NZ627530B - Combination adjuvant formulation - Google Patents

Abstract

Disclosed is an adjuvant composition comprising (a) a host defence peptide selected from ILPWKWPWWPWRR, VQLRIRVAVIRA; VQRWLIVWRIRK; VRLIVAVRIWRR; and Ile-Dhb-Ala-Ile-Dha-Leu-Ala-Abu-Pro-Gly-Ala-Lys-Abu-Gly-Ala-Leu-Met-Gly-Ala-Asn-Met-Lys-Abu-Ala-Abu-Ala-Asn-Ala-Ser-Ile-Asn-Val-Dha-Lys; (b) an immunostimulatory sequence selected from (i) a CpG oligonucleotide selected from TCCATGACGTTCCTGACGTT; TCGTCGTTGTCGTTTTGTCGTT; TCGTCGTTTTGTCGTTTTGTCGTT; and TCGTCGTTTTCGGCGCGCGCCG; and (ii) poly (I:C); (c) a bovine viral diarrhoea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di-4-oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP), wherein said adjuvant composition is capable of enhancing an immune response to the BVDV antigen as compared to the immune response elicited by an equivalent amount of the BVDV antigen when delivered without the adjuvant composition. immunostimulatory sequence selected from (i) a CpG oligonucleotide selected from TCCATGACGTTCCTGACGTT; TCGTCGTTGTCGTTTTGTCGTT; TCGTCGTTTTGTCGTTTTGTCGTT; and TCGTCGTTTTCGGCGCGCGCCG; and (ii) poly (I:C); (c) a bovine viral diarrhoea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di-4-oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP), wherein said adjuvant composition is capable of enhancing an immune response to the BVDV antigen as compared to the immune response elicited by an equivalent amount of the BVDV antigen when delivered without the adjuvant composition.

Description

COMBINATION ADJUVANT FORMULATION STATEMENT OF CORRESPONDING APPLICATIONS This application is based on the complete specification filed in relation to New Zealand Patent application no. 591925; the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD The present invention pertains generally to compositions for enhancing immune responses. In particular, the invention relates to combination adjuvant compositions including a host defense peptide, an immunostimulatory sequence and a polyanionic polymer, for use as vaccine adjuvants.

BACKGROUND Bovine viral diarrhea (BVD) continues to be one of the top two most important indigenous viral diseases in cattle, the other being infectious bovine rhinotracheitis (IBR) caused by bovine herpesvirus-1. Both are major components of bovine respiratory disease, which still is a cause of tremendous loss to the beef industry [1].

BVDV has been classified into type 1 and type 2 viruses, and a new group of viruses has been classified as BVDV-3 [2]. Acute BVDV infections, caused by type 1 strains, are very common in cattle and result in mild disease of short duration characterized by fever, increased respiratory rate, diarrhea and a reduction in white blood cells.

Although animals generally recover, the effect of BVDV on the immune cells reduces the host’s resistance to disease; thus, BVDV is an important pathogen for stressed cattle entering a feedlot. Type 2 strains can cause acute infections in herds that are characterized by high fever, hemorrhaging, diarrhea, reduction of white blood cells and platelets, and death. In addition, BVDV infections in pregnant cows can result in abortions, malformations, poor-doers and persistently infected calves, which are a major source of new infections in herds [3].

Despite the availability of several modified live and killed vaccines, the morbidity and mortality due to BVDV has not significantly decreased, which suggests that more effective vaccines need to be developed. The goal of this study was to develop a vaccine that induces a robust, balanced immune response and protection from BVDV infection. The BVDV E2 protein was selected as the protective antigen and formulated with a novel adjuvant platform consisting of poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP), a toll-like receptor agonist, and an innate defense regulator (IDR) peptide. PCEP is a synthetic water-soluble polymer with immunostimulatory properties that forms non-covalent complexes of micro/nanoparticle size with antigens, which enhances their stability and allows multimeric presentation. Either CpG ODN or poly (I:C), which are ligands for TLR9 and TLR3 respectively, is used in this adjuvant formulation. Both of these TLR agonists significantly improve immune responses through increased dendritic cell maturation. The IDR peptide is a derivative of a natural host defense peptide, Bac2a, which like all host defense peptides is cationic, amphipathic and has microbicidal, chemotactic and/or immunomodulatory properties [4]. This adjuvant system has been shown to induce protective immunity in murine, cotton rat and porcine disease models [5] [6], but not yet in cattle.

We first demonstrated that two doses of the E2 protein formulated with various versions of this adjuvant platform elicit both humoral and cell-mediated immune (CMI) responses in lambs. The optimal E2-adjuvant formulation was tested in cattle and shown to induce strong virus neutralizing (VN) antibody and cytotoxic T lymphocyte (CTL) responses and to provide protection from a high challenge dose of a virulent BVDV type 2 strain.

It is an object of the present invention to address the foregoing disadvantages or at least or provide the public with a useful choice.

DISCLOSURE OF THE INVENTION According to one aspect of the invention, there is provided an adjuvant composition comprising (a) a host defense peptide; (b) an immunostimulatory sequence; (c) a bovine viral diarrhea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP), wherein said adjuvant composition is capable of enhancing an immune response to the BVDV antigen as compared to the immune response elicited by an equivalent amount of the BVDV antigen when delivered without the adjuvant composition.

