WO2018049188A1 - Potent broad spectrum agonist for enhancing vaccine efficacy in livestock - Google Patents

Abstract

Monoclonal antibodies, and fragments and derivatives thereof, are provided that increase the immunogenicity of an antigen in a variety of species. Compositions and pharmaceutical compositions comprising the monoclonal antibodies, as well as methods for increasing the immunogenicity of an antigen in a variety of species, are also provided.

Description

TITLE OF INVENTION

POTENT BROAD SPECTRUM AGONIST FOR ENHANCING VACCINE EFFICACY IN LIVESTOCK

REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of United States provisional application No. 62/385,631, filed September 9, 2016, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to vaccine adjuvants. More specifically, the invention relates to methods and compositions for improving vaccine efficacy in livestock and companion species, including, but not limited to, cattle, swine, sheep, goats, horses and dogs.

INCORPORATION OF SEQUENCE LISTING

[0003] The sequence listing that is contained in the file named "TAMC047WO_ST25.txt", which is 3.28 kilobytes as measured in Microsoft Windows operating system and was created on September 8, 2017, is filed electronically herewith and incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0004] Immunization with whole killed pathogens or recombinant antigens alone is not effective at eliciting immune responses and thus requires formulation with an adjuvant. Currently, there are a limited number of safe, affordable, and effective adjuvants that are licensed for use in cattle and swine, as well as other livestock species. Activation of antigen presenting cells or APCs (dendritic cells, B cells, and macrophages), which are needed for inducing immune responses, has been shown to require signaling via a surface receptor called CD40. Activation of these APCs is an innate response that adjuvants as well as live vaccines stimulate through Pathogen- Associated Molecular Patterns (PAMPs), chemokines and cytokines. Importantly, activated helper T cells (CD4 T cells) provide APC activation by expressing CD40 ligand (CD40L) and thereby stimulate production of inflammatory cytokines by the APCs. Signaling via the CD40 receptor is also critical for antibody isotype switching and for effective priming of helper CD4 T cells as well as cytotoxic T lymphocytes (CTLs).

[0005] Cluster of differentiation 40 (CD40) receptor belongs to the tumor necrosis factor superfamily and it is expressed on B cells, macrophages, dendritic cells (DCs), endothelial cells and fibroblasts (Fries, et al, Clin. Immunol. Immunopathol. 77:42-51, 1995; Galy and Spits, J. Immunol. 149:775-782, 1992). CD40 is also expressed on several types of human cancer cells including bladder, breast, and ovarian (Paulie, et al., Cancer Immunol. Immunother. 20:23-28, 1985; Noelle, et al, Immunol. Today 13:431-433, 1992; Foy, et al, J. Exp. Med. 178: 1567-1575, 1993; van Kooten and Banchereau, J. Leukoc. Biol. 67:2-17, 2000). CD40L (CD154), a natural ligand for CD40, is a cytokine expressed either on the surface or secreted by activated CD4⁺ T cells (Clark and Ledbetter, Proc. Natl. Acad. Sci. USA 83:4494-4498, 1986; Hill, et al., J. Immunol. 174:41-50, 2005). The CD40L interacts with CD40 by crosslinking multiple CD40 molecules and thereby provides a critical signal for APC activation (Noelle, et al., 1992, supra; Foy, et al., 1993, supra). The CD40-CD40L interaction stimulates B cells to undergo somatic hypermutation, class switch recombination, clonal expansion, and upregulation of major histocompatibility complex II (MHCII) and secretion of proinflammatory cytokines. For example, humans suffering from X-linked hyper-IgM syndrome are deficient in either CD40 or CD40L, and thus do not undergo class switch recombination or somatic hypermutation. The X-linked hyper IgM syndrome leads to high proportions of IgMs and low levels of IgA, IgE, and IgG present in the serum, absence of germinal centers, and the inability to mount a T-cell- dependent humoral response (Aruffo, et al, Cell 72:291-300, 1993). When CD40L interacts with CD40 on macrophages, it stimulates the cells to synthesize and release nitric oxide, and upregulate MHCII and proinflammatory cytokines (Grewal and Flavell, Immunol. Today 17:410- 414, 1996; Noelle, Immunity 4:415-419, 1996).

[0006] Naive T cells require two distinct signals from APCs for proper activation and induction of differentiation: signal 1 is provided by peptide antigens in the context of MHC molecules, while signal 2 is delivered by costimulatory molecules such as CD80 or CD86 present on DCs (Haase, et al., Scand. J. Immunol. 59:237-245, 2004). For antigen-loaded DCs to provide these signals effectively, they require activation to up-regulate surface expression of MHC -peptide complexes and costimulatory molecules, and to secrete pro-inflammatory molecules such as IL-12 (Fujii, et al., J. Exp. Med. 199, 1607-1618, 2004). DC activation is an innate response that adjuvants as well as live vaccines stimulate through pattern recognition receptor (PRR)-ligand signaling, chemokine and cytokine secretion (Gallucci, et al., Nat. Med. 5: 1249-1255, 1999). [0007] Expression of CD80/CD86 is upregulated by PRR ligands, TNF-a and IFN-γ, as well as interaction between CD40 on APCs and CD40L (Gallucci, et al., 1999, supra; Fujii, et al., 2004, supra; Haase, et ah, 2004, supra). However, DCs from CD40^_/~ or CD40L^"7" mice do not elicit CD4⁺ and CD8⁺ T cell immunity, even though the DCs present antigens on MHC class I and II molecules and express high levels of CD80/86 (Fujii, et ah, 2004, supra). A distinct CD40/CD40L signal that functions together with antigen presentation and co-stimulation is required to generate functional CD4⁺ T helper and CD8⁺ CTLs (Fujii, et ah, 2004, supra). This signaling requires APC-T cell contact, CD40L expression, or an agonistic anti-CD40 antibody (Staveley-O'Carroll, et ah, J. Immunol. 171:697-707, 2003; Bonifaz, et ah, J. Exp. Med. 199:815-824, 2004; Huang, et al., Int. J. Cancer 108:696-703, 2004). Furthermore, this signaling results in increased production of pro-inflammatory cytokines such as IL-12, which is a powerful inducer of IFN-γ production and Thl differentiation (Koch, et al., J. Exp. Med. 184:741-746, 1996.)· More importantly, DC activation through CD40 signaling overcomes tolerance and may release immature DCs from the control of regulatory CD4⁺CD25⁺ T cells (Serra, et al., Immunity 19:877-889, 2003).

