Immunogenic fusion proteins against infectious animal diseases

Abstract

Immunogenic fusion proteins against infectious animal diseases. Disclosed are fusion proteins comprising a CD40 binding domain, an antigen of a pathogen, 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 uses of the fusion proteins of the invention to induce an antigen-specific cellular immune response or to reduce, inhibit, treat, and / or ameliorate an infectious animal disease caused by a pathogen in an animal in need thereof.

Description

[Technical Field]

[0001] Electronic Sequence Listing Reference Electronic sequence listing (10040-004PCT_sequence listing_ST26.xml, size 84KB, and creation date November 14, 2022 ), the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to fusion proteins, and more particularly to immunogenic fusion proteins for eliciting antigen-specific cellular immune responses against infectious animal diseases. [Background technology]

[0003] The adaptive immune system consists of humoral and cellular immunity, both of which destroy invading pathogens. B- and T-lymphocytes are responsible for antibody and cellular immune responses, respectively. Adaptive immunity to a pathogen leads to enhanced immune responses against future encounters with the pathogen. Several adaptive immunotherapies have been evaluated in clinical settings. However, there is still a need for the development of new therapeutic vaccines to treat infectious animal diseases caused by pathogens. Summary of the Invention

[0004] In one aspect, the present invention provides a fusion protein comprising: (a) a CD40 binding domain; (b) an antigen of a pathogen; (c) a translocation domain located between the CD40 binding domain and the antigen; and (d) a translocation domain located between the CD40 binding domain and the antigen. metastasisand a furin and / or cathepsin L cleavage site located between the domains, wherein the pathogen is at least one selected from the group consisting of classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), Mycoplasma hyopneumoniae, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), parvovirus, poxvirus, rotavirus, and influenza virus.

[0005] In another aspect, the present invention relates to an expression vector comprising a DNA fragment or a DNA fragment encoding a fusion protein of the present invention.

[0006] The present invention further relates to a pharmaceutical or vaccine composition comprising a fusion protein of the invention and a pharmaceutically acceptable carrier and / or adjuvant.

[0007] In yet another aspect, the invention relates to the use of a fusion protein, pharmaceutical composition, or vaccine composition of the invention in the manufacture of a medicament for eliciting an antigen-specific cellular immune response or for reducing, inhibiting, treating, and / or ameliorating an infectious animal disease caused by a pathogen in an animal in need thereof.

[0008] The present invention also relates to the fusion proteins, pharmaceutical compositions, or vaccine compositions of the invention for use in eliciting an antigen-specific cellular immune response or for use in reducing, inhibiting, treating, and / or ameliorating an infectious animal disease caused by a pathogen in an animal in need thereof.

[0009] Alternatively, the present invention relates to a method for inducing an antigen-specific cellular immune response or for reducing, inhibiting, treating, and / or ameliorating an infectious animal disease caused by a pathogen in an animal in need thereof, said method comprising administering to the animal in need thereof an effective amount of a fusion protein, pharmaceutical composition, or vaccine composition of the present invention. [Brief explanation of the drawings]

[0010] [Figure 1A-E] 1A to 1E are vector diagrams of the present invention. [Figures 2A-E] 2A to 2E are vector diagrams of the present invention. [Figure 3A-H] 3A-H are schematic diagrams illustrating immunogenic fusion proteins according to various embodiments of the present invention. [Figure 4-6] 4 to 6 are graphs showing the induction of IFN-γ, IL-2, and TNF-α in CD4+ memory T cells from each group of animals, respectively. [Figure 7A-B] 7A-B are graphs showing IFN-γ+ immunospots in splenocytes from each animal group. [Figure 8A-B] 8A-B are graphs showing serum antigen-specific antibody levels in each animal group on day 21. Antibody levels were measured by ELISA using CD40L-TPE-E2-NS3p fusion protein as the coating protein. [Figure 9A-B] 9A-B are graphs showing serum antigen-specific antibody levels in each animal group on day 21. Antibody levels were measured by ELISA using E2-NS3p peptide as the coating protein. [Figure 10A-C] 10A-C are graphs showing the induction of IFN-γ, IL-2, and TNF-α, respectively, in CD4+ memory T cells from each group of animals on an extended dosing schedule. [Figure 11] FIG. 11 is a graph showing IFN-γ+ immunospots in splenocytes from each group of animals on an extended dosing schedule. [Figure 12]FIG. 12 is a graph showing serum antigen-specific antibody levels in each animal group over an extended dosing schedule. DETAILED DESCRIPTION OF THE INVENTION

[0011] definition Professional and non-professional APCs display endogenous peptides on the cell membrane using MHC class I molecules. These peptides are derived from the cell itself, in contrast to foreign antigens displayed by professional APCs using MHC class II molecules. Cytotoxic CD8 + T cells can interact with antigens presented by MHC class I molecules.

[0012] CD40 is a costimulatory protein expressed on antigen-presenting cells (e.g., dendritic cells, macrophages, and B cells). Binding of CD40L to CD40 activates antigen-presenting cells and induces various downstream effects. CD40 is a drug target for cancer immunotherapy.

[0013] The term "CD40 binding domain" refers to a protein capable of recognizing and binding to CD40. The CD40 binding domain may be selected from one of the following: "CD40 ligand (CD40L) or a functional fragment thereof," "anti-CD40 antibody or a functional fragment thereof."

[0014] The terms "CD40L," "CD40 ligand," and "CD154" are interchangeable. CD40L binds to CD40 (a protein) on antigen-presenting cells (APCs) and exerts various effects depending on the type of target cell. CD40L plays an important role in costimulation and regulation of immune responses through priming of T cells and activation of CD40-expressing immune cells. U.S. Pat. No. 5,962,406 discloses the nucleotide and amino acid sequences of CD40L.

[0015] The terms "anti-CD40 antibody," "CD40-specific antibody," and "specific antibody against CD40" are interchangeable.

[0016] When the term "consisting essentially of" is used to describe the amino acid sequence of a polypeptide, it means that the polypeptide may or may not have the initiating amino acid "M" (translated from the initiation codon AUG) at the N-terminus, depending on the translation requirements of the protein. For example, when a second antigen is fused to a first antigen, the initiating amino acid "M" of the second antigen can be omitted or retained.

[0017] As used herein, a "translocation domain" refers to a polypeptide that has the biological activity of translocating a fused or linked antigen across the endosomal membrane into the cytoplasm of a cell. The translocation domain induces or promotes the redirection of the antigen into the class I major histocompatibility complex (MHC-I) pathway (i.e., the cytotoxic T cell pathway) for antigen presentation.