Preferably, the host defense peptide comprises the sequence of SEQ ID NO:19, the immunostimulatory sequence is poly (I:C), and the polyanionic polymer is poly(di- 4-oxyphenylproprionate)phosphazene (PCEP).

According to another aspect of the invention, there is provided a use of (a) a host defense peptide; (b) an immunostimulatory sequence; (c) a bovine viral diarrhea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP) in the manufacture of a medicament for enhancing an immune response to the BVDV antigen.

According to a further aspect of the invention, there is provided a use of (a) a host defense peptide; (b) an immunostimulatory sequence; (c) a bovine viral diarrhea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP) for enhancing an immune response to the BVDV antigen in a non-human subject.

Preferably, the use of the host defense peptide comprises the sequence of SEQ ID NO:19; the immunostimulatory sequence is poly (I:C); and the polyanionic polymer is poly(dioxyphenylproprionate)phosphazene (PCEP).

Preferably, the use of the host defense peptide, the poly (I:C), the PCEP and the BVDV antigen are present in the same composition.

Preferably, at least one of the host defense peptide, the poly (I:C), the PCEP and the BVDV antigen are present in a different composition.

In one aspect the invention provides an adjuvant composition comprising (a) a host defense peptide selected from SEQ ID NO:1, SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; and SEQ ID NO:22; (b) an immunostimulatory sequence selected from (i) a CpG oligonucleotide selected from SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:12; and (ii) poly (I:C); (c) a bovine viral diarrhea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP), wherein said adjuvant composition is capable of enhancing an immune response to the BVDV antigen as compared to the immune response elicited by an equivalent amount of the BVDV antigen when delivered without the adjuvant composition.

In one aspect the invention provides the use of (a) a host defense peptide selected from SEQ ID NO:1, SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; and SEQ ID NO:22; (b) an immunostimulatory sequence selected from (i) a CpG oligonucleotide selected from SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:12; and (ii) poly (I:C); (c) a bovine viral diarrhea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP) in the manufacture of a medicament for enhancing an immune response to the BVDV antigen.

In one aspect the invention provides the use of (a) a host defense peptide selected from SEQ ID NO:1, SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; and SEQ ID NO:22; (b) an immunostimulatory sequence selected from (i) a CpG oligonucleotide selected from SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:12; and (ii) poly (I:C); (c) a bovine viral diarrhea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP) for enhancing an immune response to the BVDV antigen in a non-human subject.

BRIEF DESCRIPTION OF THE FIGURES The invention will now be described by way of example only and with reference to any one of the accompanying drawings in which: Figure 1 shows an analysis of purified E2 protein: the pure E2 protein was resolved in a 10% polyacrylamide gel and visualized by staining with Coomassie Brilliant Blue. Molecular mass markers (x 10 ) are shown as arrows in the left margin; Figure 2 shows BVDV-specific immune responses of lambs vaccinated with BVDV-2 E2: BVDV E2 protein was formulated in PBS or with CpG ODN and PCEP (Db CpG), CpG ODN, IDR and PCEP (Tri CpG), poly(I:C) and PCEP [Db poly(I:C)], or poly(I:C), IDR and PCEP [Tri poly(I:C)]. Groups of five lambs were immunized twice IM at a three- week interval with the E2 protein formulations or with PBS (Placebo).

BVDV-2 E2-specific titers were determined after one (A) and two (B) immunizations. ELISA titers were calculated as the highest dilution resulting in a reading of two standard deviations above the value of a negative control serum. The number of E2-induced IFN-γ secreting cells was determined after one (C) and two (D) immunizations by ELISPOT assay, and expressed as the difference between the number of spots in the BVDV E2-stimulated wells and the number of spots in the medium control wells. Significance of differences is shown as **: P < 0.01; Figure 3 shows BVDV-specific antibody responses of calves vaccinated with BVDV-2 E2, and challenged with BVDV-2: Two groups of five calves each were immunized twice IM at a four-week interval with BVDV E2 protein formulated with poly(I:C), IDR and PCEP or with PBS. The calves were challenged with BVDV strain 1373 four weeks after the last vaccination. (A): E2-specific serum IgG titers. ELISA titers were calculated as the highest dilution resulting in a reading of two standard deviations above the value of a negative control serum. (B) BVDV specific serum VN titers. The VN titers were reported as the reciprocal of the highest dilution that completely inhibited viral infection in the two replicate samples. Median values with interquartile range are shown.

Significance of differences is shown as *: P < 0.05; **: P < 0.01; Figure 4 shows BVDV E2-induced IFN-γ secreting CD4 and CD8 T cells in calves vaccinated with BVDV-2 E2, and challenged with BVDV-2: Calves were immunized and challenged as described in the legend to + + + + Figure 3. (A) Gating strategy. Number of CD4 IFN-γ and CD8 IFN-γ T cells after one (B) and two (C) immunizations , and after BVDV-2 + + + + challenge (D); numbers of activated (CD25 ) CD4 IFN-γ and CD8 IFN-γ T cells (E). Median values with interquartile range are shown.

Significance of differences is shown as *: P < 0.05; **: P < 0.01; and Figure 5 shows clinical signs and leukocyte counts after challenge of calves with BVDV-2: Calves were immunized and challenged as described in the legend to Figure 3. Weights and rectal temperatures were recorded daily.

