CD40.

[0057] In one implementation, the CD40-specific antibody includes a heavy chain variable domain (V... H ) and light chain variable domain (V L ), where V H Contains the amino acid sequence of SEQ ID NO: 22; and V L The amino acid sequence containing SEQ ID NO: 23.

[0058] In another embodiment, the CD40-specific antibody is selected from the group consisting of single-chain variable fragments (scFv), biantibodies (dscFv), triantibodies, tetraantibodies, bispecific-scFv, scFv-Fc, scFc-CH3, single-chain antigen-binding fragments (scFab), antigen-binding fragments (Fab), Fab2, microantibodies, and complete antibodies.

[0059] In another embodiment, the CD40 binding domain is a CD40-specific scFv (anti-CD40scFv) that includes a heavy chain variable domain (V H ), light chain variable domain (V L ) and connection V H and V L The flexible connector (L). In one embodiment, the CD40-specific scFv contains SEQ ID NO: 20 or 21.

[0060] 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 contains V H and V L , where: (a) V H Includes SEQ ID NO: 22; and (b) V LIncludes SEQ ID NO: 23.

[0061] In another embodiment, the CD40-specific antibody or CD40-specific scFv contains V H and V L V H Includes V H CDR1, V H CDR2 and V H CDR3; and V L Including V L CDR1, V L CDR2 and V L CDR3, where: (i)V H CDR1, V H CDR2 and V H CDR3 contains SEQ ID NO: 24, 25 and 26 respectively; and (ii) V L CDR1, V L CDR2 and V L CDR3 contains SEQ ID NO: 27, 28 and 29 respectively.

[0062] In another embodiment, the CD40 binding domain is a domain containing V H and V L CD40-specific scFv, where: (a) V H Includes SEQ ID NO: 22; and (b) V L Includes SEQ ID NO: 23.

[0063] In another embodiment, the fusion protein of the present invention further comprises an endoplasmic reticulum (ER) retention sequence located at the C-terminus of the antigen, provided that the translocation domain contains a PE translocation peptide (T). PE The ER retention sequence may include SEQ ID NO: 30, 31, 32, 33 or 34. In one embodiment, the ER retention sequence is SEQ ID NO: 30.

[0064] In another embodiment, the fusion protein of the present invention further comprises a CD28 activating peptide located between a CD40 binding domain and a furin and / or cathepsin L cleavage site.

[0065] In one embodiment, the CD28 activating peptide consists of 28 to 53 amino acid residues. In another embodiment, the CD28 activating peptide comprises the minimum functional fragment of SEQ ID NO: 35. In yet another embodiment, the CD28 activating peptide consists of 28 to 53 amino acid residues, and the CD28 activating peptide comprises the minimum functional fragment of SEQ ID NO: 35.

[0066] In another embodiment, the CD28 activating peptide comprises at least 85%, 88%, 90%, 95%, 97%, or 99% of the same amino acid sequence as 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 yet another embodiment, the CD28 activating peptide is SEQ ID NO: 35, 36, or 37.

[0067] In one embodiment, the antigen comprises at least 80%, 85%, 90%, 95%, or 99% of the same amino acid sequence as SEQ ID No: 39, 40, 41, or 42. In another embodiment, the antigen is a peptide that is at least 80%, 85%, 90%, 95%, or 99% of the same amino acid sequence as SEQ ID No: 39, 40, 41, or 42. In yet another embodiment, the antigen is a peptide having the amino acid sequence of SEQ ID No: 39, 40, 41, or 42.

[0068] In one embodiment, the antigen contains at least one epitope for inducing the desired immune response, preferably containing 1 to 50 epitopes, more preferably containing 1 to 20 epitopes.

[0069] An antigen may be a single antigen or a fragment thereof, or a fusion antigen comprising at least two antigenic peptides, which may or may not be fused by a linker.

[0070] Fusion antigens can have rigid linkers (EAAAAK). n The rigid linker connects two different antigenic polypeptides, wherein n is 0 to 12, preferably 2 to 6, and more preferably an integer of 3 to 4. In other words, the rigid linker contains 0 to 12, 2 to 6, or 3 to 4 repeats of the sequence EAAAAK (SEQ ID NO: 38).

