AU693440B2 - Antigenic polypeptides of (T. ovis) and vaccines containing such polypeptides - Google Patents

Description

WO 94/22913 PCT/NZ94/00029 ANTIGENIC POLYPEPTIDES OF T. OVIS AND VACCINES CONTAINING SUCH POLYPEPTIDES FIELD OF THE INVENTION This invention relates to protective antigens, to antigenic preparations containing such antigens and to the use of the preparations as vaccines for control and eradication of cysticercosis resulting from Taenia ovis infection in susceptible hosts such as ruminants.

BACKGROUND OF THE INVENTION The Taenia ovis tapeworm exists in adult form in the small intestine of its primary host, the dog. The cystic stage is carried in the musculature of its secondary or intermediate hosts, notably sheep and goats. Current control measures include prevention of feeding of infected carcases to dogs and treatment of dogs with cestocidal drugs, notably praziquantel (Droncit, Bayer) to prevent transmission of the parasite to ruminants. These control measures are costly to implement and are not effective in eradicating T. ovis.

Accordingly, as an adjunct to current control measures and to effect eradication of the disease, it would be preferable to immunise the secondary hosts to protect them from infection and also to preserve carcase quality for the meat industry.

It is an object of the present invention to provide a protective antigen for use in vaccines for the protection of ruminants against T. ovis infection or at least to provide the public with a useful choice.

Previous investigations conducted into vaccination against T. ovis infection with oncosphere antigens are reviewed by Rickard, M D and Williams J F, Hydatidosis/Cysticercosis: Immune mechanisms and Immunisation against infection, Adv Parasitoloqy 21 230-296 (1982). However, in the work reviewed no attempt was made to identify which antigenic component of the oncospheres was responsible for the immune response. As will be appreciated, T. ovis contains a large number of antigenic components, most of which are not immunologically effective against infection.

Earlier attempts have been made to identify a host protective antigen for T. ovis (Howell M J and Hargreaves J J, Mol Biochem Parasitol 28 21-30 (1988)). A cDNA library was prepared using mRNA extracted from adult T.

ovis tape worms. Recombinants expressing antigenic determinants as

I

~(Oi~rrm~ ~slP~sDO~ I~a~-rarlli ra~-- WVO 94/22913 PCTINZ94/00029 2 B-galactosidase fusion proteins were selected using antibodies in serum from sheep infected with T. ovis but trials of the host-protective nature of purified fusion proteins were not reported.

More recent reports by the applicants identified a 47-52 kDa antigenic component which subsequently was prepared as a recombinant fusion protein and shown to be protective (Johnson et al., Nature 338 585-587 (1989)).

This molecule was initially selected because of its immunodominance on Western Blots of native oncosphere antigens reacted with antibody from immune sheep. Immunogenicity of this antigen was demonstrated by immunisation experiments using antigen-containing gel slices cut from isoelectric focusing (IEF) and polyacrylamide gels (SDS PAGE) (Harrison et al., Int J Parasitol, 23:1 41-50 (1993)).

SUMMARY OF THE INVENTION The applicants have now identified a further protective antigenic component of T. ovis, having a molecular weight of approximately 16 kDa as calculated by SDS PAGE.

It is broadly to this antigen, to the methods and means of producing the antigen and to the use of the antigen that the present invention is directed.

Accordingly, in one aspect the present invention may broadly be said to consist of a purified antigenic polypeptide having an amino acid sequence which comprises the amino acid sequence of Figure 5 and which is capable of generating a protective immunological response against T. ovis infection in a susceptible host, or a peptide fragment or variant of said polypeptide having substantially equivalent protective immunological activity thereto.

Preferably, the polypeptide has a molecular weight of about 16 kDa calculated by SDS-PAGE.

Conveniently, the protective polypeptide or peptide fragment or variant of the invention is obtained by expression of the DNA sequence coding therefor in a host cell or organism.

In still a further aspect, the invention consists of a composition of matter capable of generating a protective immunological response against T. ovis infection in a susceptible host which essentially consists of: a polypeptide having the amino acid sequence of Figure ~-~L~slr 71~~SI I WO 94/22913 PCT/NZ94/00029 3 a peptide fragment of the polypeptide having substantially equivalent protective immunological activity thereto; or a variant of or which has been modified by the insertion, substitution or deletion of one or more amino acids and which has at least substantia2', equivalent protective immunological activity thereto.