(A) Temperatures. (B) Cumulative weight changes. WBCs (C), monocytes (D), lymphocytes (E) and segregates (F), counted and expressed as counts × 109 L-1. Data are shown as the mean of five calves. Significance of differences is shown as *: P < 0.05.

DETAILED DESCRIPTION OF THE INVENTION The practice of the present invention will employ, unless otherwise indicated, conventional methods of microbiology, chemistry, biochemistry, recombinant DNA techniques and immunology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Handbook of Experimental Immunology, Vols. I-IV (D.M. Weir and C.C. Blackwell eds., Blackwell Scientific Publications); T.E.

Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

The following amino acid abbreviations are used throughout the text: Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid: Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E) Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L) Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Proline: Pro (P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr (Y) Valine: Val (V) Dehydroalanine (Dha) Dehydrobutyrine (Dhb) The following sequences are presented herein: SEQ ID NO SEQUENCE NAME 1 ILPWKWPWWPWRR indolicidin 2 VFLRRIRVIVIR JK1 3 VFWRRIRVWVIR JK2 4 VQLRAIRVRVIR JK3 VQLRRIRVWVIR JK4 6 VQWRAIRVRVIR JK5 7 VQWRRIRVWVIR JK6 8 TCCATGACGTTCCTGACGTT CpG 1826 9 TCGTCGTTGTCGTTTTGTCGTT CpG 2007 TCGTCGTTTTGTCGTTTTGTCGTT CpG 7909 or 10103 11 GGGGACGACGTCGTGGGGGGG CpG 8954 12 TCGTCGTTTTCGGCGCGCGCCG CpG 2395 or 10101 13 AAAAAAGGTACCTAAATAGTATGTTTCTGAAA non-CpG ISS 14 GRFKRFRKKFKKLFKKLSPVIPLLHLG BMAP27 GGLRSLGRKILRAWKKYGPIIVPIIRIG BMAP28 16 RLARIVVIRVAR Bactenicin 2a (Bac2a) 17 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES human LL-37 18 VQLRIRVAVIRA HH2 19 VQRWLIVWRIRK 1002 VRLIVAVRIWRR 1018 21 IWVIWRR HH18 22 Ile-Dhb-Ala-Ile-Dha-Leu-Ala-Abu-Pro-Gly-Ala-Lys-Abu- Nisin Z Gly-Ala-Leu-Met-Gly-Ala-Asn-Met-Lys-Abu-Ala-Abu-Ala- Asn-Ala-Ser-Ile-Asn-Val-Dha-Lys 23 V**R*IRV*VIR, * = any amino acid conserved motif 24 ILKWKWPWWPWRR HH111 ILPWKKPWWPWRR HH113 26 ILKWKWPWWKWRR HH970 27 ILRWKWRWWRWRR HH1010 1. DEFINITIONS In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a CpG oligonucleotide” includes a mixture of two or more CpGs, and the like.

By “host defense peptide” or “HDP” is meant any of the various host defense peptides that have the ability to enhance an immune response to a co-administered antigen. The DNA and corresponding amino acid sequences for various host defense peptides are known and described in detail below. Host defense peptides for use in the present methods include the full-length (i.e., a prepro sequence if present, the entire prepro molecule) or substantially full-length proteins, as well as biologically active fragments, fusions or mutants of the proteins. The term also includes postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes of the present invention, a “host defense peptide” refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. It is readily apparent that the host defense peptides may therefore comprise an entire leader sequence, the mature sequence, fragments, truncated and partial sequences, as well as analogs, muteins and precursor forms of the molecule.

The term also intends deletions, additions and substitutions to the reference sequence, so long as the molecule retains the desired biological activity.

By “CpG oligonucleotide” or “CpG ODN” is meant an immunostimulatory nucleic acid containing at least one cytosine-guanine dinucleotide sequence (i.e., a 5' cytidine followed by 3' guanosine and linked by a phosphate bond) and which activates the immune system. An “unmethylated CpG oligonucleotide” is a nucleic acid molecule which contains an unmethylated cytosine-guanine dinucleotide sequence (i.e., an unmethylated 5' cytidine followed by 3' guanosine and linked by a phosphate bond) and which activates the immune system. A “methylated CpG oligonucleotide” is a nucleic acid which contains a methylated cytosine-guanine dinucleotide sequence (i.e., a methylated 5' cytidine followed by a 3' guanosine and linked by a phosphate bond) and which activates the immune system. CpG oligonucleotides are well known in the art and described in, e.g., U.S. Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068; PCT Publication No. WO 01/22990; PCT Publication No.

WO 03/015711; US Publication No. 20030139364.

By “poly(I:C) oligonucleotide” or “poly(I:C)” is meant a synthetic viral-like double stranded immunostimulatory ribonucleic acid containing strands of polyriboinosinic acid and polyribocytidylic acid that are held together by hydrogen bonds between purine and pyrimidine bases in the chains. Poly I:C has been found to have a strong interferon-inducing effect in vitro and is therefore of significant interest in infectious disease research.