[0008] Agonistic mAbs against CD40 directly mimic CD4⁺ T-cell help in vivo in response to T-cell dependent antigens (Banchereau, et al., Annu. Rev. Immunol. 12:881-922, 1994; Banchereau, et al., Adv. Exp. Med. Biol. 378:79-83, 1995; Barr, et al., Immunology 109:87-92, 2003; Bishop, In: Seminars in Immunology, Vol. 21, p. 255, 2009). Using CD40-targeted antigen delivery, up to 1000-fold increased antibody responses was reported (Barr and Heath, Immunology 102:39-43, 2001; Barr et al., 2003, supra). In vitro stimulation of APCs using various forms of CD40 agonists like membrane-associated CD40L, soluble CD40L (sCD40L), or anti-CD40 antibodies evokes distinct functional responses (Fanslow, et al., Semin. Immunol. 6:267-278, 1994). Conjugation of an agonistic anti-CD40 mAb to a peptide-based vaccine, a whole killed virus vaccine, or a commercially produced split influenza virus vaccine significantly enhanced antigen- specific antibody and T cell responses (Hatzifoti and Heath, Immunology 122:98-106, 2007). Antibody class switching is also attributed to such agonistic anti-CD40 antibodies following interaction with CD40 on B cells.

[0009] The livestock industry is an economically important sector that generates revenue and jobs globally, but disease control is partly hindered by lack of safe and effective adjuvants. In addition, livestock serve as models for infectious and non-infectious human diseases. Therefore, development and optimization of an adjuvant that increases vaccine immunogenicity in a plurality of species would represent a significant advance in the art.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention overcomes one or more deficiencies in the art by providing an anti-swine CD40 mAb that has agonistic activity in a variety of livestock species.

[0011] The present invention provides an anti-swine CD40 monoclonal antibody, or a fragment thereof. In certain embodiments, the anti-swine CD40 monoclonal antibody or fragment thereof increases immunogenicity of the antigen in a plurality of species. In particular embodiments, the plurality of species are selected from the group consisting of pigs, cattle, sheep and goats. In further embodiments, the anti- swine CD40 antibody or fragment thereof is defined as a recombinant diabody, an scFv, an Fab fragment, an F(ab')2 fragment, a disulfide linked Fv or a whole immunoglobulin molecule. In one embodiment, the anti-swine CD40 antibody or fragment thereof is from clone 2E4E4.

[0012] In various embodiments, the anti-swine CD40 antibody or fragment thereof is comprised in a composition comprising an antigen. In certain embodiments, the antigen is a whole killed virus, inactivated virus or an attenuated virus. In some embodiments, the antigen is conjugated to the anti-swine CD40 monoclonal antibody, or a fragment thereof. In other embodiments the anti-swine CD40 antibody or fragment thereof comprises a pharmaceutically acceptable carrier. In yet other embodiments the anti- swine CD40 antibody or fragment thereof comprises an additional adjuvant that is distinct from said anti-swine CD40 monoclonal antibody, or a fragment thereof.

[0013] In certain embodiments, the anti- swine CD40 antibody or fragment thereof has a first light-chain variable domain having an amino acid sequence comprising SEQ ID NO:2, a second light-chain variable domain having an amino acid sequence comprising SEQ ID NO:3, a third light-chain variable domain having an amino acid sequence comprising SEQ ID NO:4, a first heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:5, a second heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:6, and a heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:7, or any combination of combinations thereof. [0014] The present invention additionally provides a pharmaceutical composition, comprising an antigen, a pharmaceutically acceptable carrier, and an anti-swine CD40 monoclonal antibody, or a fragment thereof. In some embodiments, the composition increases immunogenicity of the antigen in a plurality of species. In various embodiments, the composition is administered intravenously, intra-arterially, intra-peritoneally, intramuscularly, intradermally, orally, dermally, nasally, buccally, rectally, vaginally, by inhalation, or by topical administration.

[0015] The present invention also provides a composition comprising an anti-swine CD40 monoclonal antibody, or a fragment thereof. In particular embodiments, the anti-swine CD40 monoclonal antibody or fragment thereof increases immunogenicity of an antigen in a plurality of species. In further embodiments, the anti-swine CD40 monoclonal antibody is from clone 2E4E4.

[0016] The present invention further provides a method of increasing the immunogenicity of an antigen, comprising combining the antigen with an anti- swine CD40 monoclonal antibody, or a fragment thereof. In certain embodiments, the anti- swine CD40 monoclonal antibody or fragment thereof increases immunogenicity of the antigen in a plurality of species. In some embodiments, the antigen is conjugated to the anti-swine CD40 monoclonal antibody, or a fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0018] FIG. 1. Alignment of swine, bovine, caprine, and ovine CD40 amino acid sequences. The signal sequence is shown where the consensus sequence is highlighted in green color (amino acid 1-19), whereas the consensus sequence of the transmembrane domain is highlighted in red color (amino acid 192-215). The percentage identity of the extracellular domains of bovine, ovine, and caprine CD40 protein sequences to that of swine is 74%, 75%, and 75% respectively.

[0019] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E and FIG. 2F. Reactivity of the mAb 2E4E4 against HEK 293A cells expressing swine or bovine CD40. Evaluation of the mAb 2E4E4 specificity against swine and bovine CD40 was performed by immunocytometric and flow cytometric analysis. FIG. 2 A. Analysis of purified mAb 2E4E4 and a defined IgGl isotype control mAb by PAGE. FIG. 2B. HEK 293A cells transfected with a construct expressing full length swine CD40 and probed with mAb 2E4E4. FIG. 2C. HEK 293A cells transfected with a construct expressing full length bovine CD40 and probed with the mAb 2E4E4. FIG. 2D. Sham treated HEK 293 A cells probed with the mAb 2E4E4. FIG. 2E. Flow cytometric analysis of HEK 293A cells transfected with a construct encoding full length swine CD40 probed with either the mAb2E4E4 (red) or IgGl isotype control (blue). FIG. 2F. Flow cytometric analysis of HEK 293A cells transfected with a construct a encoding full length bovine CD40 probed with either the mAb2E4E4 (red) or IgGl isotype control (blue).

