AU783744B2 - Vaccine antigens of moraxella - Google Patents

Description

WO 01/16172 PCT/AU00/01048 1 Vaccine untigens of Moraxella FIELD OF THE INVENTION The present invention relates to antigens of Moraxella, in particular, Moraxella bovis, nucleic acid sequences encoding these antigens and formulations for use in raising an immune response against Moraxella.

BACKGROUND OF THE INVENTION Infectious bovine keratoconjunctivitis (IBK) is an economically important disease of cattle caused by the Gram-negative coccobacillus Moraxella bovis. More commonly known as pinkeye, IBK is a highly contagious ocular infection which may range from mild conjunctivitis to severe ulceration, corneal perforation and blindness. Therapeutic and preventative measures have limited success in controlling IBK and a vaccine which will prevent the disease is required. A number of factors contribute to the virulence of the organism, the two most important attributes so far identified are the expression of pili, and the ability to produce haemolysin.

Seven different serogroups of M. bovis strains isolated in Australia, Great Britain and the USA have been characterised, based on pilus types An efficacious pilus-based vaccine must contain a sufficient quantity of pili from all seven serotypes to be fully protective, because of a lack of cross protection between serotypes Expression of all seven pilus serotypes at levels high enough to be useful for commercial vaccine preparation has not been achieved.

The ideal vaccine candidate to stimulate protection against M. bovis would be a molecule that is highly-conserved among all strains of this species. Possible candidates are haemolysin, protease, lipase and/or phospholipase enzymes produced by M. bovis. For example, a partially purified cell-free supernatant from one haemolytic strain of M. bovis has been shown to confer significant protection against heterologous, wild-type challenge The possibility that a haemolysin could be conserved across all seven serotypes of M. bovis makes it a potential vaccine candidate against IBK. However, researchers have so far been unable to either clone the gene encoding the haemolysin or purify the protein to homogeneity. Nevertheless, any or all of these molecules, alone or in combination, could prove useful for the generation of an effective vaccine against IBK.

WO 01/16172 PCT/AU00/01048 2 SUMMARY OF THE INVENTION In a first aspect the present invention consists in a polypeptide, the polypeptide having an amino acid sequence as set out in SEQ. ID. NO. 1 from amino acid 37 to 1114, or a sequence having at least 50% identity thereto, or a functional fragment thereof.

In a preferred embodiment the polypeptide has a sequence of at least more preferably at least 80% and most preferably at least 90% identity with the sequence shown in SEQ. ID. NO. 1.

In a further preferred embodiment of the first aspect of the present invention the polypeptide has protease activity.

In a second aspect the present invention consists in a nucleic acid molecule, the nucleic acid molecule encoding the polypeptide of the first aspect of the present invention.

In a third aspect the present invention consists in a nucleic acid molecule comprising a sequence as set out in SEQ. ID. NO. 2 or a sequence having at least 60% identity thereto, or a sequence which hybridises thereto under stringent conditions.

In a preferred embodiment the nucleic acid molecule has a sequence of at least 70%, more preferably at least 80% and most preferably at least identity with the sequence shown in SEQ. ID. NO. 2.

In a fourth aspect the present invention consists in a composition for use in raising an immune response in an animal, the composition comprising the polypeptide of the first aspect of the present invention or the nucleic acid sequence of the second aspect of the present invention and optionally a carrier and/or adjuvant.

In a fifth aspect the present invention consists in a polypeptide, the polypeptide having an amino acid sequence as set out in SEQ. ID. NO. 3 from amino acid 26 to 616, or a sequence having at least 50% identity thereto, or a functional fragment thereof.

In a preferred embodiment the polypeptide has a sequence of at least more preferably at least 80% and most preferably at least 90% identity with the sequence shown in SEQ. ID. NO. 3 from amino acid 26 to 616.

In a further preferred embodiment of the fifth aspect the polypeptide has lipase activity.

WO 01/16172 PCT/AU00/01048 3 In a sixth aspect the present invention consists in a nucleic acid molecule, the nucleic acid molecule encoding the polypeptide of the fifth aspect of the present invention.

In a seventh aspect the present invention consists in a nucleic acid molecule comprising a sequence as set out in SEQ. ID. NO. 4 or a sequence having at least 60% identity thereto, or a sequence which hybridises thereto under stringent conditions.

In a preferred embodiment the nucleic acid molecule has a sequence of at least 70%, more preferably at least 80% and most preferably at least identity with the sequence shown in SEQ. ID. NO. 4.

In an eighth aspect the present invention consists in a composition for use in raising an immune response in an animal, the composition comprising the polypeptide of the fifth aspect of the present invention or the nucleic acid sequence of the sixth aspect of the present invention and optionally a carrier and/or adjuvant.

In a ninth aspect the present invention consists in a polypeptide, the polypeptide having an amino acid sequence as set out in SEQ. ID. NO. 5, or a sequence having at least 60% identity thereto, or a functional fragment thereof.

In a preferred embodiment the polypeptide has a sequence of at least more preferably at least 80% and most preferably at least 90% identity with the sequence shown in SEQ. ID. NO. In a further preferred embodiment of the ninth aspect the polypeptide has haemolysin activity.

In a tenth aspect the present invention consists in a nucleic acid molecule, the nucleic acid molecule encoding the polypeptide of the ninth aspect of the present invention.

In an eleventh aspect the present invention consists in a nucleic acid molecule comprising a sequence as set out in SEQ. ID. NO. 6 or a sequence having at least 60% identity thereto, or a sequence which hybridises thereto under stringent conditions.

In a preferred embodiment the nucleic acid molecule has a sequence of at least 70%, more preferably at least 80% and most preferably at least identity with the sequence shown in SEQ. ID. NO. 6.

In a twelfth aspect the present invention consists in a composition for use in raising an immune response in an animal, the composition comprising WO 01/16172 PCT/AU00/01048 4 the polypeptide of the ninth aspect of the present invention or the nucleic acid sequence of the tenth aspect of the present invention and optionally a carrier and/or adjuvant.

The term "functional fragment" as used herein is intended to cover fragments of the polypeptide which retain at least 10% of the biological activity of the complete polypeptide. In particular this term is used to encompass fragments which show immunological cross-reactivity with the entire polypeptide, eg ligands which react with the fragment also react with the complete polypeptide.

In a thirteenth aspect the present invention consists in a composition for use in raising an inmune response in an animal directed against Moraxella, the composition comprising at least one polypeptide selected from the group consisting of the polypeptides of the first, fifth and ninth aspects of the present invention and optionally including an adjuvant or carrier.

In a preferred embodiment the composition includes the polypeptide of the ninth aspect of the present invention and either one of, or preferably both, the polypeptides of the first and fifth aspects of the present invention.

In a preferred embodiment the Moraxella is M. bovis or M. catarrhalis, most preferably M. bovis.

In a fourteenth aspect the present invention consists in an antibody raised against a polypeptide selected from the group consisting of the polypeptides of the first, fifth and ninth aspects.

As will be readily appreciated by the person skilled in this field the polypeptides and antibodies of the present invention and probes derived from the nucleotide sequences can be used as diagnostic reagents in determining Moraxella, in particular, M. bovis infection. For example, the polypeptides and antibodies can be used in ELISA based assays whilst the probes can be used in PCR based assays. The probes will be of a length to provide the required level of specificity and will typically be at least 18 nucleotides in length.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

WO 01/16172 PCT/AU00/01048 BRIEF DESCRIPT'ION OF THE FIGURES Figure 1: Nucleotide and amino acid sequence of a protease from M. bovis Dalton 2d. A putative promoter sequence is singly underlined. A putative ribosome binding site is shown in bold and underlined. A putative start codon is shown in bold. Putative transcription terminator sequences are indicated by inverted arrows. Numbering for both the nucleotide and amino acid sequences are shown on the left hand side.

Figure 2: Nucleotide and amino acid sequence of a lipase from M. bovis Dalton 2d. A putative promoter sequence is singly underlined. A putative ribosome binding site is shown in bold and underlined. A putative start codon is shown in bold. Putative transcription terminator sequences are indicated by inverted arrows. Numbering for both the nucleotide and amino acid sequences are shown on the left hand side.

Figure 3: Heat stability of the lipase from M.bovis when expressed in its recombinant form (pMB1/MC1061). (Heating carried out at Figure 4: Comparison of growth rate and expression levels of the lipase of M.bovis when in its native form and (ii) recombinant form. The growth rate is shown as solid bars and the lipase expression levels as open diamonds.

Figure 5: Nucleotide and amino acid sequence of the A subunit of the RTX toxin from M. bovis Dalton 2d. A putative ribosome binding site is shown in bold and underlined. A putative start codon is shown in bold. Upstream of the A subunit open reading frame is a portion of the coding region for the C subunit (nucleotide 1 to 195) (corresponding amino acid sequence shown in SEQ ID NO:8) and downstream of the A subunit is a small portion of the B subunit coding region (nucleotide 3080 to 3250) (corresponding amino acid sequence shown in SEQ ID NO:9). Numbering for both the nucleotide and amino acid sequences are shown on the left hand side.

WO 01/16172 PCT/AU00/01048 6 DETAILED DESCRIPTION OF THE INVENTION In order that the nature of the present invention may be more clearly understood preferred forms thereof will now be described with reference to the following non-limiting Examples.

General Molecular Biology Unless otherwise indicated, the recombinant DNA techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A.

Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hanes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present) and are incorporated herein by reference.

Protein Variants Amino acid sequence variants can be prepared by introducing appropriate nucleotide changes into DNA, or by in vitro synthesis of the desired polypeptide. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final protein product possesses the desired characteristics. The amino acid changes also may alter post-translational processes such as altering the membrane anchoring characteristics, altering the intra-cellular location by inserting, deleting or otherwise affecting the transmembrane sequences of the native protein, or modifying its susceptibility to proteolytic cleavage.

In designing amino acid sequence variants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in WO 01/16172 PCT/AU00/01048 7 series, by substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or inserting residues of other ligands adjacent to the located site.

A useful method for identification of residues or regions for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (Science (1989) 244: 1081-1085). Here, a residue or group of target residues are identified charged residues such as Arg.

Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions theii are refined by introducing further or other variants. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.

For example, to optimise the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed variants are screened for the optimal combination of desired activity.

There are two principal variables in the construction of amino acid sequence variants; the location of the mutation site and the nature of the mutation. These may represent naturally occurring alleles or predetermined mutant forms made by mutating the DNA either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the characteristic to be modified.

Amino acid sequence deletions generally range from about 1 to residues, more preferably about 1 to 10 residues and typically about 1 to contiguous residues.

Amino acid sequence insertions include amino and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Other insertional variants include the fusion of the N- or C-terminus of the proteins to an immunogenic polypeptide e.g. bacterial polypeptides such as betalactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, bovine serum albumin, and chemotactic polypeptides. C-terminal fusions with proteins having a WO 01/16172 PCT/AU00/01048 8 long half-life such as iinmnunoglobulin constant regions (or other inununoglobulin regions), albumin, or ferritin, are included.

Another group of variants are amino acid substitution variants. These variants have at least one amino acid residue in the protein molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as the active site(s). Other sites of interest are those in which particular residues obtained from various species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala val; leu: ile val Arg lys; gin; asn lys Asn gin; his; lys: arg gln Asp glu glu Cys ser ser Gin asn asn Glu asp asp Gly pro pro His asn; gin; lys; arg arg lie leu; val; met; ala; phe leu norleucine Leu norleucine, ile; val; met; ala; ile phe WO 01/16172 PCT/AUOO/01048 Original Exemplary Preferred Residue Substitutions Substitutions Lys arg; gin; asn arg Met leu; phe; ile; leu Phe leu; val; ile; ala leu Pro gly gly Ser(S) thr thr Thr (T ser ser Trp tyr tyr Tyr trp; phe; thr: ser phe Val ile; leu; met; phe; ala; leu norleucine Mutants, Variants and Homology Proteins Mutant polypeptides will possess one or more mutations which are deletions, insertions, or substitutions of amino acid residues. Mutants can be either naturally occurring (that is to say, purified or isolated from a natural source) or synthetic (for example, by performing site-directed mutagensis on the encoding DNA). It is thus apparent that polypeptides of the invention can be either naturally occurring or recombinant (that is to say prepared using recombinant DNA techniques).

