WO2002038593A1 - Haemophilus antigen - Google Patents

Abstract

Isolated proteins and isolated nucleic acids encoding said proteins of Haemophilus paragallinarum are provided. The isolated proteins, in particular as an antigen, and isolated nucleic acids may be useful for diagnosis of, and immunization against, infectious coryza in an avian, in particular chickens.

Description

TITLE

HAEMOPHILUS ANTIGEN

FIELD OF THE INVENTION

THIS INVENTION relates to novel proteins of Haemophilus paragallinarum and to nucleic acids encoding same. More particularly, this invention relates to diagnosis of, and immunization against, infectious coryza in chickens using novel protein antigens and/or encoding nucleic acids, although without being limited thereto.

BACKGROUND OF THE INVENTION Haemophilus paragallinarum is a causative organism responsible for infectious coryza of chickens. Infectious coryza is an acute upper respiratory tract disease of chickens, which is of worldwide economic significance and affects both broiler and layer flocks. The disease is manifested primarily by a decrease in egg production (10-40%; Thornton & Blackall, 1984, Aust. Vet. J. 61 251) in layer flocks and retardation of growth due to decreased feed and water consumption in breeder and broiler flocks. The most common clinical symptoms include nasal discharge, facial oedema, lacrimation, anorexia and diarrhoea (Blackall, 1989, Clin. Microbiol. Rev. 2 270).

At present, inactivated whole cell vaccines against H. paragallinarum are available and are considered relatively effective (Blackall, 1989, supra). However, killed whole cell vaccines have limitations. The major problem with the current whole cell inactivated vaccines is that they do not provide cross serovar protection, i.e. they only protect against the serovar(s) present in the vaccine. Another limitation of whole cell inactivated vaccines is that since only limited serovar protection is afforded by those serovars in the vaccine, the introduction of new strains/serovars into a particular locality can produce antigenic pressure on the vaccine (Yamamoto, 1984, In: Diseases of

Poultry, 8th Ed. Hofstad et al, Eds pp 178- 186). This can result in uncontrolled

Substitute Sheet (Rule 26) RO/AU infection of infectious coryza in that particular locality. Therefore, the use of local strains in vaccines is highly recommended (Bragg et al., 1996, Onderspoort J. Vet. Res. 63 217).

The haemagglutinin antigen (HA) of H. paragallinarum plays a significant role in pathogenicity and several attempts have been made to isolate the HA protein and encoding nucleic acid (Takagi et al, 1991a, J. Vet Med. Sci. 53 917; Iritani et al, 1981, Avian Dis. 25 479; Kume et al, 1980, Am. J. Vet. Res. 41 97; Iritani et al, 1980, Am. J. Vet. Res. 41 2114; United States Patent No. 4,247,539; European Patent 870828; United States Patent No. 5,240,705). SUMMARY OF THE INVENTION

In attempting to isolate a nucleic acid encoding H. paragallinarum, haemagglutinin, the present inventors have unexpectedly isolated novel nucleic acids and encoded proteins distinct from haemagglutinin, but having properties useful in H. paragallinarum vaccination and detection. In a first aspect, the invention provides an isolated protein comprising an amino acid sequence PNFKKQ [SEQ ID NO: 1].

Preferably, the isolated protein further comprises amino acid sequence FQSASNR [SEQ ID NO: 2].

In a second aspect, the invention provides an isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID

NOS: 3-13 as shown in FIG. 1.

In a third aspect, the invention provides a homolog, fragment, variant or derivative of an isolated protein selected from the group consisting of SEQ ID NOS: 3-13 as shown in FIG. 1. It will be understood that the isolated proteins of FIG. 1 [SEQ ID

NOS: 3-13] are examples of "ORF 3 proteins of the invention".

Preferably, the isolated proteins of the first, second and third aspects, which when administered to an animal, are capable of eliciting an

Substitute Sheet (Rule 26) RO/AU immune response in said animal.

Preferably, said immune response provides protection against one or more strains of Haemophilus paragallinarum in said animal.

Preferably, the animal is an avian. More preferably, the avian is a chicken.

In a fourth aspect, the invention provides an isolated nucleic acid encoding a protein according to the first, second or third aspects.

Preferably, the isolated nucleic acid comprises a nucleotide sequence 5'-CCT AAT TTT AAG AAA CAA-3' [SEQ ID NO: 14]. More preferably, the isolated nucleic acid further comprises a nucleotide sequence 5'-TTT CAA TCG GCA TCT AAT CGC-3' [SEQ ID NO: 15].

In one embodiment of the fourth aspect, the isolated nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 16-26 as shown in FIG. 2.

In another embodiment of the fourth aspect, the isolated nucleic acid encodes a homolog, fragment, variant or derivative of an isolated nucleic acid selected from the group consisting of SEQ ID NOS: 16-26 as shown in FIG. 2. In a further embodiment of the fourth aspect, the isolate nucleic acid is a primer or probe derived from any one of SEQ ID NOS: 16-26.

Preferably, the nucleic acid primer or probe is selected from the group consisting of SEQ ID NOS: 27-45.

In a fifth aspect, the invention provides an expression construct comprising an expression vector and an isolated nucleic acid according to the fourth aspect, wherein said nucleic acid is operably linked to one or more regulatory nucleic acids in said expression vector.

In a sixth aspect, the invention provides a host cell comprising an

Substitute Sheet (Rule 26) RO/AU expression construct according to the fifth aspect. Preferably, the host cell is a bacteria. More preferably the bacteria is E. coli.

In a seventh aspect, the invention provides a method of producing a recombinant protein according to the first, second or third aspects, said method including the steps of:

(i) culturing a host cell according to the sixth aspect such that said recombinant protein is expressed in said host cell; and (ii) isolating said recombinant protein. In an eighth aspect, the invention provides an antibody or antibody fragment that binds to a protein of the first, second or third aspects.

In a ninth aspect, the invention provides a method of detecting H paragallinarum bacteria in a biological sample suspected of comprising said bacteria, said method including the steps of:- (a) isolating the biological sample;

(b) combining the abovementioned antibody or antibody fragment with the biological sample; and

(c) detecting specifically bound antibody or antibody fragment which indicates the presence of said bacteria. In a tenth aspect, the invention provides a method of detecting H. paragallinarum bacteria in a biological sample suspected of comprising said bacteria, said method including the steps of:-

(I) isolating the biological sample;

(II) detecting a nucleic acid comprising a nucleotide sequence according to the fourth aspect in said sample which indicates the presence of said bacteria. In an eleventh aspect, the invention provides a method of diagnosing infection of an avian by H paragallinarum bacteria, said method

Substitute Sheet (Rule 26) RO/AU including the steps of:-

(A) contacting a biological sample from said avian with a protein of the first, second or third aspect; and

(B) determining the presence or absence of a complex between said protein and H. paragallinarum-specific antibodies in said sample, wherein the presence of said complex is indicative of said infection. Preferably, the biological sample of the abovementioned methods is a nasal swab sample. In a twelfth aspect, the invention provides a kit for detecting H. paragallinarum bacteria in a biological sample or diagnosing H. paragallinarum bacteria infection in an avian, wherein said kit comprises one or more proteins according to the first, second or third aspects.

In a thirteenth aspect, the invention provides a kit for detecting H. paragallinarum bacteria in a biological sample or diagnosing H. paragallinarum bacteria infection in an avian, wherein said kit comprises one or more nucleic acids according to the fourth aspect.

In a fourteenth aspect, the invention provides a kit for detecting H. paragallinarum bacteria in a biological sample or diagnosing H. paragallinarum bacteria infection in an avian, wherein said kit comprise one or more antibody or antibody fragment of the eighth aspect.

In a fifteenth aspect, the invention provides a pharmaceutical composition comprising at least one isolated protein according to the first, second or third aspects in combination with a pharmaceutically acceptable carrier or diluent.

In a sixteenth aspect, the invention provides a pharmaceutical composition comprising at least one isolated nucleic acid according to the fourth aspect in combination with a pharmaceutically acceptable carrier or diluent.

Substitute Sheet (Rule 26) RO/AU Preferably, the pharmaceutical composition of the sixteenth and seventeenth aspects is a vaccine.

In a preferred embodiment, the vaccine is administered using Salmonella or Mycoplasma bacterium, the bacterium expressing at least one protein of the first, second or third asepcts.

In a seventeenth aspect, the invention provides a method of immunizing an avian against H. paragallinarum infection, including the step of administering a pharmaceutically effective amount of the abovementioned vaccine to the avian. Preferably, the avian is a chicken.

In an eighteenth aspect, the invention provides a method of identifying an immunogenic fragment of a protein of the first, second or third aspects, including the steps of:-

(a') producing a fragment of said protein; (b') administering said fragment to a mammal or avian; and

(c') detecting an immune response in said mammal or avian, which response includes production of elements which specifically bind H. paragallinarum and/or said protein, and/or a protective effect against H. paragallinarum infection.

Preferably the avian is a chicken. Preferably, the mammal is a mouse or rabbit. Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Substitute Sheet (Rule 26) RO/AU BRIEF DESCRIPTION OF THE FIGURES AND TABLES TABLE 1 : Conservative amino acid substitutions.