In still a further aspect, the invention provides a DNA molecule which is selected from the group consisting of: a nucleotide sequence encoding the antigenic polypeptide defined above; a nucleotide sequence encoding a peptide fragment of the antigenic polypeptide of which fragment has substantially equivalent protective immunological activity to the polypeptide of and a nucleotide sequence encoding a variant of the polypeptide of (a) or a variant of the peptide fragment of in which the amino acid sequence of the polypeptide or peptide fragment has been modified by the insertion, substitution or deletion of one or more amino acids, which variant has at least substantially equivalent protective immunological activity to the polypeptide of or the peptide fragment In yet further aspects, the invention provides recombinant expression vectors which contain a DNA molecule as defined above, host cells transformed with such vectors and capable of expressing the polypeptide or peptide fragment or variant thereof which is encoded, and methods of producing an antigenic polypeptide or a peptide fragment or variant thereof comprising culturing a host cell as defined above and recovering the expressed product.

In still a further aspect, the invention consists in a vaccine against infection by a cestode parasite which comprises the antigenic polypeptide, peptide fragment or variant defined above in combination with an immunologically appropriate carrier and/or adjuvant therefor.

Alternatively, the invention provides a recombinant viral vaccine which includes nucleic acid encoding an antigenic polypeptide, peptide fragment or variant as defined above and which is capable of expressing said encoded polypeptide, peptide fragment or variant in vivo in a host susceptible to infection by a cestode parasite.

In still a further aspect, the invention may be said to consist in a method of protecting a susceptible host against infection by a cestode parasite, comprising administering to said host an amount of polypeptide, ~a WO 94/22913 PCTINZ94/00029 4 peptide fragment or variant defined above which is protective against such infection.

Conveniently, the polypeptide, peptide fragment or variant is administered to said host in the form of a vaccine as defined above.

In a further aspect, the invention provides an antibody specific for the antigenic polypeptide, peptide fragment or variant defined above.

Other embodiments of the invention will become apparent from the description which follows.

DESCRIPTION OF THE FIGURES Although the invention is broadly as described above, it will be appreciated by those persons skilled in the art that the invention is not limited to the foregoing but also includes embodiments of which the following gives examples. In particular, certain aspects of the invention will be more clearly understood by having reference to the accompanying drawings in which: Figure 1 is a silver stain of the 16 kDa protein contained within a fraction cut from an SDS PAGE gel which provides immunity to T. ovis infection. Lane 1: Pharmacia Low Molecular Weight markers; Lane 2: T.

ovis oncosphere proteins; Lane 3: Proteins in Fraction A; Lane 4: Proteins in Fraction B; Lane 5: Proteins in Fraction C.

Figure 2 is a silver stain of the 16 kDa protein contained within a defined fraction cut from an SDS PAGE gel which provides immunity to T.

ovis infection. Lane 1: Proteins in Fraction C1; Lane 2: Proteins in Fraction C2; Lane 3: Proteins in Fraction C3; Lane 4: Pharmacia Low Molecular Weight markers.

Figure 3 is an immunoblot demonstrating that antibodies raised against the recombinant protein GST-16 recognise the native T. ovis oncosphere 16 kDa protein. Lane 1: Sheep antibody to T. ovis oncosphere antigens; Lane 2: Sheep antibody to Fraction C2 antigens; Lane 3: Sheep antibody to Fraction C3 antigens; Lane 4: Rabbit antibody to T. ovis 16 kDa antigen; Lane 5: Sheep antibody to GST-16 Fusion protein.

Figure 4 shows the results obtained from analysis of GST-16 fusion protein by SDS-PAGE. Lane 1: Pharmacia Low Molecular Weight markers; Lane 5: GST-16 Fusion protein.

Figure 5 represents the nucleotide sequence of T. ovis 16 cDNA and the predicted amino acid sequence of the polypeptide encoded.

B N WO 94/22913 PCTINZ94/00029 DETAILED DESCRIPTION OF THE INVENTION As defined above, in its primary aspect, the present invention is directed to the provision of an antigen which is host-protective against at least T. ovis infection. Hosts which are susceptible to T. ovis infection include ruminants. Accordingly, examples of hosts to which the invention has application are ovine and caprine hosts.

From their investigations, the applicants have identified a T. ovis polypeptide as being involved in protection against T. ovis infection in a susceptible host. This T. ovis polypeptide is that having a molecular weight approximately 16 kDa as determined by SDS-PAGE. This polypeptide further has an amino acid sequence which comprises the amino acid sequence shown as SEQ ID NO. 3.