[0071] The fusion protein of the present invention may further include a rigid linker between the CD40 binding domain and the furin and / or cathepsin L cleavage site. The rigid linker comprises 0 to 12 repeating amino acid sequences EAAAAK (SEQ ID NO: 38). The rigid linker may be (EAAAAK). nOr (SEQ ID NO: 38) n Where n is 0 to 12, preferably 2 to 6, and more preferably an integer of 3 to 4. In one embodiment, the rigid connector comprises 2 to 6 repeats or 3 to 4 repeats of SEQ ID NO: 38.

[0072] In another embodiment, the fusion protein of the present invention comprises or is substantially composed of an amino acid sequence that is at least 85%, 88%, 90%, 95%, or 99% identical to that of SEQ ID NO: 43. Further in another embodiment, the fusion protein of the present invention comprises or is substantially composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 43. In one embodiment, the fusion protein of the present invention is 300 to 1500 amino acids in length.

[0073] This invention also relates to a fusion protein for manufacturing drugs that induce antigen-specific cell-mediated immune responses and / or antigen-specific humoral immune responses, or for reducing, inhibiting, treating, and / or improving infectious animal diseases caused by FCoV in animals in need. In one embodiment, the animals are domestic felines and non-domestic felines. Domestic felines are domestic cats. Non-domestic felines include, but are not limited to, cheetahs (…). Acinonyx jubatus ), European wildcat ( Felis silvestris ),tiger( Panthera tigris ), cougar ( Puma concolor ) and lion ( Panthera leo ).

[0074] The pharmaceutical composition / vaccine of the present invention The present invention also relates to a pharmaceutical composition or vaccine comprising: (a) The fusion protein of the present invention; and (b) Pharmaceutically acceptable carriers and / or adjuvants.

[0075] The vaccine of the present invention is a prophylactic and / or therapeutic vaccine. In one embodiment, the fusion protein is used to induce antigen-specific cell-mediated immune responses and antigen-specific humoral responses against FCoV in uninfected or infected animals.

[0076] The term "carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (such as antibacterial and antifungal agents), isotonic agents, absorption delayers, salts, preservatives, pharmaceuticals, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, and combinations thereof, as known to those skilled in the art (e.g., see Remington's Pharmaceutical Sciences, 18).th Ed. MackPrinting Company, 1990, pp. 1289-1329).

[0077] Suitable adjuvants include, but are not limited to: (1) oil-in-water (O / W) adjuvants (e.g., MONTANIDE™ ISA 15AVG, MONTANIDE™ ISA 35 VG, MONTANIDE™ ISA 28R VG, and SUMMIT). TM (2) Water-in-oil (W / O) adjuvants (e.g., MONTANIDE™ ISA 61 VG, MONTANIDE™ ISA 71 VG, MONTANIDE™ ISA 71R VG, MONTANIDE™ ISA 761 VG, MONTANIDE™ ISA 78 VG, MONTANIDE™ ISA 763B VG, MONTANIDE™ ISA 660 VG and Freund's incomplete adjuvant); (3) Water-in-oil (W / O / W) adjuvants (e.g., MONTANIDE™ ISA 201 VG, MONTANIDE™ ISA 206 VG and MONTANIDE™ ISA 207 VG); (4) Aluminum salt adjuvants (e.g., aluminum hydroxide and aluminum phosphate); (5) Saponin-based adjuvants (e.g., GPI-0100, Quil A). (6) Toll-like receptor (TLR) agonist adjuvants (e.g., polyinosinic acid (Poly I:C), monophospholipid A and CpG oligonucleotides); (7) chitosan-based adjuvants (e.g., ViscoGel and ZenoParticle CH-100) and (8) combinations of two or more of the above adjuvants.

[0078] CpG oligonucleotide adjuvants include, but are not limited to, class A CpGs (i.e., CpG1585, CpG2216, or CpG2336), class B CpGs (i.e., CpG1668, CpG1826, CpG2006, CpG2007, CpG BW006, or CpG D-SL01), and class C CpGs (i.e., CpG2395, CpG M362, or CpG D-SL03). Another suitable CpG adjuvant is CpG1018. In one embodiment, the adjuvant is a CpG oligonucleotide (ODN).