[0018] Pseudomonas exotoxin A (PE) transit peptide (T PE The term "PE domain II peptide" refers to a PE domain II peptide or a functional fragment thereof that has biological activity in metastasis.

[0019] Shiga toxin (Stx) transit peptide (T Stx The term "Stx translocation domain" refers to a Stx translocation domain or a functional fragment thereof that has biological activity in metastasis.

[0020] The terms "furin and / or cathepsin L" or "furin / cathepsin L" are interchangeable.

[0021] 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 susceptible to furin and / or cathepsin L proteases. It may also be a peptide linker containing the cleavage site introduced into the fusion protein. Furthermore, the furin and / or cathepsin L cleavage site may be a PE or Stx endogenous protease cleavage site present in or adjacent to the translocation domain of the fusion protein.

[0022] The terms "antigen" and "immunogen" are interchangeable. An antigen refers to an antigenic protein or polypeptide derived from a pathogen. The antigen comprises at least one epitope for inducing a desired immune response. In some embodiments, the antigen is a polypeptide of at least 8 amino acids in length derived from a pathogen selected from the group consisting of porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine circovirus 2 (PCV2), foot-and-mouth disease virus (FMDV), porcine epidemic diarrhea virus (PEDV), swine vesicular disease virus (SVDV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), Mycoplasma hyopneumoniae, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), parvovirus, poxvirus, rotavirus, and influenza virus.

[0023] CD28 (Cluster of Differentiation 28) is a protein expressed on T cells that provides costimulatory signals necessary for T cell activation and survival. T cell stimulation via CD28 in addition to the T cell receptor (TCR) provides a powerful signal for the production of various interleukins (especially IL-6). CD28 is a receptor for the CD80 (B7.1) and CD86 (B7.2) proteins. Upon activation by Toll-like receptor ligands, CD80 expression is upregulated on antigen-presenting cells (APCs). CD28 is the only B7 receptor constitutively expressed on naive T cells. When the TCR of naive T cells binds to an MHC:antigen complex without the CD28:B7 interaction, the T cell becomes apathetic.

[0024] The term "effective amount" refers to the amount of active fusion protein required to confer a therapeutic effect on a treated subject. Effective doses will vary depending on the route of administration, excipient usage, and the possibility of co-administration with other therapeutic treatments, as will be recognized by those skilled in the art.

[0025] The term "treating" or "treatment" refers to administering an effective amount of the fusion protein to a subject in need thereof having cancer or an infectious disease, or symptoms or predisposition to such a disease, for the purpose of curing, alleviating, mitigating, treating, ameliorating, or reducing or inhibiting the disease, its symptoms, or predisposition thereto. Such subjects may be identified by a medical professional based on the results of any appropriate diagnostic method.

[0026] "0 to 12 repeats" or "2 to 6 repeats" means that all integer unit amounts within the range of "0 to 12" or "2 to 6" are specifically disclosed as part of the present invention. Thus, unit amounts of "0, 1, 2, 3, 4, ... 10, 11 and 12" or "2, 3, 4, 5 and 6" are included in embodiments of the present invention.

[0027] Abbreviations: MCS, multiple cloning site; Rap1, Ras-proximate-1 or Ras-related protein 1; CD40, cluster of differentiation 40; CDR, complementarity-determining region; sc, subcutaneous; aa, amino acid.

[0028] Fusion proteins The present invention provides a fusion protein comprising: (a) a CD40 binding domain; (b) an antigen of a pathogen; (c) a translocation domain located between the CD40 binding domain and the antigen; and (d) a translocation domain located between the CD40 binding domain and the antigen. metastasis and a furin and / or cathepsin L cleavage site located between the domains, wherein the pathogen is selected from the group consisting of classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), Mycoplasma hyopneumoniae, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), parvovirus, poxvirus, rotavirus, and influenza virus.

[0029] The fusion proteins of the present invention can induce antigen-specific T cell immune responses via the MHC class I antigen presentation pathway. The fusion proteins share a common mechanism of action: CD40L-T PE Using -Ag (where Ag represents any suitable antigen) as an example, the mechanism of action is as follows: (1) CD40L-T PE -Ag binds to cells expressing CD40 (e.g., dendritic cells and macrophages) and is taken up by CD40-mediated endocytosis. (2) CD40L-T PE-Ag is cleaved by furin and / or cathepsin L proteases in endosomes, and the CD40L fragment is released into T PE -removed from Ag fragments, (3) T PE -Ag fragments cross the endosomal membrane and enter the cytoplasm. (4) T PE -Ag fragments are digested by the cytoplasmic proteasome, generating smaller antigens containing the epitope. (5) Small antigens are delivered via the MHC class I pathway for antigen presentation; and (6) CD8 + T cell-specific immune responses are induced or enhanced by T cells recognizing these presented antigens.

[0030] The same mechanism of action is Ag-T Stx - This also applies to CD40L, where furin and / or cathepsin L protease cleavage results in Ag-T fragments from the CD40L fragment. Stx The fragment is removed. Thus, Ag-T Stx The fragments cross the endosomal membrane into the cytoplasm, where they are digested by the cytoplasmic proteasome, yielding small antigens containing the epitope. These small antigens are delivered via the MHC class I pathway for antigen presentation and are expressed by CD8 T cells that recognize the presented antigen. + A T cell-specific immune response is induced or enhanced.

[0031] In the fusion proteins of the present invention, there is no furin and / or cathepsin L cleavage site between the antigen and translocation domains. The presence of the furin and / or cathepsin L cleavage site and its position in the fusion protein allows for removal of the CD40 binding domain from the fusion protein after furin and / or cathepsin L cleavage.

[0032] In one embodiment, the furin and / or cathepsin L cleavage site comprises or consists 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 or consists of RX 1 X 2 R or RX 1 RX 2 X 3 R (where X 1 and X 2 is any amino acid residue, and X 3 is K, F or R) Comprises, consists of, or is.

[0033] In another embodiment, the fusion protein of the present 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 the peptide linker. The peptide linker may be: (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 of 0 to 12, preferably 2 to 6, more preferably 3 to 4, and the furin and / or cathepsin L cleavage site is RX 1 X 2 R or RX 1 RX 2 X 3 R (where X 1 and X 2 is any amino acid residue, and X 3 is K, F or R) In one embodiment, the peptide linker may comprise a cleavable linker comprising (EAAAAK)3 and RX 1 RX 2 X 3 R (childSo, X 1 A, X 2 is Y, X 3 In another embodiment, the peptide linker comprises R 1 X 2 R (child So, X 1 is V, X 2 includes A) and (EAAAAK)3.