An allelic variant will be a variant that is naturally occurring within an individual organism.

Protein sequences are homologous if they are related by divergence from a common ancestor. Consequently, a species homologue of the protein will be the equivalent protein which occurs naturally in another species.

Within any one species a homologue may exist as numerous allelic variants, and these will be considered homologues of the protein. Allelic variants and species homologues can be obtained by following standard techniques known to those skilled in the art. Preferred species homologues include those obtained from representatives of the same Phylum, more preferably the same Class and even more preferably the same Order.

WO 01/16172 PCT/AUOO/01048 A protein at least 50% identical, as determined by methods well known to those skilled in the art (for example, the method described by Smith, T.F.

and Waterman, M.S. (1981) Ad. Appl Math., 2: 482-489, or Needleman, S.B.

and Wunsch, C.D. (1970) J. Mol. Biol., 48: 443-453), to that of the present invention are included in the invention, as are proteins at least 70% or and more preferably at least 90% identical to the protein of the present invention. This will generally be over a region of at least 20, preferably at least 30, contiguous amino acids.

Mutants. Variants and Homology Nucleic Acids Mutant polynucleotides will possess one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagensis on the DNA).

It is thus apparent that polynucleotides of the invention can be either naturally occurring or recombinant (that is to say prepared using recombinant DNA techniques).

An allelic variant will be a variant that is naturally occurring within an individual organism.

Nucleotide sequences are homologous if they are related by divergence from a common ancestor. Consequently, a species homologue of the polynucleotide will be the equivalent polynucleotide which occurs naturally in another species. Within any one species a hoinologue may exist as numerous allelic variants, and these will be considered homologues of the polynucleotide. Allelic variants and species homologues can be obtained by following standard techniques known to those skilled in the art. Preferred species homologues include those obtained from representatives of the same Phylum, more preferably the same Class and even more preferably the same Order.

A polynucleotide at least 70% identical, as determined by methods well known to those skilled in the art (for example, the method described by Smith, T.F. and Waterman, M.S. (1981) Ad. Appl Math., 2: 482-489, or Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol., 48: 443-453), to that of the present invention are included in the invention, as are proteins at least 80% or 90% and more preferably at least 95% identical to the WO 01/16172 PCT/AU00/01048 11 polynucleotide of the present invention. This will generally be over a region of at least 60, preferably at least 90, contiguous nucleotide residues.

Antibody Production The term "antibody" should be construed as covering any specific binding substance having a binding domain with the required specificity.

Thus, the term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide including an irnnunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules including an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included.

Antibodies, either polyclonal or monoclonal, which are specific for a protein of the present invention can be produced by a person skilled in the art using standard techniques such as, but not limited to, those described by Harlow et al. Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory Press (1988), and D. Catty (editor), Antibodies: A Practical Approach, IRL Press (1988).

Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of a protein. For the production of polyclonal antibodies, a number of host animals are acceptable for the generation of antibodies by inununization with one or more injections of a polypeptide preparation, including but not limited to rabbits, mice, rats, etc.

Various adjuvants may be used to increase the immunological response in the host animal, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin. pluronic polyols, polyanions, oil emulsions, keyhole lyipet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.

A monoclonal antibody to an epitope of a protein may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 493-497), and the more recent human B-cell hybridoma technique (Kesber et al. 1983, Immunology Today 4:72) and EBV-hybridoma technique (Cole et al. 1985, Monoclonal Antibodies and WO 01/16172 PCT/AU00/01048 12 Cancer Therapy, Alan R. Liss. Inc. pp. 77-96). In addition, techniques developed for the production of "chimeric antibodies" by splicing the genes from an antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity may be used (Morrison et al. 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neuberger et al. 1984 Nature 312:604-608; Takeda et al. 1985 Nature 31:452-454). Alternatively, techniques described for the production of single chain antibodies Patent 4,946,778) can be adapted to produce 4-specific single chain antibodies.

Recombinant human or humanized versions of monoclonal antibodies are a preferred embodiment for human therapeutic applications. Humanized antibodies may be prepared according to procedures in the literature (e.g.

Jones et al. 1986, Nature 321:522-25; Reichman et al. 1988, Nature 332:323-27; Verhoeyen et al. 1988, Science 239:1534-36). The recently described "gene conversion mutagenesis" strategy for the production of humanized monoclonal antibody may also be employed in the production of humanized antibodies (Carter et al. 1992 Proc. Natl. Acad. Sci. U.S.A.

89:4285-89). Alternatively, techniques for generating the recombinant phage library of random combinations of heavy and light regions may be used to prepare recombinant antibodies Huse et al. 1989 Science 246:1275-81).

Antibody fragments which contain the idiotype of the molecule such as Fu F(ab') and F(ab 2 may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab) E2 fragment which can be produced by pepsin digestion of the intact antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and the two Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.

Alternatively, Fab expression libraries may be constructed (Huse et al. 1989, Science 240:1275-1281) to allow rapid and easy cloning of a monoclonal Fab fragment with the desired specificity to a protein.

Adjuvants and Carriers Pharmaceutically acceptable carriers or diluents include those used in compositions suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. They are WO 01/16172 PCT/AU00/01048 13 non-toxic to recipients at the dosages and concentrations employed.

Representative examples of pharmaceutically acceptable carriers or diluents include, but are not limited to water, isotonic solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline) and can also contain one or more of, mannitol, lactose, trehalose, dextrose, glycerol, ethanol or polypeptides (such as human serum albumin). The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

As mentioned above the composition may include an adjuvant. As will be understood an "adjuvant" means a composition comprised of one or more substances that enhances the immunogenicity and efficacy of a vaccine composition. Non-limiting examples of suitable adjuvants include squalane and squalene (or other oils of animal origin); block copolymers; detergents such as Tween®-80; Quil® A, mineral oils such as Drakeol or Marcol, vegetable oils such as peanut oil; Corynebacteriuni-derived adjuvants such as Corynebacterium parvum; Propionibacterium-derived adjuvants such as Propionibacterium acne; Mycobacterium bovis (Bacille Calmette and Guerin or BCG); interleukins such as interleukin 2 and interleukin 12; monokines such as interleukin 1; tumour necrosis factor; interferons such as gamma interferon; combinations such as saponin-aluminium hydroxide or Quil-A aluminium hydroxide; liposomes; ISCOM adjuvant; mycobacterial cell wall extract; synthetic glycopeptides such as murarmyl dipeptides or other derivatives; Avridine; Lipid A derivatives; dextran sulfate; DEAE-Dextran or with aluminium phosphate; carboxypolymethylene such as Carbopol' EMA; acrylic copolymer emulsions such as Neocryl A640 U.S. Pat. No.

5,047,238); vaccinia or animal poxvirus proteins; sub-viral particle adjuvants such as cholera toxin, or mixtures thereof.

Gene/DNA Isolation The DNA encoding a protein may be obtained from any cDNA library prepared from tissue believed to express the gene mRNA and to express it at a detectable level. DNA can also be obtained from a genomic library.

Libraries are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries, suitable probes include monoclonal or polyclonal WO 01/16172 PCT/AU00/01048 14 antibodies that recognize and specifically bind the protein; oligonucleotides of about 20-80 bases in length that encode known or suspected portions of cDNA from the same or different species; and/or complementary or homologous cDNAs or fragments thereof that encode the same or a hybridizing gene. Appropriate probes for screening genomic DNA libraries include, but are not limited to, oligonucleotides; cDNAs or fragments thereof that encode tile same or hybridizing DNA including expressed sequence tags and the like; and/or homologous genomic DNAs or fragments thereof.

Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures as described in chapters 10-12 of Sambrook et al.

An alternative means to isolate a gene encoding the protein of interest is to use polymerase chain reaction (PCR) methodology as described in section 14 of Sambrook et al. This method requires the use of oligonucleotide probes that will hybridize to the gene.

The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The actual nucleotide sequence(s) is usually based on conserved or highly homologous nucleotide sequences or regions of the gene. The oligonucleotides may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is known. The oligonucleotide must be labelled such that it can be detected upon hybridization to DNA in the library being screened. The preferred method of labelling is to use (a- 2 dATP with polynucleotide kinase, as is well known in the art, to radiolabel the oligonucleotide. However, other methods may be used to label the oligonucleotide, including, but not limited to, biotinylation or enzyme labelling.

DNA encompassing all the protein coding sequence is obtained by screening selected cDNA or genomic libraries, and if necessary, using conventional primer extension procedures as described in section 7.79 of Sambrook et al., to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.

Another alternative method for obtaining the gene of interest is to chemically synthesize it using one of the methods described in Fingels et al.

(Agnew Chem. Int. Ed. Engl. 28: 716-734, 1989). These methods include WO 01/16172 PCT/AUOO/01048 triester, phosphite, phosphoramidite and H-Phosphonate methods, PCR and other autoprimer methods, and oligonucleotide syntheses on solid supports.

These methods may be used if the entire nucleic acid sequence of the gene is known, or the sequence of the nucleic acid complementary to the coding strand is available, or alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.

Substantially Purified By "substantially purified" we mean a polypeptide that has been separated from lipids, nucleic acids, other polypeptides, and other contaminating molecules.

Hybridisation The polynucleotide sequence of the present invention may hybridise to the respective sequence set out SEQ. ID. NOS. 2, 4, or 6 under high stringency. As used herein, stringent conditions are those that employ low ionic strength and high temperature for washing after hybridization, for example, 0.1 x SSC and 0.1% SDS at 50°C; (ii) employ during hybridization conditions such that the hybridization temperature is lower than t.e duplex melting temperature of the hybridizing polynucleotides. for example 1.5 x SSPE, 10% polyethylene glycol 6000, 7% SDS, 0.25 mg/ml fragmented herring sperm DNA at 65C; or (iii) for example, 0.5M sodium phosphate, pH 7.2, 5mM EDTA, 7% SDS and 0.5% BLOTTO at 70°C; or (iv) employ during hybridization a denaturing agent such as formamide, for example, 50% formamide with x SSC, 50mM sodium phosphate (pH 6.5) and 5 x Denhardt's solution (32) at 42 0 C; or employ, for example, 50% formamide, 5 x SSC, sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50gg/ml) and dextran sulphate at 42°C.

WO 01/16172 PCT/AUOO/01048 16 EXAMPLE 1 This example describes the cloning and characterisation of a protease from Moraxella bovis.

Bacteria and construction of a genomic library Moraxella bovis strain Dalton 2d was a field isolate collected from a bovine eye and characterised by CSIRO Animal Health, Parkville, Australia Escherichia coli strain DH5a has been previously described 8).

All enzymes were purchased from Promega (Madison, WI, USA) except where otherwise noted.

General cloning and DNA techniques were as described unless otherwise noted.

A genomic library was constructed by carrying out partial Sau3A digests on genomic DNA extracted from M. bovis strain Dalton 2d using a CTAB method which is outlined below. This DNA was size fractionated using a NaCl gradient (10) and ligated with the cosmid cloning vector pHC79 (11) which had been previously digested with BamHI. This DNA was packaged into lambda bacteriophage heads using the Packagene Lambda DNA packaging system (Promega, Madison,WI, USA) and this was used to transduce the E. coli strain DH5a. The library was stored in 96 well trays glycerol luria broth ampicillin (50pg/ml)) at -70 0

C.