TABLE 2: Oligonucleotide primers used for PCR and DNA sequencing [SEQ ID NOS: 27-45]. TABLE 3: Summary of H. paragallinarum reference strains. ^a(P) = reference strain for Page serotyping scheme (Page, 1962, Am. J. Vet. Res. 23 85); (K) = reference strain for Ku e serotyping system (Kume et al, 1983, J. Clin. Microbiol. 17 1958). FIG. 1: Amino acid sequences [SEQ ID NOS: 3-13] for ORF3 proteins from strains of H. paragallinarum listed in Table 3. Amino acid residues conserved between the ORF3 sequences are indicated thus *. Minimal consensus sequences unique to the ORF3 proteins are underlined. The following sequences are shown: HP1 [SEQ ID NOS: 3], HP137 [SEQ ID NOS: 4], HP90 [SEQ ID NOS: 5], HP144 [SEQ ID NOS: 6], HP14 [SEQ ID NOS: 7], HP2 [SEQ ID NOS: 8], HP147 [SEQ ID NOS: 9], HP3 [SEQ ID NOS: 10], HP146 [SEQ ID NOS:

11], HP145 [SEQ ID NOS: 12] and HP60 [SEQ ID NOS: 13]. FIG. 2: ORF3 nucleotide sequences [SEQ ID NOS: 16-26] encoding the

ORF3 proteins of H. paragallinarum shown in FIG. 1. Nucleotides conserved between the ORF3 sequences are indicated thus *. Minimal consensus sequences unique to the ORF3 nucleic acids are underlined. The following sequences are shown: HP1 [SEQ ID NOS: 16], HP137 [SEQ ID NOS: 17], HP90 [SEQ ID

NOS: 18], HP144 [SEQ ID NOS: 19], HP14 [SEQ ID NOS: 20], HP2 [SEQ ID

NOS: 21], HP147 [SEQ ID NOS: 22], HP3 [SEQ ID NOS: 23], HP146 [SEQ ID NOS: 24], HP 145 [SEQ ID NOS: 25] and HP60 [SEQ ID NOS: 26]. FIG. 3: Western blotting of proteins using MAb 4D (at 1/1,000 dilution)

Substitute Sheet (Rule 26) RO/AU and H paragallinarum serovar A polyclonal antiserum (at 1/100,000 dilution).

The expected migration position of the ~39 kD haemagglutinin antigen is

indicated by <- ΗA. (A): Lanes 1-3 are prestained protein ladder, H. paragallinarum serovar A (positive control) and clone 8.2 encoded protein

immunoblotted with MAb 4D. (B): Lanes 1-3 are prestained protein ladder, H paragallinarum serovar A (positive control) and clone 8.2 encoded protein respectively, immunoblotted with H. paragallinarum serovar A polyclonal antiserum. (C): Lanes 1-4 are prestained protein ladder, H. paragallinarum serovar A (positive control), clone 7.2 encoded protein and vector without insert

(negative control) respectively, immunoblotted with MAb 4D. (D): Lanes 1-4 are prestained protein ladder, H. paragallinarum serovar A (positive control), clone

7.2 encoded protein and vector without insert (negative control) respectively, immunoblotted with H paragallinarum serovar A polyclonal antiserum. (E):

Lanes 1-4 are prestained protein ladder, H. paragallinarum serovar A (positive control), clone 14.2 encoded protein and vector without insert (negative control) respectively, immunoblotted with H paragallinarum serovar A polyclonal

antiserum. (F): Lanes 1-10 are prestained protein ladder, H. paragallinarum

serovar A (positive control), clone 8.2 encoded protein, clone 17.1 encoded protein, clone 17.2 encoded protein, vector without insert (negative control), clone 8.2 encoded protein, clone 17.1 encoded protein, clone 17.2 encoded protein, and vector without insert (negative control) respectively, immunoblotted

with H. paragallinarum serovar A polyclonal antiserum. (G): Lanes 1-15 are

vector without insert (negative control), clone 18.1 encoded protein, clone 16.1 encoded protein, clone 10.1 encoded protein, clone 9.2 encoded protein, clone 9.1

encoded protein, clone 8.2 encoded protein, clone 1.2 encoded protein, H.

Substitute Sheet (Rule 26) RO/AU paragallinarum serovar A (positive control) prestained protein ladder, H.

paragallinarum serovar A (positive control) clone 8.1 encoded protein, clone 13.1 encoded protein, clone 16.2 encoded protein and vector without insert (negative control) respectively, immunoblotted with H. paragallinarum serovar A polyclonal antiserum. All serovar A positive controls were derived from strain

ΗP1.

FIG. 4: Coomassie blue stained 12% SDS-PAGE gel of purified protein with 10 μl of sample loaded per lane. Lane 1 is a molecular mass ladder

(Benchmark, GibcoBRL); lane 2 is M15/pQE30/ORF3 lysed cell supernatant; lane 3 is ORF3 post-binding to resin; lanes 4-5 are respective washes 1-2; lane 6

is column flow-through; lane 7 is column wash; and lanes 8-13 are respective elutions 1-6 (lOμL from 2 mL fraction).

FIG. 5: Antibody response of chickens immunised with 100 μg rORF3 per

chicken. Titre of sera obtained 21 days after injection of either rORF3 (100

μg)/Alum or PBS/Alum is expressed as the reciprocal of the last dilution which

showed reactivity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, at least in part, on the isolation of Haemophilus paragallinarum proteins and encoding nucleic acids by the present inventors. The proteins set forth in FIG. 1 correspond to novel and unexpected proteins from several distinct serovars of Haemophilus paragallinarum. Unexpectedly, these novel proteins and nucleic acid were isolated following attempts to isolate haemagglutinin (HA) proteins and nucleic acids. The present inventors therefore have provided isolated proteins and nucleic acids which provide hitherto unsuspected molecules useful for the purposes of

Substitute Sheet (Rule 26) RO/AU Haemophilus paragallinarum detection and large-scale production of recombinant vaccines for mass immunization against infectious coryza in chickens.

The term "recombinant" as used herein means artificially produced through human manipulation of genetic material, such as involving techniques generally falling within the scope of "recombinant DNA technology" as is well understood in the art.

By "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in recombinant or native form.

A "protein" refers to a "polypeptide" or "peptide", either term referring to an amino acid polymer which may include natural and/or non-natural amino acids as are well known in the art.

A "peptide" is a protein having no more than fifty (50) amino acids.

A peptide is an example of a polypeptide "fragment". In one embodiment, a "fragment" includes an amino acid sequence which constitutes less than 100%, but at least 20%, preferably at least 50%, more preferably at least 80% or even more preferably at least 90%> of said protein.

In another embodiment, a "fragment" is a peptide, for example of at least 6, preferably at least 10 and more preferably at least 20 amino acids in length, which comprises one or more antigenic determinants or epitopes. Larger fragments comprising more than one peptide are also contemplated, and may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For

Substitute Sheet (Rule 26) RO/AU example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis " by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines " edited by Nicholson and published by Blackwell Scientific Publications. Alternatively, peptides can be produced by digestion of a protein of the invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcins V8- protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques.

As used herein, "variant " proteins are proteins of the invention in which one or more amino acids have been replaced by different amino acids. It is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the protein (conservative substitutions). Exemplary conservative substitutions in the protein may be made according to TABLE 1. Substantial changes in function are made by selecting substitutions that are less conservative than those shown in TABLE 1. Other replacements would be non-conservative substitutions and relatively fewer of these may be tolerated. Generally, the substitutions which are likely to produce the greatest changes in a protein's properties are those in which (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val); (b) a cysteine or proline is substituted for, or by, any other residue; (c) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g. , Glu or Asp) or (d) a residue having a bulky side chain (e.g., Phe or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

The term "variant" also includes ORF3 proteins produced from allelic variants of the sequences exemplified in this specification. Accordingly, the term variant also includes nucleic acid variants, for example allelic variants.

Substitute Sheet (Rule 26) RO/AU Protein variants fall within the scope of the term "protein homologs".

The proteins of the invention show homology to other members.

Therefore, in an embodiment, protein homologs of the invention share at least 60%, preferably at least 80% and more preferably at least 90% sequence identity with the amino acid sequences set forth in FIG. 1 [SEQ ED NOS: 3-13].

As generally used herein, a "homolog" shares a definable nucleotide or amino acid sequence relationship with a nucleic acid or protein of the invention as the case may be.

Included within the scope of homologs are "orthologs", which are functionally-related proteins and their encoding nucleic acids, isolated from organisms other than Haemophilus paragallinarum, such as other Haemophilus species. Terms used herein to describe sequence relationships between respective nucleic acids and proteins include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because respective nucleic acids/proteins may each comprise (1) only one or more portions of a complete nucleic acid/protein sequence that are shared by the nucleic acids/proteins, and (2) one or more portions which are divergent between the nucleic acids/proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically at least 6 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (t.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window

Substitute Sheet (Rule 26) RO/AU may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by 5 inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. 0 A detailed discussion of sequence analysis can be found in Unit

19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

The term "sequence identity" is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an 5 appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield o the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, "sequence identity" may be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available 5 from Hitachi Software engineering Co., Ltd., South San Francisco, California,

USA).

Thus, it is well within the capabilities of the skilled person to prepare protein homologs of the invention, such as variants as hereinbefore

Substitute Sheet (Rule 26) RO/AU defined, by recombinant DNA technology. For example, nucleic acids of the invention can be mutated using either random mutagenesis for example using transposon mutagenesis, or site-directed mutagenesis. The resultant DNA fragments are then cloned into suitable expression hosts such as E. coli using conventional technology and clones that retain the desired activity are detected.

Where the clones have been derived using random mutagenesis techniques, positive clones would have to be sequenced in order to detect the mutation.

As used herein, "derivative " proteins are proteins of the invention which have been altered, for example by conjugation or complexing with other chemical moieties or by post-translational modification techniques as would be understood in the art. Such derivatives include amino acid deletions and/or additions to proteins of the invention, or variants thereof, wherein said derivatives elicit an immune response.

"Additions" of amino acids may include fusion of the proteins or variants thereof with other proteins. Particular examples of such proteins include

Protein A, glutathione S-transferase (GST), maltose-binding protein (MBP), hexahistidine (HIS₆) and epitope tags such as FLAG and c-myc tags.

Other derivatives contemplated by the invention include, but are not limited to, modification to side chains, incorporation of unnatural amino acids and/or their derivatives during protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteins, fragments and variants of the invention. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidination with methylacetimidate; carbamoylation of amino groups with cyanate; pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBFL;; reductive alkylation by reaction with an aldehyde followed by reduction with

Substitute Sheet (Rule 26) RO/AU NaBH₄; and trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS).

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, by way of example, to a corresponding amide.

The guanidine group of arginine residues may be modified by formation of heterocyclic condensation products with reagents such as 2,3- butanedione, phenylglyoxal and glyoxal.