[0034] The translocation domain may be selected from Pseudomonas exotoxin A (PE) or Shiga toxin (Stx). metastasis The domain is Pseudomonas exotoxin A (PE). metastasis Peptide (T PE In another embodiment, the translocation domain comprises or is a Shiga toxin (Stx) translocation peptide (T Stx ), where the antigen is located at the N-terminus of the fusion protein.

[0035] In one embodiment, the fusion protein of the invention comprises, in order (from N-terminus to C-terminus): (a) a CD40 binding domain; (b) a furin and / or cathepsin L cleavage site; (c) a PE transit peptide (T PE ) a translocation domain comprising the nucleotide sequence of the target gene, and (d) an antigen of a pathogen.

[0036] In another embodiment, the fusion protein of the invention comprises, in order (from N-terminus to C-terminus), (a) a CD40 binding domain, (b) a peptide linker comprising a furin and / or cathepsin L cleavage site, (c) a PE transit peptide (T PE ) a translocation domain comprising the nucleotide sequence of the target gene, and (d) an antigen of a pathogen.

[0037] In another embodiment, the fusion protein of the invention comprises, in order (from N-terminus to C-terminus), (a) an antigen of a pathogen, (b) an Stx transit peptide (T Stx ), (c) a translocation domain comprising a furin and / or cathepsin L cleavage site, and (d) a CD40 binding domain.

[0038] In another embodiment, the fusion protein of the invention comprises, in order (from N-terminus to C-terminus), (a) an antigen of a pathogen, (b) an Stx transit peptide (T Stx ), (c) a peptide linker comprising a furin and / or cathepsin L cleavage site, and (d) a CD40 binding domain.

[0039] T PE or T Stx teeth, metastasis The furin and / or cathepsin L cleavage site is a functional site having biological activity in RX 1 X 2 R or RX 1 RX 2 X 3 R (where X 1 and X 2 is any amino acid residue, and X 3 is K, F or R) or an endogenous furin cleavage site within or derived from PE or Stx.

[0040] In one embodiment, the PE transit peptide (T PE ) is domain II (aa residues 253 to 364, SEQ ID NO: 9) of Pseudomonas exotoxin A protein (full-length PE, SEQ ID NO: 4) or a functional portion thereof.

[0041] In another embodiment, PE metastasis Peptide (T PE ) consists of 26 to 112 aa residues in length. metastasis Peptide (T PE ) contains the minimal functional fragment GWEQLEQCGYPVQRLVALYLAARLSW (SEQ ID NO: 5).

[0042] In one embodiment, PE metastasis Peptide (T PE) comprises an amino acid sequence that is at least 95%, 97%, or 99% identical to SEQ ID NO: 5, 6, 7, 8, or 9. In other embodiments, T PE comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8 and 9. In other embodiments, T PE 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).

[0043] In one embodiment, Stx metastasis Peptide (T Stx ) is a functional fragment of Shiga toxin (Stx) subunit A (SEQ ID NO: 10) or Shiga-like toxin I (Slt-I) subunit A (SEQ ID NO: 11). metastasis Peptides are metastasis It has a function but lacks the cytotoxic effect of subunit A. The sequence identity of Shiga toxin (Stx) subunit A and Slt-I subunit A is 99%, and the two proteins have only a single amino acid difference.

[0044] In other embodiments, Stx metastasis Peptide (T Stx ) consist of 8 to 84 aa residues in length. metastasis Peptide (T Stx ) contains the smallest functional fragment, LNCHHHAS (SEQ ID NO: 12).

[0045] In one embodiment, Stx metastasis Peptide (T Stx ) comprises an amino acid sequence that is at least 95%, 97%, or 99% identical to SEQ ID NO: 12, 13, 14, 15, or 16. In other embodiments, T Stx comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 13, 14, 15 and 16. In other embodiments, T Stx is Stx of Stx subunit A 240-247(SEQ ID NO: 12), Stx 240-251 (SEQ ID NO: 13), Stx 211-247 (SEQ ID NO: 14), Stx 211-251 (SEQ ID NO: 15) or Stx 168-251 (SEQ ID NO: 16).

[0046] The CD40 binding domain enables the fusion protein of the invention to bind to the CD40 receptor on a CD40-expressing cell (e.g., a dendritic cell or macrophage). The 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, the functional fragment of CD40L is a full-length CD40L. 1-261 It is a truncated CD40L that substantially lacks the transmembrane and cytoplasmic regions of the protein (SEQ ID NO: 17).

[0047] In another embodiment, CD40L or a functional fragment thereof is 154 to 261 aa residues in length. In another embodiment, CD40L comprises a minimal functional fragment of SEQ ID NO: 19. In yet another embodiment, CD40L or a functional fragment thereof is 154 to 261 aa residues in length, and said CD40L comprises a minimal functional fragment of SEQ ID NO: 19.

[0048] In one embodiment, CD40L comprises an amino acid sequence that is at least 95%, 97%, or 99% identical to SEQ ID NO: 17, 18, or 19. In other embodiments, CD40L comprises 1-261 (SEQ ID NO: 17), CD40L 47-261 (SEQ ID NO: 18) and CD40L 108-261 (SEQ ID NO: 19).

[0049] 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 CD40-expressing cells.

[0050] In one embodiment, the CD40-specific antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH consists of the amino acid sequence of SEQ ID NO: 22 and the VL comprises the amino acid sequence of SEQ ID NO: 23.

[0051] In other embodiments, the CD40-specific antibody is selected from the group consisting of a single-chain variable region fragment (scFv), a dimer (dscFv), a trimer (triabody), a tetramer (tetrabody), a bispecific antibody (bispecific-scFv), scFv-Fc, scFc-CH3, a single-chain antigen-binding fragment (scFab), an antigen-binding fragment (Fab), Fab2, a minibody, and a complete antibody.

[0052] In other embodiments, 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 VL. In one embodiment, the CD40-specific scFv comprises SEQ ID NO: 20 or 21.

[0053] In other embodiments, the CD40 binding domain according to the invention is (i) a CD40-specific antibody or binding fragment thereof, or (ii) a CD40-specific single-chain variable fragment (scFv) or binding fragment thereof, wherein said CD40-specific antibody or said CD40-specific scFv comprises a VH and a VL, wherein (a) the VH comprises SEQ ID NO: 22, and (b) the VL comprises SEQ ID NO: 23.