CTAB genomic DNA extraction from M. bovis A 5ml brain heart infusion (BHI) (Oxoid Ltd., Basingstoke, Hampshire, broth was inoculated with a colony of Dalton 2d taken from a fresh overnight culture on horse blood agar and incubated with shaking at 37 0 C for 6 hours. This culture was used to inoculate 50ml of BHI broth which was grown with shaking at 37 0 C overnight. 40ml of the culture was transferred to an SS34 tube and the cells pelleted at 3000 x g for 10 minutes. Following resuspension of the pellet in 9.5ml of 2596 sucrose in TE buffer (10mM Tris, 1mM EDTA 500pl of 10% SDS, 50pl of 20mg/ml proteinase K and of 10mg/ml RnaseA were added and this mixture incubated in an orbital shaker for 1 hour at 37 0 C. To this mixture, 1.8ml of 5M NaCI and 1.5ml of a CTAB (N-Cetyl-N,N,N-trimethyl-ammonium bromide) NaCl solution was added and incubation continued for 20 minutes at 65°C. The DNA was WO 01/16172 PCT/AU00/01048 17 extracted using phenol/chloroform and precipitated with 0.6 volumes of isopropanol. The resulting DNA was washed in 70% ethanol, dried and resuspended in 2ml of TE buffer.

Screening of genomic library for enzyme activity The genomic library was cultured on skim milk agar to screen for the presence of a clone displaying protease activity (double strength Columbia agar base (Oxoid Ltd., Basingstoke, Hampshire, 10% skim milk) for 24 hours at 37 0 C followed by refrigeration at 4°C for one to two days.

A single clone from the genomic library was detected as having activity against skim milk agar. DNA analysis confirmed that the clone contained a fragment of M. bovis Dalton 2d genomic DNA approximately 40 kilobases in size. The construct was designated pJF1.

Nuclootide sequence of the M. bovis prutease clone pJF1 Plasmid and cosmid DNA for automated sequencing was extracted using the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI, USA) and the Qiagen Plasmid Midi Kit (Qiagen Pty. Ltd., Clifton Hill, Vic, Australia), respectively.

The nucleotide sequence of the insert DNA was determined using the process of "primer walking" This was achieved using synthetic oligonuclcotides (Bresatec Geneworks, Thebarton, SA, Australia) and the dye terminator cycle sequencing ready reaction (Perkin Elmer Corporation, Norwalk, CT, USA). The resulting sequence was analysed on an Applied Biosystems 373A DNA sequencer.

Automated sequencing revealed an open reading frame of 3345bp capable of encoding a protein of 1115 amino acids. The sequence is written in the 5' to 3' direction and is shown in Figure 1 together with the corresponding amino acid sequence which is predicted to encode a protein with a molecular weight of 120kDa. The amino acid sequence is shown in SEQ. ID. NO. 1 and the DNA sequence is shown in SEQ. ID. NO. 2.

The putative start codon for the mature protease protein was identified by the presence of a possible ribosome binding site upstream. This RBS was identified by its similarity to the consensus sequence for the E. coli RBS and that previously identified for the M. bovis pilin genes (AGGAG) (27) WO 01/16172 PCT/AU00/01048 18 Due to the secreted nature of the protease, it was assumed that it would contain in its N-terminal sequence a signal peptide which would be used in the secretion of the protein. This analysis was carried out using a prediction program (SignalP) available through the Expasy website (http://www.expasy.ch/tools/), which allows for the identification of prokaryotic signal peptides and predicts possible cleavage sites. This analysis only identified a signal peptide using the start codon indicated in the accompanying protein/DNA sequence.

Sequence comparisons Comparisons of the deduced amino acid sequence with those in the database were carried out using the BlastX and BlastP programs (13) which are available at http://www.ncbi.nin.nih.gov.

At the amino acid level, the protease cloned from Dalton 2d displayed the following similarity and identity to the proteins listed.

Organism Protein Similarity Identity Serratia marcescens ssp-h2 serine protease 39% 23% autotransporter Serratia marcescens ssp-hl serine protease 37% 22% autotransporter Pseudomonas serine protease 34% flourescens homologue Pseudomonas tolaasii serine protease 35% 21% More generally the 5' domain of the M. bovis protease displays homology to a family of subtilisins (serine proteases) while the 3' region resembles a number of outer membrane proteins.

The M. bovis sequence was found to contain a highly proline rich region which distinguished it from all other proteins to which it was closely related.

Protease type encoded by pJF1 In order to identify the type of protease activity encoded by pJF1, a range of specific protease inhibitors were examined for their effect on the expression of the M. bovis protease.

WO 01/16172 PCT/AU00/01048 19 The method of Bourgeau et al., (1992) (14) was used to determine inhibitor activity with the following modifications. 1004l of cell free supernatant from a fresh overnight broth culture was mixed with 650ld of 100nm Tris (pH 7.2) and a suitable volume of inhibitor PMSF (phenylmethylsulfonyl fluoride) 5mM; EDTA 5mM; leupeptin 100lg/ml; pepstatin 50Lg/ml]. Distilled water was used to make the volume up to Iml.

The mixture was incubated at 370C for 30 minutes and 10mg of azocoll (Calbiochem, Alexandria, NSW, Australia) was then added. The suspensions were incubated at 37 0 C for 16 hours and the optical density read at 520nm.

In this way it was confirmed that the activity attributable to the protease encoded by pJF1 was that of a serine protease since PMSF (a serine protease inhibitor) reduced the protease activity of both Dalton 2d and pJF1 to zero.

Conservation of protease in M. bovis Southern hybridisation using an internal fragment of the protease coding region as a probe was carried out to investigate whether the protease was present in strains representing the known M. bovis pili serotypes.

Genomic DNA extracted from the representative strains of M. bovis was digested with Xbal and EcoRI and separated using agarose gel electrophoresis. The DNA was transferred to a Hybond N+ filter (Amersham, Little Chalfont, Buckinghamshire, using the method described The probe used in the southern hybridisation was a PCR amplified fragment which was internal to the protease coding region. This fragment was labelled with n P-dATP using the Megaprime labelling system (Amersham, Little Chalfont, Buckinghamshire, according to the manufacturers instructions. High stringency conditions were used (hybridisation temperature 68 0 C; 2 washes at room temperature in 2 x SSC 0.1% SDS; 1 wash at 68 0 C in 0.1 x SSC 0.1% SDS) and the resulting filters were exposed to autoradiographic film (Kodak. Rochester, New York, USA) for 5 to 24 hours before developing.

Results showed that the protease gene cloned in pJF1 is present in all strains of M. bovis examined.

WO 01/16172 PCT/AUOO/O1048 EXAMPLE 2 This example describes the cloning and characterisation of a lipase from Moraxella bovis.

Bacteria and construction of a plasmid library Moraxella bovis strain Dalton 2d was a field isolate collected from a bovine eye and characterised by CSIRO Animal Health, Parkville, Australia Escherichia coli strain MC1061 has been previously described (16).

All enzymes were purchased from Promega (Madison, WI, USA) except where otherwise noted. General cloning and DNA techniques were as described unless otherwise noted.

A plasmid library was constructed in the cloning vector pBR322 (17).

This was done by partially digesting genomic DNA extracted from Dalton 2d (using the CTAB method described in Example 1) with Sau3A under conditions that maximised the amount of DNA in the range of 1 to 2kb. This DNA was ligated with pBR322 which had been previously digested with Banil. The ligated DNA was electroporated (2.5kV, 200f and 200F, for a theoretical time constant of 4.7) into electrocompetent E. coli MC1061 cells.

Screening of plasmid library for lipase expression Following electroporation of the ligated DNA into MC1061 cells, recombinant clones displaying lipase activity were detected by culturing the library for 24 hours at 37 0 C on media containing Tween 80 [10ml Tween (Sigma, St Louis, MO, USA), 5g NaC1, 3g agar No.1 (Oxoid Ltd., Basingstoke, Hampshire, 10g peptone, 0.lg CaCl,.H 2 0 litre].

Twenty eight out of 24,000 clones screened were found to be displaying lipase activity. DNA analysis confirmed that all of these clones contained one 5.4kb fragment of DNA in common. One clone was chosen to continue work with and this was designated pMB1.

In some experiments (below), a photometric assay of extracellular lipase activity was performed with p-nitiophenylpalmitate as the substrate (18, 19). Strains of E. coli and/or M. bovis were grown at 370C for the required time points. Cell free culture supernatant (100ll) was mixed with 2.4ml of enzyme buffer (19) to assay secreted lipase activity. After minutes incubation at 37°C, the optical density at 410nm was determined.

WO 01/16172 PCT/AUOO/01048 21 One enzyme unit was defined as the amount of enzyme that releases 1 nmol of p-nitrophenyl from p-nitrophenylpalmitate ml min'. Under the conditions described by Stuer et al., an optical density at 410nm of 0.041 is equivalent to 1 enzyme unit.

Nucleotide sequence of the M. bovis lipaso clone pMB1 Plasmid pMB1 was subjected to automated DNA sequencing using the methodology described in Example 1.

This analysis revealed an open reading frame of 1851bp capable of encoding 617 amino acids. The sequence is written in the 5' to 3' direction and is shown in Figure 2 together with the corresponding amino acid sequence that is predicted to encode a protein with a molecular weight of 65.8kDa. The amino acid sequence is shown in SEQ. ID. NO. 3 and the DNA sequence is shown in SEQ. ID. NO. 4.

The techniques set out above in respect of the protease were used to identify the potential start codon for the lipase protein.

Sequence comparisons Sequence comparison; were made using the methodology described in Example 1.

At the amino acid level, the lipase cloned from M. bovis Dalton 2d was shown to display the following similarity and identity to the proteins listed.

Organism Protein Similarity Identity Xenorhabdus triacylglycerol lipase 36% 24% luminescens Pseudunonas putida hypothetical protein 36% 24% Salmonella typhiminuium outer membrane 35% 23% esterase Pseudomonas aeruginosa lipase esterase 36% 23% The M. bovis lipase was identified as being a possible new member of the GDSL family (20) of lipolytic enzymes.

N-terminal sequencing carried out on the lipase mature protein WO 01/16172 PCT/AUOO/01048 22 The required strains of E. coli were cultured overnight with shaking at 37°C in 500nls of luria broth. The cells were pelleted at 5,000 rpm for inins and the supernatant filtered through a 0.45lum filter. Solid ammonium sulfate was added to the supernatant to 60% saturation (180g 500ml). and dissolved at 4 0 C with stirring for 30 minutes. This mixture was left at 4°C overnight and the precipitated proteins pelleted at 7,000 rpm for 30 mins.

The proteins were resuspended in 3ml of double distilled water and the solubilised proteins dialysed against double distilled water overnight to remove any salt. The resulting mixture was filtered through a 0.454m filter and stored at -20 0

C.

Following separation of the proteins by SDS-PAGE, the proteins were transferred to PVDF membrane and excised. The protein was subjected to automated (Edman degradation) sequence analysis (28) with vapour phase delivery of critical reagents (29) in an automated sequenator (model 470A; Applied Biosystems) (Applied Biosystems Division, Foster City, CA, USA) in conjunction with a PTH amino acid separation system (model 120A PTH analyzer; Applied Biosystems).

Using this technique 17 amino acids with two gaps were identified KEFSQVIIFGDSLXDXG (SEQIDNO:7) which corresponds exactly with amino acids 26 through to 42 shown on the accompanying sequence. This result also indicated that the protein most likely includes an amino terminal signal peptide which is involved in the secretion of the protein. This amino terminal corresponds to amino acids 1 through to 25 in the accompanying sequence.

Raising antibodies to the lipase in rabbits Antibody to the recombinant lipase was raised in rabbits by injecting ammnonium sulfate precipitated supernatant from E. coli MC1061/pMB4. Prior to vaccination, the lipase preparation was inactivated by heating to 90 0 C for 90min. 30utg of this protein was injected at 2 weekly intervals for 4 weeks.

The primary inoculum was emulsified with Freunds complete adjuvant and subsequent vaccinations with Freunds incomplete adjuvant.

Heat stability of M. bovis lipase The recombinant lipase cloned from M. bovis Dalton 2d was found to be very heat stable since it required heating at 90 0 C for 105 minutes for the WO 01/16172 PCT/AU00/01048 23 activity to be reduced by 97%. Figure 3 illustrates this phenomenon with enzyme activity expressed as "lipase enzyme units" as determined in the extracellular lipase assay.