By “polyphosphazene is meant a high-molecular weight, water-soluble polymer, containing a backbone of alternating phosphorous and nitrogen atoms and organic side groups attached at each phosphorus atom. See, e.g., Payne et al., Vaccine (1998) 16:92-98; Payne et al., Adv. Drug. Deliv. Rev. (1998) 31:185-196. A number of polyphosphazenes are known and described in more detail below.

By “antigen” or “immunogen” is meant a molecule, which contains one or more epitopes (defined below) that will stimulate a host's immune system to make a cellular antigen-specific immune response when the antigen is presented, or a humoral antibody response. The terms denote both subunit antigens, i.e., proteins which are separate and discrete from a whole organism with which the antigen is associated in nature, as well as killed, attenuated or inactivated bacteria, viruses, parasites or other microbes.

Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which can mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used herein. Similarly, an oligonucleotide or polynucleotide which expresses a therapeutic or immunogenic protein, or antigenic determinant in vivo, such as in gene therapy and nucleic acid immunization applications, is also included in the definition of antigen herein. Further, for purposes of the present invention, antigens can be derived from any of several known viruses, bacteria, parasites and fungi, as well as any of the various tumor antigens.

The term “derived from” is used to identify the original source of a molecule (e.g., bovine or human) but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

The terms “analog” and “mutein” refer to biologically active derivatives of the reference molecule, that retain desired activity as described herein. In general, the term “analog” refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy activity and which are “substantially homologous” to the reference molecule as defined below. The term “mutein” refers to peptides having one or more peptide mimics (“peptoids”), such as those described in International Publication No. WO 91/04282. Preferably, the analog or mutein has at least the same desired activity as the native molecule. Methods for making polypeptide analogs and muteins are known in the art and are described further below.

The terms also encompass purposeful mutations that are made to the reference molecule. Particularly preferred analogs include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acidic -- aspartate and glutamate; (2) basic -- lysine, arginine, histidine; (3) non-polar -- alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar -- glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably predictable that an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid, will not have a major effect on the biological activity.

For example, the molecule of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-20 conservative or non-conservative amino acid substitutions, or any integer between 5-20, so long as the desired function of the molecule remains intact. One of skill in the art can readily determine regions of the molecule of interest that can tolerate change by reference to Hopp/Woods and Kyte-Doolittle plots, well known in the art.

By “fragment” is intended a molecule consisting of only a part of the intact full-length polypeptide sequence and structure. The fragment can include a C-terminal deletion, an N-terminal deletion, and/or an internal deletion of the native polypeptide.

A fragment will generally include at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains the ability to elicit the desired biological response.

By “immunogenic fragment” is meant a fragment of a parent molecule which includes one or more epitopes and thus can modulate an immune response or can act as an adjuvant for a co-administered antigen and/or is capable of inducing an adaptive immune response. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715.

Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci USA (1981) 78:3824-3828 for determining antigenicity profiles, and the Kyte-Doolittle technique, Kyte et al., J. Mol.

Biol. (1982) 157:105-132 for hydropathy plots.

Immunogenic fragments, for purposes of the present invention, will usually be at least about 2 amino acids in length, more preferably about 5 amino acids in length, and most preferably at least about 10 to 15 amino acids in length. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes of the protein in question.

The term “epitope” refers to the site on an antigen or hapten to which specific B cells and T cells respond. The term is also used interchangeably with “antigenic determinant” or “antigenic determinant site.” Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

An “immunological response” to a composition is the development in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display a protective immunological response to the microorganism in question, e.g., the host will be protected from subsequent infection by the pathogen and such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host or a quicker recovery time.

The term “immunogenic” molecule refers to a molecule which elicits an immunological response as described above. An “immunogenic” protein or polypeptide, as used herein, includes the full-length sequence of the protein in question, including the precursor and mature forms, analogs thereof, or immunogenic fragments thereof.

An adjuvant composition comprising a host defense peptide, a polyphosphazene and an immunostimulatory sequence “enhances” or “increases” the immune response, or displays “enhanced” or “increased” immunogenicity vis-a-vis a selected antigen when it possesses a greater capacity to elicit an immune response than the immune response elicited by an equivalent amount of the antigen when delivered without the adjuvant composition. Such enhanced immunogenicity can be determined by administering the antigen and adjuvant composition, and antigen controls to animals and comparing antibody titers against the two using standard assays such as radioimmunoassay and ELISAs, well known in the art.

“Substantially purified” generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion- exchange chromatography, affinity chromatography, metal chelation chromatography, reversed phase chromatography, hydrophobic interaction chromatography, and sedimentation according to density.

By “isolated” is meant that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type. The term “isolated” with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.

“Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% , preferably at least about 75%, more preferably at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules (the reference sequence and a sequence with unknown % identity to the reference sequence) by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353-358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm.

These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.

“Recombinant” as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term “recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

The terms “effective amount” or “pharmaceutically effective amount” of a composition, or a component of the composition, refers to a nontoxic but sufficient amount of the composition or component to provide the desired response, such as enhanced immunogenicity, and, optionally, a corresponding therapeutic effect. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, and the particular components of interest, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

By “vertebrate subject” is meant any member of the subphylum chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The invention described herein is intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.