Sulphydryl groups may be modified by methods such as performic acid oxidation to cysteic acid; formation of mercurial derivatives using 4- chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate; 2-chloromercuri-4- nitrophenol, phenylmercury chloride, and other mercurials; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; carboxymethylation with iodoacetic acid or iodoacetamide; and carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified, for example, by alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides or by oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified by N- carbethoxylation with diethylpyrocarbonate or by alkylation with iodoacetic acid derivatives.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include but are not limited to, use of 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4- amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/or D-isomers of amino

Substitute Sheet (Rule 26) RO/AU acids.

The invention also contemplates covalently modifying an ORF3 protein, fragment or variant of the invention with dinitrophenol, in order to render it immunogenic in chickens ORF3 proteins of the invention (inclusive of fragments, variants, derivatives and homologs in general) may be prepared by any suitable procedure known to those of skill in the art.

For example, a recombinant ORF3 protein may be prepared by a procedure including the steps of: (i) preparing an expression construct which comprises a ORF3 nucleic acid of the invention, operably linked to one or more regulatory nucleotide sequences; (ii) transfecting or transforming a suitable host cell, for example E.coli, with the expression construct; and (iii) expressing the protein in said host cell.

Preferably, the ORF3 nucleic acid at step (i) has a nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in FIG. 2 [SEQ ID NOS: 16-26].

It will also be appreciated that the abovementioned method is suitable for producing recombinant proteins from nucleic acid homologs, as will be described in more detail hereinafter.

Suitable host cells for expression may be prokaryotic or eukaryotic.

Preferably, the host cell is prokaryotic. Preferably, the prokaryotic cell is a bacterium.

Preferred bacteria are E. coli, or bacteria of the genus Salmonella and the genus Mycoplasma.

For the purposes of host cell expression, the ORF3 nucleic acid is

Substitute Sheet (Rule 26) RO/AU operably linked to one or more regulatory sequences in an expression vector.

An "expression vector" may be either a self-replicating extra- chromosomal vector such as a plasmid, or a vector that integrates into a host genome. By "operably linked" is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the ORF3 nucleic acid of the invention or homolog thereof, to initiate, regulate or otherwise control transcription of the nucleic acid .

Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression, as regulatory sequences and host cell are often interdependent. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences.

Constitutive or inducible promoters as known in the art are contemplated by the invention. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.

In a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

The expression vector may also include a fusion partner (typically provided by the expression vector) so that the recombinant protein of the invention is expressed as a fusion protein with said fusion partner. The main advantage of fusion partners is that they assist identification and/or purification of said fusion protein.

Substitute Sheet ⁽Rule 26) RO/AU In order to express said fusion protein, it is necessary to ligate the

ORF3 nucleic acid or homolog into the expression vector so that the translational reading frames of the fusion partner and the operably linked nucleic acid coincide.

Well known examples of fusion partners include, but are not limited to, glutathione-S-transferase (GST), Fc portion of IgG, maltose binding protein (MBP) and hexahistidine (HIS₆), which are particularly useful for isolation of the fusion protein by affinity chromatography. For the purposes of fusion protein purification by affinity chromatography, relevant matrices for affinity chromatography are glutathione-, amylose-, and nickel- or cobalt- conjugated resins respectively. Many such matrices are available in "kit" form, such as the QIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners as described herein. Using this system, a HIS₆ fusion protein may be isolated using, for example, Ni-NTA resin. Other kits include the Pharmacia GST purification system. Another fusion partner well known in the art is green fluorescent protein (GFP). This fusion partner serves as a fluorescent "tag" which allows the fusion protein of the invention to be identified by fluorescence microscopy or by flow cytometry. The GFP tag is useful when assessing subcellular localization of the fusion protein of the invention, or for isolating cells which express the fusion protein of the invention. Flow cytometric methods such as fluorescence activated cell sorting (FACS) are particularly useful in this latter application.

Preferably, the fusion partners also have protease cleavage sites, such as for Factor X_a or Thrombin, which allow the relevant protease to partially digest the fusion protein of the invention and thereby liberate the recombinant protein of the invention therefrom. The liberated protein can then be isolated from the fusion partner by subsequent chromatographic separation.

Fusion partners according to the invention also include within their scope "epitope tags", which are usually short peptide sequences for which a

Substitute Sheet (Rule 26) RO/AU specific antibody is available. Well known examples of epitope tags for which specific monoclonal antibodies are readily available include c-myc, influenza virus haemagglutinin and FLAG tags.

As hereinbefore described, ORF3 proteins of the invention may be produced by culturing a host cell transformed with the aforementioned expression construct. The conditions appropriate for protein expression will vary with the choice of expression vector and the host cell. For example, the induction system used for protein system varies from one vector to another. This is easily ascertained by one skilled in the art through routine experimentation and reference to the appropriate product literature.

The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook, et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), incorporated herein by reference, in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. 1995-1999), incorporated herein by reference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. 1995-1999) which is incorporated by reference herein, in particular Chapters 1, 5, 6 and 7. Nucleotide sequences

The invention provides an isolated nucleic acid that encodes a ORF3 protein of the invention.

In preferred embodiments, said isolated nucleic acid has a nucleotide sequence selected from the group consisting of the sequences set forth in FIG. 2 [SEQ ID NOS : 16-26] .

The term "nucleic acid" as used herein designates single-or double-stranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomic DNA.

Substitute Sheet (Rule 26) RO/AU In one embodiment, a nucleic acid "fragment" comprises a nucleotide sequence that constitutes less than 100% of a nucleic acid of the invention. A fragment includes a polynucleotide, oligonucleotide, probe, primer and an amplification product, eg. a PCR product. Examples of fragments are primers [SEQ ID NOS: 27-45] as used herein.

A "polynucleotide " is a nucleic acid having eighty (80) or more contiguous nucleotides, while an "oligonucleotide" has eight (8) to eighty (80) contiguous nucleotides.

A "probe" may be a single or double-stranded oligonucleotide or polynucleotide, suitably labeled for the purpose of detecting complementary sequences in Northern or Southern blotting, for example.

A "primer" is usually a single-stranded oligonucleotide, preferably having 15-50 contiguous nucleotides, which is capable of annealing to a complementary nucleic acid "template" and being extended in a template- dependent fashion by the action of a DNA polymerase such as Taq polymerase,

RNA-dependent DNA polymerase or Sequenase™. Examples of primers used herein include SEQ ID NOS: 27-45.

The present invention also contemplates homologs of ORF3 nucleic acids of the invention. In one embodiment, nucleic acid homologs encode protein homologs of the invention, inclusive of variants, fragments and derivatives thereof.

In another embodiment, nucleic acid homologs share at least 60%>, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% sequence identity with the nucleotide sequences of FIG. 2.

In yet another embodiment, nucleic acid homologs hybridize to the nucleotide sequences of FIG. 2 [SEQ ID NOS: 16-26] under at least low stringency conditions, preferably under at least medium stringency conditions and

Substitute Sheet ⁽Rule 26) RO/AU more preferably under high stringency conditions.

"Hybridize and Hybridization" is used herein to denote the pairing of at least partly complementary nucleotide sequences to produce a DNA-DNA,

RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementary nucleotide sequences occur through base-pairing between complementary purines and pyrimidines.

Modified purines (for example, inosine, methylinosine and methyladenosine) and modified pyrimidines (thiouridine and mefhylcytosine) may also engage in base pairing. "Stringency" as used herein, refers to temperature and ionic strength conditions, and presence or absence of certain organic solvents and/or detergents during hybridisation. The higher the stringency, the higher will be the required level of complementarity between hybridizing nucleotide sequences.

"Stringent conditions " designates those conditions under which only nucleic acid having a high frequency of complementary bases will hybridize.

Reference herein to low stringency conditions includes and encompasses :-

(i) from at least about 1% v/v to at least about 15 > v/v formamide and from at least about 1 M to at least about 2 M salt for hybridisation at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C; and

(ii) 1%) Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M

NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C, and

(i) 2xSSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at room temperature. Medium stringency conditions include and encompass:- (i) from at least about 16%> v/v to at least about 30%> v/v

Substitute Sheet (Rule 26) RO/AU formamide and from at least about 0.5 M to at least about

0.9 M salt for hybridisation at 42°C, and at least about 0.5

M to at least about 0.9 M salt for washing at 42°C; and

(ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C and

(a) 2 x SSC, 0.1% SDS; or (b) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at 42°C. High stringency conditions include and encompass:- (i) from at least about 31% v/v to at least about 50%> v/v formamide and from at least about 0.01 M to at least about

0.15 M salt for hybridisation at 42°C, and at least about

0.01 M to at least about 0.15 M salt for washing at 42°C;

(ii) 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C, and (a) 0.1 x SSC, 0.1% SDS; or (b) 0.5% BSA, lmM EDTA, 40 mM NaHPO₄ (pH 7.2), 1%

SDS for washing at a temperature in excess of 65°C for about one hour; and (iii) 0.2 x SSC, 0.1% SDS for washing at or above 68°C for about 20 minutes. In general, the T_m of a duplex DNA decreases by about 1°C with every increase of 1% in the number of mismatched bases.

Notwithstanding the above, stringent conditions are well known in the art, such as described in Chapters 2.9 and 2.10 of. Ausubel et al, supra, which are herein incorporated be reference. A skilled addressee will also recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization.

Typically, complementary nucleotide sequences are identified by

Substitute Sheet ⁽Rule 26) RO/AU blotting techniques that include a step whereby nucleotides are immobilized on a matrix (for example, a synthetic membrane such as nitrocellulose), a hybridization step, and a detection step. Southern blotting is used to identify a complementary DNA sequence; northern blotting is used to identify a complementary RNA sequence. Dot blotting and slot blotting can be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNA polynucleotide sequences. Such techniques are well known by those skilled in the art, and have been described in Ausubel et al, supra, at pages 2.9.1 through 2.9.20.

A microarray also uses hybridization-based technology that, for example, may allow detection and/or isolation of a nucleic acid by way of hybridization of complementary nucleic acids. A microarray provides a method of high throughput screening for a nucleic acid in a sample that may be tested against several nucleic acids attached to a surface of a matrix or chip. In this regard, a skilled person is referred to Chapter 22 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Eds. Ausubel et al. John Wiley & Sons NY, 2000).

Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridizing the membrane bound DNA to a complementary nucleotide sequence. In dot blotting and slot blotting, DNA samples are directly applied to a synthetic membrane prior to hybridization as above.