Relative expression levels of native versus recombinant lipase An experiment was performed to plot growth rate with lipase production and to compare production of the recombinant lipase from MC1061/pMB1 with that of the native form of the lipase from M. bovis Dalton 2d. Figure 4 illustrates that the two strains grow at approximately the same rate but they do not reach the same cell density, with Dalton 2d substantially lower after 9 hours than MC1061/pMB1. Lipase expression levels were greatest from the pMB1 construct in E. coli compared to native lipase expression from M. bovis Dalton 2d.

This result was further substantiated when proteins from cell-free supernatants of either the E. coli clone or M. bovis Dalton 2d were ammonium sulfate precipitated and analysed by SDS-PAGE and western blot using antisera to the recombinant heat-deactivated lipase.

Ammonium sulfate precipitated supernatants were prepared from overnight cultures of E. coli or M. bovis that had been shaken at 37 0 C in either 500mls of Luria broth or brain heart infusion broth, respectively. Cells were pelleted at 5000 x g for 15 minutes and the supernatant filtered through a 0.45lm filter. Solid ammonium sulfate was added to the supernatant to saturation (180g 500ml) and dissolved at 4 0 C with stirring for minutes. This mixture was left at 4"C overnight and the precipitated protein pelleted at 7000 x g for 30 minutes. Proteins were resuspended in 3ml of double distilled water and the solubilised proteins dialysed against double distilled water overnight to remove any salt. The resulting mixture was filtered through a 0.45pmn filter and stored at -20 0

C.

Protein samples (100gl) were prepared for SDS-PAGE by resuspension in 100l of 2x sample buffer (5ml 0.5M Tris (pH6.8), 8ml 10% SDS, 4ml glycerol, 0.8ml P-mercaptoethanol, 1ml double distilled HzO, bromophenol blue) and heating to 100 0 C for 5 minutes. The proteins were separated on a 12.5% polyacrylamide gel using the buffer system of Laemlli (21).

Western blots were carried out according to the method of Towbin et al., (22) and following separation of proteins by SDS-PAGE and transfer to nitrocellulose using the Bio-Rad minicell (Bio-Rad, Hercules, CA, USA) WO 01/16172 PCT/AU00/01048 24 transfer system. Filters were iinmunoblotted with the recombinant lipase antiserum (at a concentration of 1/100) which had been adsorbed against MC1061 cells. The antiserum was raised against ammonium sulfate precipitated recombinant lipase which had been heat deactivated (1 hour minutes at 90 0 C) and used to inoculate rabbits (three doses of 50g each) at 4 week intervals. Blood samples were collected from the marginal ear vein prior to immunisation and at each vaccination time point.

The results showed a prominent band present in the recombinant lipase positive construct MC1061/pMB1 that is detectable in relatively minor amounts in M. bovis Dalton 2d preparation. The protein detected with the antisera was approximately the same size as that of the predicted molecular weight for the M. bovis lipase (65.8kDa).

Lipase type encoded by pMB1 Thin layer chromatography (TLC) was used to determine whether the lipase of M. bovis Dalton 2d displayed phospholipase activity.

Characterisation of phospholipase type essentially followed a previously described method (23) except that the results of separation on Silica Gel plates were visualised by developing with a 10% ethanolic solution of molybdophosphoric acid at 100°C. All reagents used were purchased from Sigma (Sigma, St Louis, MO, USA).

TLC determined that the M. bovis lipase displayed the same enzyme specificity as that of a commercially-available phospholipase B when lysophosphatidylcholine and phosphatidylcholine were used as substrates (data not shown).

Conservation of lipase among M. bovis A southern blot using an internal fragment of the Dalton 2d lipase coding region was used to investigate whether the lipase gene was present in strains of M. bovis representing the known pilus serotypes.

Genomic DNA extracted from the strains of M. bovis representing each of the known pilus serotypes (15) was digested with HindII and separated using agarose gel electrophoresis. The DNA was transferred to a Hybond N+ filter (Amersham, Little Chalfont, Buckinghamshire, using a previously described method The probe used in the southern hybridisation was a HindIII fragment that contained sequence internal to the lipase coding WO 01/16172 PCT/AUOO/01048 region. This fragment was labelled with o3P-dATP using the Megaprime labelling system (Amersham, Little Chalfont, Buckinghamshire, U.K.) according to the manufacturers instructions. High stringency conditions were used (hybridisation temperature 68 0 C; 2 washes at room temperature in 2 x SSC 0.1% SDS; 1 wash at 68 0 C in 0.1 x SSC 0.1% SDS) and the resulting filters were exposed to autoradiographic film (Kodak, Rochester, New York, USA) for 5 to 24 hours before developing.

Results showed that the lipase gene is present in all strains of M. bovis examined.

To confirm whether or not the lipase gene was expressed in each of the serotype representative strains, antisera raised against recombinant heat deactivated lipase was used in a western blot analysis of whole cell preparations. Results showed that the lipase was indeed being expressed by all of these M. bovis strains.

EXAMPLE 3 Bacteria and construction of a haemolysin clone Moraxella bovis strain Dalton 2d was a field isolate collected from a bovine eye and characterised by CSIRO Animal Health, Parkville, Australia All of the M. bovis strains representative of the known pilus serotypes express a haemolytic activity that is detected on horse blood agar.

Eschericuia coli strain degP4::Tn5 has a leaky outer membrane and is defective in proteolysis and has been previously described (24).

All enzymes were purchased from Promega (Madison, WI, USA) except where otherwise noted.

General cloning and DNA techniques were as described unless otherwise noted.

A phoA fusion technique that allows for the identification of exported proteins (25) was utilised with some modifications. Genomic DNA from M.

bovis Dalton 2d (prepared using the CTAB method described in Example 1) was partially digested with Sau3A. Restricted DNA was ligated with a series of vectors that allow fusions with an alkaline phosphatase gene in three different reading frames. The ligated DNA was electroporated into E. coli and the resulting clones screened on Luria agar containing WO 01/16172 PCT/AUOO/01048 26 ampicillin (50 g/ml) and X-P (200pg/ml) (5-bromo-3-chloro-indolyl phosphate). Selection of clones relies on the observation that if the fragment is clonedin frame and contains an export sequence the resulting colony will be blue in colour. The leaky E. coli strain allows the outer membrane-bound proteins and secreted proteins (both fused with phoA) to be distinguished from non-secreted fusion proteins.

Sequencing of the M. bovis haemulysin determinant Clones selected for the presence of a secreted or outer membrane protein gene sequence were subjected to automated DNA sequencing using the methods described in Example 1. One of these clones, pMbhl, was found to contain 200bp of DNA which displayed high homology to the A subunit of other RTX toxins. Inverse PCR and degenerate oligonucleotides were utilised to obtain the sequence of the entire A subunit. The open reading frame of 2784bp was capable of encoding 928 amino acids. The sequence is written in the 5' to 3' direction and is shown in Figure 5 together with the corresponding amino acid sequence that is predicted to encode a protein with a molecular weight of 98.8kDa. The amino acid sequence is shown in SEQ. ID. NO. 5 and the DNA sequence is shown in SEQ. ID. NO. 6.

The putative start codon was identified using the RBS technique outlined above. A signal peptide analysis was not carried out as the A subunit is not secreted on its own. However as the protein sequence of these proteins (RTX) is quite highly conserved, on amino acid homologies alone this start codon was the one of choice.

Sequence homology At the amino acid level the M. bovis Dalton 2d haemolysin gene product shows striking similarity to the A subunit of the of several RTX and other haemolysins as shown in the following table.

WO 01/16172 PCT/AU00/01048 Organism Protein Similarity Identity Pasteurella haemolytica LktA protein (leukotoxin) 68% Actinobacillus RTX toxin determinant 68% 48% pleurolp eumoniae Escherichia coli Haemolysin plasmid 58% 43% E. coli Haemolysin 58% 43% chromosomal Functional complementation by the M. bovis haemolysin A construct which expressed the chroinosomal-borne haemolysin of E. coli was obtained (pLG900; generated by combining the two plasmids pLG575 (26) and pLG816 (hlyC and hlyA cloned into pBluescriptSK). pLG900 comprises the four genes of the RTX operon, hlyC, hlyA, hlyB, hlyD, cloned into pBluescriptSK and is capable of conferring a haemolytic phenotype on E.

coli cells that were previously non-haemolytic. The A subunit (hlyA) of this construct was mutated such that it was no longer able to be expressed but the other genes involved in the operon (hlyB, hlyC and hlyD) remained intact.

The E. coli strain containing this construct (pLG900 hlyA negative) was no longer haemolytic. However, the haemolytic phenotype was restored by providing in trans the cloned haemolysin subunit gene from M. bovis Dalton 2d. Thus it was confirmed that the cloned M. bovis haemolysin gene encoded a structural subunit that was most probably a member of the RTX family of haemolytic enzymes.

Further sequence analysis has established that, like other members of the family, the M. bovis RTX A subunit gene is flanked by DNA sequences capable of encoding the RTX B.C and D proteins.

Conservation of the RTX A subunit among M. bovis To determine whether the gene for the RTX A subunit was present in M. bovis strains representing the known pilus serotypes, a southern hybridisation analysis was performed using the coding region of the RTX A subunit as a probe.

Genomic DNA extracted from the seven serotype strains of M. bovis was digested with EcoRV and separated using agarose gel electrophoresis. The DNA was transferred to a Hybond N+ filter (Amersham, WO 01/16172 PCT/AUOO/01048 28 Little Chalfont, Buckinghamshire, using a previously described method The probe used was a PCR amplified product that contained all of the coding region from the A subunit of the RTX haemolysin of M. bovis. This fragment was labelled with O 2 P-dATP using the Megaprime labelling system (Amersham, Little Chalfont, Buckinghamshire, according to the manufacturers instructions. High stringency conditions were used (hybridisation temperature 68 0 C; 2 washes at room temperature in 2 x SSC 0.1% SDS; 1 wash at 68 0 C in 0.1 x SSC 0.1% SDS) and the resulting filters were exposed to autoradiographic film (Kodak, Rochester, New York, USA) for 5 to 24 hours before developing.

Results showed that the gene encoding the RTX A haemolysin subunit was conserved in all seven representative strains of M. bovis examined.

Interestingly, each of these strains is known to display the haemolytic phenotype on horse blood agar. In contrast, the non-haemolytic M. bovis strain Gordon 26L3 did not hybridise to the RTX A gene probe possibly suggesting that M. bovis contains only a single structural gene responsible for the haemolytic phenotype detected on horse blood agar.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

WO 01/16172 WO 0116172PCT/AUOO/01048 29 References 1. Moore L. and A. W. D. Lepper. (1991). Vet Microbial. 29:75-83.

2. Lehr, C. G. Jayappa and R. A. Goodnow. (1985). Cornell Vet. 75:484- 492.

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9. Sambrook, E. F. Fritsch, and T. Maniatis. (1989). Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York.

Dillela, A. and S. L. C. Woo. (1985). Focus. 7(2):1-5.

11. Hahn, and J. Collins. (1980). Gene. 11(3-4):291-298.

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13. Altschul, S. T. L. Madden, A. A. Schaffer, Z. jinghui, Z. Zhang, W.

Miller, and DJ. J. Lipman. (1997). Nucleic Acids Res. 25:3389-3402.

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A. Smith, and K. St~ruhl. (1994). Current Protocols in Molecular Biology.

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Heynecker, and H. W. Boyer. (1977). Gene. 2(2):95-113.

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19. Winikler, U. and M. Stuckrnan. (1979). J Bacteriol. 138:663-670.

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is Miller, and M. A. Lovett. (1993). J Bacteriol. 175:4129-4136.

26. Macknian, J-M. Nicaud, L. Gray, and 1. B. Holland. (1985). Mol Genet. 201:282-288.

27. Atwell, J. J. M. Tennent, A. W. Lepper and T. C. Elleman. (1994). J Bacteriol 176(16):4875-82.

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29. Hewick, R. M. W. H-unkapillar, L. E. Hood, and W. J. Dreyer. 1981. J Biol Chemn. 256: 7990-7997 EDITORIAL NOTE APPLICATION NUMBER 68116/00 The following Sequence Listing pages 1/14 to 14/14 are part of the description. The claims pages follow on pages 31 to WO 01/16172 WO 0116172PCT/AUOOIOI 048 1/14 SEQUENCE LISTING <110> The University of Melbourne Commonwealth Scientific and industrial Research Organisation <120> Vaccine antigens of Moraxella <160> 9 <170> Patentln Ver. 2.1 <210> 1 <211> 1114 <212> PRT <213> Moraxella bovis <400> 1 Met Ser Leu Gin Thr Gin

I.