The term “treatment” as used herein refers to either (1) the prevention of infection or reinfection (prophylaxis), or (2) the reduction or elimination of symptoms of the disease of interest (therapy). 2. Materials and Methods 2.1 Cells and virus: Madin Darby bovine kidney (MDBK) cells were grown in Eagle’s minimal essential medium (MEM) (Sigma-Aldrich) with 10 mM HEPES, 50 μg/ml gentamicin and 10% horse serum at 37 C in a CO atmosphere. The MDBK cells were confirmed to be BVDV-free by Prairie Diagnostic Services, Saskatoon, SK, Canada.

The BVDV type 2 strain 1373 was grown in MDBK cells as described previously [7, 2.2 Production and formulation of the BVDV-2 E2 protein: An ORF encoding a truncated version of the BVDV-2 E2 protein (strain Q140; accession number AAB01811; amino acids 41-383) with the transmembrane domain deleted, was codon- optimized for expression in mammalian cells and synthesized by GeneArt. The ORF was additionally modified to express a protein with a N-terminal signal peptide of tissue plasminogen activator (accession number AAO34406) and a C terminal his tag.

The ORF was cloned downstream of the human CMV promoter and intron A contained within an episomal vector containing the EBNA-1 antigen ORF and P origin; the ORF was proceeded by a Kozak sequence, and elements downstream of the E2 protein ORF included a woodchuck hepatitis post-transcriptional regulatory element and bovine growth hormone poly-adenylation site. Transfected HEK293 cells were grown in suspension in SFM4HEK medium (Thermo Fisher Scientific); his-tagged E2 protein was purified from the culture supernatants using ProBond (Life Technologies) according to the manufacturer’s instructions. Plasmid maps and sequences are available upon request. 2.3 E2 protein formulations, immunizations and viral challenge: For lambs, 50 µg of E2 protein was formulated with: 1) PBS, 2) 250 µg CpG ODN + 500 µg IDR; 3) 250 µg poly(I:C) + 500 µg IDR, 4) 250 µg CpG ODN + 500 µg IDR + 250 µg PCEP or 5) 250 µg poly(I:C) + 500 µg IDR + 250 µg PCEP. CpG ODN [9] and Poly (I:C) were formulated with IDR 1002 [5] at a 1:2 ratio at 37°C; PCEP along with E2 protein was added after 30 min to make a final 1:2:1 ratio of CpG ODN or poly(I:C), IDR and PCEP. Three month-old lambs (n = 5) were immunized twice intramuscularly (IM) at a three-week interval, with 1 ml of the E2 vaccine formulations or PBS (placebo).

For cattle, 50 µg of E2 protein was formulated in PBS or with 500 µg poly(I:C) + 1000 µg IDR + 500 µg PCEP. Seven to eight month-old BVDV seronegative Angus- Hereford crossbred calves were randomly allocated to two groups of five animals each and immunized twice IM at a one-month interval with either the E2 protein formulation or PBS in 2 ml. One month after the second immunization, the calves were challenged with BVDV strain 1373 (6 x 10 50% TCID50 in PBS; 2 ml into each nostril) using an intranasal cannula (Pfizer Canada Inc.). Sera and peripheral blood were collected at the start of the trial, and after each immunization and BVDV challenge. Nasal swabs and blood for isolation of white blood cells (WBCs) were collected three days prior to challenge, on the day of challenge, and on days 2, 4, 6, 8, 10, and 12 post-challenge.

Temperatures and weights were recorded daily prior to and for 14 days after challenge.

All procedures were approved by the University Council for Animal Care and Supply in accordance with the standards stipulated by the Canadian Council on Animal Care. 2.4 Serology: E2-specific IgG titers were determined in serum by enzyme-linked immunosorbent assay (ELISA). Immulon 2HB microtiter plates (Thermo Fisher Scientific) were coated with E2 protein overnight at 4°C. After washing, serially diluted samples were applied to the coated plates and incubated overnight at 4°C.

Bound antibodies were detected using alkaline phosphatase (AP)-conjugated anti-ovine or anti-bovine IgG (Kirkegaard & Perry Laboratories). The enzymatic reaction was detected with p-nitrophenyl phosphate substrate (Sigma-Aldrich). Absorbance was read R 384 on a model Spectramax 340 PC Microplate Spectrophotometer (Molecular Devices Corp.) at 405 nm, with a reference wavelength of 490 nm.

Virus neutralization titers were determined as previously described [9]. Sera were inactivated for 30 min at 56°C. BVDV strain 1373 was incubated for 1.5 h at 37°C with serially diluted bovine sera, and these samples were added to MDBK cells.

The plates were incubated for 1.5 h at 37°C in a CO incubator, MEM with 2% horse serum was added, and the plates were further incubated at 37°C for five days. The cells were fixed, permeabilized, washed, and blocked with PBS containing 10% horse serum. Subsequently, the cells were incubated with type 2 E2-specific rabbit antibody, followed by AP-conjugated goat anti-rabbit IgG. 5-bromochloro- indolylphosphate/nitroblue tetrazolium (BCIP/NBT) (Bio-Rad) was used for detection. 2.4 Enzyme-linked immunosorbent (ELISPOT) assay and flow cytometry: Ovine and bovine peripheral mononuclear cells (PBMCs) were isolated as previously described [5, 10], and suspended at 1×10 cells per ml of Eagle’s MEM (Sigma- Aldrich) containing 10% FBS, 10 mM HEPES, 1mM sodium pyruvate, 100 mM non- essential amino acids, 50 μg/ml gentamycin and 5×10 M 2-mercaptoethanol.