An alternative blotting step is used when identifying complementary nucleic acids in a cDNA or genomic DNA library, such as through the process of plaque or colony hybridization. Other typical examples of this procedure is described in Chapters 8-12 of Sambrook et al, supra which are herein incorpoated by reference.

Typically, the following general procedure can be used to determine hybridization conditions. Nucleic acids are blotted/transferred to a

Substⁱtute Sheet ⁽Rule 26) RO/AU synthetic membrane, as described above. A wild type nucleotide sequence of the invention is labeled as described above, and the ability of this labeled nucleic acid to hybridize with an immobilized nucleotide sequence analyzed.

A skilled addressee will recognize that a number of factors influence hybridization and detection of hybridized nucleic acids. The specific activity of radioactively labeled polynucleotide sequence should typically be greater than or equal to about 10⁸ dpm/μg to provide a detectable signal. A radiolabeled nucleotide sequence of specific activity 10 to 10 dpm/μg can detect approximately 0.5 pg of DNA. It is well known in the art that sufficient DNA must be immobilized on the membrane to permit detection. It is desirable to have excess immobilized DNA, usually 10 μg. Adding an inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) or polyethylene glycol 6000 during hybridization can also increase the sensitivity of hybridization (see Ausubel et al, supra at 2.10.10). To achieve meaningful results from hybridization between a nucleic acid immobilized on a membrane and a labeled nucleic acid, a sufficient amount of the labeled nucleic acid must be hybridized to the immobilized nucleic acid following washing. Washing ensures that the labeled nucleic acid is hybridized only to the immobilized nucleic acid with a desired degree of complementarity to the labeled nucleic acid.

Methods for detecting labeled nucleic acids hybridized to an immobilized nucleic acid are well known to practitioners in the art. Such methods include autoradiography, chemiluminescent, fluorescent and colorimetric detection. In an embodiment, nucleic acid homologs of the invention may be prepared according to the following procedure:

(i) obtaining a nucleic acid extract from a bacterium;

(ii) creating primers which each comprise a distinct portion of

Substitute Sheet (Rule 26) RO/AU a nucleotide sequence according to [SEQ ID NOS: 16-26] shown in FIG. 2; and (iii) using said primers to amplify, via nucleic acid amplification techniques, one or more amplification products from said nucleic acid extract.

Preferably, the bacterium is of the genus Haemophilus such as Haemophilus paragallinarum.

More preferably, the bacterium is of the species Haemophilus paragallinarum . Suitably, the primers may be degenerate or non-degenerate.

Specific examples of PCR primers useful according to this embodiment are provided in Table 2 [SEQ ID NOS: 27-45].

Suitable nucleic acid amplification techniques are well known to the skilled addressee, and include polymerase chain reaction (PCR) as for example described in Chapter 15 of Ausubel et al supra, which is incorporated herein by reference; strand displacement amplification (SDA) as for example described in United States Patent No. 5,422,252 which is incorporated herein by reference; rolling circle replication (RCR) as for example described in Liu et al,

1996, J. Am. Chem. Soc. 118 1587 and International Publication WO 92/01813) and Lizardi et al, (International Publication WO 97/19193) which are incorporated herein by reference; nucleic acid sequence-based amplification

(NASBA) as for example described by Sooknanan et α/.,1994, Biotechniques 17

1077) which is incorporated herein by reference; ligase chain reaction (LCR) as for example described in International Publication WO 89/09385 which is incorporated by reference herein; and Q-β replicase amplification as for example described by Tyagi et al, 1996, Proc. Nail. Acad. Sci. USA 93 5395) which is incorporated herein by reference.

The preferred nucleic acid sequence amplification technique is

Substitute Sheet ⁽Rule 26) RO/AU PCR, as will be described in detail hereinafter.

As used herein, an "amplification product " refers to a nucleic acid product generated by nucleic acid amplification techniques. Antibodies The invention also contemplates antibodies against the ORF3 proteins, fragments, variants and derivatives of the invention. Antibodies of the invention may be polyclonal or monoclonal. Well-known protocols applicable to antibody production, purification and use may be found, for example, in Chapter 2 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D. Antibodies: A Laboratory

Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, which are both herein incorporated by reference.

Generally, antibodies of the invention bind to or conjugate with a protein, fragment, variant or derivative of the invention. For example, the antibodies may comprise polyclonal antibodies. Such antibodies may be prepared for example by injecting a protein, fragment, variant or derivative of the invention into a production species, which may include mice or rabbits, to obtain polyclonal antisera. Methods of producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols which may be used are described for example in Coligan et al. , CURRENT PROTOCOLS IN IMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.

In lieu of the polyclonal antisera obtained in the production species, monoclonal antibodies may be produced using the standard method as for example, described in an article by Kδhler & Milstein, 1975, Nature 256, 495, which is herein incorporated by reference, or by more recent modifications thereof as for example, described in Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with one or

Substitute Sheet (Rule 26) RO/AU more of the proteins, fragments, variants or derivatives of the invention.

The invention also includes within its scope antibodies which comprise Fc or Fab fragments of the polyclonal or monoclonal antibodies referred to above. Alternatively, the antibodies may comprise single chain Fv antibodies (scFvs) against the peptides of the invention. Such scFvs may be prepared, for example, in accordance with the methods described respectively in United States Patent No 5,091,513, European Patent No. 239400 or the article by Winter & Milstein, 1991, Nature 349 293, which are incorporated herein by reference.

The antibodies of the invention may be used for affinity chromatography in isolating native or recombinant ORF3 proteins. For example reference may be made to immunoaffinity chromatographic procedures described in Chapter 9.5 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra.

For example, the anti-ORF3 antibodies may be used for serological analysis such as by ELISA, as described in more detail in Example 13.

However, it will be appreciated that any suitable technique for determining formation of antibody complex may be used. For example, an antibody or antibody fragment according to the invention having a label associated therewith may be utilized in immunoassays. Such immunoassays may include, but are not limited to, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic techniques (ICTs) which are well known to those of skill in the art.

For example, reference may be made to Chapter 7 of Coligan et al, CURRENT PROTOCOLS IN IMMUNOLOGY, supra which discloses a variety of immunoassays that may be used in accordance with the present invention. Immunoassays may include competitive assays as understood in the art.

The label associated with the antibody or antibody fragment may

Substitute Sheet (Rule 26) RO/AU include the following:

(A) direct attachment of the label to the antibody or antibody fragment;

(B) indirect attachment of the label to the antibody or antibody fragment; i.e., attachment of the label to another assay reagent which subsequently binds to the antibody or antibody fragment; and

(C) attachment to a subsequent reaction product of the antibody or antibody fragment. The label may be selected from a group including a chromogen, a catalyst, an enzyme, a fluorophore, a chemiluminescent molecule, a lanthanide ion such as Europium (Eu³⁴), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

A large number of enzymes suitable for use as labels is disclosed in United States Patent Specifications United States Patent No. 4,366,241, United States Patent No. 4,843,000, and United States Patent No. 4,849,338, each of which is herein incorporated by reference. Suitable enzyme labels useful in the present invention include alkaline phosphatase, horseradish peroxidase, luciferase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase and the like. The enzyme label may be used alone or in combination with a second enzyme in solution. Fluorophores may be selected from a group including fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), allophycocyanin (APC), Texas Red (TR), Cy5 or R-Phycoerythrin (RPE). Examples of useful fluorophores may be found, for example, in United States

Substitute Sheet (Rule 26) RO/AU Patent No. 4,520,110 and United States Patent No. 4,542,104 which are herein incorporated by reference. Pharmaceutical compositions

A further feature of the invention is the use of the ORF3 proteins, fragments, variants or derivatives of the invention (^immunogenic agents ") as actives in a pharmaceutical composition. Suitably, the pharmaceutical composition comprises a pharmaceutically-acceptable carrier.

By "pharmaceutically-acceptable carrier" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

Any suitable route of administration may be employed for providing a chicken with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intradermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Intramuscular and subcutaneous injection is appropriate, for example, for administration of immunogenic compositions, vaccines and DNA vaccines.

Preferred administration routes in chickens include intramuscular, intranasal, oral, in ovo, intraocular and subcutaneous. Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms

Substitute Sheet (Rule 26) RO/AU of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmefhyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

Pharmaceutical compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more immunogenic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. Vaccines The above compositions may be used as therapeutic or prophylactic vaccines. Accordingly, the invention extends to the production of vaccines containing as actives one or more of the immunogenic agents of the invention. Any suitable procedure is contemplated for producing such vaccines. Exemplary procedures include, for example, those described in NEW GENERATION VACCINES (1997, Levine et al, Marcel Dekker, Inc. New

York, Basel Hong Kong) which is incorporated herein by reference.

In this regard, reference is made to United States Patent 5 ,770, 213, Australian Patent 704882, Reid & Blackall, 1987, 31 59 and Webb & Cripps,

Substitute Sheet (Rule 26) RO/AU 2000, Infect. Immun. 68 377 (which are each incorporated herein by reference) which describe immunization methods which may be applicable to immunogenic agents of the present invention.

An immunogenic agent according to the invention can be mixed, conjugated or fused with other antigens, including B or T cell epitopes of other antigens. In addition, it can be conjugated to a carrier as described below.

When an haptenic protein of the invention is used (i.e., a protein which reacts with cognate antibodies, but cannot itself elicit an immune response), it can be conjugated with an immunogenic carrier. Useful carriers are well known in the art and include for example: thyroglobulin; albumins such as human serum albumin; toxins, toxoids or any mutant crossreactive material (CRM) of the toxin from tetanus, diptheria, pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus; polyamino acids such as poly(lysine:glutamic acid); influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis B virus recombinant vaccine and the like. Alternatively, a fragment or epitope of a carrier protein or other immnogenic protein may be used. For example, a haptenic protein of the invention can be coupled to a T cell epitope of a bacterial toxin, toxoid or CRM. In this regard, reference may be made to United States Patent No 5,785,973 which is incorporated herein by reference.

In addition, an ORF3 protein, fragment, variant or derivative of the invention may act as a carrier protein in vaccine compositions directed against Haemophilus paragallinarum, or against other bacteria or viruses.