Leu Tyr Lys Pro Val Pro Ser Asn Tyr 145 T yr Asn Met As n Ser Pro Pro Pro Ser 115 Lys Val1 Thr Ile Al a Ser Phe Thz Th r Ile 100 GI u Arg Met Thr Asp 180 5 C ys Ala Tyr Pro Pro 8 5 Ser Val As n Asp GI u 165 Th r Met Pro Asp Val 70 Al a Gly Arg Gin Th r 150 Asp Axrg Pro Leu Met Ty r Pro Pro Gly Gin Pro 135 Ser GI y Gln Ala Lys Val Ile Ile Val Tyr Leu Ser Pro Ile Pro Ile Ser 105 Pro Asp 120 Ala Pro Asn Asn Tyr Al a Ala Lys 185 Gly Phe Ala Ser Ser Gin Phe Leu Arg Pro Pro Thr Ser Tyr Thr Arg Ala Gly 140 Asn Leu 155 Arg Leu Gly Val Lys Val1 Ser Phe Glu Pro Pro Gl n Th r Lys Leu 175 Th r Ile Asn Arg 195 Phe Asn Arg Asp Leu Val Gly Ala Asn Val His Asp Thr WO 01116172 WO 0116172PCT/AUOO/OI 048 2/14 Gly Arg Ser Thr Cys Tyr 220 Gin Ile Giu Cys Val Ser Ala 210 215 Thr Pro Giu Giu Ile Pro Asn Ala Lys Ilie Gi y Lys Tyr 305 Pro Val Th r Le u Arg 385 C ys Leu Thr Ile Ala~ 465 Asn Ilie 290 Asp Asn Ile Gly Met 370 Giu Val Ser Ala Ser 450 Asn UiS 275 Phe Alia Pro His As n 355 Asn As p Ser Ser Val1 435 Gin Ser 260 Phe ;k n Gin Tyr Asp 340 Giu Ser P he Al a Tyr 420 Leu Thr P ro Met Ala Ala 245 Tyr Gly Ser Leu Met Met Asn Ser Trp 295 Arg Leu Asn 310 Arg Thr Ser 325 Leu Ile Met Gly Leu Asn Asn Phe Lys 375 Gly Lys Ala 390 Thr Ser Ser 405 Lys Gly Thr Vai Gin Ser Sle Leu Giy 455 Asn Gly Tyr 470 *Tyr Gly Ser 485 ValI A\sp Prg 280 Giy Tyr Ilie Asn Asp 360 Lys As n Th r Ser Ala 440 Thr Gin Tyr lie Ser 265 Lys Ser Asnf Th r A-rg 345 Al a Gly His Gin Pro 425 Tyr Ala Gi y Tyr Thr Thr 235 Ala Gly 250 Ile Asp2 Leu Asn Asn Asn Pro Thr 315 Asn Al a 330 Asp Ser His Asp Phe Ile Cys Gly 395 Asa Tyr 410 Ala Thr Pro Trp Lys Asp Leu Arg 475 Thr Asp 490 5er Ala ~n Asn %rg Arg Gin Asp 285 Thr Asp 300 Thr Gly Glu Vai Leu Ile Giu Asn 365 Thr Vai 380 Arg Thr Al1a Asn Ala Azg Met Lys 445 Phe Ser 460 Lys Val Asn Gin Ser Gi y Ser 270 His Gin Gin Thr Ile 350 Leu Ser Al a Asp Vali 430 Asn Giu Ser Gly Giy Met 255 Asn Giy Trp Ile Leu 335 Lys Al a Ser GI u Gi y 415 Se r Glu Ile Arg Asn 495 Ser 240 Th r Gly Val1 Tyr As n 320 Pro Ala Pro Pro Trp 400 Arg Giy As n Th r Leu 480 Phe Pro Ser Gly Tyi Tyr Vai Pro Gly Asn 500 Val Asn Trp Asn Arg Arg Ile Val Ala Asn 510 WO 0 1116172 PCT/AUOO/01048 3/14 Glu Asp Gly Trp Gly Leu Leu Asp His Asn Gly 515 Lys Asn lie Thr Trp 520 Ala Ala Ala Thr Gly Asn Asn Ser Leu G Gly Gly 625 lie Asn Leu Ser Tyr 705 Pro Asn Tyr Asp Leu 785 ;lu ;lu 610 rrP Asn Met Thr Thr 690 Arg Giu AsF Gly Lys 77C Th Val I 595 Leu Val Thr Leu Gly 675 Val Ser Val Val Ile 755 Val r Ser 580 sn rhr Asn Gin Thr 660 Glu Leu Ser Asp Gin 740 Ser Leu Asp Gly Gly Asn Arg 645 Val Thr Arg Asn Arg 725 Val Met As n Glt Lys Gly Tyr G 535 Gly Thr Pro I 550 Phe Thr Lys I Tyr Lys Gly I Asn Asn Gly 600 Tyr Gly Asn 615 Glu Gly Asn 630 Gly Val Asp Asp Gly Lys Lys Asp Gly 680 Ala Lys Arg 695 Pro Leu Phe 710 Asn Gly Arg Thr Ala Lys Asn Asp Ser 760 Asp Leu Asp 775 a Lys Gin Phe 790 ;ly .eu Lys ksp 585 Gly Val Leu Al a Ala 665 Ile Gly Glu Val Arg 745 Gly Lys Ala ;ly Ser Gly 570 Ser Ser Al a Asn Gly 650 Lys lie Leu Val Val 730 Leu Ser Lys Asn Phe 1I Val 555 Glu Val Thr I Asn Ile 635 Leu Leu Ser Glu Thr 715 Gly Ser Arg Gin Arg 795 yr Phe ;i y Ile Miet Met Val 620 Arg Lys Gly Lys Gly 700 Asn Gi Ala Val Git 78( Val 525 Trp Tyr Lys Glu Val 605 Arg Gly Al a Gly Ser 685 Gin Val Ser Gly Ala 765 a Thr Phe sp Asn Leu Gly 590 Val Gin Asp Gin Thr 670 Gly Phe Glu Arg Asn 750 Gin Gin Thr Asn Asp Va1 575 Gly Lys Thr Tyr Phe 655 Leu Ser Asp Tyr Thr 735 Val Asn Gly Gly Val Leu 560 Phe Ser Giy Gly Asn 640 Gly Asn Arg Asn Thr 720 Asn Va1 Leu Ser Phe 800 Glu Asn Met Asn Ser Gly Ala Glu Ser Lys Leu Ser Thr Val Ser Thr WO 01/16172 PCT/AU0/01048 4/14 Asn Arg Glu Leu Tyr Lys Leu Asp Pro Thr Phe Tyr Ala Asp Ser Ala 820 825 830 Leu Asn Ala Val Glu Asp Ser Ala Asn His Ala Thr Glu Phe Gly Lys 835 840 845 Arg Val Ser Ala Pro Arg Gly Val Trp Gly Asn Ile Ser His His Asp 850 855 860 Tyr Asp Val Glu Leu Glu His Ala Thr Ser Ala Arg Lys Gly Asn Asn 865 870 875 880 Ile Ser Val Gly Ala Ser Thr Gin Thr Ala Ala Asp Ile Ser Val Gly 885 890 895 Ala Gin Leu Asp Val Ser Lys Leu Asp Leu Glu Glu Ser Val Tyr Gly 900 905 910 Ile Gly Asn Lys Thr Lys Thr Asp Ser Ile Gly Leu Thr Val Gly Ala 915 920 925 Ser Lys Lys Leu Gly Asp Ala Tyr Leu Ser Gly Trp Val Lys Gly Ala 930 935 940 Lys Val Asp Thr Glu Ala Asn Arg Gly Glu Asn Ser Asn Lys Val Glu 945 950 955 960 Tyr Asn Gly Lys Leu Tyr Gly Ala Gly Ile Gin Ala Gly Thr Asn Ile 965 970 975 Asp Thr Ala Ser Gly Val Ser Val Gin Pro Tyr Ala Phe Val Asn His 980 985 990 Gin Gin Tyr Lys Asn Asp Gly Ser Phe Asn Asp Gly Leu Asn Val Val 995 1000 1005 Asp Asp Ile Glu Ala Lys Gin Thr Gin Val Gly Val Gly Ala Asp Met 1010 1015 1020 Val Phe Gin Ala Thr Pro Ala Leu Gin Leu Thr Gly Gly Val Gin Val 1025 1030 1035 1040 Ala His Ala Val Ser Arg Asp Thr Asn Leu Asp Thr Arg Tyr Val Gly 1045 1050 1055 Thr Ala Thr Asp Val Gin Tyr Gly Thr Trp Asp Thr Asp Lys Thr Lys 1060 1065 1070 Trp Ser Ala Lys Val Gly Ala Asn Tyr Asn Val Thr Pro Asn Ser Gin 1075 1080 1085 Val Gly Leu Asn Tyr Ser Tyr Thr Gly Ser Gly Asp Ser Asp Ala Ser 1090 1095 1100 Gin Val Gly Val Ser Phe Thr Ser Lys Phe 1105 1110 WO 01/16172 WO 0116172PCT/AUOO/01048 5/14 <210> 2 <211> 4384 <212> DNA <213> Moraxella <400> 2 ttc-tcatgtt ttgctaacgc caccgtcacc gcgggatatc tgcgttgatg ccgcccagtc cacacccgtc ttattcgtaa tttgtaaata aatcttattt tttgattaaa atcaatattg aataacttgt gaggtgtaat aagtatggct tccaatgatt agatttcctt tcctgaactc ggcaccaatt gagacagcct aagtgctggc atctaaattt caccattgat ccgagacttg ttccacctgc tagtggtagt cgccaaaatt gatgatgcg t taacaacact acagattaat cattcatgat cttgaacgat tttcattact tgccgaatgg gagtagctat gcaatctgct caaggatttc tagtagaitg tgttcctggc cattacatgg tggtttctat caatgaccta t gg taat aat taacaacggt agctaatgtt tgactacaaC catgcttacc caaagatggt tcttgaaggt tgacagctta agtcaggcac ctgga tgctg gtccattCCg caatttctat ctgctcgctt ctgtggatca tattgccata ataaacattt ttgaaaatac attgccaaaa caacatggta t-2tggatgat atgtcattac tgcatgctgg gttgattcac aaacgtttta gttcgtccga agtggcggta gatitacaaa acacgtacag tatggcacaa acacgtcaag gttggtgcaa tatacgccag catggcaacc tacggcagtg aagctgaacc gaccaatggt ccaaatcctt cttattatga gctcatgatg gtctcctcgc tcjtgtatccg aagggtacat tatccttgga icagagatta ccatctggtt aatgtcaatt gaagatggtt tgggataatg aaaggtgata *agctataaag *ggttcaacca cgtcaaacag atcaacactc gtggacggta atcatcagca caatttgaca tcatcga Laa cgtgtatgaa taggcatagg acagcatcgc gcgcacccgt cgctacttgg ataattaatg aaaataatac gtttatctaa aataatactg cttgtgtaaa aatgattgct tgatggcaat a aact caa cc taattagtgc agtacaa tag gaccaactcc ccccagcccc tatcaggtag gacgcgttca gttatagtgt ccgaagatgg ccaaagtagg atgtgcatga aaaatgattc aaatggCggc acagtattga aagaccatgg actacgatgc acagaaccag atcgtgactc aaaacctagc ctagagaaga caacatcatc cacctgcaac tgaaaaatga ctgccaattc attacggctc gggaaaaccq ggggtttgtt tgga at taga aaggctttac gcgactctgt tggttgttaa *gtggttgggt aacgtqgcgt *aggccaaat *aatcaggtac *attatcgttc gctttaatgc atctaacaat cttggttatg cagtcactat tctcggagca agccactatc aacatatata at tat ttcta aaaaataaat caattgctta taagtttcac atgttgttgg gataaactta tgccaagaga tagtagtacg ttctaaatac aactccagtg gattccggct c tat attg ct agccaatcta catggatacg ttatgccgag tgtgattgat tacacagatt aggcattgtt tgtcatcgct tcgacgttca tgt ca agat t tcagcgccta tattaccaat gcttatcatt accgctcatg ttttggtaaa tacccaaaat *cgctcgtgtg aaatatctct *acctaatggc *ttattacact tcgaattgtC *agatccagaa Lcactaaaggc *caaaaaaggt *catcgagggt iaggtggtgaa *taacaacgaa -ggatgctggt *aggtggtaca ccgtagcact -aagcaaccca ggtagtttat gcgctcatcg ccggtactgc ggcgtgctgC ctgtccgac gactatgCga ctctatttaa tattaactaa aatataaatC atctagacat cgaattgata gcattgcata gtgacaatga ggttctatg gtaagttatg tctttctacg ccaagccctg ccaacgcctg ccagtatcgC aaacgCaac tcaaataatt cgtcttgaca acaggcatta gagtgtgttt gaaatcccaa ggtaacaacg aatggtggCa tttaacaact aattacaatc gctgaagtga aaagcaaCag aacagcaact gcgaatcatt ta cgccaacg tccggcacgg caaaccattt taccaaggac gacaatcagg gctaatcata gcggccgcta acgcctttat gaaggtaaaC ggttcactag ctaacaggtt ggtaacctaa ctaaaagctc ctaaatctaa gtacttcgtg ttatttgaag cacagttaaa tcatcctcgg 120 cgggcctctt 180 tagcgctata 240 gctttggccg 300 tcatggcgac 360 tatttcttat 420 actgttaata 480 aagcaattac 540 taagtttatt 600 ctttaagggt 660 aattgtctat 720 taaacqcaaa 780 ttaagccttt 840 ccaactcagc 900 attactattt 960 tgagaccggc 1020 tgccaacacc 1080 catcagaggt 1140 aacctgcacc 1200 ctaatttgac 1260 acctaaagaa 1320 accgcttcaa 1380 ctgctggacg 1440 caacctctgc 1500 gcatgaccaa 1560 accatttctt 1620 cttggggttc 1680 ctactacagg 1740 ctttgcctgt 1800 gtaacgaagg 1860 tcaaaaaagg 1920 gtggtcgaac 1980 atggcagact 2040 cagtgctcgt 2100 tgggtactgc 2160 taagaaaggt 2220 gtaatttcta 2280 acggcaagaa 2340 aaggttatgg 2400 ctgtattcta 2460 ttgtctttac 2520 aagtaaatgg 2580 atggtaatgt 2640 acatcagagg 2700 aatttggcaa 2760 ctggtgagac 2820 ctaagcgtgg 2880 taacaaatgt 2940 W001/16172 WO 0116172PCT/AUOO/01048 tgaatatacg tgacgtgcaa.