ELISPOT plates (Millipore) were coated overnight with bovine IFN-γ-specific monoclonal antibody [11]. PBMCs were stimulated with E2 protein (2 µg/ml) or medium and incubated overnight at 37°C. IFN-  secreting cells were detected with a bovine IFN- -specific rabbit serum [11] followed by AP-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories), and spots were visualized with BCIP/NBT (Sigma-Aldrich).

To identify IFN-γ secreting CD4 and CD8 T cells, PBMCs were isolated, suspended in RPMI (Life Technologies) supplemented with 0.1 mM non-essential amino acids, 10 mM HEPES buffer, 1 mM sodium pyruvate, 2 mM L-glutamine, antibiotic/antimycotic (Life Technologies), 50 μg/mL gentamicin and 10% FBS, and then added at 1 x 10 cells per well in a 200 µl volume of a 48-well round-bottom microtiter plate. The cells were cultured at 37°C in the presence of E2 protein (2 µg/ml) for 4 days. GolgiStop was added and left for 12 h before harvesting the cells. Cells were washed twice with, and resuspended in, FACS buffer, and then incubated with mouse anti-bovine CD4 or CD8 MAb (FITC) (AbD Serotech) and mouse anti-bovine CD25 Mab (PE) (AbD Serotech) for 30 min at 4°C. Intracellular staining of IFN-γ was accomplished using the Cytofix/Cytoperm kit (BD Biosciences). Cells were washed, permeabilized at 4°C for 30 min, then again washed, and resuspended in BD Perm/Wash. Mouse anti-bovine IFN-γ (APC) (AbD Serotech) was added and left for 30 min at 4°C. Subsequently, cells were washed and acquired by flow cytometry. Data were analyzed using Kaluza Software. Cells were gated for live cells, singlets and lymphocytes and then analyzed for the indicated markers. 2.5 Virus isolation: Nasal secretions were collected with cotton swabs in 1 ml MEM supplemented with antibiotic-antimycotic. WBCs were isolated from blood with 0.83% ammonium chloride (Sigma-Aldrich) and resuspended in 1 ml Eagle’s MEM containing 10% FBS, 10 mM HEPES, 1mM sodium pyruvate, 100 mM non-essential amino acids, 50 μg/ml gentamycin. All samples were stored at -80°C. Nasal swabs and WBCs were serially diluted, added to duplicate wells of a microtiter plate with MDBK cells, and incubated for 1.5 h at 37 C. Subsequently, MEM with 2% FBS was added, and the plates were further incubated. After five days, the cells were blocked with 10% horse serum, fixed with 75 EtOH/25% acetic acid and permeabilized with 0.01% Triton X-100 in PBS. After washing, the cells were incubated with type 2 E2-specific rabbit antibody (in-house). AP-conjugated goat anti-rabbit IgG (Kirkegaard & Perry Laboratories), followed by BCIP/NBT (Bio-Rad) were used for detection. The reciprocal of the highest dilution still showing virus in replicate wells was reported as the virus titer. 2.6 Hematological analysis: Blood samples were analyzed on the day of challenge and on days 2, 4, 6, 8, 10 and 12 post-challenge by Prairie Diagnostic Services (Saskatoon, SK, Canada). Total WBC counts and differential leukocyte counts including lymphocytes, monocytes, and segregates were quantified and expressed as counts (× 9 -1 L ). 2.7 Statistical analysis: Data were analyzed with the aid of a software program (GraphPad Prism 5.0). As sample sizes were small, outcome variables were assumed not to be normally distributed. Therefore, differences among all groups were examined using the Kruskal-Wallis test. If a significant difference was found among the groups, median ranks between pairs of groups were compared using the Mann-Whitney U test.

Differences were considered significant if P < 0.05. 3. Results and Discussion 3.1 Production and purification of BVDV-2 E2 protein As the challenge model with BVDV-2 is more robust and thus a more rigorous test of vaccine efficacy, a type 2 E2 protein was tested in this study. A secreted version of the BVDV-2 E2 protein with a C-terminal his tag was generated and produced in HEK293 cells. The E2 protein was produced at about 10 mg per liter and purified from the supernatant of HEK293 culture with ProBond resin. The resulting protein had an apparent molecular weight of ~53kD as expected and was >90% pure making it suitable as subunit antigen (Fig. 1). The identity of the E2 protein was further confirmed by Western blotting (data not shown). 3.2 Immune responses induced in lambs immunized with E2-adjuvant formulations To evaluate the potential of various adjuvant combinations to enhance immune responses induced by the BVDV E2 protein, three month-old lambs were immunized twice IM at a three-week interval with E2 formulated in PBS or PCEP with either CpG ODN or poly(I:C), in the presence or absence of IDR peptide. However, no combination of IDR and PCEP was evaluated as this was not very effective in mice (data not shown). While signaling through both TLRs is known to mediate the induction of balanced to Th1-biased immune responses, TLR3, but not TLR9, is highly expressed on myeloid dendritic cells, which are most capable at cross-presenting antigens.