[0079] Research and development of chitosan is underway to enhance the efficacy of various vaccines through optimized formulation. Chitosan-based adjuvants are known for their ability to enhance vaccine antigenicity and promote innate and adaptive immune responses (Aishwarya Rathore et al. (2022)). Biomed J Sci & Tech Res, 45(1)-2022. BJSTR. MS.ID.007140.). Chitosan adjuvants can be prepared by known methods, such as those disclosed in patent CN109833474B. In one embodiment, the adjuvant is a chitosan-based adjuvant.

[0080] In one embodiment, the fusion protein of the present invention can be formulated with at least one adjuvant selected from water-in-oil (W / O) adjuvants, Toll-like receptor (TLR) agonist adjuvants, or chitosan-based adjuvants. In one embodiment, the fusion protein of the present invention can be formulated with a water-in-oil (W / O) adjuvant. In one embodiment, the fusion protein of the present invention can be formulated with a Toll-like receptor (TLR) agonist adjuvant. In one embodiment, the fusion protein of the present invention can be formulated with a CpG oligonucleotide (ODN) adjuvant. In one embodiment, the fusion protein of the present invention can be formulated with a chitosan-based adjuvant. The formulated pharmaceutical compositions or vaccines are expected to exhibit a potent immune response against FCoV and prevent and / or treat FIP.

[0081] The pharmaceutical composition / vaccine may be in enteral or parenteral formulation, suitable for transdermal, transmucosal, nasopharyngeal, pulmonary, or direct injection, or for systemic (e.g., parenteral) or local (e.g., intratumoral or intralesional) administration. Parenteral injection may be administered intravenously ( iv ), intraperitoneal ( IP ), intramuscular ( im ), subcutaneous ( sc ) or intradermal ( id This pharmaceutical composition can also be administered orally, for example in the form of tablets, coated tablets, sugar-coated pills, hard gelatin capsules, and soft gelatin capsules.

[0082] The dosage of the fusion protein may vary depending on the disease to be controlled, the age and individual condition of the animal, and the method of administration. The dosage can be adjusted according to individual needs in each case to obtain a therapeutically effective amount of the fusion protein of the present invention to achieve the desired therapeutic response.

[0083] For infected animals, a single dose considered is approximately 0.01 to 10 mg, particularly approximately 0.01 to 5 mg. Depending on the severity of the disease and precise pharmacokinetic characteristics, the fusion protein may be administered weekly, bi-weekly, or monthly, with a total of 1 to 6 doses per cycle to meet this type of treatment.

[0084] In one embodiment, the present invention provides a kit or packaged pharmaceutical composition comprising the fusion protein of the present invention and instructions for use for treating one or more symptoms of an infectious disease caused by FCoV in animals in need.

[0085] Example Methods and Materials Table 1 shows the SEQ ID number and its corresponding polypeptide / fusion protein.

[0086] Table 1

[0087] Flow cytometry. Spleen cells were treated with stimulants at 37°C for 2 hours, then incubated with Brefeldin A and Monensin for 2 hours, and harvested simultaneously for immunostaining 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. Spleen cells were washed, permeabilized, fixed, and simultaneously stained with antibodies PE-conjugated anti-IFN-γ, PE / Cy7-conjugated anti-IL-2, and eFluor450-conjugated anti-TNF-α. Intracellular cytokines (IFN-γ, IL-2, or TNF-α) induced in CD8+ and / or CD4+ memory T cells were analyzed using Gallios flow cytometry and Kaluza software.

[0088] Enzyme-linked immunospot (ELISpot) assay. Spleen cells (2 × 10⁻⁶) were used. 5 96-well CTL IMMUNOSPOT® plates were seeded with mouse IFN-γ cells (1 cell / well) in triplicate. Cells were incubated at 37°C for 24 hours with or without irritant, and then discarded. Captured IFN-γ was detected using a biotin-conjugated anti-mouse IFN-γ antibody. IFN-γ immunospots were developed according to the manufacturer's instructions. The number of IFN-γ immunospots was scanned and counted using a CTL IMMUNOSPOT® S5 Micro analyzer. Results were expressed as IFN-γ per million spleen cells. + Representation of the number of immune spots.