The present invention also includes within its scope antigens derived from the native T. ovis polypeptide identified above where such derivatives have host-protective activity. These derivatives will normally be peptide fragments of the native polypeptide which include the protective epitope, but can also be functionally equivalent variants of the native polypeptide modified by well known techniques such as site-specific mutagenesis (see Adelman et al., DNA 2 183 (1983)). For example, it is possible by such techniques to substitute amino acids in a sequence with equivalent amino acids. Groups of amino acids known normally to be equivalent are: Ala Ser Thr Pro Gly; Asn Asp Glu Gln; His Arg Lys; Met Leu Ile Val; and Phe Tyr Trp.

The protective antigen of the invention can be produced by isolation from the native T. ovis oncosphere complement using conventional purification techniques. However, it is recognised that for production of the antigen in commercial quantities, production by synthetic routes is desirable. Such routes include the stepwise solid phase approach described by Merryfield (J Amer Chem Soc 85 2149-2156 (1963)) and production using recombinant DNA techniques. The latter route in particular is being employed by the applicants.

In a further aspect, the invention accordingly relates to the recombinant production of the antigenic polypeptide or peptide defined above.

"-LBL I~~-P~~rdl WO 94/22913 PCT/NZ94/00029 6 Stated generally, the production of the protective antigen of the invention by recombinant DNA techniques involves the transformation of a suitable host organism or cell with an expression vector including a DNA sequence coding for the antigen, followed by the culturing of the transformed host and subsequent recovery of the expressed antigen. Such techniques are described generally in Sambrook et al., "Molecular Cloning", Second Edition, Cold Spring Harbour Press (1987).

An initial step in the method of recombinantly producing the antigen involves the ligation of a DNA sequence encoding the antigen into a suitable expression vector containing a promoter and ribosome binding site operable in the host cell in which the coding sequence will be transformed.

The most common examples of such expression vectors are plasmids which are double stranded DNA loops that replicate autonomously in the host cell.

However, it will be understood that suitable vectors other than plasmids can be used in performing the invention.

Preferably, the host cell in which the DNA sequence encoding the polypeptide is cloned and expressed is a prokaryote such as E.coli. For example, E.coli DH5 (Raleigh E A et al., Nucleic Acid Research 16 (41 1563- 1575 (1988)), E. coli K12 strain 294 (ATCC 31446), E. coli B, E. coll X1776 (ATCC 31537), E. coli strain ST9 or E. coli JM 101 can be employed. Other prokaryotes can also be used, for example bacilli such as Bacillus subtilis and enterobacteriaceae such as Salmonella typhimurium, Serratia marcesans or the attenuated strain Bacille Calmette-Guerin (BCG) of Mycobacterium bovis.

In general, where the host cell is a prokaryote, expression or cloning vectors containing replication and control sequences which are derived from species compatible with the host cell are used. The vector may also carry marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli has commonly been transformed using pBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene 2 95 (1977)). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.

For use in expression, the plasmid including the DNA to be expressed contains a promoter. Those promoters most commonly used in recombinant DNA construction for use with prokaryotic hosts include the B-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275 615 (1978); Itakura et al., Science 198 1056 (1977); Goeddel et al., Nature 281 ill I I WO 94/22913 PCT/NZ94/00029 7 544 (1979)) and a tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res 8 4057 (1980); EPO Publ No. 0036776). While these are the most commonly used, other microbial promoters such as the tac promoter (Amann et al., Gene 25 167-178 (1983)) have been constructed and utilised, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally in operable relationship to genes in vectors (Siebenlist et al., Cell 20: 269 (1980)).

In addition to prokaryotes, eukaryotic microbes, such as yeast may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 (1980)) is commonly used. This plasmid already contains the trDl gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977)). The presence of the tr1l lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J Biol Chem 255, 2073 (1980)) or other glycolytic enzymes (Hess et al., J Adv Enzyme Rea 2 149 (1968); Holland et al., Biochemistry 17 4900 (1978). Other promoters, which have the additional advantage of transcription controlled by growth conditions, are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilisation. Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms such as mammals and insects may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure (Tissue Culture, Academic Press, ~dLL- ~b ._I WO 94/22913 PCT/NZ94/00029 8 Kruse and Patterson, editors (1973)). Examples of such useful host cell lines are VERO and HeLa cells and Chinese hamster ovary (CHO) cells.

Expression vectors ior such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream from the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional termination sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by v:ral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40(SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273, 113, (1978)). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindIII site toward the BgII site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

Upon transformation of the selected host with an appropriate vector, the antigenic polypeptide or peptide fragment encoded can be produced often in the form of a fusion protein by culturing the host cells. The fusion protein including the polypeptide or peptide fragment is then recovered and purified as necessary. Recovery and purification can be achieved using any of those procedures known in the art, for example by adsorption onto and elution from an anion exchange resin. As will be apparent from the specific examples provided, the carrier portion of the fusion protein can prove useful in this regard.