[0020] FIG. 3A and FIG. 3B. The mAb 2E4E4 recognized CD40 on stimulated swine and bovine PBMCs. FIG. 3A Flow cytometry performed on non-stimulated (red) or LPS-stimulated (orange) swine PBMCs and then probed with the mAb 2E4E4. IgGl isotype control (blue) was also used to probe LPS-stimulated swine PBMCs. FIG. 3B Flow cytometry performed on non-stimulated (red) or LPS-stimulated (orange) bovine PBMCs and probed with the mAb 2E4E4. IgGl isotype control (blue) was also used to probe LPS-stimulated bovine PBMCs.

[0021] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F, FIG. 4G and FIG. 4H.

Validation of the specificity of the mAb 2E4E4 against CD40 expressed on cells in swine, bovine, ovine, and caprine spleen. Immunohistochemistry performed on swine (FIG. 4A), bovine (FIG. 4B), ovine (FIG. 4C), and caprine (FIG. 4D) spleen tissues probed with the mAb 2E4E4. Background reactivity was tested by probing swine (FIG. 4E), bovine (FIG. 4F), ovine (FIG. 4G), and caprine (FIG. 4H) spleen tissues with an IgGl isotype control mAb.

[0022] FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D. Proliferation of swine and bovine PBMC by mAb 2E4E4 stimulation. Agonistic activity of the mAb 2E4E4 on swine, bovine, ovine, and caprine PBMCs was evaluated by H- Thymidine incorporation. Data is shown for n=3 (blue, green and red boxes), or an IgGl isotype control (blue, green and red triangles). Each point represents the mean stimulation index from triplicate wells + SD; * P<0.001. FIG. 5A. Swine PBMC response after incubation with 2E4E4 FIG. 5B. Bovine PBMC response after incubation with mAb 2E4E4. FIG. 5C. Ovine PBMC response after incubation with 2E4E4. FIG. 5D. Caprine PBMC response after incubation with 2E4E4. [0023] FIG. 6. Nitric oxide production by macrophages following stimulation by mAb 2E4E4. Agonistic effect of 2E4E4 was verified by nitric oxide assay using bovine macrophages incubated with graded amount of the mAb 2E4E4 (black) or IgGl isotype control (grey). Each column represents mean μΜ of N0₂ ^~ of triplicate wells stimulated with the mAb 2E4E4 at each concentration + SD; * P<0.0001.

[0024] FIG. 7A, FIG. 7B, FIG. 7C and FIG. 7D. Pro-inflammatory cytokine response upregulation by mAb 2E4E4. Intracellular cytokine staining was used to evaluate the ability of mAb 2E4E4 to upregulate pro-inflammatory cytokines in PBMCs. Swine PBMCs were incubated with the mAb 2E4E4, LPS, or media alone, harvested at 12 hours (grey) and 24 hours (black), and then probed with mAbs against IL-la (FIG. 7A), TNF-a (FIG. 7B), IL-6 (FIG. 7C) or IL-8 (FIG. 7D). Each column represents the mean florescent intensity of two wells + SD.

[0025] FIG. 8. Amino acid sequence of 2E4E4 light and heavy chain variable domains (SEQ ID NO: l).

[0026] FIG. 9. Amino acid (SEQ ID NO: l) sequence of 2E4E4 diabody. Shown are light chain V_K CDR1 (SEQ ID NO:2), light chain V_K CDR2 (SEQ ID NO:3), light chain V_K CDR3 (SEQ ID NO:4), heavy chain V_H CDR1 (SEQ ID NO:5), heavy chain V_H CDR2 (SEQ ID NO:6), and heavy chain V_H CDR3 (SEQ ID NO:7).

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention provides agonistic anti-CD40 monoclonal antibodies (mAbs) that are potent at activating bovine and porcine antigen presenting cells. One of the agonistic anti-CD40 mAb is termed 2E4E4, and the amino acid sequence of the light and heavy chain variable domains is shown in FIG. 8. A recombinant version of the agonist (diabody) has also been generated (nucleotide and amino acid sequence of the 2E4E4 diabody is shown in FIG. 9). This mAb is used to generate an adjuvant for enhancing or increasing the immune response to a wide variety of antigens in these two food animal species, as well as other livestock species, including, but not limited to, sheep and goats. The mAb or its recombinant versions can be used as a standalone stimulant in a vaccine formulation or conjugated to an antigen for targeting to APCs as well as adjuvant activity. In certain embodiments, the anti-CD40 monoclonal antibody, or a fragment thereof, is present in an immunologically effective amount. [0028] The agonist can be used to enhance efficacy of whole killed virus vaccines, live attenuated vaccines, inactivated virus vaccines, purified subunit antigen or engineered chimeric polypeptide containing defined protective B cell, CD4 T cell, and cytotoxic T lymphocyte (CTL) epitopes from bovine, porcine, ovine and/or caprine pathogens. To reduce host immune response against the agonist scaffold, the mAb is modified to replace the constant regions of the mouse IgG heavy and light chains with cognate bovine or swine IgG components.

[0029] Vaccine Compositions

[0030] As used herein, the term "vaccine composition" includes at least one immunogenic antigen or immunogen, along with one or more of the presently described agonistic anti-CD40 monoclonal antibodies, or fragments thereof, in a pharmaceutically acceptable carrier useful for inducing an immune response in a host. Vaccine compositions can be administered in dosages and by techniques well known to those skilled in the medical or veterinary arts, taking into consideration such factors as the age, sex, weight, species and condition of the recipient animal, and the route of administration. As used herein, the term "host cell" refers to any mammalian cell, whether located in vitro or in vivo. For example, host cells may be located in a transgenic animal.