[0089] Indirect enzyme-linked immunosorbent assay (ELISA). To capture antigen-specific antibodies of interest, the corresponding antigen protein or peptide was synthesized and plated at a concentration of 1 μg / well on a 96-well plate at 4°C overnight. After washing, the plate was blocked with BSA in PBS. Serum samples were serially diluted, and 100 μl of the dilution was incubated on the antigen-coated culture plate at 37°C for 2 hours. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was reacted at 37°C for 30 minutes, followed by color development with TMB (tetramethylbenzidine), and the relative titer of antigen-specific antibodies in the serum samples was measured to detect the captured antigen-specific antibodies.

[0090] Reverse transcription polymerase chain reaction (RT-PCR). Fecal samples were collected for RNA analysis. RNA was extracted from a 10% fecal suspension in 200 μl PBS using a nucleic acid detection kit (BioGuard, Taiwan) according to the manufacturer's instructions. After extraction, the extracted RNA was amplified using a BioGuard real-time PCR analyzer. Results are expressed as Ct values, reflecting the amount of RNA in the sample.

[0091] Quantitative assay for feline coronavirus (FCoV). A commercial rapid fluorescent immunochromatographic assay (Dawnsail Biotech, China) was used to detect the pathogen FCoV. Fecal swab samples were dissolved in preservation buffer, and then 80 μl aliquots were added to the test strip of the Dawnsail fluorescence reader for analysis. Results were collected after 10 minutes and expressed as IU values, reflecting viral load.

[0092] ALT and AST activity assays. The enzyme activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by substrate detection. Both enzymes convert NADH to NAD. + Measurements. The reduction in NADH was measured by the decrease in absorbance at 340 nm. Results were calibrated to IFCC reference materials and expressed in units per liter (U / L), where one unit represents 1 μmol of substrate converted per minute.

[0093] Statistical analysis. A t-test was used; a result was considered significant when p < 0.05. (Symbol) Indicates statistical significance, p < 0.05.

[0094] Example 1 Construction of expression carriers The vector CD40L-T, used to generate a fusion protein containing the FCoV-N antigen, was developed. PE -FCoV ( Figure 1Plasmid pTAC-MAT-Tag-2 (catalog number E5405, Sigma-Aldrich) Hin dIII / Sal I digest. Encoding. HindIII CD40L-Connector-PE NcoI, XhoI, SalI DNA fragments that have CD40L 108-261 A cleavable linker and a PE translocation peptide (PE) 280-305 This fragment was synthesized by PCR and... Hin dIII / Sal I digested the sample and inserted plasmid pTAC-MAT-Tag-2 to generate the intermediate vector P07-His-pNC ( Figure 2 The DNA fragment encoding the FCoV-N protein (SEQ ID NO: 42) carrying the His tag was inserted into the intermediate vector P07-His-pNC to obtain the final vector CD40L-T. PE -FCoV, which is used to express a fusion protein with the FCoV N antigen ( Figure 1 ).

[0095] Other FCoV antigens of interest, such as the spike protein S1, membrane protein M, or antigenic peptide of FCoV antigens, can be inserted into the intermediate vector P07-His-pNC (Figure 2) to express alternative fusion protein antigens. Figure 1 and Figure 3 ).

[0096] Carrier CD40L-T PE -FCoV. Construction of the vector CD40L-T PE -FCoV ( Figure 1 To express the fusion protein CD40L-T PE -FCoV (SEQ ID NO: 43; Figure 3 ), which includes: (a) a truncated CD40 ligand CD40L 108-261 (SEQ ID NO: 19); (b) a cleavable peptide linker comprising (EAAAAK)3 (SEQ ID NO: 3) and RX 1 RX 2 X 3 R (where X) 1 Let A and X be the two numbers. 2 Let Y and X be the numbers. 3 (c) PE translocation peptide; and (d) antigen FCoV N protein (SEQ ID NO: 42).

[0097] The cleavable linker allows furin and / or cathepsin L protease to cleave the fusion protein of the present invention to release T from the fusion protein. PE -FCoV fragment.