The purification procedure adopted will of course depend upon the degree of purity required for the use to which the polypeptide or peptide is to be put. For most vaccination purposes, separation of the fusion protein from most of the remaining components of the cell culture is s I, _I WO 94/22913 PCT/NZ94/00029 9 sufficient as the antigen can be incorporated into a vaccine in a relatively crude form. However, in cases where a greater degree of purity is desired, the carrier component of the fusion protein can be cleaved from the antigenic component. As will again be apparent from the specific examples provided, this can be easily achieved through the provision of an appropriate enzyme cleavage site between the carrier component and the antigen.

Where as is preferred, recombinant techniques are used to produce the antigenic peptide, the first step is to obtain DNA encoding the desired product. Such DNA molecules comprise still a further aspect of this invention.

The DNA molecule of the invention preferably comprises at least the nucleotide sequence shown as SEQ ID NO. 2 (nucleotides 10 to 369 of the Figure 5 sequence). It is however most preferred that the DNA molecule comprise nucleotide sequence shown as SEQ ID NO. 1 (the entire nuc'eotide sequence of Figure The DNA molecule of the invention can be obtained contained within a DNA molecule isolated from an appropriate natural source or can be produced as intron-free cDNA using conventional techniques such as those used in the specific description set out hereinafter. cDNA is preferred.

However, as indicated above, the invention also contemplates variants of the polypeptide which differ from the native amino acid sequences by the insertion, substitution or deletion of one or more amino acids. Where such a variant is desired, the nucleotide sequence of the native DNA molecule is altered appropriately. This alteration can be made through elective synthesis of the DNA using an appropriate synthesizer such as the Applied Biosystems DNA Synthesizer or by modification of the native DNA by, for example, site specific or cassette mutagenesis.

Once obtained, the DNA molecule is treated to be suitable for insertion together with the selected control sequence into the appropriate cloning and/or expression vector. To this end the DNA is cleaved, tailored and religated as required.

Cleavage is performed by treating with restriction enzyme(s) in a suitable buffer. Any of the large number of commercially available restriction enzymes can be used as specified by the manufacturer. After cleavage, the nucleic acid is recovered by, for example, precipitation with ethanol.

I ~l~dl-~ WO 94/22913 PCTI/NZ94/00029 10 Tailoring of the cleaved DNA is performed using conventional techniques. For example, if blunt ends are required, the DNA may be treated with DNA polymerase I (Klenow), phenol and chloroform extracted, and precipitated by ethanol.

Re-ligation can be performed by providing approximately equimolar amounts of the desired components, appropriately tailored for correct matching, and treatment with an appropriate ligase (eg T 4 DNA ligase).

In addition to the protective antigens of the invention and the method of producing these, the present invention provides a vaccine against T. ovis infection. Such a vaccine normally includes as the essential component a host protective amount of the polypeptide, peptide fragment or variant referred to above, together with an immunologically appropriate adjuvant or carrier.

Examples of appropriate adjuvants known to those skilled in the art are saponins (or derivative or related material), muramyldipeptice, trehalose dimycollate, Freund's complete adjuvant, Freund's incomplete adjuvant, other water in oil emulsions, double emulsions, dextran, diethylaminoethyldextran, potassium alum, aluminium phosphate, aluminium hydroxide, bentonite, zymosan, polyelectrolytes, retinol, calcium phosphate, protamine, sarcosine, glycerol, sorbitol, propylene glycol, fixed oils and synthetic esters of higher fatty acids. Saponins in particular have been found to be effective adjuvants.

In still further embodiments, the vaccine may also be formulated to further include other host-therapeutic agents. Such therapeutic agents include anthelmintics or other vaccines, or immunostimulants such as interferons or interleukins.

The protective antigen of the invention may also be treated in any conventional way to enhance its stability or to conserve or potentiate its immunogenic efficiency. For example, the antigen may be treated with a suitable inhibitor, modifier, crosslinker or denaturant in such a way as to enhance its immunogenicity.

The vaccine described above can be administered to the host by any of those methods known in the art. However, the preferred mode of administration of the vaccine is parenteral. The term "parenteral" is used herein to mean intravenous, intramuscular, intradermal and subcutaneous injection. Most conveniently, the administration is by subcutaneous injection.