gaatgacagt aaaacaagaa cactggtttt ccqgagcta agacagtgct gggtaatatc aggcaacaac acaacttgat caaaactgac atcaggttgg caaagttgag tactgcatcg cgatggtagc ggtaggtgtg tgtgcaagtt agcgacagat tggtgctaac tagtggcgat taataaggca aaaattttcc gtattgcatt aatcggtgct cgac ccagaagtag gtaactgcca ggtagccgtg acacaaggtt gaaaa ca tga tacaagcttg aaccatgcaa agtcaccatg attagtgttg gtaagtaaac agcattggct gtaaaaggtg tacaatggta ggcgtgagtg ttcaatgacg ggtgctgata gctcacgctg gtacagtatg tataatgtga tcagatgctt acaaaaaaca caaaaaaagc tatgggttgt aacttgtttt acagaaatgg aacgtctaag ttgcacaaaa cactgaccag attctggtgc acccaacttt ccgaatttgg attatgatgt gtgcaagcac ttgacttgga tgactgttgg ccaaagttga agctatatgg tacaacctta gtcttaacgt.

tggtgttcca ttagccgtga gcacttggga caccaaacag cccaagtggg qcacaatttc gtgataatta taagcaaccc gccacaggct 6/14 cagagtggta tgcaggcaat cctagacaaa tgatgagaag agaatctaaa ctatgctgac taagcgtgtt agaactagag tcaaa ctgca agaatctgtt tgcttctaag tacagaagcg tgctggtatc tgcctttgtt tgttgacgac agcaacacct caccaaccta tactgacaaa ccaagtgggt tgtgagcttc ggttgtgctg ccacgctttt gtccaaatac cgtcaatgtg ggtggttcac gttgtttatg gtacttaatg caatttgcta ctttctacag agtgcattaa agcgccccaa catgctacaa gccgacatta tatggtattg aagttgggtg aaccgtggtg caagcgggta aaccatcagc atcgaagcaa.

gctctacagc gacactcgct accaaatggt ctaaactaca accagcaagt ttttttgtga ttattgcata cccctaaaca tcggcatcat gcacgaacaa 3000 gcatcagcat 3060 atttagataa 3120 accgtgtatt 3180 taagcaccaa 3240 acgcagtaga 3300 gaggtgtttg 3360 gtgcacgtaa 3420 gtgttggtgc 3480 gcaacaaaac 3540 atgcctatct 3600 aaaactctaa 3660 caaacattga 3720 agtacaaaaa 3780 aacaaactca 3840 ttactggtgg 3900 atgttggtac 3960 cagccaaggt 4020 gctacacagg 4080 tctaattcat 4140 tgccgagcgt 4200 ttgcaaaata 4260 actccacccc 4320 caaccaitac 4380 4384 <210> 3 <211> 616 <212> PRT <213> moraxella bovis <400> 3 Met Lys Lys

I

Ser Ala Phe Ala Lys Tyr 5 Ala Leu Ala Leu Met Val Gly Met Cys Leu His Thr Ala Tyr A-la 25 Lys Glu Phe Ser Gin Val Ile Asp Met Val Ile Phe Gly Ala Arg Lys Asp Set Leu Ser Thr Gly Arg Leu Asp Gly Thr Leu Gly Asn Thr Leu Gin Pro Ser Phe Thr 55 Trp Ser Ser Leu Phe Ala Gin ser Tyr Gly Asn Pro Asp Pro Lys Thr Ala Ser Ala Asn Thr Pro Tyr Pro Thr Gly Thr Asn Tyr Ala Val Gly Ala Arg Ser Gly Ser 105 Glu Val Asn Trp Asn Gly Phe 110 Val Asn Val Pro Se: Thr Lys Thr Gin Ile Thr Asp His Leu Thr Ala WO 01/16172 PCT/AUOO/01048 Thr Gly Gly Lys Ala Asp Pro Asn 130 135 7/14 Thr Leu Tyr Ala Ile Trp Ile Gly 140 Ser Asn Asp Leu Ile Ser Ala Ser Gin Ala

I

145 Gin A Thr I Asp I Gly Leu 225 Thr Phe Gly Leu His 305 Ile Lys Leu Arg Ser 385 Asp Ls n .eu ,eu /al 210 ?he Phe Lys Ala lie 290 Pro Met Thr Ser Thr 370 His Asp Ala Asn Ser 195 Gin Glu Ala Asn Asp 275 Glu Ser Asp GIy GIy 355 Asp Thr Thr Ile Gin 180 Leu Asp Ala Leu Thr 260 Asp Asn Gly Ala Ser 340 Ser Pro Gly Leu Asp 420 Lys 165 a rhr Lys Leu Leu 245 Gin Val Gly A.r g Pro 325 Ala Gin Thr Ala Ser 405 150 Gly Gly I Pro I Ala Asn 230 Gin Gly Ala Ala Thr 310 Thr His His Thr Tyr 390 Ser daa kla krg Lys 215 Gln Glu Val Ser Asn 295 His His Asp Ser Gin 375 Leu Asp Val Thr Ala 200 Leu Ser Ala Al a Thr 280 Asp Arg Met Arg Ile 360 lie Sex Val Thr Thr 185 Ile Ala Thr Thr Cys 265 Ser Thr Ile Gly His 345 Trp Gly H His Lys L Arg 425 %rg 170 Ile ryr Ser Aila Thr 250 Gin Leu Tyr Leu Lys 330 Val Ala Leu Gin Thr Thr 155 Thr Leu Gly Ser Asn 235 Asn Met Alla Ala Ala 315 Leu Tyr Asr Asp Asr 395 Ili Thr Val Val Glu Leu 220 lie Lys Pro Cys Phe 300 Gin Ser Arg Vai Val 380 Gin i Gly rhr Ile Pro Ser 205 Tyr Ile Glu Ala Thr 285 Ala Tyr GIy Gin His 365 Ala Asp Met Ala Glu Asp Ile 175 Asn Val 190 Leu Met Asn Ser Pro Ala Ala Phe 255 Arg Thr 270 Lys Ala Asp Asp Tyr Arg Glu Leu 335 Leu Asp 350 Ala Ser Gly Ser Tyr Val Gly Leu 415 Ala 160 Glu Pro Ala Gly Asn 240 Gly Thr Asn Ile Ser 320 Val Arg Asp Ser Leu 400 Tyr His Arg His Ile Gly Asn Va Leu Lys Gly Val Ala Gly Ile WO 01/16172 WO 0116172PCT/AUOO/01048 8/14 Arg His Ile Asp Arg Leu 435 Ser Val Asp Thr Asp Trp 445 Arg Phe 460 Giu Gly Ala His Ala Gly Ser Arg 450 Ser His Thr Ala Asp 455 Thr Thr Ala Arg Gin Ala Ser Tyr Ile Asp Met Gly Lys Ala Thr Val Arg 475 Leu Ile Gly Val Ala Gin Lys Val Val Arg Asp Leu Val Giu 495 Asn Glu Pro Lys Ser Leu 515 Leu Ser Thr Ala Arg Phe Gly Giu Gin Giu Gin 510 Pro Ile Ser Gin Gly Glu Ile Val Asp Val Ala Pro Ala 530 Leu Thr Leu Thr Giy Ile Ala His Ala His Glu Phe Asn 540 Asp 545 Asp Giu Arg Thr Asn Ala Thr Leu Ser Ile Arg Glu Thr Lys Gly Phe Thr Ser Vai Ser Asp Lys Ser His Al a Thr 575 Thr Ala His Ala Gly Val 595 Gly Val Gin Gly Gin Leu Gly Lys Ala 585 Asn Ile His 590 Val Gly Gly His Ala Thr His Asp 5cr Asp Thr Ser Leu 610 Gly Val Arg Leu Met Phe 615 <210> 4 <211> 2110 <212> DNA <213> Moraxeila bovis <400> 4 tgacaaataa aaaaaaacac aagcaactca taatgqttgg ttggggacag cucttggcaa tatttgccca ctaactatgc atgtaccctC accctaatac ccaccacaac acatcgaaaC tgagcctcac ttgggcattg gagagtaata gatgtgcctg cttgtccgat caccttacagaagttatggc cgtgggcgga caccaaaacg cctgtatgc agccgaagcc act caatcaa gccccgagcc ggcagataac tgtaatatct atgaaaaaat cacaccgctt acaggtcgcc ccatctttta aaaaccgcca gctcgctctg caaatcaccg atttggattg caaaacgcca gcaggggcga atctatggcg ccatcaaaga tgttacactt ccgcctttgc acgccaagga taaaagatat ccaccaaccc gtgccaacac gctcggaggt accatttgac gctctaatga ttaaaggtgc caaccatttt aaagcctcat cccaaagcaa tacaagtgtt caaatactca gtttagccaa ggtcgcccga cqaccctgta gccctacaat ca at tgga at cgccacaggt cttaatttca ggtaactcgc ggtgccaaat ggcaggcgtg cccataaatc tttactttga 120 gcacttgccc 180 gtcatcattt 240 aaagatggca 300 tggtcaagct 360 cccactggca 420 ggttttgtga 480 ggcaaagccg 540 gcttctcaag 600 accgtgatag 660 gtgcctgatt 720 caagacaaag 780 WO 0 1/16172 WO 0116172PCT/AUD0101048 ccaaactcgc ccaacatcat cctttggttt cggatgatgt caaatgacac tggcacagta agcttgtcaa gtggctcaca cccaaatcgg aaaaccaaga ggctgtatca gacttagcgt cagacaccac gcaaagccac tggtagagaa ccctacaagg cgggcggtat taacctccat acgccaccac gcgttcacgc tgatgttttg aaaatctatg caaacatgag ctcaagtctg ccctgccaaC taaaaacacg ggcttctact ctacgccttt ttaccgttct aacaggttca gcacagcatt cttggacgtg ttatgtgctg tcgccatgac ggatacgcac cgccagacgt cgtgcgtcCg tgagcctac cgagattggc cgctcacgct tcgtgaatac cgctcatctg cacccaccaa attggctttt tttgagtaca tataatagcg acctttgccc caaggcgtgg tccttggcat gccgatgaca atcatggacg gcccacgacc tgggcaaacg gcaggttcat gatgacaccc a tcggcaatg cgccatatcg tttcatgcag cttatcggcg ctatccaccg gtcgatgtgg catgagttta acgaagggct ggcgtacaag gacagcgata aaagataaaa tcaaagcctt 9/14 gtctgtttga tactccaaga cgtgtcaaat gtaccaaagc ttcacccatc cccctactca gtcatgttta tccatgccag caagccatac tatcatcaga iccgtctaaa actgggaggg ggctacaagc tacatgccca ccatgcgttt cttatccgat acgatgatga ttaatacaag ggcaacttgg cagacgtggg agtggtatca tcacatcatc agcattaaat agcgaccaca gcccgctcgt caatcttata gggacgcacg catgggtaaa ccgtcagctt cgaccgtacc aggggcgtat tgtcaaaacc aggcgtggca ggcaagccgt cagctatggc aaaagtcaaa tggcgagcaa tagccctgct acgcaccatt cgttagcacc caaggcaaat tggttcgctt tgccactttt gccatgcgat caatccaccg aataaagaag accacagggg gaaaacgggg caccgcattt ctctcaggcg gacaggctta gaccccacca ctgagccacc attggcatgg ggtatcgacc tcgcacacgg atagacatgg gtgcgtgatt gaacaaaagt ttgactctga aatgccactt gacaaatctc attcatgcag ggggttcgct tattttgcca gataagctgt 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 19B0 2040 2100 2110 <210> <211> 927 <212> PRT <213> Moraxelia bovis <400> Met Ser 1 Asn Ile Asn Val Ile Lys Set 5 Ile Gln Ala Gly Leu Asn Ser Thr Lys Tyr Asp Pro Gly Leu Lys Asn Tyr Leu Ala Ile Pro Lys Asp Lys Ala Ala Gin Lys Gly Gly Leu Asn Asp Phe Asp Glu Leu Gly Ile Ala Arg Leu Ala Glu Giu Asn His Thr Giu Ala Lys Lys Ser Asp Thr Val Asn Phe Leu Ser Leu Gin Thr Gly Ile Ile Ser Ala Thr Leu GiU Lys Phe Leu Gin Lys His Ser Thr Asn Lys Leu Ala 100 Gly Leu Asp Ser Val Giu Asn 110 Thr Leu Ser Ile Asp Arg 115 Lys Leu Gly Lys Ser Asn Val Leu Ser Phe Leu Gly Thr 130 Ala Leu 135 Ala Gly Ile Giu Asp Ser Leu Ile WO 01/16172 PCT/AUOO/01048 10/14 Lys Lys Gly Asp Ala Ala Pro 145 150 Leu Ile Asn Giu Ile Ile Gly Glu Ala Ser Ile 225 Lys Thr Leu Leu His 305 Tyr lle Ala Ala Al a 385 Lys Lys la Lys Lys 210 Ser Val Tys Ser Ala 290 Ala Asp Glu Gly Leu 37 Sez lie Gi Phe I Gly 195 Thr Ala Ala Ala Thr 275 Ile Asn Gly SAla Val 355 Leu r Lys i Leu y Tyr er ?he Asn Gly Ala Ile 260 Thr Ser Ala Asp Ser 340 Ser Val Gin Gl.