[0098] Using a similar approach, any other antigen of interest from FCoV (e.g., spike protein, membrane protein, or desired antigenic peptide) can replace the FCoV N protein and insert into... Figure 2 In plasmids, to produce, as Figure 1 The expression vector shown is used to express a fusion protein containing the antigen of interest.

[0099] Example 2 Protein expression Cultured with expression vector CD40L-T PE -FCoV was synthesized in E. coli BL21 cells, and the fusion protein was expressed by IPTG induction. The fusion protein was purified and refolded from inclusion bodies dissolved in guanidine hydrochloride.

[0100] Example 3 Both humoral and cell-mediated immune responses were enhanced after inoculation with the fusion protein. The fusion protein CD40L-TPE-FCoV constructed as described above was used in the following studies. It was dissolved in 5 mM citrate buffer at pH 4.0. Unless otherwise specified, 0.5% chitosan was used as an adjuvant. The final vaccine formulation contained 0.2 mg / mL of the fusion protein.

[0101] Female C57BL / 6NCrlBltw mice aged 5 to 6 weeks were randomly assigned to a placebo group and vaccination groups A and B (n=5 in each group). Following the dosing regimen in Table 2, the placebo group received treatment on days 0, 7, and 14. sc Received PBS; Group A on day 0 sc Group B received the vaccine containing 100 μg of fusion protein on day 14. sc Mice received a vaccine containing 100 μg. Serum was collected on days 0, 7, 14, and 21 to determine antigen-specific humoral immune responses. Mice were sacrificed on day 21.

[0102] The levels of antigen-specific antibodies in serum were analyzed using ELISA. The thioredoxin-FCoV nucleocapsid (N) fusion protein was used as a capture antigen for coating ELISA plates.

[0103] Spleen cells harvested from day 21 mice were cultured and stimulated with an antigenic stimulus consisting of a pool of short peptides covering the FCoV N protein sequence to determine antigen-specific cell-mediated immune responses by detecting IFN-γ secretion via ELISpot assay. Flow cytometry was also used to analyze intracellular cytokine induction (IFN-γ, IL-2, and TNF-α) in spleen cells within memory T cells. The frequency of IFN-γ-secreting spleen cells was analyzed by enzyme-linked immunospot (ELISpot) assay.

[0104] Table 2 shows the dosing schedule.

[0105] “V”: Vaccinated; “-”: Not vaccinated.

[0106] Table 2

[0107] The levels of antigen-specific antibodies in the serum of vaccinated group A were significantly higher than those in the placebo and group B on days 14 and 21. Antibody levels in group B serum were not different from those in the placebo group. The data indicate that 7 days is insufficient to induce significant antibody titers (group B), but 14 days is sufficient to induce significant antibody titers lasting at least two weeks (group A). Figure 4 ).

[0108] Both groups A and B showed a significant increase in the frequency of spleen cells secreting IFN-γ. Data from group B (administered on day 14) showed that the vaccine of the present invention could induce a highly increased cellular immune response as early as day 7 post-immunization. Data from group A (administered on day 0) showed that the highly increased cellular immune response induced by the vaccine of the present invention could persist for at least 21 days post-immunization. Figure 5 ).

[0109] Compared with the placebo group, CD4 counts in groups A (vaccinated on day 0) and B (vaccinated on day 14) were significantly lower. + The levels of IFN-γ, IL-2, and TNF-α induced in memory T cells all increased. The results show that the fusion protein vaccine of this invention can elicit a specific cell-mediated immune response. Figures 6 to 8 ).

[0110] Therefore, the fusion protein of this invention can promote both humoral and cell-mediated immune responses. A rapid cellular immune response occurs early and may be beneficial for treating cats with FIP in the early stages of the disease.

[0111] Example 4 Cats with FIP showed reduced viral load after receiving a fusion protein vaccine. Two cats (C1 and C2) with FIP were subcutaneously administered the fusion protein CD40L-T formulated as described in Example 3 on days 0, 7, 14, and 21. PE -FCoV. Fecal samples were collected on days 7, 14, 21, 28, and 35, and FCoV levels were determined using a quantitative assay for feline coronavirus (FCoV). The results of the quantitative assay are shown below. Figure 9 As shown. The results from day 7 and day 14 were integrated and compared with the integrated results from day 21, day 28, and day 35 using an unpaired t-test. The two integrated results were statistically significant compared with each other (P < 0.05). This indicates that inoculation with the CD40L-T fusion protein... PE -FCoV viral load decreased statistically significantly after administration.