I WO 94/22913 PCTNZ94/00029 11 The amount of the vaccine administered to the ,ost to be treated will depend on the type, size and body-weight of the host as well as on the immunogenicity of the vaccine. Conveniently, the vaccine is formulated such that relatively small dosages of vaccine (1-5 ml) are sufficient to be protective.

In another embodiment, the vaccine may also be in the form of a live recombinant viral vaccine including nucleic acid encoding the polypeptide, peptide fragment or variant. The vaccine is administered to the host in this form and once within the host expresses the encoded polypeptide, peptide fragment or variant to induce a host-protective response.

A number of such live recombinant viral vaccine systems are known. An example of such a system is the Vaccinia virus system (US Patent 4603112; Brochier et al., Nature 354 520 (1991)).

In still a further aspect, the invention provides a method of protecting a host susceptible to infection by a cestode parasite. The method of invention includes as its essential step the administration to the host of either the antigenic polypeptide or peptide fragment or variant per se, or of a vaccine as described above.

While the specific examples provided hereinafter describe only the use of the antigens of the invention in protecting against 7. ovis infection, those persons skilled in the art will appreciate that cross-species protection can be achieved by the use of antigens derived from one parasite species (see, for example, Lightowlers, Acta Leidensia 57 135-142 (1989)).

It will therefore be understood that the antigens of the invention are candidate protective antigens against at least the following cestode parasites other than T. ovis: E. multilocularis, E. vogelii, E.

granulosus, T. saginata, T. solium, T. multiceps and T. hydatigena.

As yet a further aspect of the invention, the use of the DNA molecule described above or a subsequence thereof as a probe is contemplated. In this aspect, the DNA molecule is used to identify by hybridisation DNA of a cestode parasite such as T. saginata, T. hydatigena or E. granulosus which encodes an immunogenic antigen of that parasite. In this way, further parasite antigens suitable for use in a vaccine can be identified.

The method of use of the DNA molecule of the invention as a probe will be well understood by those persons skilled in the art. For example, those techniques set out in Maniatis et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbour (1982) could be used.

~I p- PCT/NZ94/00029 12 Alternatively, DNA amplification techniques such as the polymerase chain reaction (PCR) (Saiki et al., Science 239 487 (1988)) can be employed to detect homologous DNA of other parasites, with the PCR primers being based upon the nucleotide sequence of SEQ ID NO. 2.

Similarly to the use of the DNA molecules of the invention to identify DNA encoding the corresponding protective antigen of other parasites, antibody probes specific for the protective antigens o- the invention can be used to screen the antigens expressed by organisms transformed by the DNA of the parasite in question. The location of a positive clone (one expressing an antigen recognised by the antibody) will allow identitication of both the protective antigen itself and the DNA which encodes it. Such antibody probes can be either polyclonal or monoclonal and can be prepared by any of those techniques known in the art. For example, a suitable procedure by which polyclonal antibody probes can be prepared is set out in 15 Example 3.

If required, monoclonal antibodies can be prepared in accordance with the procedure of Kohler and Milstein (Kohler G and Milstein C, "Continuous cultures of fused cells secreting antibody of predefined specificity", Nature 256 495-497 (1975)).

Antibody binding fragments can be prepared by controlled protease digestion of whole immunoglobulin molecules as described by Tjissen P, Practice and Theory of Enzyme Immunoassays in Laboratory Techniques in S. Biochemistry and Molecular Biology, Elsevier, Amsterdam, New York. Oxford, 117-121 (1990). Alternatively, the production of antigen binding Lragments 25 (Fv, ScFv, Fab etc) can be achieved by recombinant means (Hodgson J, "Making Monoclonals in Microbes", Biotechnology 9 4231-325 (1991)).

The immunogenicity of the antigenic polypeptide of the invention will be appreciated from the following non-limiting examples.

Example 1 Investigations of the immunogenicity of immunodominant oncosphere antigens have been reported (Harrison et Int J Parasitol, (1993) supra). In these studies sections of gels containing the immunodominant antigens were tested for their protective ability, resulting in the identification of protective antigens in the molecular weight range 47-52 kDa and 32-34 kDa. In an extension of this approach, fractions of gels 4-Cj~II WO 94/2213 P'r/NZ94/00029 13 containing non-immunodominant antigens were tested for their ability to provide protective immunity in sheep.