[0031] Antigens and Immunogens

[0032] A peptide, polypeptide, protein or polynucleotide coding for such a molecule is "immunogenic" (and thus an "antigen" or "immunogen" within the present invention), if it is capable of inducing an immune response. In the case of the present invention, immunogenicity is more specifically defined as the ability to induce a T-cell response. Thus, an "antigen" or an "immunogen" would be a molecule that is capable of inducing an immune response, and in the case of the present invention, a molecule capable of inducing a T-cell response.

[0033] The term "peptide" is used herein to generally designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The peptides are generally about 8 or about 9 amino acids in length, but can be as long as about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29 or about 30 amino acids in length. The term "polypeptide" is used herein to generally designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. The length of the polypeptide is not critical to the invention as long as the correct epitopes are maintained. In contrast to the term peptide, the term polypeptide is meant to refer to molecules containing more than about 30 amino acid residues, for example about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 250, about 300, about 400, about 500 amino acids or greater. The term "protein" is used herein to generally designate a full length amino acid sequence.

[0034] The antigen in the vaccine may be an antigenic determinant. An "antigenic determinant" or "epitope" as used herein refers to a portion of an antigen that contacts a particular antibody. When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies that bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants.

[0035] The term "isolated" means that the material is removed from its original environment (e.g. , the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

[0036] Viral Antigens

[0037] In some aspects the invention is directed to a vaccine that is composed of an isolated viral antigen. A viral "antigen" or "immunogen" as used herein refers to a non-infectious virus or immunogenic portion, fragment or derivative thereof. The antigen may be a nucleic acid antigen and/or a peptide antigen and optionally may include lipids, such as those found in viral lipid envelopes. For instance an antigen or immunogen may comprise a viral like particle (VLP), whole organism, killed, attenuated, inactivated or live; a subunit or portion of an organism; a recombinant vector containing an insert with immunogenic properties; a piece or fragment of DNA capable of inducing an immune response upon presentation to a host animal; a protein, a glycoprotein, a lipoprotein, a polypeptide, a peptide, an epitope, a hapten, or any combination thereof.

[0038] In some instances the antigen, and thus the vaccine, is composed of an inactivated or attenuated virus. In certain embodiments, the virus may be inactivated by, for instance, heat, formalin, or a detergent. In some embodiments, the virus is attenuated by virtue of one or more mutation in the viral genome.

[0039] A viral antigen may also be a nucleic acid encoding a viral peptide. In order to effect expression the nucleic acid may be delivered in a vector and/or operably linked to a heterologous promoter and transcription terminator. As used herein, a "vector" may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids, and virus genomes.

[0040] A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably linked to regulatory sequences and may be expressed as an RNA transcript.

[0041] As used herein, a coding sequence and regulatory sequences are said to be "operably linked" when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably linked to a coding sequence if the promoter region is capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide. [0042] As used herein, reference to a DNA sequence includes both single stranded and double stranded DNA. Thus, the specific sequence, unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded DNA) and the complement of such sequence. The term "coding region" refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene. The coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.

[0043] Pharmaceutical Compositions

[0044] Pharmaceutically acceptable carriers are well known and are usually liquids, in which an active therapeutic agent is formulated. The carrier generally does not provide any pharmacological activity to the formulation, though it may provide chemical and/or biological stability, release characteristics, and the like. Exemplary formulations can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams & Wilkins, 2000, and include, but are not limited to, saline, water, buffered water, 0.3% glycine, hyaluronic acid, dextrose and the like.

[0045] The pharmaceutical compositions can be used for parenteral administration, such as subcutaneous, intradermal, intramuscular, or intraperitoneal, or intravenous, intra-arterial, oral, dermal, nasal, buccal, rectal, vaginal administration, by inhalation, or by topical administration. For this, the peptides and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. The peptides can also be administered together with immune stimulating substances, such as cytokines.

[0046] In addition, the composition can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. An extensive listing of excipients that can be used in such a composition, can be, for example, taken from Handbook of Pharmaceutical Excipients, 5th ed., edited by Raymond Rowe, Paul Sheskey and Sian Owen, Pharmaceutical Press, 2006. Suitable excipients include, but are not limited to, antioxidants like ascorbic acid or glutathione, preserving agents such as phenol, m-cresol, methyl- or propylparaben, chlorobutanol, thiomersal (thimerosal) or benzalkoniumchloride, stabilizer, framework former such as saccharose, lactose, maltose, trehalose, mannitose, mannitol and/or sorbitol, mannitol and/or lactose and solubilizer such as polyethyleneglycols (PEG), i.e., PEG 3000, 3350, 4000 or 6000, or cyclodextrins, i.e., hydroxypropyl-P-cyclodextrin, sulfobutylethyl-P-cyclodextrin or γ-cyclodextrin, or dextrans or poloxamers, i.e., poloxamer 407, poloxamer 188, Tween 20, or Tween 80. In certain embodiments pharmaceutical compositions of the present invention include one or more well tolerated excipients, selected from the group consisting of antioxidants, framework formers and stabilizers.

[0047] As used herein, "a pharmaceutically acceptable salt" refers to a derivative of antigenic or immunogenic agent modified by making acid or base salts of the agent. For example, acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral — NH₂ group) involving reaction with a suitable acid. Suitable acids for preparing acid salts include both organic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid phosphoric acid and the like. Conversely, preparation of basic salts of acid moieties which may be present on a peptide are prepared using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like.

[0048] The pharmaceutical compositions of the present invention may also include sugars, sugar alcohols, amino acids such as glycine, arginine, glutamic acid and others as framework former. The sugars may be mono-, di- or trisaccharide. These sugars may be used alone, as well as in combination with sugar alcohols. Examples of sugars include glucose, mannose, galactose, fructose or sorbose as monosaccharides, saccharose, lactose, maltose or trehalose as disaccharides and raffinose as a trisaccharide. A sugar alcohol may be, for example, mannitose. Preferred ingredients are saccharose, lactose, maltose, trehalose, mannitol and/or sorbitol.