Sera and PBMCs were collected to determine E2-specific humoral and cellular immune responses. As shown in Fig. 2A, after one immunization only lambs immunized with E2 and the triple poly(I:C) adjuvant combination developed enhanced serum IgG titers, significantly higher than those induced by the double combination of PCEP and poly (I:C), or any of the other formulations. Even after the second immunization, no immune responses were detected in lambs immunized with E2 protein in PBS. In contrast, the E2-specific IgG titers were significantly higher in lambs immunized with PCEP and either CpG or poly(I:C); furthermore, lambs that received the E2 protein with the triple combinations developed even higher titers, up to 100-fold higher than lambs immunized with just the E2 protein (Fig. 2B). Additionally, the lambs that received the E2 protein with either of the triple combinations developed significantly increased numbers of E2-specific IFN-γ secreting cells in the blood (Fig. 2C) when compared to E2 protein with the double adjuvants or PBS. After the second immunization the IFN-γ production also increased in the double adjuvant groups, but remained stable in the triple adjuvant groups (Fig. 2D). These results demonstrate the efficacy of formulation with PCEP, IDR and either CpG or poly(I:C), the poly(I:C) - containing combination being slightly superior. 3.3 Immune responses elicited by E2 formulated with the poly(I:C)-containing triple adjuvant formulation in cattle Since the immune responses induced by the E2 protein formulated with the combination of poly(I:C), IDR and PCEP were robust and superior to those induced by the other formulations in lambs, calves were immunized twice IM at a four-week interval, with E2 formulated with poly(I:C), IDR and PCEP. Sera and PBMCs were collected to determine E2-specific humoral and cell-mediated immune responses. As shown in Fig. 3A and B, after two immunizations the E2-specific IgG and BVDV- specific VN titers were significantly higher in vaccinated calves than in control calves that received PBS. Interestingly, both E2-specific IFN-γ secreting CD4 and CD8 cells were identified in the PBMCs after the first and the second vaccination (Fig. 4B,C), and the CD8 cells numbers increased upon challenge as expected (Fig. 4D). Based on the increased expression of the activation marker, CD25 , the CD8 T cells can be considered CTLs (Fig. 4E). 3.4 Protection from BVDV challenge in E2 vaccine-immunized calves To evaluate whether the immune responses were robust enough to provide protection, all calves were challenged with BVDV-2, and changes in temperatures, weights and WBCs were examined. The temperatures in the placebo-treated calves increased from day 6 onwards and then returned to pre-challenge values on day 14, while their weights decreased from day 9 and at the end of the trial were on average 20 kg lower than at the start. While experiencing a slight change in temperature and weight for about two days, the type 2 E2 vaccine-immunized calves had lower temperatures than PBS-immunized animals from days 8 to 12 post-challenge, and these differences were significant on days 9 and 11; they also experienced less weight loss from days 9 to 13 (Fig. 5A,B), and at the end of the study their weights had slightly increased. In addition, the total WBCs counts were higher in the E2-immunized calves than in the PBS-treated animals between days 6 and 12 (Fig. 5C), while the monocyte counts were higher on days 2, 8 and 12, the lymphocytes between days 8 and 12, and the PMNs on day 8 (Fig. 5 D, E and F). With the exception of one animal with virus in the nasal fluids for one day, the vaccinated animals did not show evidence of viral replication (Table 1), while the placebo-treated animals shed virus for three to five days, demonstrating the induction of significant protection with this strategy. 4. Conclusions The BVDV E2 protein, when formulated with the triple adjuvant, induced robust virus neutralizing antibodies as well as cell-mediated immune responses, including CD8 CTL responses. Importantly, to our knowledge this is the first time CTL responses have been demonstrated in calves vaccinated with an E2 protein subunit vaccine, indicating that the adjuvant formulation promotes cross-presentation.

Furthermore, upon BVDV-2 challenge, all calves showed significant protection from viral infection. These data are the first to demonstrate the efficacy of this novel adjuvant combination in a vaccination-challenge trial in cattle. To protect additionally against BVDV-1 strains, type 1 E2 protein could be added to the vaccine formulation.

This E2 subunit vaccine would also allow differentiation of infected from vaccinated animals (DIVA). The high level of protection from BVDV-2 challenge afforded by this E2 vaccine contrasts with results for a previous E2 formulation; this may be due to the difference in adjuvant [12], and is possibly correlated to the induction of CTL responses observed in the current study.

These and other results show that the BVDV E2 protein is a valid candidate for a subunit vaccine. Recently, alphavirus replicon particles containing the gene encoding type 1 BVDV E2 protein were shown to induce cross-neutralizing titers, and reduce clinical disease and leukopenia post challenge [13]. In another recent report, a truncated version of the E2 protein from BVDV was fused to APCH, which targets to antigen presenting cells, and expressed in plants. The partially purified APCH-tE2 protein was found to induce neutralizing antibodies in bovines [14]. Both humoral and cell- mediated immune responses have been demonstrated to E. coli-expressed BVDV E2 protein when formulated with mesoporous silica nanoparticles in mice; however no protection studies in cattle were carried out [15]. These studies were all carried out with the type 1 BVDV challenge strains. The robust and balanced immune responses and complete protection from virulent viral challenge suggest that this E2 subunit vaccine formulation is a very promising vaccine candidate against BVDV-2.