T. ovis oncospheres were prepared from bleach-hatched eggs as described previously (Harrison et al., supra) and solubilised in 1% sodium dodecyl sulphate in 10 mM tris-HCl buffer pH 7.5 containing 1% dithiothreitol, 5 mM MgCl 2 and a cocktail of enzyme inhibitors, at a concentration of 4 x 106 oncosphere equivalents per mL. 4 mL of this antigen preparation were applied to a preparative 3mm thick SDS PAGE gel containing a 12-18% polyacrylamide gradient.

After electrophoresis for 16h, the proteins were visualised by negative staining with CuC1, (Lee et al., Analytical Biochem 166, 308-312, (1987)).

Fractions were cut from the preparative gel with reference to Pharmacia Low Molecular Weight marker proteins run on the outside lanes of the same gel. Three fractions were cut as follows: Fraction A 67 kDa to 40 kDa Fraction B 40 kDa to 28 kDa Fraction C 28 kDa to 16 kDa The fractions were placed.in separate containers and allowed to dry slightly before being homogenised with a glass rod.

A small sample of each fract.on was removed and boiled in the minimum volume of SDS PAGE sample buffer. The solubilised proteins were recovered from the gel particles by centrifugation and analysed by SDS PAGE.

The protein content of each fraction is shown in Figure 1.

The remainder of the homogenised polyacrylamide gel fractions were divided into two lots and blended with an equal volume of STM oil adjuvant (Bokhout et al., Vet Immunol and Immunopathol 2 491-500 (1981)).

Groups of 5 Romney sheep were immunised by injecting half the antigen in a 1 mL dose subcutaneously over the rib area. Two weeks later the remaining antigen was injected intramuscularly into the rear leg. Five sheep were injected in a similar manner but with adjuvant only as a control.

Three weeks after the second immunisation all sheep were infected orally with ImL saline dose containing 2000 mature T. ovis eggs.

Three weeks after infection all sheep were humanely slaughtered and carcases examined for the number of developing cysticerci by finely slicing the musculature.

Cyst counts found at post mortem are shown in Table 1.

-c I WO 94/22913 PCTINZ94/00029 14 Table 1 Cyst counts at post mortem of sheep immunised with Fractions A, B or C.

Group Cyst No. in Mean Significance* individual sheep s.d. Protection Adjuvant 13 62 63 82 130 70 42.1 Control Fraction A 0 3 24 52 121 40 49.8 43 NSD Fraction B 0 0 0 0 36 7.2 16.1 90 P<0.05 Fraction C 0 0 0 0 2 0.4 0.9 99 P<0.01 Mann-Whitney Test NSD, not significantly different (P>0.05) The results show that the immunodominant antigens contained in Fraction A have (surprisingly) not provided significant protection in this experiment, whereas the immunodominant antigens in Fraction B eg 32-34 kDa antigens, have given significant protection. The results further show that Fraction C also contains highly effective immunogens even though they are not immunodominant on Western Blots.

The nature of protective antigens in Fraction C was further examined in Example 2.

Example 2 23 million oncosphere equivalents were applied to a 3 mm preparative SDS PAGE gel containing a 12-18% polyacrylamide gradient. After electrophoresis for 17 h the proteins were visualised by the copper staining method described in Example 1. The region of the gel between kDa and 14 kDa was located by reference to Pharmacia Low Molecular Weight marker proteins run on the same gel. Three fractions were cut as follows: Fraction C1 29-24 kDa Fraction C2 24-18 kDa Fraction C3 18-12 kDa Gel fractions were allowed to dry slightly and were then homogenised with a glass rod. A sample was taken from each fraction, boiled in SDS PAGE sample buffer and centrifuged to remove gel particles. The solubilised proteins were analysed by SDS PAGE (see Figure 2).

WO 94/22913 PCTINZ94/00029 15 The remainder of the homogenised fractions were divided into 2 lots and blended with an equal volume of STM adjuvant.

Groups of 4 or 5 Romney sheep were immunised with a 2 mL dose, 1 mL was injected subcutaneously over the rib area and 1 mL was injected intramuscularly into the rear leg. Four weeks later the immunisation procedure was repeated. Two weeks after the second immunisation all sheep were infected orally with 3000 mature T. ovis eggs.

Sheep were humanely slaughtered two weeks after infection and carcases examined for the number of developing cysticerci.

Cyst counts at post mortem are shown in Table 2.

Table 2 Cyst counts at post mortem of sheep immunised with Fractions C1, C2 and C3.