[0049] Further Adjuvants

[0050] Known vaccine adjuvants for use in addition to the presently described agonistic anti-CD40 monoclonal antibodies include, but are not limited to, oil and water emulsions, oil-in-water emulsions, water-in-oil emulsions, water-in-oil-in-water emulsions, saponin, aluminum hydroxide, dextran sulfate, carbomer, sodium alginate, (N,N-dioctadecyl-N',Nt-bis(2- hydroxyethyl)-propanediamine), paraffin oil, muramyl dipeptide, cationic lipids, DMRIE, DOPE, and TLR ligands such as CpG oligonucleotides. Additional adjuvants for use with the presently described agonistic anti-CD40 monoclonal antibodies include, but are not limited to, 1018 ISS, aluminium salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, Mologen's dSLIM, GM CSF, IC30, IC31, Imiquimod, ImuFact IMP321, interferon-a or -β, IS Patch, ISS, ISCOMs, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, and other non-toxic LPS derivatives, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM- 174, OM- 197-MP-EC, ONTAK, PepTel® vector system, PLG microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, MA, USA) which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics. Also cytokines may be used, including, but not limited to, TNF-a, IL-1, IL-4 and IL- 12.

[0051] Kits

[0052] Kits of the present invention preferably comprise a formulation of one or more of the presently described agonistic anti-CD40 monoclonal antibodies in a suitable container and instructions for its use. Suitable containers include, for example, bottles, vials (e.g. , dual chamber vials), syringes (such as dual chamber syringes) and test tubes. The container may be formed from a variety of materials such as glass or plastic.

EXAMPLES

[0053] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1 - Generation and characterization of a cross-reactive anti-CD40 agonistic monoclonal antibody

[0054] Cluster of Differentiation 40 (CD40), a member of the tumor necrosis factor receptor superfamily, is a receptor expressed on the surface of Antigen Presenting Cells (APCs) of the immune system. The CD40 ligand (CD40L) is expressed on activated CD4+ T cells, and upon interaction with CD40 leads to enhanced antigen presentation, nitric oxide (NO) expression, proinflammatory cytokine expression by APCs, and stimulation of B cells to undergo somatic hypermutation, immunoglobulin class switching, and proliferation. Anti-CD40 monoclonal antibodies (mAbs) capable of crosslinking CD40 molecules act as agonists and mimic native CD40-CD40L interactions. An anti-swine CD40 mAb was developed, designated 2E4E4, and its agonistic activity on cells from livestock species was characterized. Immunocytometric analysis of HEK 293A cells transfected with plasmids expressing swine or bovine CD40 genes demonstrated the specificity of the mAb 2E4E4. Recognition of CD40 by the mAb 2E4E4 was validated by flow cytometry on primary swine and bovine peripheral blood mononuclear cells (PBMCs). Further validation was provided by immunohistochemistry on swine, bovine, caprine, and ovine spleen sections. Swine PBMCs stimulated with the mAb 2E4E4 revealed a significant increase in expression of MHC-II, TNF-a, IL-6, and IL-8 by intracellular flow cytometry. In addition, the mAb 2E4E4 stimulated robust swine, bovine, caprine, and ovine PBMC proliferation, and activated bovine macrophages to upregulate significant NO synthesis. These outcomes showed that the mAb 2E4E4 is broadly agonistic and therefore can be utilized as a powerful adjuvant for use in livestock species.

Materials and Methods

[0055] Alignment of CD40 sequences. Swine CD40 (AAL92924.1), Bovine CD40 (NP 001099081.1), Ovine CD40 (NP_001068569.1), and Caprine CD40 (XP_005688676.1) protein sequences were obtained from GenePept. Sequences were aligned using Jalview 2.9.0b2 and analyzed by Clustal X. Homology between these CD40 protein sequences was calculated by Protein BLAST to determine percentage identity to swine CD40 sequence.

[0056] Cell Culture. Hybridomas, human embryonic kidney (HEK) 293A cells, and fresh peripheral blood mononuclear cells (PBMCs) were grown in an atmosphere of 5% C0₂ at 37°C. Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, Glutamax™ (2 mM), HEPES (0.01 M), penicillin- streptomycin (100 U/mL), non-essential amino acid (0.1 M), sodium pyruvate (1 mM), and 2-mercaptoethanol (0.1 mM) was used to grow hybridomas. HEK 293A cells were grown in DMEM supplemented with 10% FBS, Glutamax™, HEPES (0.01 M), non-essential amino acids (0.1 M), and 2-mercaptoethanol (0.1 M). Swine, bovine, caprine, and ovine PBMCs were cultured in RPMI 1640 supplemented with 10% FBS, Glutamax™, non-essential amino acids (0.1 M), HEPES (0.01 M), 2-mercaptoethanol (0.1 M), and Penicillin- Streptomycin (100 U/mL). FreeStyle™ 293-F cells were grown in Freestyle™ Expression Media (Thermoscientific) in an atmosphere of 8% C0₂ at 37°C.

[0057] Generation and purification of recombinant swine CD40. Total RNA was isolated from swine spleen using Trizol® (Invitrogen, Carlsbad, CA) and used for cDNA synthesis using Superscript II reverse transcriptase (Invitrogen). Sequences encoding full length swine CD40 (swCD40) or the extracellular domain (swCD40ED) was PCR amplified with Accuprime Pfx DNA Polymerase (Invitrogen) using primers based on GenBank sequence AF248545.1. The PCR product encoding the swCD40ED was cloned into PCR-TOPO vector (Invitrogen), and following colony screening and DNA sequencing of positive clones, one construct encoding the authentic swCD40ED was modified by overlap extension PCR to incorporate a secretory signal sequence at the 5' terminus and the FLAG-tag at the 3'-terminus. The resultant gene and the PCR product encoding full length swCD40 were sub-cloned into the eukaryotic expression vector pcDNA2.2-TOPO (Invitrogen) and verified by sequencing. A construct encoding full length bovine CD40 (boCD40) was similarly generated. Recombinant swCD40ED was expressed as a FLAG-tagged protein by transfecting HEK 293 Free-Style cells (Invitrogen) and affinity purified using anti-FLAG M2-agarose affinity chromatography (Sigma) as previously described (Hope, et al., J. Immunol. Methods 301: 114-123, 2005; Mwangi, et al., J. Leukoc. Biol. 78:401-411, 2005).