[0054] In other embodiments, the CD40-specific antibody or CD40-specific scFv comprises a VH and a VL, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3, wherein: (i) the V H CDR1, V H CDR2 and V H CDR3 comprises SEQ ID NOs: 24, 25, and 26, respectively; and (ii) V L CDR1, V L CDR2 and V L CDR3 comprises SEQ ID NOs: 27, 28 and 29, respectively.

[0055] In another embodiment, the CD40 binding domain is a 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.

[0056] In another embodiment, the fusion protein of the invention further comprises an endoplasmic reticulum (ER) retention sequence located C-terminal to the antigen, with the proviso that the translocation domain is a PE translocation peptide (T PE ) 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.

[0057] In another embodiment, the fusion protein of the invention further comprises a CD28 activation peptide positioned between the CD40 binding domain and the myfurin and / or cathepsin L cleavage site.

[0058] In one embodiment, the CD28 activation peptide is 28 to 53 aa residues in length. In another embodiment, the CD28 activation peptide comprises the minimum functional fragment SEQ ID NO: 35. In another embodiment, the CD28 activation peptide is 28 to 53 aa residues in length, and the above-mentioned CD28 activation peptide comprises the minimum functional fragment SEQ ID NO: 35.

[0059] In other embodiments, the CD28 activation peptide comprises an amino acid sequence that is at least 95%, 97%, or 99% identical to SEQ ID NO: 35, 36, or 37. In other embodiments, the CD28 activation peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 36, and 37. In other embodiments, the CD28 activation peptide is SEQ ID NO: 35, 36, or 37.

[0060] In one embodiment, the pathogen is selected from the group consisting of PRRSV, ASFV, CSFV, PCV2, FMDV, PEDV, SVDV, PRV, TGEV, Mycoplasma hyopneumoniae, NDV, IBV, IBDV, parvovirus, poxvirus, rotavirus, and influenza virus. In another embodiment, the pathogen is selected from the group consisting of PRRSV, ASFV, CSFV, PCV2, FMDV, and PEDV.

[0061] The antigen may comprise one or more antigenic polypeptides derived from or selected from PRRSV, ASFV, CSFV, PCV2, FMDV, PEDV, SVDV, PRV, TGEV, Mycoplasma hyopneumoniae, NDV, IBV, IBDV, parvovirus, poxvirus, rotavirus, and influenza virus.

[0062] In one embodiment, the antigen is a pathogenic antigen selected from the group consisting of ASFV CP204L protein, ASFV E183L protein, CSFV E2 protein, CSFV NS3p protein, PCV2 ORF2 protein, PEDV S1 protein, PRRSV ORF6 protein, PRRSV ORF5 protein, PRRSV ORF7 protein, and antigenic polypeptides thereof. The antigen may be a fusion antigen comprising at least two antigenic polypeptides. Examples include a fusion antigen of ASFV CP204L and E183L, a fusion antigen of CSFV E2 and NS3p, and a fusion antigen derived from PRRSV and PCV2.

[0063] In one embodiment, the antigen comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51. In another embodiment, the antigen is a peptide that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51. In another embodiment, the antigen is a peptide having the amino acid sequence of SEQ ID NO: 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 51. In yet another embodiment, the antigen is a peptide of SEQ ID NO: 39, 40, 41, 42, 43, or 44.

[0064] In one embodiment, the above-mentioned antigen comprises at least one epitope to induce a desired immune response, preferably contains 1 to 50 epitopes, more preferably contains 1 to 20 epitopes.

[0065] The antigen may be a single antigen or antigenic fragment thereof, or may be a fusion antigen comprising at least two antigenic polypeptides fused with or without a linker between the two antigenic polypeptides.

[0066] The fusion antigen may have a rigid linker, (EAAAAK)n, connecting two different antigenic polypeptides, where n is an integer from 0 to 12, preferably from 2 to 6, and more preferably from 3 to 4. 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).

[0067] The fusion proteins of the present 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 is: (EAAAAK) n , or (SEQ ID NO: 38) nwhere n is an integer of 0 to 12, preferably 2 to 6, more preferably 3 to 4. In one embodiment, the rigid linker comprises 2 to 6 repeats or 3 to 4 repeats of SEQ ID NO:38.

[0068] In another embodiment, a fusion protein of the invention comprises or consists essentially of an amino acid sequence that is at least 90%, 95%, or 99% identical to SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, or 59. In yet other embodiments, a fusion protein of the invention comprises or consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 52, 53, 54, 55, 56, 57, 58, or 59.

[0069] The present invention further relates to the fusion protein in the manufacture of a medicament for eliciting an antigen-specific cellular and / or humoral immune response, or for reducing, inhibiting, treating, and / or ameliorating an infectious animal disease caused by a pathogen in an animal in need thereof. In one embodiment, the animal is a non-human animal. In another embodiment, the animal is not infected with a pathogen.

[0070] Pharmaceutical Compositions / Vaccines The present invention further comprises: (a) a fusion protein of the invention, and (b) a pharmaceutically acceptable carrier and / or adjuvant The present invention relates to a pharmaceutical composition or vaccine comprising:

[0071] The vaccines of the present invention are prophylactic and / or therapeutic vaccines. In one embodiment, the fusion proteins are for use in eliciting antigen-specific cellular and humoral immune responses against a pathogen of interest in uninfected animals.

[0072] The term "carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonicity agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, and the like, and combinations thereof, which will be known to those skilled in the art (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329).

[0073] Suitable adjuvants include: (1) oil-in-water (o / w) adjuvants (e.g., MONTANIDE TM ISA 15A VG, MONTANIDE TM ISA 35 VG and MONTANIDE TM ISA 28R VG), (2) water-in-oil (w / o) adjuvants (e.g., MONTANIDE TM ISA 61 VG, MONTANIDE TM ISA 71 VG, MONTANIDE TM ISA 71R VG, MONTANIDE TM ISA 761 VG, MONTANIDE TM ISA 78 VG, MONTANIDE TM ISA 763B VG, MONTANIDE TM (3) water-in-oil-in-water (w / o / w) adjuvants (e.g., MONTANIDE TM ISA 201 VG, MONTANIDE TM ISA 206 VG and MONTANIDE TM(ISA 207 VG), (4) aluminum salt adjuvants (e.g., aluminum hydroxide and phosphate), (5) saponin-based adjuvants (e.g., GPI-0100, Quil A, or QS-21), (6) Toll-like receptor (TLR) agonist adjuvants (e.g., poly I:C, monophosphoryl lipid A, CpG oligonucleotides), and (7) mixtures of the above adjuvants. CpG oligonucleotide adjuvants include, but are not limited to, Class A CpG (i.e., CpG1585, CpG2216, or CpG2336), Class B CpG (i.e., CpG1668, CpG1826, CpG2006, CpG2007, CpG BW006, or CpG D-SL01), and Class CpG (i.e., CpG2395, CpG M362, or CpG D-SL03). Another suitable CpG adjuvant is CpG1018. In one embodiment, the adjuvant is a CpG oligonucleotide. In another embodiment, the adjuvant is Freund's incomplete adjuvant (FIA).