[0112] The CD40L-T fusion protein was evaluated using C3, C4, C5, and C6 in cats diagnosed with FCoV infection but without FIP ​​symptoms. PE - The effect of FCoV in clearing the virus. The fusion protein CD40L-T prepared according to Example 3 was used. PE -FCoV was administered subcutaneously. Cats C3 received two injections on days 0 and 14; cats C4 received a single injection on day 0. Cats C5 and C6, serving as control groups, were not vaccinated. Fecal samples were collected on days 0, 14, 21, and 35 for reverse transcription polymerase chain reaction (RT-PCR) analysis of viral levels. Figure 10A ).

[0113] On day 0, RT-PCR testing showed detectable FCoV RNA levels in cats C3, C4, C5, and C6. By day 21, vaccinated cats C3 and C4 were still alive, and their FCoV RNA levels had decreased. In contrast, control group cats C5 and C6 had died, making it impossible to measure FCoV viral RNA on day 21.

[0114] In another experiment, cats C7 diagnosed with FCoV infection but without FIP ​​symptoms were injected with the same fusion protein prepared as described above on days 0 and 14, and fecal virus levels were detected by fluorescent immunoassay. On day 0, cat C7 already had detectable viral levels. By day 14, fecal virus levels had decreased below the detection limit. Figure 10B ).

[0115] Both RT-PCR and fluorescence immunoassay showed that inoculation with the fusion protein CD40L-T... PE -FCoV-infected cats with FCoV showed fecal viral loads reduced to below detectable levels and survived. In contrast, unvaccinated control animals died before the end of the experiment. This demonstrates the effectiveness of the CD40L-T fusion protein. PE-FCoV vaccination is effective in maintaining the life of animals.

[0116] Example 5 Vaccinate cats with fusion protein vaccines to prevent fever, weight loss, liver damage, and death caused by FCoV. Cats (4 to 10 months old) were randomly assigned to a placebo group and a vaccination group (n=3 in each group). Placebo group sc Receive PBS treatment. Vaccination groups will be categorized as previously described. sc Receive the fusion protein. On days 21 and 42 following PBS or fusion protein treatment, administer type II FIPV 79-1146 (10 2 TCID50) for cats IP Challenges included routinely measuring body temperature and weight, and collecting serum to determine ALT and AST activities.

[0117] After being challenged with the virus, the vaccinated group showed relatively stable body temperature and weight for up to 35 days, while the placebo group showed a sustained increase in body temperature, peaking above 39°C, and weight loss. Figures 11 to 12 Following viral challenge, serum ALT and AST liver enzyme levels in the vaccinated group remained stable for at least 20 days without change. The placebo group showed elevated ALT and AST levels starting on day 7 post-viral challenge. Figures 13 to 14 During the entire 35-day observation period, all animals in the vaccinated group survived. All cats in the placebo group died before day 21 after the viral challenge. Figure 15 ).

[0118] The results showed that inoculation with the fusion protein of the present invention could prevent fever, weight loss, liver damage and death caused by FIPV 79-1146 virus.

[0119] Example 6 Adjuvants enhance cell-mediated immune responses to fusion proteins Multiple adjuvants were evaluated to enhance the efficacy of fusion protein vaccines in providing active immunity against infectious diseases caused by FCoV. Five adjuvants were tested, including: (1) chitosan: chitosan adjuvant (0.5%); (2) ODN: CpG1826 (200 µg / dose); (3) oil-in-water (O / W): SUMMIT. TM S350 (20%); (4) Water-in-oil (W / O): ISA 660 VG (50%); and (5) Mineral salt: Aluminum phosphate (10%).

[0120] The constructed fusion protein CD40L-T PE-FCoV was dissolved in 5 mM citrate buffer (pH 4.0) and formulated into a suspension with adjuvant. Five- to six-week-old female C57BL / 6NCrlBltw mice were randomly assigned to either a placebo group or an adjuvant group (n=5 per group). On day 0, the placebo group received no treatment, while the adjuvant group received the fusion protein vaccine with added adjuvant. On day 7, the mice were sacrificed, and spleen cells were harvested and cultured to determine the antigen-specific cell-mediated immune response.