[0058] Monoclonal antibody production. Monoclonal antibodies (mAbs) against swCD40ED were produced as previously described (Waghela, et al., Vet. Parasitol. 94: 133-139, 2000). Briefly, three female BALB/c mice were inoculated subcutaneously three times every 2 weeks with 50 μg of recombinant swCD40ED in RIBI adjuvant. Seroconversion was monitored on a weekly basis by ELISA using plates coated with recombinant swCD40ED. Three and five days prior to splenocyte harvest, the mouse with the best anti-swCD40ED antibody response was stimulated by intraorbital injection of 50 μg of the recombinant swCD40ED without adjuvant. On the day of fusion, the spleen was harvested for preparing single cell suspension for electrofusion with Sp2/0 myeloma cells (ATCC, Manassas, VA). Hybridomas were plated in 96-well cell culture plates (Nunc) and grown in hypoxanthine- aminopterin-thymidine (HAT) medium. Primary screening was performed by ELISA on day 14 post-fusion using ELISA plates coated with recombinant swCD40ED (100 ng/well). Proliferation assay was used to test ELISA positive hybridomas for agonistic effect on swine PBMCs. Positive hybridoma clones identified by proliferation assay were subcloned by limiting dilution and retested by ELISA and proliferation assays. The leading candidate, clone 2E4E4, was selected for further analysis.

[0059] Immunocytometric analysis. HEK 293 A cell monolayers were transfected with constructs encoding either swCD40 (pcDNAswCD40) or boCD40 (pcDNAboCD40) using Polyethylenimine (Polyscience) as previously described (Hopkins, et ah, In: L.J. Hartley (Ed.) Protein Expression in Mammalian Cells: Methods and Protocols. Humana Press, Totowa, NJ, p. 251-268, 2012). Following 48 hour incubation, the monolayers were fixed with cold methanol, rinsed with PBS, blocked with 10% FBS/TBS solution, and incubated for 1 hour at room temperature with 5 μg of the mAb 2E4E4 or 5 μg of an IgGl isotype control (Biolegends). The cell monolayers were washed 3X with blocking buffer and then incubated for 1 hour with Alkaline Phosphatase AffiniPure F(ab')₂ Fragment Donkey Anti-Mouse IgG (H+L) (Jackson Immuno Research Laboratories, Inc.). Following washes as described above, Fast Red TR- Naphthol AS-MX substrate (Sigma, F4523) was used to detect alkaline phosphatase activity. Photos were captured using Spot RT3 camera on Olympus 1X70 microscope.

[0060] Flow cytometry. Transfection of HEK 293A cells. The pcDNAswCD40 and pcDNAboCD40 constructs were used to transfect HEK 293A cell monolayers using Polyethylenimine (Polyscience) as previously described (Hopkins, et ah, 2012, supra). Following 48 hour incubation, one million transfected cells were added to each well of a 96 well V-bottom plate and stained with Zombie Red™ Fixable Viability Kit (Biolegend) following the manufacturer's protocol. The pcDNAswCD40 and pcDNAboCD40 transfected HEK 293A cells were incubated with 5 μg of the mAb 2E4E4 or 5 μg of IgGl isotype control for 30 minutes and washed 3X with blocking buffer (cDMEM with 0.05% sodium azide/20% bovine serum). The cells were incubated for 30 minutes with AffiniPure F(ab')₂ Fragment Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.), washed 3X with block buffer, and stored in FACS fixer (12.5% formaldehyde/PBS). Data was collected using BDfacscalibur (Becton Dickinson) and data analysis was done using FlowJo 10 software (FlowJo).

[0061] LPS stimulated swine and bovine PBMCs. Swine and bovine PBMCs were added to a 12 well plate (4 million PBMCs per well) and incubated for 24 hours in 1 mL of cRPMI alone or in cRPMI containing LPS (10 μg/ml). Half a million swine or bovine PBMCs from either treatment was added to each well of a 96 well v-bottom plate (Axygen), stained with zombie red™ viability kit (Biolegends) following manufacturer's protocol, and blocked using either swine blocking buffer (20% swine serum in FACS media) or bovine blocking buffer (20% bovine serum in FACS media). The swine and bovine PBMCs were incubated for 30 minutes on ice with either 5 μg of the mAb 2E4E4-FITC or 5 μg of IgGl isotype control (Biolegends) conjugated to FITC, washed 3X with blocking buffer, and fixed using FACS Fixer. FACS data was collected and analyzed as above.

[0062] MHCII expression. Swine PBMCs were seeded in a 12 well plate at 4 million PBMCS per well in 1 mL of cRPMI media alone, media containing LPS (10 μg/ml), or media containing graded amounts of the mAb 2E4E4 (0.5, 1.0, 2.5, or 5.0 μg/ml). After a 24 hour incubation, half a million PBMCs were added to each well of a 96 well v-bottom plate (Axygen), blocked (20% swine serum/FACS media) for 30 minutes on ice, and then incubated for 30 minutes with 5 μg of mouse anti-swine MHCII-FITC (Monoclonal Antibody Center Washington State University, clone MSA3). The PBMCs were washed 3X with blocking buffer, and then fixed and stored in FACS fixer. FACS data was collected and analyzed as above.

[0063] Immunohistochemistry. Swine, bovine, caprine, and ovine spleen tissue (donated by Texas A&M Veterinary Medical Diagnostic Laboratory) were used to prepare histology slides as previously described (Whiteland, et ah, J. Histochem. Cytochem. 43, 313-320, 1995). Slides were incubated for 20 minutes with Peroxidazed 1 (Biocare Medical), washed with TBS IX for 15 seconds, and incubated with Background Sniper (Biocare Medical) for 20 minutes. After washing as described above, the slides were incubated with 5 μg of the mAb 2E4E4 or 5 μg of IgGl isotype control. Following 1 hour incubation, the slides were washed IX with TBS and incubated for 1 hour with Immpress™ Goat Anti-Mouse IgG Serum-HRP (Vector) secondary antibody. After washing the slides as described above, Horseradish peroxidase activity was tested using Nova red (Vector Labs) and then counter-stained with crystal violet. Photos were captured using Spot RT3 camera on Olympus 1X70 microscope.