Group Cyst No. in Mean Significance individual sheep s.d. Protection Adjuvant 0 9 12 21 10.5 8.7 Control Fraction C1 0 8 11 14 18 10.2 6.8 3 NSD Fraction C2 0 0 1 1 0.5 0.6 95 NSD Fraction C3 0 0 0 0 0 0 100 P<0.01 Mann-Whitney Test NSD, not significantly different. (P>0.05) The results show that Fraction C3 induced statistically-significant protection while Fraction C2 greatly reduced the number of cysts when compared to the control and Fraction C1. It is therefore clear that Fractions C2 and C3 contain protective antigens.

Western Blots of sera from groups immunised with Fraction C3 revealed that the sheep had made antibodies to antigens at 18,16, and 12 kDa (Fraction C3) (Fig The 16 kDa protein was selected for further investigation.

Example 3 A preliminary step in the technique used in the present invention involved the production of antibody specific to the 16 kDa antigen of the

I

WO 94/22913 PCT/NZ94/00029 16 invention. As will be appreciated, such antibodies are commonly used as probes for screening the products of expression of a population of host organisms or cells transformed by an expression vector to allow identification of the organisms or cells expressing the required product.

The specific antibody probes of the invention was formed as follows: Twelve million oncospheres solubilised in 1% SDS were separated on a 12-18 gradient SDS PAGE gel and stained with copper chloride as described.

The 20-14 kDa region was located by reference to Pharmacia Low Molecular Weight Marker proteins run on the same gel. Strips containing the 16kDa protein and the regions imnuediately adjacent to the 16 kDa protein were excised, homogenised and dialysed against TBS to remove excess copper.

After dialysis the gel particles and liquid inside the dialysis tubing were recovered, blended with STM oil adjuvant and injected into three rabbits on three occasions spread over two months. Two weeks after the third injection a 20mL blood sample was collected from each rabbit and the sera were analysed on Western Blots of native oncosphere antigens.

One rabbit produced antibody that reacted predominantly with the 16 kDa protein (Figure 3, lane 4).

This antibody was subsequently used to screen the T. ovis cDNA library.

Example 4 The T. ovis onco.'phere Agt11 cDNA library (Johnson et al., Nature 338 585-587 (1989)) was screened using a 1/50 dilution of rabbit anti-16 kDa serum following standard methods (Glover, DNA Cloning: A Practical Approach; IRL Press, Oxford (1985)). Positive clones were detected using alkaline phosphatase (AP) conjugated goat anti-rabbit IgG or 125 I-labelled protein A. Positive clones were subjected to two further rounds of purification and screening, followed by high titre phage stock preparation.

Antibodies specific for individual clones were affinity purified from the rabbit anti-16 kDa serum by low pH elution from nitrocellulose filters impregnated with Agt11 expressed fusion protein. These antibodies were used in plaque arrays to determine which clones were related (sibling analysis) and in immunoblots to confirm that antibody to the recombinant protein recognised the 16 kDa oncosphere protein.

DNA from the selected positive clone was prepared from liquid cultures and restriction digested with Eco R1. The insert DNA was recovered after 11I I 4 WO 94/22913 PCT/NZ94/00029 17 electrophoresis in 1% agarose gel and was ligated to pGEX-1T plasmid DNA which had been prepared as described below.

To allow direct cloning of insert DNA from Agt11 into the GST fusion protein expression system, the vector pGEX-1T was constructed to allow inframe expression of subcloned DNA together with the thrombin cleavage site to enable subsequent removal of the GST fusion partner.

Plasmid DNA from pGEX-2T was cut wi'l EcoRi and BamH1 and the linear plasmid isolated by agarose gel electrophoresis followed by purification using a GENECLEAN Kit (Bio 101). pUC19 plasmid DNA was also cut with EcoR1 and BamH1 and the small linker fragment purified by electrophoresis on a polyacrylamide gel in tris-Borate-EDTA buffer. The linker band was visualised with ethidium bromide staining, cut out and DNA eluted into d.H 2 0 overnight. The linker was ligated to the linear pGEX-2T which was then used to transform E. coli JM101. Resultant colonies were grown in L-broth and plasmid DNA isolated. Colonies containing the linker sequence were identified by the appearance of new restriction sites for Kpn 1 and Sac 1 in the plasmid DNA. The new plasmid was named pGEX-1T.

pGEX-1T plasmid DNA was cut with EcoRI, treated with phosphatase and ligated to the insert cDNA's derived from the selection of Agt11 clones described above. Ligations were transformed into E. coli JM101 and clones expressing the GST-16 kDa fusion protein were identified by colony immunoassay or by immunoblotting of protein extracts from the bacteria.