As; 165 Ser C Ser Leu Phe Gly 245 Ser Gly Pro Leu His 325 Leu Ala Ala Ala Trp 405 Ser 1in ksn ;ly kla 230 Phe Ser Ala Leu Asp 310 Leu Thr Ala Gly Met 390 Glu Arg Leu Ile Leu 215 Leu Glu Tyr Val Al a 295 Glu Leu Thr Ala Val 375 Phe Lys Tyi Asp Ala Asn Leu Ala Lys 185 Gly Asn 200 Giu Ile Ala Asp Leu Ser Val Leu 265 Ala Ala 280 Phe Met Phe Ala Ala Glu Ile Sez 345 Val Gly 360 Thr Gi Glu Set Gin Asr Ala Ai 42! Leu Ala I 155 Ser Gin 170 Leu Gly Lys Leu Ile Thr Lys Asn 235 Asn Gin 250 Ala Gin Leu 1Ie Asn A-la Lys Gin 315 Tyr Gin 330 Thr Aa Set Ala Leu Ile Vai Ala 395 Gly Gly 410 i Tyr Leu Lys Ser Set Gin Gly 220 Al a Val Arg Thr Ala 300 Phe Arg Leu Val Ser 380 Asn Gin Al a Ala Thr Thr Asn 205 Leu Ser Ile Val Ser 285 Asp Arg Gly Gly Gly 365 Gly Arg Asn Asn Ser Ile Asp 160 Gin Thr Ile 175 Ile Set Gin 190 Leu Asn Phe Leu Ser Gly Thr Gly Lys 240 Gly Asn Val 255 Ala Ala Gly 270 Ser Ile Met Lys Phe Asn Lys Phe Gly 320 Val Gly Thr 335 Ala Val Set 350 Thr Pro Ile Ile Leu Glu Leu Gin Gly 400 Tyr Phe Asp 415 Asn Leu Lys 430' Val Ile Ala 420 Phe Leu Set 435 Glu Leu Asn Lys Leu Glu Ala Glu WO 01/16172 PCT/AUOO/01048 11/14 Ile Thr Gin Gin Arg Trp 450 Thr 465 Phe Ala Gin Arg Phe 545 Lys Thr Ile Asp Asp 625 Val Lys Arg Glu Phe 705 Gly Lys Leu Gly Glu lu Lys Al a Glu 530 Gly Leu Asp Phe Arg 610 Gly Al a Val Lys Glu 690 Asn Ala Asp Thr Leu I 515 Arg Arg Asp Glu Vai 595 Val Thr Arg Gly Val 675 Vai Asp Gly 3iy Gly 500 His Leu Val rhe Ile 580 Gly Phe Ser Gly Lys 660 Giy lie Ile Asp Lys 485 Ile Phe Thr Lys Ser 565 Gly Gin Tyr Al a Asp 645 Arg Tyr Gly Phe Asp 725 Arg 470 Lys Ile Thr Asn Asn 550 Lys Leu Gly Ser Thr 630 Ile Thr Gly Ser His 710 Arg Asp 455 Ile Val Asp Ser Gly 535 Trp Vai Ile Lys Lys 615 Glu Tyr Glu Tyr Gin 695 Ser Leu Lys Ser Glu Ala Ile Ser 505 Pro Leu 520 Lys Tyr Gin Vai Ile Gin Vai Asn 585 Met Asn 600 Asp Gly Ala Gly His Glu Thr Ile 665 Gin Ser 680 Phe Asn Gly Glu Phe Gly Gly Gly 490 Asn Leu Ser Thr Arg 570 Ala Ile Giy Ser Val 650 Gin Thr Asp Gly Gly 730 1 Lys 475 Ser Ser Thr Tyr Asp 555 Val Lys Asp Phe Tyr 635 Val Tyr Asp Va1 Asp 715 Lys 460 Ala Asn Asn Ala Ile 540 Giy Ala Ala Gly Gly 620 Thr Lys Arg Asn Phe 700 Asp Gly Tyr Ile Gly Gly 525 Asn Glu Glu Gly Gly 605 As n Vai Arg Asp Leu 685 Lys Leu As n Ala Thr Lys 510 Thr Lys Ala Thr Asn 590 Asp Ile Asn Gin Tyr 670 Lys Giy Leu Asp ksn Asn Ile Giy Giu Leu Ala Gly Ile Ala 480 Asp Thr Ser Lys Ser 560 Gly Asp His Val Lys 640 Thr Leu Val Lys Gly 720 Leu Ser Gly Asp Giu Gly Asp Asp Leu Leu Asp Giy Gly Ser Gly Asp Asp I An 745 750 WO 01/16172 WO 0116172PCTAUOO/0I 048 12/14 Val Leu Asn '755 Gly Gly Ala Gly Asn Asp Val 760 Tyr Ile Phe 765 Arg Lys Gly Asp Gly 770 Asn Asp Thr Leu Asp Gly Thr Gly Asn Asp Lys Leu Ala 780 Giu Arg Thr Lys Glu Ala Asp Ala Asn Ile 790 Ser Asp Ile Met Gly Ile Ilie Val Arg Asn Asp His Gly Ser Ile Asn Ile Pro 815 Arg Trp Tyr Asp His Lys 835 Thr Ser Asn Leu Asn Tyr Gin Ser Asri Lys Thr 830 Tyr Ile Thr Ile Giu Gin Leu Gly Lys Asp Gly Ser Asp 850 Gin Ile Asp Lys Leu Gin Asp Lys Asp Giy Thr Val Ile Thr Ser Gin Glu 865 Lys Leu Ser Aia Ser 885 Lys Lys Leu Ala Giu Asn Lys Ser Asp Ile Ala Ser Leu Asn Lys Leu Val Gly 895 Ser Met Ala Gin Pro Ilie 915 Phe Gly Thr Ala Ser Val Ser Ser Asn Ala Leu 910 Thr Gin Pro Thr Gin 920 Gly Ile Leu Ala Pro Ser Val 925 <210> 6 <211> 3231 <212> DIIA <213> Moraxelia bovis <400> 6 atgagaacgt catggtaaga tttgcacaat tctaaagata ataaatgtaa aatctt tact tttattaaag gaaacagaaa attgctattt gccaaagggt ttatcaacat atcaaaaaag gaga taattg gcaaagttag caaaacttaa tattttcaga.