Thus novel methods for treating and preventing infectious diseases are disclosed.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications, additions or equivalents may be made thereto without departing from the scope thereof as defined in the appended claims.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it) or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

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Ridpath J. The contribution of infections with bovine viral diarrhea viruses to bovine respiratory disease. The Veterinary clinics of North America Food animal practice. 2010;26:335-48.

Kindrachuk J, Jenssen H, Elliott M, Townsend R, Nijnik A, Lee SF, et al. A novel vaccine adjuvant comprised of a synthetic innate defence regulator peptide and CpG oligonucleotide links innate and adaptive immunity. Vaccine. 2009;27:4662-71.

Garlapati S, Garg R, Brownlie R, Latimer L, Simko E, Hancock RE, et al. Enhanced immune responses and protection by vaccination with respiratory syncytial virus fusion protein formulated with CpG oligodeoxynucleotide and innate defense regulator peptide in polyphosphazene microparticles. Vaccine. 2012;30:5206-14.

Polewicz M, Gracia A, Buchanan R, Strom S, Halperin SA, Potter AA, et al.

Influence of maternal antibodies on active pertussis toxoid immunization of neonatal mice and piglets. Vaccine. 2011. [7] Liang R, Babiuk LA, van Drunen Littel-van den Hurk S. Compatibility of plasmids encoding bovine viral diarrhea virus type 1 and type 2 E2 in a single DNA vaccine formulation. Vaccine. 2007;25:5994-6006.

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Immunization with plasmid DNA encoding a truncated, secreted form of the bovine viral diarrhea virus E2 protein elicits strong humoral and cellular immune responses.

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Van Drunen Littel-van den Hurk S, Braun RP, Lewis PJ, Karvonen BC, Babiuk LA, Griebel PJ. Immunization of neonates with DNA encoding a bovine herpesvirus 40 glycoprotein is effective in the presence of maternal antibodies. Viral Immunol. 1999;12:67-77.

Raggo C, Habermehl M, Babiuk LA, Griebel P. The in vivo effects of recombinant bovine herpesvirus-1 expressing bovine interferon-gamma. J Gen Virol. 2000;81 Pt 45 11:2665-73.

Liang R, van den Hurk JV, Babiuk LA, van Drunen Littel-van den Hurk S.

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Claims (11)

1. An adjuvant composition comprising (a) a host defense peptide selected from SEQ ID NO:1, SEQ ID NO:18; SEQ ID 5 NO:19; SEQ ID NO:20; and SEQ ID NO:22; (b) an immunostimulatory sequence selected from (i) a CpG oligonucleotide selected from SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:12; and (ii) poly (I:C); (c) a bovine viral diarrhea virus (BVDV) antigen; and 10 (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP), wherein said adjuvant composition is capable of enhancing an immune response 15 to the BVDV antigen as compared to the immune response elicited by an equivalent amount of the BVDV antigen when delivered without the adjuvant composition.

2. The adjuvant composition of claim 1, wherein the host defense peptide comprises the sequence of SEQ ID NO:19, the immunostimulatory sequence is poly 20 (I:C), and the polyanionic polymer is poly(dioxyphenylproprionate)phosphazene (PCEP).

3. The adjuvant composition of claim 1 or claim 2 wherein the BVDV antigen is BVDV E2 antigen.

4. Use of (a) a host defense peptide selected from SEQ ID NO:1, SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; and SEQ ID NO:22; (b) an immunostimulatory sequence selected from (i) a CpG oligonucleotide 30 selected from SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:12; and (ii) poly (I:C); (c) a bovine viral diarrhea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP) in the manufacture of a 5 medicament for enhancing an immune response to the BVDV antigen.

5. Use of (a) a host defense peptide selected from SEQ ID NO:1, SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; and SEQ ID NO:22; 10 (b) an immunostimulatory sequence selected from (i) a CpG oligonucleotide selected from SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:12; and (ii) poly (I:C); (c) a bovine viral diarrhea virus (BVDV) antigen; and (d) a polyanionic polymer, wherein the polyanionic polymer is poly[di(sodium 15 carboxylatophenoxy)phosphazene] (PCPP), poly(di oxyphenylproprionate)phosphazene (PCEP), or a PCPP polymer comprising 90% PCPP copolymer with 10% hydroxyl groups (90:10 PCPP) for enhancing an immune response to the BVDV antigen in a non-human subject. 20

6. The use of claim 4 or claim 5, wherein the host defense peptide comprises the sequence of SEQ ID NO:19; the immunostimulatory sequence is poly (I:C); and the polyanionic polymer is poly(dioxyphenylproprionate)phosphazene (PCEP).

7. The use of any one of claims 4 to 6, wherein the host defense peptide, the 25 immunostimulatory sequence, the polyanionic polymer and the BVDV antigen are present in the same composition.

8. The use of any one of claims 4 to 7, wherein at least one of the host defense peptide, the immunostimulatory sequence, the polyanionic polymerand the BVDV 30 antigen are present in a different composition.

9. The use of any one of claims 4 to 8 wherein the BVDV antigen is BVDV E2 antigen.

10. An adjuvant composition as claimed in any one of claims 1 to 3 and substantially as herein described with reference to any one of the examples and/or figures.

11. The use as claimed in any one of claims 4 to 9 and substantially as herein described with reference to any one of the examples and/or figures.