[0064] Proliferation assay. Swine, bovine, caprine, and ovine PBMCs (250,000 cells/well) were cultured in triplicate wells of 96 well round bottom plates for 24 hours in a total volume of 100 μΐ of cRPMI containing graded amounts of the mAb 2E4E4 (0.5, 1.0, 2.5, 5.0, or 10 μg/ml), IgGl (0.5, 1.0, 2.5, 5.0, or 10 μg/mL), PMA (1 μg/ml)/Ionomycin (0.5 μg/ml), or media alone. The cells were labeled for 12 hours with 0.3 μθ of H-thymidine and incorporation of the isotope by the cells was determined using liquid scintillation counter (Becton-Dickinson). The stimulation index (SI) was calculated for both 2E4E4 and IgGl by dividing the treatment (2E4E4 or IgGl control) by the media control.

[0065] Nitric oxide assay. The level of Nitrite (N0₂ ^~) released by activated bovine macrophages was measured by a Griess assay using monocyte-derived macrophages generated as previously described (Shoda, et ah, J. Interferon Cytokine Res. 19: 1169-1177, 1999). Briefly, the macrophages (200,000 cells/well) were added in triplicate wells of a 96 well flat bottom plate containing graded amounts of the mAb 2E4E4 (0.5, 1.0, 2.5, 5.0, or 10 μg/ml), IgGl isotype control (0.5, 1.0, 2.5, 5.0, or 10 μg/ml), LPS (10 μg/ml), or media alone. Following a 24 hour incubation, macrophage supernatants were tested for nitrite concentration using Nitric Oxide Assay kit (ThermoFisher) following manufacturer's protocol. Nitrite released was presented as μΜ N0₂ ^".

[0066] Intracellular cytokine staining. For intracellular cell cytokine staining, swine PBMCS (4 million cells) were added to each well of a 12 well plate containing graded amounts of the mAb 2E4E4 (1.0, or 2.5 μg/ml), LPS (1 μg/ml) or media alone. The cells were incubated for 12 or 24 hours and twelve hours before the PBMCs were harvested, Brefeldin A was added to each well at a final concentration of ^g/ml. The PBMCs were plated in 96 well v-bottom plate (5 x 10⁵ cells/well), incubated for 15 minutes in PERM/WASH™, blocked (20% porcine serum in IX PERM/WASH™ buffer), and further incubated with 5 μg of mouse anti-swine TNF-a clone 103314 (R&D Systems), mouse anti-swine IL-la clone 85733.11 (R&D Systems), mouse anti- swine IL-6 clone 77830 (R&D Systems), or mouse anti-swine IL-8 Clone 105115 (R&D Systems) for 1 hour. After 3 washes with blocking buffer, the cells were fixed and stored in FACS fixer. Flow data was collected, and analyzed as above.

[0067] Statistics. All analyses were performed using GraphPad 6.05 software. Data from the nitric oxide assay and proliferation assays were analyzed by two way ANOVA with post HSD Tukeys multiple comparison tests comparing similar concentrations of 2E4E4 to IgGl isotype control. Intracellular cytokine data was also analyzed by two way ANOVA with post HSD Tukeys multiple comparison except treatments and media control were compared. A value of p< 0.05 was considered statistically significant. mAb 2E4E4 recognized cell-surface expressed recombinant CD40

[0068] A mouse anti-swine CD40 mAb, designated 2E4E4, was generated by immunizing mice using the extracellular domain of swine CD40 (FIG. 1). The mAb 2E4E4 was shown to be an IgGl with a kappa light chain (FIG. 2A). Immunocytometric analysis of HEK 293 A cells transfected with a construct expressing full length swine CD40 confirmed that the mAb 2E4E4 recognized CD40, whereas sham treated cells were negative (FIG. 2B and FIG. 2D, respectively). This outcome was confirmed by performing flow cytometry on similarly transfected cells. The mAb 2E4E4, but not IgGl isotype control, strongly recognized surface- expressed swine CD40 (FIG. 2E). Since swine and bovine CD40 protein sequences are highly conserved (FIG. 1), mAb 2E4E4 was tested for binding to bovine CD40. Indeed, immunocytometric and flow cytometric analysis of HEK 293A cells transfected with a construct expressing full length bovine CD40 yielded similar results (FIG. 2C and FIG. 2F). These outcomes showed that the mAb 2E4E4 can bind to both swine and bovine CD40, and hence all further experiments were designed to evaluate the interaction of the mAb 2E4E4 with bovine CD40 as well. mAb 2E4E4 recognized native swine and bovine CD40

[0069] Flow cytometric analysis showed that the mAb 2E4E4 bound to the CD40 expressed on LPS-stimulated swine (FIG. 3A) and bovine (FIG. 3B) PBMCs. The mAb 2E4E4 showed significant fluorescence on stimulated PBMCs (FIG. 3A and FIG. 3B, orange) as compared to non-stimulated controls (FIG. 3A and FIG. 3B, red). A specific signal was also detected on non-stimulated PBMCs probed with the mAb 2E4E4, but not on cells probed with an IgGl isotype control (FIG. 3A and FIG. 3B, blue). These results showed that the mAb 2E4E4 binds to the CD40 expressed on the cell surface of stimulated PBMCs. In addition, immunohistochemistry (IHC) showed that mAb 2E4E4, but not an IgGl isotype control, reacted strongly to swine and bovine spleen tissues (FIG. 4 A and FIG. 4B, respectively, compared to FIG. 4E and FIG. 4F). Taken together, the flow cytometry and IHC data showed that the mAb 2E4E4 recognized CD40 expressed on swine and bovine cells. In addition, IHC data also showed cross-reactivity of the mAb 2E4E4 to caprine and ovine CD40, but not an IgGl isotype control (FIG. 4C and FIG. 4D, respectively, compared to FIG. 4G and FIG. 4H). These results indicate that the mAb 2E4E4 can be used for assaying levels of expression and distribution of swine, bovine, ovine, and caprine CD40. Agonistic effect of mAb 2E4E4

[0070] The mAb 2E4E4 showed significant agonistic effects on swine, bovine, caprine, and ovine PBMCs. The mAb 2E4E4 stimulated significant (P<0.001) proliferation of swine and bovine PBMCs compared to the IgGl isotype control (FIG. 5A and FIG. 5B). Interestingly, the mAb 2E4E4 also had significant stimulatory effect on ovine and caprine PBMCs in a dosage dependent manner (FIG. 5C and FIG. 5D). Further evidence of the agonistic effect of mAb 2E4E4 was shown by demonstrating that mAb 2E4E4, but not an IgGl isotype control, stimulated significant (p<0.001) NO release by bovine macrophages (FIG. 6). NO production by swine macrophages was also tested, and the outcome was negative as expected.