tatctgaatt atcacgaaga attaatacaa ttaaatctaa tggctattCC ctgctgatga aaaaatctgt ctgcaacaaa tagacagtgt taagctcttt gtgatgctga gtaatctatc gttctactat atttttctaa tgaattgttt ttatggaaag tttgacgagc ccttttctaa tattcaagca caaagattat attaggtatt tgacacagta attagaaaag agaaaatatt tttgggcact acctgatgct tcagagtact atcgcaggct aacaaatct agagcgattc tctgttgatt aaattgtcaa cacaacgagg ggcttgaatt gatccgcaaa gctcgtttag aatcagtttc ttcttacaaa gatcgtaaat gcattagcgg ttggctaaag caaacgattg aaaggcttct ggtttggaaa gtgtagatgg caaaattagc ctcagaataa agagacatat caacaaagtc aaggtgggac cagaagagcc tctctctcac aacattctac taggtaaagc gtatagaact ctagtattga aagcattttc ctaatatagg taattactgg aaattcatcg ctcaagaata ttttattata tatgtccaat tggattaaaa tttaaatgat taatcacact acaaactggt caataagtta aagtaatgta tgattcttta cttgatiaat ttcacagtta aaacaagttg tttgctatca WO 01/16172 WO 0116172PCT/AUOO/01048 13/14 ggcatttctg gcaggttttg gttttagcac acttcatcga aatcatgcta gatcatttat atiagtacgg gttggtacac gaagcgtcta gagtgggaaa gctgcttatt gaacgtgtta attaccaaat ggcaagaaag attagtaatt gcaggaactg aaattcggac ttctctaaag gtaaa tgcaa ggtggagatg gtagatggta ggtgatatct actatccagt aatttgaaat aaattcaacg gacgaccgct ttactcgatg atctttcgga gcatttgcag gttaaacgaa ttacaaaatt ggtagttata gtaattacat gcttcggaca gcaaatagtq gctccaagtc~ agattatgaa ccttaatagt tatccttgcc caggctttgC t aattaagcaat aacgtgttgc ttatgttggC atgctcttgaI tggctgaataI cattaggtgc cgattgcact aacaggcaat agcaaaatgg tagctaataa ttgcaatcac tgggtgaacg ttgaagctgg caaatgggaa aatcacgtga gtgtaaaaaa ttattcagcg aagctggcaa gacacgatcg cgaqtgcaac accatgaagt atcgtgatta cagtagaaga acatattcca tgtttggtgg gcggtt ctgg aaggtgatgg atgcaaatat atgatcattc atcaaagtaa tcacttccga ctcaagaatt ttgcaagtag tgagttctaa tttagtgatt tactagtgata aatttagtta aaatactatg :ttagcggat :caagttatt :gctggtcta iattagtcct tgagtttgca tcagcgtggt agtttctgct attagttgca gtttgaaagt cggtcagaac cttaaaattt ccaacaacgt cattaagagc ttccaatatt aaaaacgcaa acgtttaact c tggca a gt t tgtagccgag tgacgatatc tgtcttctat agaagcaggc tgtgaagcgt tgaattaaga agtaattggt tagtggtgaa taaaggcaac tgatgatgta taatgatact atctgatatt aggtagtatt taaaacagat tcaaattgat gaaaaagctt cttaaataag cgccttacag taatttacta tgggtggtga tgatagatta gtatttctgc aaaaatgcat ggtaatgtaa tcaactactg ttggcattta aaacaaitcc gtgggtacta ggtgtttccg ggtgttacag gttgctaacc tattttgata ttgtctgagc tgggataata ggaaaagctt actttggatg gcgttgcatt aatggtaaat acagatggag acagaaggca tttgttggtc agtaaagacg agttatacag caagaaacca aaagttgggt tctcaattta ggtgatgatt gatcgacttt ttaaatggtg ttgtacgatg atgattgaac aacataccaa cataaaattg aaaattttgc gctgatgaga ctagttgggt ccaattacac gacaa tat ca tacttcttta tgctcaacaa aagtccagca cgactggcaa caaaagcaat gtgctgttgc tgaatgcagc gaaaatttgg ttgaagcttc ctgctgCtgt gattgatctc gtttacaagg aaggctatga taaataaaga atattggtga atgcagatgc ctaaaactgg tcacttcgcc actcttatat aggctagttc cagacgagat aaggtaaaat gaggatttgg ttaatcgtaa aggtgggtaa atggttatca atgatgtatt tact cgatgg ctggagatga gtgctggtaa gcacgggcaa gtaccaaaga gatggtacat agcaactaat aagataagaa ataagagcca caatggcact aaccaactca ccacccatat attagactta cctgctctat gacattatgc aaaagttgct 960 ttcttcatat 1020 tgctttaatt 1080 agataaattc 1140 ctatgatggg 1200 attaactaca 1.260 aggatctgct 1320 tggaatttta 1380 taaaatttta 1440 ttctcgttat 1500 gttggaagct 1560 gttagcaggt 1620 ttttgaagat 1680 tatcatagac 1740 tttgttaaca 1800 taataagtta 1860 taaattagat 1.920 tggtctaata 1.980 gaatattgat 2040 taatattact 2100 ggttgctcga 2160 acgtactgaa 2220 gtctaccgat 2280 caaaggttct 2340 tggtgctggt 2400 aggcgatgat 2460 tgatgtctat 2520 tgataaatta 2580 gggtattata 2640 aacatcaaat 2700 tIggtaaagat 2760 agatggtaca 2820 aaaartatct 2880 atttggtaca 2940 aggaattttg 3000 cattggttat 3060 atttacaaac 31.20 ctgctctggt 31.80 a 3231 <210> 7 <211> 17 <21.2> PRT <21.3> moraxella bovis <220> <221> misc feature <223> Xaa unknown <400> 7 Lys Giu Phe Ser Gin Val Ile Ile Phe Gly Asp Ser Leu Xaa Asp Xaa 1 5 10 WO 01/16172 WOOI/6172PCT/AUOO/01 048 14/14 Gly <210> 8 <211> 64 <212> PRT <213> Moraxelia bovis <400> 8 Met Arg Thr Let' Phe Set Asp Git' Leu Phe Azg Ala Ile Arg Val Asp 1 5 10 Gly Asn Ser set His Gly Lys Ile Ser Giu Phe Tyr Gly Lys Ser Val 25 Asp Ser Lys Let' Ala Ser Arg Ile Phe Ala Gin Tyr His Git' Asp Leu 40 Thr Ser Lys Leu Set Thr Gin Asn Asn Phe Ile Ile Ser Lys Asp Asn 55 <210> 9 <211> 57 <212> PRT <213> Mozaxelia bovis <400> 9 Met Gly Gly Asp Thr Ser Le' Ile Arg Let' Asn Let' Gin Thr Let' Asn 1 5 10 Ser Asn Let' Val Met Ile Asp Tyr Ala Gin Gin Pro Ala Let' Ser Ala 25 Let' Val Ile Leu Ala Lys Tyr Tyr Gly Ile Ser Ala Ser Pro Ala Asp 40 Ile Met His Arg Let' Ala Lys Lys Let'

Claims (34)

1. A polypeptide, the polypeptide having an amino acid sequence as set out in SEQ. ID. NO. 1 from amino acid 37 to 1114, or a sequence having at least 50% identity thereto, or a functional fragment thereof.

2. A polypeptide as claimed in claim 1, the polypeptide having a sequence of at least 70% identity with the sequence shown in SEQ. ID. NO:1 from amino acid 37 to 1114.

3. A polypeptide as claimed in claim 1, the polypeptide having a sequence of at least 80% identity with the sequence shown in SEQ. ID. NO:1 from amino acid 37 to 1114.

4. A polypeptide as claimed in claim 1, the polypeptide having a sequence of at least 90% identity with the sequence shown in SEQ. ID. NO:1 from amino acid 37 to 1114. A polypeptide as claimed in any one of claims 1 to 4, the polypeptide having protease activity.

6. A nucleic acid molecule, the nucleic acid molecule comprising a sequence encoding a polypeptide as claimed in any one of claims 1 to

7. A nucleic acid molecule comprising a sequence as set out in SEQ. ID. NO:2 or a sequence having at least 60% identity thereto, or a sequence which hybridises thereto under stringent conditions.

8. A nucleic acid molecule as claimed in claim 7, the nucleic acid molecule comprising a sequence having least 70% identity with the sequence shown in SEQ. ID. NO:2.

9. A nucleic acid molecule as claimed in claim 7, the nucleic acid molecule comprising a sequence having least 80% identity with the sequence shown in SEQ. ID. NO:2. WO 01/16172 PCT/AU00/01048 32 A nucleic acid molecule as claimed in claim 7, the nucleic acid molecule comprising a sequence having least 90% identity with the sequence shown in SEQ. ID. NO:2.

11. A composition for use in raising an immune response in an animal, the composition comprising the polypeptide as claimed in any one of claims 1 to or a nucleic acid sequence as claimed in claim 6 and optionally a carrier and/or adjuvant.

12. A polypeptide, the polypeptide having an amino acid sequence as set out in SEQ. ID. NO:3 from amino acid 26 to 616, or a sequence having at least identity thereto, or a functional fragment thereof.

13. A polypeptide as claimed in claim 12, the polypeptide having a sequence of at least 70% identity with the sequence shown in SEQ. ID. NO:3 from amino acid 26 to 616.

14. A polypeptide as claimed in claim 12, the polypeptide having a sequence of at least 80% identity with the sequence shown in SEQ. ID. NO:3 from amino acid 26 to 616. A polypeptide as claimed in claim 12, the polypeptide having a sequence of at least 90% identity with the sequence shown in SEQ. ID. NO:3 from amino acid 26 to 616.

16. A polypeptide as claimed in any one of claims 12 to 15, the polypeptide having lipase activity.

17. A nucleic acid molecule, the nucleic acid molecule comprising a sequence encoding a polypeptide of any one of claims 12 to 16.

18. A nucleic acid molecule comprising a sequence as set out in SEQ. ID. NO:4 or a sequence having at least 60% identity thereto, or a sequence which hybridises thereto under stringent conditions. WO 01/16172 PCT/AU00/01048 33

19. A nucleic acid molecule as claimed in claim 18, the nucleic acid molecule comprising a sequence having at least 70% identity with the sequence shown in SEQ. ID. NO:4.

20. A nucleic acid molecule as claimed in claim 18, the nucleic acid molecule comprising a sequence having at least 80% identity with the sequence shown in SEQ. ID. NO:4.

21. A nucleic acid molecule as claimed in claim 18, the nucleic acid molecule comprising a sequence having at least 90% identity with the sequence shown in SEQ. ID. NO:4.

22. A composition for use in raising an immune response in an animal, the composition comprising a polypeptide as claimed in any one of claims 12 to 16 or a nucleic acid sequence as claimed in claim 17 and optionally a carrier and/or adjuvant.

23. A polypeptide, the polypeptide having an amino acid sequence as set out in SEQ. ID. NO:5, or a sequence having at least 60% identity thereto, or a functional fragment thereof.

24. A polypeptide as claimed in claim 23. the polypeptide having a sequence of at least 70% identity with the sequence shown in SEQ. ID.

25. A polypeptide as claimed in claim 23, the polypeptide having a sequence of at least 80% identity with the sequence shown in SEQ. ID.

26. A polypeptide as claimed in claim 23, the polypeptide having a sequence of at least 90% identity with the sequence shown in SEQ. ID.

27. A polypeptide as claimed in any one of claims 23 to 26, the polypeptide having haemolysin activity.

28. A nucleic acid molecule, the nucleic acid molecule comprising a sequence encoding a polypeptide of any one of claims 23 to 27. WO 01/16172 PCT/AU00/01048 34

29. A nucleic acid molecule comprising a sequence as set out in SEQ. ID. NO:6 or a sequence having at least 60% identity thereto, or a sequence which hybridises thereto under stringent conditions.

30. A nucleic acid molecule as claimed in claim 29, the nucleic acid molecule comprising a sequence having at least 70 identity with the sequence shown in SEQ. ID. NO:6.

31. A nucleic acid molecule as claimed in claim 29, the nucleic acid molecule comprising a sequence having at least 80 identity with the sequence shown in SEQ. ID. NO:6.

32. A nucleic acid molecule as claimed in claim 29, the nucleic acid molecule comprising a sequence having at least 90 identity with the sequence shown in SEQ. ID. NO:6.

33. A composition for use in raising an immune response in an animal, the composition comprising a polypeptide of any one of claims 23 to 27 or a nucleic acid sequence of claim 28 and optionally a carrier and/or adjuvant.

34. A composition for use in raising an immune response in an animal directed against Moraxella, the composition comprising at least one polypeptide selected from the group consisting of a polypeptide as claimed in any one of claims 1 to 5, a polypeptide as claimed in any one of claims 12 to 16, and a polypeptide as claimed in any one of claims 23 to 27, and optionally including an adjuvant or carrier. A composition as claimed in claim 34, the composition comprising a polypeptide as claimed in any one of claims 23 to 27 and either one of, or preferably both of, a polypeptide as claimed in any one of claims 1 to 5 and a polypeptide as claimed in any one of claims 12 to 16.

36. A composition as claimed in claim 34 or claim 35 wherein the Moraxella is M. bovis or M. catarrhalis. WO 01/16172 PCT/AU00/01048

37. A composition as claimed in claim 34 or claim 35 wherein the Moraxella is M. bovis.

38. An antibody raised against a polypeptide selected from the group consisting of a polypeptide as claimed in any one of claims 1 to 5, a polypeptide as claimed in any one of claims 12 to 16, and a polypeptide as claimed in any one of claims 23 to 27.