[0074] The pharmaceutical composition / vaccine may be in enteral or parenteral dosage form suitable for transdermal, transmucosal, nasopharyngeal, pulmonary, or direct injection, or for systemic (e.g., parenteral) or local (e.g., intratumoral or intraregional injection) administration. Parenteral injection may be by the intravenous (iv), intraperitoneal (ip), intramuscular (im), subcutaneous (sc), or intradermal (id) route. The pharmaceutical composition may also be administered orally, for example, in the form of tablets, coated tablets, dragees, hard gelatin capsules, and soft gelatin capsules.

[0075] The dosage of the fusion protein may vary depending on the disease to be controlled, the age and individual condition of the patient, and the mode of administration. The dosage may be adapted to the individual needs in each case to obtain a therapeutically effective amount of the fusion protein of the invention to achieve the desired therapeutic response.

[0076] For adult patients, a single dose of about 0.1 to 50 mg, particularly about 0.1 to 5 mg, is contemplated. Depending on the severity of the disease and the precise pharmacokinetic profile, the fusion protein may be administered in a single dose weekly, biweekly, or monthly, with a total of 1 to 6 doses administered per cycle to achieve such treatment.

[0077] In one embodiment, the invention provides a kit or packaged pharmaceutical composition comprising a fusion protein of the invention and instructions for its use to treat one or more symptoms of an infectious animal disease caused by a pathogen in an animal in need thereof. [Example]

[0078] Methods and Materials Table 1 shows the SEQ ID NOs and the corresponding polypeptides / fusion proteins.

[0079] [Table 1] JPEG2023081595000002.jpg254157JPEG2023081595000003.jpg177157

[0080] Flow cytometry. Spleen cells were stimulated with an antigen stimulator (to enhance the immune response to the specific antigen used in the fusion protein of the present invention) for 2 hours at 37°C, then treated with 50 μg / mL Brefeldin A and Monensin for 2 hours at 37°C. Cells were harvested, washed with PBS containing 0.5% BSA, and simultaneously stained with APC / Cy7-labeled anti-CD3 antibody, PerCP / Cy5.5-labeled anti-CD4 antibody, FITC-labeled anti-CD8 antibody, PE-labeled anti-CD44 antibody, and APC-labeled anti-CD62L antibody. After washing, cells were permeabilized, fixed, and simultaneously intracellularly stained with PE-labeled anti-IFN-γ antibody, PE / Cy7-labeled anti-IL-2 antibody, and eFluor450-labeled anti-TNF-α antibody. CD8+ or CD4+ memory T cell phenotype (CD3 + / CD44 hi CD62L loIntracellular cytokine induction (IFN-γ, IL-2, or TNF-α) of spleen cells bearing the IFN-γ, IL-2, or TNF-α antibodies was further analyzed using a Gallios flow cytometer and Kaluza software.

[0081] Enzyme-linked immunospot (ELISpot) assay. Spleen cells were plated at a cell density of 2 x 10 in pretreated mouse IFN-γ capture 96-well plates (CTL IMMUNOSPOT®) in the presence or absence of antigenic stimuli (to enhance the immune response). 5 The cells were seeded in triplicate at 1000 cells / well and cultured at 37°C for 24 hours before discarding. After washing, the captured IFN-γ was detected with a biotin-labeled anti-mouse IFN-γ antibody at room temperature for 2 hours, and IFN-γ-immunospots were formed according to the manufacturer's instructions. IFN-γ-immunospots were scanned and counted using an IMMUNOSPOT® S5 Microanalyzer (CTL). The results were expressed as IFN-γ per million splenocytes. + Shown as immunospots.

[0082] Indirect enzyme-linked immunosorbent assay (ELISA). Collected whole blood samples were incubated at 4°C for 30–60 minutes and then centrifuged at 5,000 g for 10 minutes to pellet clots. Serum samples were stored at -20°C. To capture specific antibodies to the desired antigen, corresponding antigenic proteins or peptides were synthesized and used as coating proteins. The coating proteins were diluted in PBS buffer and dispensed into a 96-well plate at 1 μg / well. After overnight incubation at 4°C, the 96-well plate was blocked with 1% BSA in PBS at 37°C for 1 hour. The serum samples were thawed and then serially diluted 10-fold in PBS containing 1% BSA. The coating proteins were then incubated with 100 μl of a 1:1000 diluted serum sample at 37°C for 2 hours. After washing four times with phosphate-buffered saline TWEEN®-20 (PBST), antigen-specific antibodies bound to the coating protein were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (dilution 1:10,000, Cat#31430, Thermo Fisher Scientific) for 30 minutes at 37°C. After washing four times with PBST, HRP-mediated color development was performed in the presence of 100 μL of TMB substrate and quenched with 100 μL of 1N hydrochloric acid. The relative titers of antigen-specific antibodies in serum samples were determined by absorbance at 450 nm.

[0083] Statistical analysis: A t-test was used, and results were considered significant when p<0.05.

[0084] Example 1 Construction of expression vectors CD40L-T PE -CP204L-E183L, CD40L-T PE -CP204L, CD40L-T PE -E183L and CD40L-T PE -E2-NS3p. Vector CD40L-T PE -CP204L-E183L (Figure 1A) was constructed, and (a) a truncated CD40 ligand, CD40L 108-261 (SEQ ID NO: 19), (b) (EAAAAK)3 (SEQ ID NO: 3) and RX 1 RX2 X 3 R (child Here X 1 A, X 2 is Y, X 3 (c) a cleavable peptide linker containing PE metastasis Peptide PE 280-305 (SEQ ID NO: 5), and (d) a CD40L-T comprising the fusion antigen ASFV CP204L-E183L (SEQ ID NO: 41), comprising the antigen ASFV CP204L (SEQ ID NO: 39) and the antigen ASFV E183L (SEQ ID NO: 40). PE A -CP204L-E183L (SEQ ID NO: 52, Figure 3A) fusion protein was generated.