The immunogenic agents of the invention may be administered as multivalent subunit vaccines in combination with antigens of Haemophilus paragallinarum, or antigens of other organisms including Mycoplasma and Salmonella species, Marek's virus and Newcastle disease virus. Alternatively or additionally, they may be administered in concert with oligosaccharide or

Substitute Sheet ⁽Rule 26) RO/AU polysaccharide components of Haemophilus paragallinarum.

The vaccines can also contain a physiologically-acceptable diluent or excipient such as water, phosphate buffered saline and saline.

The vaccines and immunogenic compositions may include an adjuvant as is well known in the art. Suitable adjuvants include, but are not limited to: surface active substances such as hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammonium bromide, N, N-dicoctadecyl-N', N'bis(2-hydroxyethyl-propanediamine), mefhoxyhexadecylglycerol, and pluronic polyols; polyamines such as pyran, dextransulfate, poly IC carbopol; proteins such as murarnyl dipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions; and mineral gels such as aluminum phosphate, aluminum hydroxide or alum; lymphokines, QuilA and immune stimulating complexes (ISCOMS).

The immunogenic agents of the invention may be expressed by attenuated viral and/or bacterial hosts. By "attenuated" is meant viruses or bacteria (for example transformed with an expression construct of the invention) that are either naturally, or have been rendered, substantially avirulent. A virus or bacterium may be rendered substantially avirulent by any suitable physical (e.g., heat treatment) or chemical means (e.g., formaldehyde treatment) or by genetic manipulation. By "substantially avirulent " is meant a virus or bacterium whose ability to cause disease has been destroyed. Ideally, the pathogenicity of the virus or bacterium is destroyed without affecting immunogenicity. From the foregoing, it will be appreciated that attenuated viral and bacterial hosts may comprise live or inactivated viruses and bacteria. Attenuated viral and bacterial hosts which may be useful in a vaccine according to the invention may comprise viral vectors inclusive of Marek's disease virus, adenovirus and cytomegalovirus and attenuated Salmonella or Mycoplasma strains. Live vaccines are particularly advantageous

Substitute Sheet (Rule 26) RO/AU because they lead to a prolonged stimulus that can confer substantially long- lasting immunity. For example, with regard to Salmonella or Mycoplasma strains, upon introduction of an attenuated bacterium harbouring an expression construct of the invention to a chicken, the ORF3 protein or fragment expressed by the bacterium will suitably elicit a host immune response. In this regard, reference is particularly made to United States Patent No. 6,001,348 for a description of such an approach using Mycoplasma synoviae.

Multivalent vaccines can be prepared from one or more microorganisms that express different epitopes of Haemophilus paragallinarum (e.g., other surface proteins or epitopes of Haemophilus paragallinarum). In addition, epitopes of other pathogenic microorganisms can be incorporated into the vaccine.

A wide variety of other vectors useful for therapeutic administration or immunization with the immunogenic agents of the invention will be apparent to those skilled in the art from the present disclosure.

In a further embodiment, the nucleotide sequence may be used as a vaccine in the form of a "naked DNA" vaccine as is known in the art. For example, an expression vector of the invention may be introduced into a chicken, where it causes production of a protein in vivo, against which the host mounts an immune response as for example described in Barry et α/.,1995, Nature 377 632 which is hereby incorporated herein by reference. Detection kits

The present invention also provides kits for the detection of Haemophilus paragallinarum in a biological sample. A biological sample may include for example a nasal swab, blood, faecal or tissue sample from an animal.

Preferably, the biological sample is a nasal swab sample. A kit may comprise one or more particular agents described above depending upon the nature of the test method employed. In this regard, the kits may include one or more of an ORF3

Substitute Sheet (Rule 26) RO/AU protein, fragment, variant, derivative, antibody, antibody fragment or nucleic acid according to the invention. The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) an ORF3 nucleic acid according to the invention (which may be used as a positive control), (ii) one or more primers according to the invention (eg. selected from the group consisting of SEQ ID NOS: 27-45), and optionally a DNA polymerase, DNA ligase etc depending on the nucleic acid amplification technique employed. A preferred method of detection comprises the steps of:

(i) obtaining a nucleic acid sample from a mammal or avian;

(ii) using one or more primers which correspond to distinct regions of an ORF3 nucleic acid, together with a nucleic acid sequence amplification technique (as hereinbefore defined) to produce one or more amplification products from the sample obtained in step (i); and (iii) detecting the one or more amplification products produced at step (ii) and coπelating the amplification products so detected with the presence or absence of H. paragallinarum.

Preferably, the nucleic acid sequence amplification technique is PCR. The primer(s) may be degenerate or non-degenerate as is well understood in the art.

Preferably, the nucleic acid sample is obtained from a chicken. Preparation of immunoreactive fragments

The invention also extends to a method of identifying an immunoreactive fragment of an ORF3 protein, variant or derivatives according to the invention. This method essentially comprises generating a fragment of the

Substitute Sheet (Rule 26) RO/AU protein, variant or derivative, administering the fragment to a chicken or mammal such as a mouse or rabbit; and detecting an immune response in the chicken. Such response will include production of elements which specifically bind Haemophilus paragallinarum and/or said protein, variant or derivative, and/or a protective effect against Haemophilus paragallinarum infection.

Prior to testing a particular fragment for immunoreactivity in the above method, a variety of predictive methods may be used to deduce whether a particular fragment can be used to obtain an antibody that cross-reacts with the native antigen. These predictive methods may be based on amino-terminal or carboxy-terminal sequence as for example described in Chapter 11.14 of Ausubel et al, supra. Alternatively, these predictive methods may be based on predictions of hydrophilicity as for example described by Kyte & Doolittle 1982, J. Mol. Biol. 157 105 and Hopp & Woods, 1983, Mol. Immunol. 20 483) which are incorporated by reference herein, or predictions of secondary structure as for example described by Choo & Fasman,1978, Ann. Rev. Biochem. 47 251), which is incorporated herein by reference.

In addition, "epitope mapping" uses monoclonal antibodies of the invention to identify cross-reactive epitopes by first testing their ability to provide cross-protection, followed by identifying the epitope recognized by said antibodies. An exemplary method is provided in Coligan et al, CURRENT

PROTOCOLS LN IMMUNOLOGY, supra.

Generally, peptide fragments consisting of 10 to 15 residues provide optimal results. Peptides as small as 6 or as large as 20 residues have worked successfully. Such peptide fragments may then be chemically coupled to a carrier molecule such as keyhole limpet haemocyanin (KLH) or bovine serum albumin (BSA) as for example described in Sections 11.14 and 11.15 of Ausubel et al, supra).

The peptides may be used to immunize an animal as for example

Substitute Sheet (Rule 26) RO/AU discussed above. Antibody titers against the native or parent protein from which the peptide was selected may then be determined by, for example, radioimmunoassay or ELISA as for instance described in Sections 11.16 and 114 of Ausubel et al, supra. Antibodies may then be purified from a suitable biological fluid of the animal by ammonium sulfate fractionation or by chromatography as is well known in the art. Exemplary protocols for antibody purification are given in Sections 10.11 and 11.13 of Ausubel et al, supra, which are herein incorporated by reference. Immunoreactivity of the antibody against the native or parent protein may be determined by any suitable procedure such as, for example, by Western blot.

In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.

EXAMPLE 1

Bacterial Strains

All Haemophilus paragallinarum strains referenced in Table 3 were obtained from Dr Patrick Blackall, Queensland Poultry Research and Development Centre, Animal Research Institute, Yeerongpilly, Queensland, Australia.

EXAMPLE 2 Antibodies Monoclonal antibody D4 recognises a 39 kDa protein and is specific for serovar A H. paragallinarum strains (Takagi et al, 1991b, Vet. Microbiol. 27 327 which is herein incorporated by reference). MAb E5C12D10 (Yamaguchi et al, 1990,

Avian Dis. 34 964) which is specific for H. paragallinarum ΗA was also used. An H. paragallinarum serovar A-specific polyclonal antiserum (Sawata et al, 1979, Am. J. Vet. Res. 40 1450; Thornton & Blackall, 1984, Aust. Vet. J. 61

Substitute Sheet (Rule 26) RO/AU 251; Eaves et al, 1989, J. Clin. Microbiol. 27 1510) was purified and used as will be described in more detail hereinafter.

Secondary antibodies were a goat anti-mouse IgG alkaline phosphatase (alkaline phosphatase) conjugate (Promega Corp) and a goat anti- rabbit IgG alkaline phosphatase conjugate (Sigma).

EXAMPLE 3

Purification of the H. paragallinarum serovar A-specific polyclonal antibody

Bacterial cell pellets of H. paragallinarum serovars B (strain HP2) and C (strain

HP3) and E. coli XL-1 Blue MRF were resuspended in 1 mL of TBST containing 0.1% (w/v) sodium azide. The OD₆oo of these cell suspensions were measured and adjusted to approximately equal concentrations in TBST. 300 μL of the cell suspensions were then mixed together, made up to a final volume of 5 mL in TBST + 0.1% (w/v) sodium azide and sonicated until the liquid cleared using the Branson Sonifier 250 (John Morris Scientific Pty Ltd). 1 mL of polyclonal antiserum was added to 4 mL TBST. The diluted antibody solution and the bacterial cell lysate mix were mixed together, incubated at 4°C for 30 min, with gentle stirring, and centrifuged at 10,000 x g for 20 min.

Affinity purification was based on the method of Beall & Mitchell 1986, J. Immunol. Methods 86 217. 150 μL of H. paragallinarum serovar A

(strain HP1) cells and 150 μL of 2x SDS sample buffer were combined and run on a 12% SDS-PAGE and then blotted onto nitrocellulose membrane (Bio-Rad). This blot was incubated with the anti-serovar A polyclonal antiserum (1/200 dilution in Blotto) at room temperature for 3-4 hours. The end strips from the blot were developed by immuno logical screening and then lined up with the rest of the blot to detect the desired HA protein band. The strip coπesponding to the HA band was cut out and washed three times with TBST for 10 min. The polyclonal antibody specific for HA was eluted by adding 1.5 mL of 0.1 M glycine

Substitute Sheet (Rule 26) RO/AU containing 0.15 M NaCl buffer, (pH 2.6) to the strip and incubating at room temperature for 10 min. The strip was removed from the solution and the eluted polyclonal antibodies were then immediately neutralised by 1 mL of 1 M Tris-

HC1, pH 8. EXAMPLE 4

Isolation of proteins and Western blotting

E. coli cells (200 μL from an overnight culture) were inoculated into 2 mL LB broth with 50 μg/mL ampicillin and incubated for 1 hr at 37°C. 15 μL of 0.5 M

IPTG was added and cultures incubated with shaking for a further 3-4 hr. Cells were centrifuged and then resuspended in 1 mL PBS. H. paragallinarum cultures were grown overnight in TMSN broth, centrifuged and resuspended in 1 mL PBS.