[0121] With IFN-γ + ELISpot assays assessed the enhancing effect of adjuvants on Th1-based immunity, expressed as fold changes. All five adjuvant groups showed a significant fold change in cell-mediated immunity compared to the placebo group. Figure 16 -#).

[0122] Each showed more than 10 times the efficacy of the placebo. Figure 16 - Compared to the O / W and mineral salt groups, the chitosan, ODN, and W / O adjuvant groups all showed more significant effects of the fusion protein on IFN-γ. + The induction effect on spleen cells was enhanced. The ODN adjuvant group showed the most significant efficacy, demonstrating almost 200 times that of the placebo group. Both the chitosan group and the W / O adjuvant group showed a nearly 100-fold increase in Th1-based immunity. Figure 16 ).

[0123] Surprisingly, the adjuvant chitosan, ODN, and W / O groups exhibited a more potent enhancement of the FCoV fusion protein vaccine efficacy than the O / W and mineral salt groups. Equally unexpectedly, the ODN group was the most potent adjuvant for enhancing the immunogenicity of the fusion protein vaccine of this invention. These findings demonstrate that the enhancing efficacy of adjuvants is unpredictable.

[0124] CD4 measured by flow cytometry + Intracellular cytokines IFN-γ, IL-2, and TNF-α within memory T cells. Compared to the placebo group, all adjuvant groups showed enhanced IFN-γ, IL-2, and TNF-α-induced activity. Figures 17 to 19 ).

[0125] Currently, there is a shortage of FCoV vaccines available. For example, the VANGUARD® feline FIP intranasal unadjuvanted vaccine, containing attenuated live virus, is approved for the prevention of FIP caused by feline infectious peritonitis virus (FIPV). However, according to the American Animal Hospital Association (AAHA), its use is generally not recommended at present because its ability to uniformly prevent the disease in North American cat populations is uncertain, making routine use unreasonable. Furthermore, the benefits and risks of vaccination remain unclear.

[0126] Our evidence shows that vaccinating cats with the fusion protein of this invention can prevent fever, weight loss, liver damage, and death caused by FCoV. In cats with FIP, vaccination with the fusion protein of this invention can reduce viral load and prevent death.

[0127] All references cited and discussed in this specification are incorporated herein by reference in their entirety, to the same extent as if each reference were incorporated individually.

Claims

1. A fusion protein comprising: (a) A CD40 binding domain located at the N-terminus of the fusion protein; (b) Feline coronavirus (FCoV) antigen; (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 contains at least 85% of the same amino acid sequence as SEQ ID NO: 5, 6, 7, 8 or 9, and has a length of 26 to 112 amino acids.

3. The fusion protein as described in claim 1, wherein, The furin and / or cathepsin L cleavage site contains the amino acid sequence RX. 1 X 2 R or RX 1 RX 2 X 3 R, where X 1 and X 2 X is any amino acid, and X 3 It can be 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 an antibody specifically targeting CD40 or a functional fragment thereof.

5. The fusion protein of claim 4, wherein, The CD40L contains at least 85% of the same amino acid sequence as SEQ ID NO: 14, 15, 16, 17, 18 or 19, and has a length of 153 to 261 amino acids.

6. The fusion protein of claim 1, wherein, The antigens are selected from the group consisting of FCoV S1, S2, M, N proteins and any antigen fragments thereof.

7. The fusion protein of claim 1, wherein, The FCoV mentioned 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 as described in claim 1; and (ii) Pharmaceutically acceptable carriers and / or adjuvants.

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

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

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

13. Use of a fusion protein as described in any one of claims 1 to 8 or a pharmaceutical composition as described in claim 9 in the manufacture of a medicament for the prevention of fever, weight loss, liver damage and death caused by FCoV in animals in need, or for reducing viral load and preventing death in animals suffering from feline infectious peritonitis.

14. The use as described in claim 13, wherein, The animal in question is a feline.

15. The use as described in claim 13, wherein, The animals were selected from a group consisting of tigers, lions, and domestic cats.

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