[0085] Briefly, CD40L 108-261 , a cleavable linker and a PE transfer peptide (PE 280-305 ) HindIII CD40L-Linker-PE NcoI、XhoI、SalI A DNA fragment encoding the CD40L-T was PCR synthesized, digested with HindIII / SalI, and ligated into the plasmid pTAC-MAT-Tag-2 (Cat. No. E5405, Sigma-Aldrich) with HindIII / XhoI cleavage sites to obtain the plasmid P07-His-pNC (Figure 1B). Another DNA fragment encoding the above-mentioned antigen ASFV CP204L-E183L with a His tag was inserted into the plasmid P07-His-pNC (Figure 1B) via the NcoI / XhoI sites to generate the expression vector CD40L-T. PE -CP204L-E183L was generated (Figure 1A).

[0086] The cleavable linker is used to separate the fusion protein from the T PE -Allows furin and / or cathepsin L proteases to cleave the fusion protein of the invention to release the CP204L-E183L fragment.

[0087] Using similar methods as described above, any other antigen(s) of interest from a pathogen may be substituted for the antigen ASFV CP204L-E183 L and inserted into the plasmid of Figure 1B to generate an expression vector as in Figure 1A for expressing a fusion protein containing the antigen(s) of interest.

[0088] By replacing the antigen ASFV CP204L-E183L with the antigen ASFV CP204L (SEQ ID NO: 39), CD40L-T PE An expression vector (FIG. 1C) was constructed to generate the CD40L-CP204L (SEQ ID NO: 53, FIG. 3B) fusion protein. The antigen ASFV CP204L-E183L was replaced with the antigen ASFV E183L (SEQ ID NO: 40). PE Another expression vector (FIG. 1D) was constructed to generate the CD40L-T-E183L (SEQ ID NO: 54, FIG. 3C) fusion protein by replacing the antigen ASFV CP204L-E183L with the fusion antigen CSFV E2-NS3p (SEQ ID NO: 44). PE Another expression vector (FIG. 1E) was constructed to generate the -E2-NS3p (SEQ ID NO: 55, FIG. 3D) fusion protein.

[0089] Vector CP204L-E183L-T Stx -CD40L, CP204L-T Stx -CD40L, E183L-T Stx -CD40L, and E2-NS3p-T Stx -CD40L. Vector CP204L-E183L-T Stx -CD40L (Figure 2A) was constructed to contain (a) the fusion antigen ASFV CP204L-E183L (SEQ ID NO: 41) comprising the antigen ASFV CP204L (SEQ ID NO: 39) and the antigen ASFV E183L (SEQ ID NO: 40), (b) the Stx transit peptide Stx 211-247 (SEQ ID NO: 14), (c) RX 1 X 2 R (where X 1 is V, X 2 A) and (EAAAAK) 3(SEQ ID NO: 3), and (d) a truncated CD40 ligand, CD40L. 108-261 (SEQ ID NO: 19), including CP204L-E183L-T Stx A fusion protein was generated to identify the IL-16-CD40L (SEQ ID NO: 56, Figure 3E).

[0090] Briefly, Stx transit peptide (Stx 211-247 ), a cleavable linker and CD40L 108-261 Contains HindIII、XhoI Stx-Linker-CD40L Sal A DNA fragment encoding the ASFV CP204L-E183L antigen was PCR synthesized, digested with HindIII / SalI restriction enzymes, and ligated into the pTAC-MAT-Tag-2 backbone plasmid with HindIII / XhoI cleavage sites, resulting in the plasmid P08(RP)-His-pNC (Figure 2B). Another DNA fragment encoding the ASFV CP204L-E183L antigen with a His tag was inserted into the plasmid P08(RP)-His-pNC (Figure 2B) via the HindIII / XhoI sites, resulting in the expression vector CP204L-E183L-T. Stx -CD40L was generated (Figure 2A).

[0091] The cleavable linker allows cleavage of the fusion protein by furin and / or cathepsin L proteases to separate the fusion protein into the fragment CP204L-E183L-T Stx This is essential for the fusion protein of the present invention as it allows the release of

[0092] Using similar methods as described above, any other antigen(s) of interest from a pathogen may be substituted for the antigen ASFV CP204L-E183 L and inserted into the plasmid of Figure 2B to generate an expression vector as in Figure 2A for expressing a fusion protein containing the antigen(s) of interest.

[0093] CP204L-T by replacing antigen ASFV CP204L-E183L with antigen ASFV CP204L (SEQ ID NO: 39). StxAn expression vector (Figure 2C) was constructed to generate the ASFV-CD40L (SEQ ID NO: 57, Figure 3F) fusion protein. The antigen ASFV CP204L-E183L was replaced with the antigen ASFV E183L (SEQ ID NO: 40), resulting in E183L-T. Stx An expression vector (FIG. 2D) for producing the E2-NS3p-T (SEQ ID NO: 58, FIG. 3G) fusion protein was similarly constructed. The E2-NS3p-T (SEQ ID NO: 44) was obtained by replacing the antigen ASFV CP204L-E183L with the antigen CSFV E2-NS3p. Stx Another expression vector (FIG. 2E) was constructed to generate a CD40L (SEQ ID NO: 59, FIG. 3H) fusion protein.

[0094] Example 2 Protein expression Protein expression vector CD40L-T PE E. coli BL21 cells carrying -E2-NS3p were grown at 37°C in ZY medium (10 g / L tryptone and 5 g / L yeast extract) containing selective antibiotics. When the culture reached early logarithmic phase (OD 600 When the fusion protein reached a pH of 2 to 5, expression of the fusion protein was induced with isopropyl-1-thio-β-D-galactopyranoside (IPTG) (0.5 to 2 mM). Four hours after IPTG induction, cells were harvested and disrupted by sonication. The overexpressed fusion protein was recovered by isolation of inclusion bodies and solubilization with solubilization buffer (6 M guanidine hydrochloride, 20 mM potassium phosphate, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, pH 7.4). After purification, the fusion protein was refolded overnight at 4°C by dialysis against a 20- to 50-fold volume of dialysis buffer (10 mM PBS). The refolded fusion protein was analyzed by SDS-PAGE under reducing (with dithiothreitol, +DTT) and non-reducing (without dithiothreitol, -DTT) conditions to assess whether it had refolded properly.

[0095] Fusion protein CD40L-T PE -CP204L-E183L, CD40L-TPE -CP204L, CD40L-T PE -E183L, CP204L-E183L-T Stx -CD40L, CP204L-T Stx -CD40L, E183L-T Stx -CD40L, and E2-NS3p-T Stx -CD40L is expressed, purified, and refolded in the same manner as above.