Before application to an SDS-PAGE gel, cells were mixed 1 : 1 in 2x SDS-PAGE sample buffer and boiled for 5 min.

Genes cloned into lambda phage virions were expressed as follows.10 μL of phage mix was added to 200 μL of XL-1 Blue MRF' cells

(OD₆oo ~1) and incubated at 37 °C for 15 min. 3 mL of LB broth was added to the cell and phage mixture and they were incubated at 37 °C with shaking for 1 hour.

Induction of protein synthesis was carried out by adding 2.5 mM IPTG to cells and incubating at 37 °C with shaking for 3 hours. The cell cultures were taken at 2 and 3 hours and the cell pellets were centrifuged down and then resuspended in

100 μL of PBS. The cell samples were then prepared for SDS-PAGE by adding

2x SDS sample buffer and boiling for 5 min.

SDS-PAGE gels (12% resolving) were run in Mini-PROTEAN®

II Electrophoresis Cell (Bio-Rad) at 150 V for ~1 hour using a Tris-glycine electrode buffer. Protein sizes were determined by comparison with the prestained

SDS-PAGE standard, low range (Bio-Rad) or BenchMark™ Prestained Protein

Ladder (GibcoBRL).

Polyacrylamide gels were stained with Coomassie Blue staining

Substitute Sheet (Rule 26) RO/AU solution for one hour with gentle agitation and then were destained with destain solution.

For Western blotting, gels transfeπed to nitrocellulose in a Trans- Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) for one hour at 15 V. The nitrocellulose membrane was then screened using the QIAGEN Western and colony blot protocol (QIAGEN). The primary antibodies, MAb 4D, MAb E5C12D10 or polyclonal H paragallinarum serovar A antiserum were used. The secondary antibodies, goat anti-mouse IgG alkaline phosphatase-conjugate or goat anti-rabbit IgG alkaline phosphatase-conjugate were subsequently used. An alkaline phosphatase staining method (BCIP/NBT) was then used for detection.

EXAMPLE 5 Extraction of bacterial genomic DNA This protocol was based on the method of Muπay & Thompson, 1980, Nucl. Acids Res. 8 4321 which is incorporated herein by reference. A 300 mL culture of H paragallinarum was grown to saturation and then pelleted by centrifugation at 6,000 rpm for 10 min and resuspended in 9.5 mL TE buffer. A 0.5 mL of 10% (w/v) SDS and 1 mg proteinase K were then added and the cell suspension incubated at 65 °C for one hour.

1.8 mL of 5 M NaCl and 1.5 mL of hexadecyltrimethyl ammonium bromide (0.68 M NaCl, 0.27 M CTAB) solution were added to the cell lysate and incubated at 65 °C for 20 min. An equal volume of chloroform/isoamyl alcohol (24:1) was added and the cell suspension was centrifuged at 7,000 rpm for 10 min to separate the two phases. The aqueous supernatant was transferred to a fresh tube, and the DNA was precipitated with 0.6 volume of isopropanol. The precipitate was then washed with 1 mL of 70%> (v/v) ethanol at 9,000 rpm for 5 min. The supernatant was removed and the pellet redissolved in 1 mL of TE buffer. The absorbance (A) of the DNA solution was measured at 260 nm using an Ultrospec® Plus UN/Visible Spectrophotometer (Pharmacia LKB Biochrom

Substitute Sheet (Rule 26) RO/AU Ltd, Cambridge, USA). The concentration of double-stranded DNA was estimated such that 50μg/mL of DNA gives an A ₆o of 1.

EXAMPLE 6 Polymerase Chain Reaction (PCR) isolation ofORF3 nucleic acids Initial attempts to isolate H. paragallinarum nucleic acids encoding ΗA antigens utilized PCR-amplification of genomic DNA, based on the method of Saiki et al, 1988, Science 239 487. Four specific oligonucleotide primers were designed from the published DNA sequence of the ΗMW gene of non-typeable Haemophilus influenzae NTΗi (Barenkamp & Leininger, 1992, Infect. Immun. 60 1302; see Table 2): ΗEM.TAN [93.4 pmol/μl], a tandem repeat upstream of the ΗMW gene, ΗEM.ΗOM [101 pmol/μl], an homologous 5 amino acid sequence, ΗEM.BEG [289.7 pmol/μl], the beginning of the ΗMW gene and ΗEM.END [152.7 pmol/μl], the end of the homologous region of the gene.

Genomic DNA preparations of Haemophilus paragallinarum serovar A (strain ΗP1), serovar B (strain ΗP2) and serovar C (strain HP3) and

NTHi were diluted to 100 μg/mL. Each PCR contained 5 μL of lOx Taq polymerase buffer, 3 μL of 1.5 mM MgCl₂, 0.4 μL of 25 mM dNTPs, 50 pmol of each of forward and reverse primers, 5 μL of diluted genomic DNA and 2 μL of Taq polymerase (1/5 dilution in lx buffer, Promega, Sydney, NSW) and sterilised distilled water making up to 50 μL. DNA was amplified using an Omn-E Thermal

Cycler (Hybaid) and the following program: 35 cycles of 94°C, 1 min; 55 °C, 1 min and 72 °C, 1 min with the final extension of 72° for 7 min. Resulting amplification fragments were checked by gel electrophoresis.

Amplification fragments were digested by restriction endonucleases (New England BioLabs, Arundel, QLD) and the methods were followed as per the manufacturer's instruction.

DNA fragments were extracted from gels using the QIAquick gel extraction kit (QIAGEN, Clifton Hill, Victoria) according to the manufacturer's

Substitute Sheet (Rule 26) RO/AU instructions. For ligation of isolated fragments, 2 μL of lOx T4 ligase buffer, 1 μL of T4 DNA ligase (New England BioLabs), 1 μL of pT7Blue, 10 μl of DNA and 6 μl sterilised distilled water were mixed together and incubated at 16°C overnight. Sometimes, 14% (w/v) polyethylene glycol (PEG) was added to enhance the ligation reaction. The only specific product which appeared promising was a 1.5 kB product amplified from serovar A (strain HP1). This product was subcloned into pT7Blue and sequenced but showed none of the required homology with HMW genes of NTHi.

EXAMPLE 7 Genomic Expression Library Construction and Screening

Given the negative PCR results, a strategy was devised employing immunoscreening of a H. paragallinarum serotype A genomic library derived from strain HP1. H. paragallinarum serotype A chromosomal DNA was partially digested by Apol and the partial digest of run on a 1% (w/v) agarose gel. The 2 to 5 kb fragments of DNA were then extracted from the gel using a QIAquick Gel

Extraction Kit (QIAGEN).

The extracted DNA was ligated into a plasmid vector as follows: 5 μl of ligation mix was prepared by adding 1 μL of Lambda ZAP II vector, 3 μL of extracted DNA, 0.5 μL of lOx T4 DNA ligase buffer and 0.5 μL of T4 DNA ligase (New England BioLab). The mixture was then incubated at 16 °C overnight.

The Lambda ZAPII Predigested EcoRI/CIAP-treated Kit (Stratagene) was used together with the Gigapack® III Gold packaging kit for packaging (Stratagene). Blue-white colour selection was used to identify recombinants.

For immunological screening to detect protein expression, the protocol from picoBlue™ Immunoscreening Kit (Stratagene) was used with the following modifications: 10,000 plaques and 200 μL of E. coli XL-1 Blue MFR'

Substitute Sheet ⁽Rule 26) RO/AU were plated out onto each 87-mm plate and 10 plates were prepared for primary immunological screening, while 1,000 plaques and 100 plaques were plated out for the secondary and tertiary screening, respectively, until a single positive plaque could be picked for the single-clone excision process. In immunological screening, MAb 4D, E5C12D10 or polyclonal antiserum was used as a primary antibody and anti-mouse IgG alkaline phosphatase-conjugate or anti-rabbit IgG alkaline phosphatase-conjugate was used as a secondary antibody. The alkaline phosphatase staining method was used to detect immunoreactive (positive) plaques. Individual positive phages were converted to plasmids using

ExAssist helper phage in a single-clone excision process. This was performed by following the protocols outlined in the Lambda ZAP II Predigested EcoRI/CIAP- treated Kit (Stratagene).

For tertiary screening, 200 μL of E. coli XL-1 Blue MRF cells (Stratagene, OD A₆oo 0.5) were added to 4 mL of NZY top agar and poured onto

NZY agar plates. After drying the top agar, 1 μl of phage mix was spotted onto the top agar and dried. The plate was then incubated at 37 °C for 6-8 hours prior to immunological screening using antibodies MAb 4D and H. paragallinarum serovar A polyclonal antiserum. A total of four libraries were constructed by this method, of which libraries #3 and #4 were combined so as to provide -18,000 plaques, a number considered necessary to identify at least one positive clone. A total of -58,000 insert-containing plaques were immunoscreened.

A number of plaques scored positive by tertiary screening and by subsequent dot blotting using antibodies MAb 4D and H. paragallinarum serovar

A polyclonal antiserum.

In FIG. 4, the results of Western blotting to detect proteins expressed by a number of the positive clones are shown. Two of the clones,

Substitute Sheet (Rule 26) RO/AU designated 7.2 and 8.2 appeared to encode -39 kD proteins that were recognized by the H. paragallinarum serovar A polyclonal antiserum. Neither clone produced a protein detected by MAb 4D. Thus clones 7.2 and 8.2 appeared not to encode H. paragallinarum ΗA. These candidate clones were then sequenced to determine their respective identities.