[0071] Flow cytometry confirmed that the mAb 2E4E4 upregulated MHCII expression on swine PBMCs. A significant increase in MHCII expression was observed when swine PBMCs were stimulated with the mAb 2E4E4 and the response was dose dependent (Table 1). Swine PBMCs were incubated with the mAb 2E4E4 or LPS for 24 hours and MHCII upregulation was determined by flow cytometric analysis. Data is presented as percentage of MHCII positive cells compared to IgGl isotype control.

Table 1

MHCII upregulation by swine PBMCs stimulated with the mAb 2E4E4

[0072] The MHCII upregulation is expected to result in enhanced antigen presentation by APCs. Further confirmation of the mAb 2E4E4 agonistic effect was validated by intracellular cytokine staining. Upregulation of TNF-a, IL-la, and IL-8 was observed at 12 hours post-stimulation (FIG. 7A, FIG. 7B and FIG. 7C). Unlike TNF-a, IL-la, and IL-8, no significant IL-6 expression was observed until 24 hours post-stimulation (FIG. 7D). These results showed that the mAb 2E4E4 stimulates the upregulation of MHCII and release of proinflammatory cytokines, indicating that the mAb 2E4E4 is capable of being used as vaccine adjuvant.

[0073] In summary, an anti-swine CD40 mAb, designated 2E4E4, was generated and characterized. The mAb 2E4E4 was shown to be specific to swine CD40, but it also had robust cross-reactivity against bovine, caprine, and ovine cells and spleen tissues. The mAb 2E4E4 had agonistic effects on swine cells and in addition, it showed broad agonistic effects against bovine, caprine, and ovine cells. These outcomes indicate that mAb 2E4E4 can be used as a broad immune modulator for use in livestock. One limitation with current immune modulators is their inability to cross-react and have broad reactivity in multiple species. Broad spectrum immune modulators that can be used in a variety of animal models are able to overcome this limitation making them a valuable resource in developing new treatments for livestock and human infectious and noninfectious diseases. By demonstrating 2E4E4 ability to stimulate PBMCs from several species indicates it utility as a broad immune modulator.

[0074] In some embodiments, numbers expressing quantities used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth herein are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0075] In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term "or" as used herein, including the claims, is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0076] The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that "comprises," "has" or "includes" one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

[0077] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. , "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0078] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [0079] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

[0080] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. It is intended that all matter contained in the foregoing description shall be interpreted as illustrative rather than limiting. The breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims appended hereto and their equivalents.

Claims

1. An anti- swine CD40 monoclonal antibody, or a fragment thereof.

2. The antibody or fragment thereof of claim 1, further defined as a recombinant diabody, an scFv, a Fab fragment, a disulfide linked Fv or a whole immunoglobulin molecule.

3. The antibody or fragment thereof of claim 1 or 2, wherein the anti-swine CD40 monoclonal antibody is from clone 2E4E4.

4. The antibody or fragment thereof of any one of claims 1 to 3, wherein the anti- swine CD40 monoclonal antibody, or a fragment thereof, is an F(ab')₂ fragment.

5. The antibody or fragment thereof of any one of claims 1 to 4, further defined as comprised in a composition comprising an antigen.

6. The antibody or fragment thereof of claim 5, wherein the antigen is a whole killed virus, inactivated virus or an attenuated virus.

7. The antibody or fragment thereof of claim 5, wherein the antigen is conjugated to the anti-swine CD40 monoclonal antibody, or a fragment thereof.

8. The antibody or fragment thereof of any one of claims 1 to 7, further comprising a pharmaceutically acceptable carrier.

9. The antibody or fragment thereof of any one of claims 1 to 8, further defined as having a first light-chain variable domain having an amino acid sequence comprising SEQ ID NO:2, a second light-chain variable domain having an amino acid sequence comprising SEQ ID NO:3, a third light-chain variable domain having an amino acid sequence comprising SEQ ID NO:4, a first heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:5, a second heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:6, and a heavy-chain variable domain having an amino acid sequence comprising SEQ ID NO:7.

10. The antibody or fragment thereof of any one of claims 1 to 9, further comprising an adjuvant distinct from said anti-swine CD40 monoclonal antibody, or a fragment thereof.

11. A pharmaceutical composition, comprising: a) an antigen; b) a pharmaceutically acceptable carrier; and c) the antibody or fragment thereof of any one of claims 1 to 10.

12. The pharmaceutical composition of claim 11, wherein said composition is administered intravenously, intra-arterially, intra-peritoneally, intramuscularly, intradermally, orally, dermally, nasally, buccally, rectally, vaginally, by inhalation, or by topical administration.

13. A composition comprising the anti-swine CD40 monoclonal antibody, or a fragment thereof, of any one of claims 1 to 10.

14. The composition of claim 13, wherein the anti-swine CD40 monoclonal antibody is from clone 2E4E4.

15. A method of increasing the immunogenicity of an antigen, comprising combining the antigen with the anti-swine CD40 monoclonal antibody, or a fragment thereof, of any one of claims 1 to 10, wherein said anti-swine CD40 monoclonal antibody or fragment thereof increases immunogenicity of the antigen in a plurality of species.

16. The method of claim 15, wherein the antigen is conjugated to the anti-swine CD40 monoclonal antibody, or a fragment thereof.