[0096] Example 3 Immunogenicity analysis of fusion proteins CD40L-T PE The E2-NS3p fusion protein was selected as a representative of the fusion proteins of the present invention and subjected to immunogenicity analysis to evaluate the biological activity of the fusion protein. Table 2 shows the animal groups, the doses of fusion proteins for vaccination, and the administration schedule. "V": vaccinated, "-": not vaccinated.

[0097] [Table 2]

[0098] Vaccine preparation: The fusion protein of the present invention dissolved in phosphate-buffered saline was mixed with an equal volume of Freund's incomplete adjuvant (FIA) to form an emulsion. The final formulation contained 0.5 mg of fusion protein / mL and 50% (v / v) Freund's incomplete adjuvant.

[0099] C57BL / 6NCrlBltw female mice (5-6 weeks old) were randomly divided into six groups (n = 5 per group): group A (placebo), group B (50 μg of test fusion protein), and groups C to E (100 μg of test fusion protein). The placebo group received sc PBS injections on days 0, 7, and 14. Mice in vaccinated groups (B to E) received sc injections of the fusion protein adjuvanted with Freund's incomplete adjuvant according to the administration schedule in Table 2. Serum was collected on days 0, 7, 14, and 21 to measure antigen-specific humoral immune responses. Animals were sacrificed on day 21. Spleen cells were collected and cultured to measure antigen-specific cellular immune responses.

[0100] The spleen cells were used to analyze intracellular cytokine induction (IFN-γ, IL-2, and TNF-α) in memory T cells using flow cytometry. Furthermore, the frequency of IFN-γ-secreting spleen cells was analyzed using an enzyme-linked immunospot (ELISpot) assay. Serum antigen-specific antibody levels in blood samples were analyzed using ELISA.

[0101] Figures 4-6 show the results of IFN-γ, IL-2, and TNF-α induction after stimulating splenocytes with or without an antigenic stimulant (a pool of short peptides covering the sequence of the antigen CSFV E2), respectively. On day 21, CD40L-T cells were stimulated with different doses or different regimens. PE -CD4 in the group receiving E2-NS3p + The induction levels of IFN-γ, IL-2 and TNF-α in memory T cells were observed and compared with the placebo group.

[0102] Figures 7A-B show the IFN-γ secretion after stimulation of spleen cells obtained from animals on day 21 with or without the in vitro antigenic stimuli described above. + Immunospot results are shown in Figure 7A, which shows a significant increase in the population of IFN-γ-secreting splenocytes obtained from animals in groups B and C on day 21, where animals in groups B and C received 50 μg and 100 μg of CD40L-T, respectively. PEThe animals were vaccinated with IFN-γ-E2-NS3p on days 0, 7, and 14. Significantly, the high-dose group showed more potent activity in inducing IFN-γ-secreting splenocytes compared to the low-dose group (p=0.006). Figure 7B shows a significant increase in the population of IFN-γ-secreting splenocytes in animal groups C, D, and E compared to the placebo group. All animals in groups C, D, and E received 100 μg of CD40L-T PE -E2-NS3p fusion protein, but on different schedules (DO-D7-D14, DO-D7, and DO-D14, respectively).

[0103] The fusion proteins of the present invention can effectively induce antigen-specific antibody responses. Figures 8A-B show serum antibody levels on day 21 measured by ELISA. CD40L-T PE The -E2-NS3p fusion protein was used to coat ELISA plates as a capture antigen.

[0104] Figure 8A shows serum antigen-specific antibody levels in animal groups A, B, and C on day 21. Animals in groups B and C received 50 μg and 100 μg of CD40L-T, respectively. PE Patients were vaccinated with E2-NS3p on days 0, 7, and 14. Serum levels of antigen-specific antibodies in groups B and C were higher than those in the placebo group, and the high-dose group induced stronger antibody responses than the low-dose group (p=0.015).

[0105] Figure 8B shows serum antigen-specific antibody levels in animal groups A, C, D, and E on day 21. All animals in groups C, D, and E received 100 μg of CD40L-T PE The three-dose vaccination group and the two-dose vaccination group both showed a significant increase in serum antigen-specific antibody titers. Notably, the third dose was able to induce significantly higher antigen-specific antibody responses than the two-dose vaccination group.

[0106] Figures 9A-B show serum antibody levels in animal groups on day 21. PE Antigen-specific antibody levels were measured by ELISA as in Figures 8A-B, except that the E2-NS3p peptide (capture antigen) was used instead of the E2-NS3p fusion protein. The results were consistent with those in Figures 8A-B. The serum levels of antigen-specific antibodies in Groups B and C on Day 21 were higher than those in the placebo group, and the high-dose group induced stronger antibody titers than the low-dose group.

[0107] Fusion protein CD40L-T PE -CP204L-E183L, CD40L-T PE -CP204L, CD40L-T PE -E183L, CP204L-E183L-T Stx -CD40L, CP204L-T Stx -CD40L, E183L-T Stx -CD40L, and E2-NS3p-T Stx Based on the same mechanism of action, the antigen-carrying fusion protein of the present invention is expected to induce a strong immune response against the target antigen, resulting in the induction of antigen-specific antibodies, activation of T cells, and induction of IFN-γ, IL-2, and TNF-α.

[0108] Example 4 Immunogenicity analysis of the fusion protein under extended administration schedules CD40L-T in the induction of immune responses PE The effect of the E2-NS3p fusion protein was also evaluated under an extended administration schedule. Table 3 shows the animal groups and the administration schedule for each group. "V": vaccinated, "-": unvaccinated.

[0109] [Table 3]

[0110] Except for the extended administration schedule and mouse groups, the vaccine preparation, mice, administration route, and analytical methods were the same as those described in Example 3.

[0111] Briefly, C57BL / 6NCrlBltw female mice (5-6 weeks old) were randomly divided into four groups (n = 5 per group): group A (placebo), group B, group C, and group D (100 μg fusion protein). The placebo group received sc PBS on days 0, 21, and 42. Mice in groups B, C, and D were sc vaccinated with the test fusion protein adjuvanted with FIA according to the respective dosing schedules for groups B to D in Table 3. Serum was collected weekly to measure antigen-specific humoral immune responses. On day 63, animals were sacrificed, and spleen cells were harvested and cultured to measure antigen-specific cellular immune responses using the analytical methods described in Example 3.

[0112] Figures 10A-C show the results of IFN-γ, IL-2, and TNF-α induction after stimulation of splenocytes obtained from animals on day 63 with or without an antigen stimulator (a pool of short peptides covering the sequence of the antigen CSFV E2). PE -CD4 in groups receiving different E2-NS3p regimens + The levels of IFN-γ, IL-2, and TNF-α induction in memory T cells were observed and compared with the placebo group.