EXAMPLE 8 Bacterial Transformation E. coli DΗ5α cells were transformed with plasmid DNA by heat shock treatment at 37°C for 5 min based on the method of Cohen et al, 1972, Proc. Nafl. Acad. Sci. 69 2110. The transformed cells were plated on LB agar plates supplemented with ampicillin (100 μg/mL), X-gal (80 μg/ml) and IPTG (0.5 mM) and incubated at 37 °C overnight for blue/white selection.

Recombinants were screened by restriction analysis of plasmid minipreparations produced by an alkaline lysis method based on the methods of Birnboim & Doly, 1979, Nucl. Acid. Res. 7 1513 and Ish-Horowicz & Burke,

1981, Nucl. Acid. Res. 9 2989. Isolated DNA was checked by gel electrophoresis.

EXAMPLE 9

DNA Sequencing

ABI Prism™ BigDye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS' (PE Applied Biosystems) was used for DNA sequencing. Briefly, 200-500 ng of double stranded DNA or 30-60 ng of PCR product, 8 μL of ready reaction premix, 3.2 pmol of primer were mixed and sterilised distilled water was added to a final volume of 20 μL. The samples were amplified using an Omn-E Thermal Cycler (Hybaid), with the following program - 96°C for 10 s, 50°C for 5 s and 60°C for 4 min for 25 cycles. The samples were then ethanol precipitated. A 20 μL of sequence reaction was added to 1/10 volume of 3 M sodium acetate, pH 5.2 and 10 x volume of 100% (v/v) ethanol and incubated on ice for 30 min. Then the samples were centrifuged at 14,000

Substitute Sheet (Rule 26) RO/AU rpm for 30 min and the supernatant removed. The pellet was washed with 500 μL of 70%) (v/v) ethanol and centrifuged at 14, 000 rpm for 5 min. The pellet was air- dried for 5-10 min. Finally, the DNA sample was sent to the Australian Genomic Research Facility (AGRF), for automatic sequencing by an ABI 373A automatic sequencer (Applied Biosystems International, Perkin Elmer).

Clones 7.2 and 8.2, subcloned into pT7Blue or pBluescript, were initially sequenced using -21 Ml 3 (forward) and reverse primers (Table 2). As new sequence was generated, new primers (designated HOI, HO2, HO3, HO4, HO5, HP1, HP2, HP3, HP4, HP5 and HP6; see Table 2) were designed and synthesized until the complete gene sequence was determined.

EXAMPLE 10

Sequence comparisons

The DNA sequences were imported into the computer program, SeqEd™ vl.0.3

(Applied Biosystems), and their sequences were compared. BLASTN and BLASTX searches in the program, MacVector v.6 (Oxford Molecular Group) or

NCBI BLAST server were used to search a non-redundant genebank. DNA sequences were aligned to form a contig by the computer program, AssemblyLIGN™ vl .0.6 (Oxford Molecular Group) or SeqEd™ vl.0.3 (Applied Biosystems). DNA sequencing and BLAST analysis revealed that the clones 7.2 and 8.2 actually contained identical genes, but that clone 7.2 (-3.6 kB) had a longer insert in the 5' end thanclone 8.2 (-2.4 kB). There were four putative open reading frames identified in clone 7.2 and three in clone 8.2 (designated ORFs 1- 4). The amino acid sequences of the 1005 bp ORF (ORF3) found in both clones were identified as having 45 % similarity and 27% identity to the E. coli. lipoprotein-34 precursor.

The 7.2 and 8.2 ORF3 sequences were then restriction mapped by MacVector v.6 (Oxford Molecular Group). Appropriate restriction sites were

Substitute Sheet chosen to delete ORF4 from the 3' end of ORF3. The expressed product was of a similar size to the -39 kD protein detected by Western blotting in FIG. 3.

Subsequent haemagglutination assays revealed that none of the products encoded by clones 7.2 or 8.2 displayed the haemagglutination which would be expected of an isolated HA protein.

It was therefore concluded that ORF3 encoded a novel protein distinct from HA, which appears to be highly immunogenic and represents a potential vaccine candidate.

Sequence analysis identified PNFKKQ [SEQ ID NO: 1] as a minimal consensus sequence common to SEQ ID NOS: 3-13. A further consensus sequence is FQSASNR [SEQ ID NO: 2]. Nucleotide sequence analysis identified 5'-CCT AAT TTT AAG AAA CAA-3' [SEQ ID NO: 14] as a minimal consensus sequence common to SEQ ID NOS: 16-26. A further consensus sequence is 5'-TTT CAA TCG GCA TCT AAT CGC-3' [SEQ ID NO: 15]. These sequences are novel to the proteins and nucleic acids of the invention and may be useful as a vaccine or production thereof and/or may be useful for detecting said proteins and nucleic acids in a sample. It will be appreciated that a vaccine comprising a protein or nucleic acid having a consensus sequence as described above may potentially provide protective immunity against all of the serovars described herein and may be specific thereto. A method of detection and a detection kit comprising the abovementioned protein or nucleic acid, or antibody or antibody fragment that binds said protein, may specifically detect H. paragallinarum.

EXAMPLE 11 Isolation ofORF3from other strains ofH. paragallinarum

Given the conclusions presented in Example 10, ORF3 nucleic acids were PCR amplified from other serovar A, B and C H paragallinarum strains, and then sequenced.

Substitute Sheet (Rule 26) RO/AU The full length ORF3 nucleotide sequences from strains HP1

(serovar A), HP2 (serovar B) HP3 (serovar C), HP 137 (serovar A), HP90 (serovar

A), HP 14 (serovar A), HP 145 (serovar C), HP 147 (serovar B) and HP60 (serovar

C) and partial nucleotide sequences from strains HP 144 (serovar A) and HP 146 (serovar C) are shown in FIG. 2 [SEQ ID NOS: 16-26].

The encoded protein sequences are shown in FIG. 1 [SEQ ED NOS: 3-13].

Comparison of sequences by methods as already described revealed a very high degree of identity between all of the isolated ORF3 DNA and encoded protein sequences.

Minimal consensus sequences common to all ORF3 proteins were

identified as PNFKKQ [SEQ ED NO: 1] and FQSASNR [SEQ ED NO: 2].

EXAMPLE 12

Expression of recombinant W. paragallinarum ORF3 protein Computer analysis of the HP 14 ORF3 sequence was used to predict the location of a putative signal sequence required for transport of the predicted protein across the bacterial inner membrane. PCR primers were designed to amplify the mature coding sequence (excluding the signal sequence) from H. paragallinarum strain

HP14 (serovar A).

The primers used were:

ORF3up incorporating a 5' BamHl into the PCR amplification product: 5'-ATA TGG ATC CTG TTC TAC CAG CAA TGA AAG C-3' [SEQ ED NO: 44]; and

ORF3down, incorporating a 3' Pstl site into the PCR amplification product: 5'- ATA TCT GCA GTT ATT TGG CTA AAA TCG CTT G -3' [SEQ ED

NO: 45].

Products were amplified using Taq DNA polymerase (Promega) using the following "hot start" conditions: 95 °C for 5 min prior to adding Taq,

Substitute Sheet ⁽R le 26) RO/AU then 30 cycles of 94 °C for 30 sec, 49 °C for 30 sec and 72 °C for 60 sec with a final incubation at 72°C for 10 min. Amplification products were analyzed by 1%> agarose gel electrophoresis and the desired product extracted using the Qiagen Qiaquick™ kit. The PCR product was digested sequentially with BamHl and Pstl before ligation into expression vector pQE30 (Qiagen N-terminal histag vector) and transformation into E. coli Ml 5 cells which harbour plasmid pREP4. Putative transformants were confirmed by restriction digestion, PCR and DNA sequencing.

ORF3 protein expression and purification were performed essentially as described in the Qiaexpressionist manual from Qiagen.

Expression and Purification: A 10 ml culture of M15 (pREP4) containing pQE30/ORF3 was grown at 37°C with shaking overnight in LB broth supplemented with 100 μg ampicillin ml^"1 and 25 μg kanamycin ml^"1. The overnight culture was sub-cultured into 500 ml LB broth supplemented with 100 μg ampicillin ml^"1 and 25 μg kanamycin ml^"1 and grown at 37°C, with shaking to an optical density of A₆oonm =0.3-0.5. Expression of rORF3 was induced at 37°C with 0.5 mM EPTG (isopropyl-β-D-thiogalactopyranoside) for 4 h. Cells were centrifuged at 870 x g and lysed by French pressing at 1000 psi. The lysed cells were centrifuged at 5000 rpm for 15 min at 4 °C to remove any unlysed cells. The supernatant was incubated at room temperature for 30 min with equal volume native buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0) comprising 20 mM imidazole and 15 mM 2-mercaptoethanol. Pre-equilibrated Ni-NTA resin (QIAGEN, Germany) was incubated with the treated supernatant at room temperature for 1 h with agitation. The Ni-NTA resin was equilibrated with 15 ml native lysis buffer comprising 20 mM imidazole for 30 min at room temperature with agitation. The resin was washed twice with 5 bed volumes of wash buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0, 20 mM imidazole). The resin was resuspended in wash buffer and packed into a 10 ml column and washed with a further 5 bed volumes of wash buffer. The His-tagged protein was eluted in three bed volumes of elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0, 500 mM

NaCl, 250 mM imidazole) in 2 ml fractions. All eluted fractions were analysed by SDS-PAGE for presence of rORF3. The combined elutions comprising rORF3

Substitute Sheet (Rule 26) RO/AU were dialysed against PBS overnight at 4°C.

FIG. 4 shows a Coomassie blue stained 12% SDS-PAGE gel of purified expressed proteins as described above.

Other exemplary protein purification methods are described in Chapter 16 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al, supra, Chapters 5-7 of CURRENT PROTOCOLS IN PROTEIN SCIENCE,

Coligan et al, supra or as described in Webb & Cripps, 1999, Protein Exp. Purif.

15 1, which is herein incorporated by reference.

EXAMPLE 13 Vaccination with purified recombinant H. paragallinarum ORF3 proteins The vaccination strategy was based on that described in Reid & Blackall, 1987, 31 59, incorporated herein by reference.