[0113] FIG. 11 shows the IFN-γ response of spleen cells obtained from animals on day 63 after treatment with or without antigenic stimuli as described above. + The results of immunospot are shown. Compared with the placebo group, CD40L-T PE A significant increase in the population of IFN-γ-secreting splenocytes was observed in all animal groups receiving different regimens of IFN-E2-NS3p. A single injection of the fusion protein of the present invention was sufficient to induce an increase in the population of IFN-γ-secreting splenocytes (Figure 11, group D).

[0114] Figure 12 shows serum antigen-specific antibody levels in animal groups A, B, C, and D, respectively. Animals in groups B, C, and D were treated with different regimens (D0-D21-D42, D21-D42, and D42, respectively) to induce CD40L-T PE The mice were vaccinated with the fusion protein CD40L-T. The coating protein (capture antigen) used in the ELISA was the antigen peptide E2-NS3p. The results were consistent with those described above. PE A single dose of E2-NS3p was sufficient to induce antigen-specific antibody responses. Remarkably, in the three-dose group, a prolonged antibody response was observed, lasting at least 21 days after the third dose on day 42.

[0115] In summary, the fusion proteins of the present invention are capable of eliciting potent T cell immune responses, increasing the expression of IFN-γ, IL-2 and TNF-α, and generating antigen-specific antibody responses.

[0116] All references cited and discussed herein are incorporated by reference in their entirety and to the same extent as if each reference was individually incorporated by reference.

Claims

1. A fusion protein comprising: (a) a CD40-binding domain, which is CD40 ligand (CD40L) or a functional fragment thereof; (b) pathogen antigens; (c) a translocation domain selected from a Pseudomonas exotoxin A (PE) translocation peptide or a Shiga toxin (Stx) translocation peptide, located between the CD40 binding domain and the antigen; and (d) a furin and / or cathepsin L cleavage site located immediately adjacent to the CD40 binding domain and translocation domain. Including, wherein the pathogen is at least one selected from the group consisting of classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), Mycoplasma hyopneumoniae, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), parvovirus, poxvirus, rotavirus, and influenza virus.

2. 2. The fusion protein of claim 1, wherein the translocation domain is a Pseudomonas exotoxin A (PE) translocation peptide and the CD40 binding domain is located at the N-terminus of the fusion protein.

3. 2. The fusion protein of claim 1, wherein the translocation domain is a PE translocation peptide consisting of 26 to 112 amino acid residues in length and comprising an amino acid sequence at least 95% identical to SEQ ID NO: 5, 6, 7, 8 or 9.

4. 2. The fusion protein of claim 1, wherein the translocation domain is a Shiga toxin (Stx) translocation peptide and the antigen is located at the N-terminus of the fusion protein.

5. 2. The fusion protein of claim 1, wherein the translocation domain is an Stx translocation peptide consisting of 8 to 84 amino acid residues in length and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 12, 13, 14, 15, or 16.

6. 2. The fusion protein of claim 1, wherein the furin and / or cathepsin L cleavage site comprises the amino acid sequence RX1X2R or RX1RX2X3R (wherein X1 and X2 are any amino acid residues and X3 is K, F or R).

7. The fusion protein of claim 1, further comprising a peptide linker comprising a furin and / or cathepsin L cleavage site located immediately adjacent to the CD40 binding domain and translocation domain.

8. A fusion protein comprising: (a) a CD40-binding domain, which is an antibody specific for CD40 (CD40-specific antibody) or a functional fragment thereof; (b) pathogen antigens; (c) a translocation domain selected from a Pseudomonas exotoxin A (PE) translocation peptide or a Shiga toxin (Stx) translocation peptide, located between the CD40 binding domain and the antigen; and (d) a furin and / or cathepsin L cleavage site located immediately adjacent to the CD40 binding domain and translocation domain. Including, wherein the pathogen is at least one selected from the group consisting of classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), Mycoplasma hyopneumoniae, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), parvovirus, poxvirus, rotavirus, and influenza virus.

9. 2. The fusion protein of claim 1, wherein the CD40 binding domain is CD40L consisting of 154 to 261 amino acid residues in length and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 17, 18, or 19.

10. The CD40 binding domain is a CD40-specific antibody or binding fragment thereof, or a single chain variable fragment (scFv), and the CD40-specific antibody or the scFv comprises: (a) a heavy chain variable region (VH) comprising SEQ ID NO: 22; and (b) a light chain variable region (VL) comprising SEQ ID NO: 23 2. The fusion protein of claim 1, comprising:

11. The CD40 binding domain is a CD40-specific antibody or binding fragment thereof, wherein the CD40-specific antibody comprises a VH and a VL, wherein the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3, and further wherein: (i) the VH CDR1, VH CDR2, and VH CDR3 comprise the amino acid sequences of SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; and (ii) the VL CDR1, VL CDR2, and VL CDR3 comprise the amino acid sequences of SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively; The fusion protein of claim 1.

12. The fusion protein of claim 8, wherein the translocation domain is a Shiga toxin (Stx) translocation peptide and the antigen is located at the N-terminus of the fusion protein.

13. The fusion protein of claim 8, wherein the translocation domain is a Pseudomonas exotoxin A (PE) translocation peptide, and the CD40 binding domain is located at the N-terminus of the fusion protein.

14. The fusion protein described in claim 8, wherein the translocation domain is a PE translocation peptide consisting of 26 to 112 amino acid residues in length and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 5, 6, 7, 8 or 9.

15. The fusion protein described in claim 8, wherein the translocation domain is an Stx translocation peptide consisting of 8 to 84 amino acid residues in length and comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 12, 13, 14, 15 or 16.

16. A fusion protein described in any one of claims 1 to 15, wherein the pathogen is selected from classical swine fever virus (CSFV) or African swine fever virus (ASFV).

17. The fusion protein of claim 16, wherein the translocation domain is the PE translocation peptide and the pathogen is classical swine fever virus (CSFV).

18. (a) a fusion protein according to any one of claims 1 to 15, and (b) a pharmaceutically acceptable carrier and / or adjuvant A pharmaceutical composition comprising:

19. The fusion protein of any one of claims 1 to 15 or the pharmaceutical composition of claim 18 for inducing an antigen-specific cellular immune response or for reducing, inhibiting, treating and / or ameliorating an infectious animal disease caused by a pathogen in an animal in need thereof.

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