Ten female 6-8 week old commercial Hyline Brown layer poults (from MacLeans Hatchery at Pittsworth), known to be free of H. paragallinarum infections were vaccinated with 100 μg of recombinant ORF3 protein (rORF3) in a total volume of 0.5 ml per bird with Alum or Alhyrdrogel 1.3% adjuvant (Superfos Biosector, Netherlands)(50% adjuvant, 50%> antigen). A control group of 10 birds was vaccinated with 0.5 ml per bird of PBS with Alum or Alhydrogel 1.3% (Superfos Biosector, Netherlands) adjuvant (50% PBS, 50% adjuvant).

Blood was collected from the birds prior to vaccination and 21 days after vaccination. Sera were stored at -20 C until required for analysis. Serial two-fold dilutions of serum samples were analysed by ELISA. Plates were coated overnight at 4 C with 100 μl of carbonate coating buffer (per L, 1.9292 g Sodium carbonate, 3.8052 g Sodium hydrogen carbonate, pH 9.6) comprising 10 μg/ml rORF3. Plates were blocked overnight at 4 C with 5%_> skim milk in PBS/0.5% Tween 20 (150 μl/well). 100 μl aliquots of serum at a 1 :100 dilution were serially diluted (in 0.5%> skim milk in PBS/0.5%) Tween 20) and incubated at

Substitute Sheet (Rule 26) RO/AU 37°C for 1 h. Plates were washed six times with PBS/0.5%_> Tween 20. After washing, plates were incubated with 100 μL/well secondary antibody (Affinity purified peroxidase labelled Goat anti-Chicken IgG (H+L), Kirkegaard & Perry Laboratories, USA, in 0.5% skim milk/PBS/0.5%) Tween 20) at 37°C for 1 h, followed by washing three times with PBS/0.5%> Tween 20. Assays were developed for 30 min at room temperature with 100 μL /well ABTS solution per well (200 μl ABTS stock (280 mg ABTS/10 mL water), 200 μL hydrogen peroxide solution (126 μL/10 mL water), 10 mL substrate buffer (substrate buffer = 200 mM Na₂HPO₄ adjusted to pH 4.2 using 100 mM citric acid). The absorbance of each well was determined at 405 nm. Titre is expressed as the reciprocal of the last dilution which showed reactivity (cut off determined as mean of the blanks plus 3 standard deviations of the mean). As shown in FIG. 5, titres of the control and rORF3 groups prior to vaccination at day 0 were below background. However, 21 days after vaccination, a significant increase in anti- rORF3 titre was observed in the rORF3 group when compared to the controls

(p=0.003).

EXAMPLE 14 Vaccination with recombinant H. paragallinarum ORF3 proteins expressed in a live attenuated vector Live attenuated Salmonella expressing an ORF3 protein under the control of an inducible promoter will be constructed. Cultures of the Salmonella strain will be grown, and the resulting cells washed with phosphate buffered saline. The resulting cell suspension will be innoculated intranasally or orally into chickens at a dose sufficient to allow colonization of the chicken. Vaccination may be repeated after 14 days with immune responses being measured regularly. The immune response will be monitored by ELISA or Western blotting to detect antobodies against H. paragallinarum or Salmonella, or against ORF3 proteins. A control group, consisting of birds vaccinated with Salmonella that have not

Substitute Sheet (Rule 26) RO/AU been modified to express the ORF3 will be included. One to 3 weeks after the final vaccination chickens will be challenged with a virulent H paragallinarum strain. Chickens will be monitored for 7 days before necroscopy. Vaccine efficacy will be monitored using clinical signs (over 7 days), and gross pathology and comparative reisolation rates of H. paragallinarum.

Throughout this specification the aim has been to describe the prefeπed embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

Substitute Sheet (Rule 26) RO/AU TABLE 1

Substitute Sheet ⁽Rule 26) RO/AU TABLE 2

Substitute Sheet (Rule 26) RO/AU TABLE 3

Strain⁸ ARI Code Source Page Kume

Country Serovar Serovar

0083 (P) HP 1 USA A A-l

0222 (P) HP 2 USA B B-l

Modesto (P,K) HP 3 USA C C-2

221 (K) HP 137 Japan A A-l

2403 (K) HP 90 Germany A A-2

E-3C (K) HP 144 Brazil A A-3

HP14 (K) HP 14 Australia A A-4

2671 (K) HP 147 Germany B B-l

H-18 (K) HP 146 Japan C C-l

SA-3 (K) HP 145 South Africa C C-3

HP60 (K) HP 60 Australia C C-4

Substitute Sheet (Rule 26) RO/AU

Claims

I . An isolated protein comprising an amino acid sequence PNFKKQ [SEQ ED NO: 1].

2. The isolated protein of claim 1 further comprising amino acid sequence FQSASNR [SEQ D NO: 2].

3. An isolated protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 3-13.

4. A homolog, fragment, variant or derivative of an isolated protein selected from the group consisting of SEQ D NOS: 3-13.

5. The isolated protein of any one of claims 1 to 4, which when administered to an animal, is capable of eliciting an immune response in said animal.

6. The isolated protein of claim 5, wherein said immune response provides protection against one or more strains of Haemophilus paragallinarum in said animal.

7. The isolated protein of claim 6, wherein said animal is an avian.

8. The isolated protein of claim 7, wherein said avian is a chicken.

9. An isolated nucleic acid encoding a protein according to any one of claims 1 to 4.

10. An isolated nucleic acid comprising a nucleotide sequence 5'-CCT AAT TTT AAG AAA CAA-3* [SEQ ED NO: 14].

I I. The isolated nucleic acid according to claim 10, wherein said nucleic acid further comprises a nucleotide sequence 5'-TTT CAA TCG GCA TCT AAT CGC-3' [SEQ ED NO: 15].

12. An isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ D NOS: 16-26.

13. An isolated homolog, fragment or variant of an isolated nucleic

Substitute Sheet (Rule 26) RO/AU acid selected from the group consisting of SEQ ED NOS: 16-26.

14. An isolated nucleic acid primer or probe derived from any one of SEQ ED NOS: 16-26.

15. The isolated nucleic acid according to claim 14 wherein said isolated nucleic acid is selected from the group consisting of SEQ ED NOS: 27-

45.

16. An expression construct comprising an expression vector and an isolated nucleic acid according to claim 9, or homolog, fragment or variant according to claim 13, wherein said nucleic acid is operably linked to one or more regulatory nucleic acids in said expression vector.

17. A host cell comprising an expression construct according to claim 16.

18. The host cell of claim 17 wherein said host cell is a bacterium.

19. A method of producing a recombinant protein according to any one of claims 1 to 4, said method including the steps of:

(i) culturing a host cell according to claim 17 such that said recombinant protein is expressed in said host cell; and (ii) isolating said recombinant protein.

20. An antibody or antibody fragment that binds to a protein of any one of claims 1 to 4.

21. A method of detecting H. paragallinarum bacteria in a biological sample suspected of comprising said bacteria, said method including the steps of:-

(a) isolating the biological sample;

(b) combining the antibody or antibody fragment of claim 20 with the biological sample; and

(c) detecting specifically bound antibody or antibody fragment which indicates the presence of said bacteria.

22. A method of detecting H. paragallinarum bacteria in a biological

Substitute Sheet (Rule 26) RO/AU sample suspected of comprising said bacteria, said method including the steps of:-

(I) isolating the biological sample;

(II) detecting a nucleic acid comprising a nucleotide sequence according to claim 9 or claim 13 in said sample which indicates the presence of said bacteria.

23. A method of diagnosing infection of an avian by H. paragallinarum bacteria, said method including the steps of:-

(A) contacting a biological sample from said avian with a protein of any one of claims 1 to 4; and (B) determining the presence or absence of a complex between said protein and H. paragallinarum-specific antibodies in said sample, wherein the presence of said complex is indicative of said infection.

24. The method of any one of claims 21 to 23 wherein said biological sample is a nasal swab sample.

25. A kit for detecting H. paragallinarum bacteria in a biological sample or diagnosing H. paragallinarum bacteria infection in an avian, wherein said kit comprises one or more isolated proteins according to any one of claims 1 to 4.

26. A kit for detecting H. paragallinarum bacteria in a biological sample or diagnosing H. paragallinarum bacteria infection in an avian, wherein said kit comprises one or more isolated nucleic acids according to claim 9 or claim 13.

27. A kit for detecting H. paragallinarum bacteria in a biological sample or diagnosing H. paragallinarum bacteria infection in an avian, wherein said kit comprises one or more isolated nucleic acids according to claim 14 or claim 15, and optionally a thermostable polymerase.

28. A kit for detecting H. paragallinarum bacteria in a biological

Substitute Sheet (Rule 26) RO/AU sample or diagnosing H paragallinarum bacteria infection in an avian, wherein said kit comprise one or more antibody or antibody fragment of claim 20.

29. A pharmaceutical composition comprising at least one isolated protein according to any one of claims 1 to 4 in combination with a pharmaceutically acceptable carrier or diluent.

30. A pharmaceutical composition comprising at least one isolated nucleic acid according to claim 9 or claim 13 in combination with a pharmaceutically acceptable carrier or diluent.

31. The pharmaceutical composition of claim 29 or claim 30 wherein said pharmaceutical composition is a vaccine.

32. The pharmaceutical composition of claim 31 wherein said vaccine is administered using Salmonella or Mycoplasma bacterium, said bacterium expressing at least one protein of claims 1 to 4.

33. A method of immunizing an avian against H. paragallinarum infection, including the step of administering a pharmaceutically effective amount of the vaccine of claim 31 to said avian.

34. A method of identifying an immunogenic fragment of a protein of any one of claims 1 to 4, including the steps of:-

(a') producing a fragment of said protein; (b') administering said fragment to a mammal or avian; and

(c') detecting an immune response in said mammal or avian, which response includes production of elements which specifically bind H. paragallinarum and/or said protein, and/or a protective effect against H paragallinarum infection.

35. The method of claim 34 wherein said avian is a chicken.

36. The method of claim 34 wherein said mammal is a mouse or rabbit.

Substitute Sheet (Rule 26) RO/AU