Soluble fusion protein was isolated from bacterial extracts by affinity purification with glutathione-agarose (Smith and Johnson, Gene 67 31-40 (1988)). Analysis of the fusion protein by SDS PAGE indicated a relative electrophoretic mobility corresponding to a molecular weight of approximately 40 kDa (Figure 4).

The nucleotide sequence of caesium chloride purified pGEX-1T-16 DNA was determined by direct dideoxy chain-termination sequencing using primers corresponding to pGEX-1 forward and reverse sequences flanking the insert site (Maniatis et al., 1989, supra). Both coding and non-coding DNA strands were sequenced in this manner. The DNA sequence and predicted amino acid sequence are shown in Figure 5. The calculated molecular weight of the T. ovis portion of the fusion protein is 13,386 Da based on the predicted amino acid sequence.

L' WO 94/22913 PCT/NZ9/00029 18 Example Three vaccination trials were undertaken to investigate the immunogenicity of the fusion protein GST-16.

Trial A Four Romney sheep were immunised by subcutaneous injection on three occasions four weeks apart, with 50 pg of GST-16 affinity purified fusion protein and 1 mg Saponin adjuvant.

Three weeks after the third injection, these sheep plus eight nonvaccinated control sheep were infected orally with 2000 mature T. ovis eggs.

Six weeks later the sheep were humanely slaughtered and carcases examined for the numbers of cysts.

Trial B Five Romney sheep were immunised by subcutaneous injection on two occasions four weeks apart, with 100 pg GST-16 affinity purified fusion protein and 1 mg Saponin adjuvant.

Three weeks after the second injection, these sheep plus seven nonvaccinated control sheep were infected orally with 2000 mature T. ovis eggs.

Six weeks later the sheep were humanely slaughtered and carcases examined for the number of cysts.

Trial C Fifteen Romney lambs were immunised by subcutaneous injection on two occasions four weeks apart, with 100 pg GST-16 affinity purified fusion protein and 10mg Saponin adjuvant.

Four weeks after the second injection, these sheep plus 11 non vaccinated control sheep were infected orally with 2000 mature T. ovis eggs.

Six weeks later the sheep were humanely slaughtered and carcases examined for the number of cysts.

The results of trials A, B and C are shown in Table 3.

II

WO 94/22913 *PCT/NZ94/00029 19 Table 3 Cyst counts at post mortem of sheep immunised with GST-16.

Gop Cyst No. in individual Mean %Significance*j ProtectionJ_ Trial Control 0 1 1 3 8 10 23.0 56 105 GST-16 0 0 1 1 0.5 98 NSD Trial B I Control 4 19 29 45 75 87 51.9 GS-6 0 0 1 4 19 4.8 91___P<0.05 Trial Control 13 16 22 27 35 35 35.5 37 48 50 52 56 GST-16 0 0 0 0 0 0 0 9.7 73 P<0.01 1 1 6 15 16 18 31 *Mann-.Whitney Test NSD, not significantly different. (P>0.05) The results, particularly of Trial A, show that the fusion protein GST-16 is a potent imxnunogen.

Serum samples from sheep immunised with GST-16 were analysed by immunoblotting against native oncosphere antigens. (Fig.3) Antibodies the recombinaint GST-16 recognised the 16 kDa native antigen indicating the recombinant protein shares one or more epitopes with the native 16 protein.

to that kDa INDUSTRIAL APPLICATION In accordance with the present invention there is provided a polypeptide antigen of ovis which is effective in generating a protective immunological response against T. ovis infection in susceptible hosts. It has been established that vaccination with this polypeptide stimulates a degree of immunity against challenge infection with avis wwm Il~eQp~"u-~r~-smarr~ a~ WO 94/22913 PCT/NZ94/00029 20 eggs. The invention also provides a recombinant method for expression of the antigen by which commercial quantities can be obtained.

It will be appreciated that the above description is provided by way of example only and that variations in both the materials and the techniques used which are known to those persons skilled in the art are contemplated.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited", and that the word "compriseo" has a corresponding meaning.

C

C..

o IrlC~IT~II(LI~ WO 94/22913 PC'T'1NZ9400029 21 SEQUENCE LISTING GENERAL INFORMATION APPLICANT: PITMAN-MOORE NEW ZEALAND LIMITED, a New Zealand company of 33 Whakatiki Street, Upper Hutt, New Zealand, NEW ZEALAND PASTORAL AGRICULTURE INSTITUTE LIMITED, a company incorporated under the Companies Act 1955 pursuant to the Crown Research Institutes Act 1992 and having its registered office at Peat Marwick Tower, 85 Alexandra Street, Hamilton, New Zealand, THE UNIVERSITY OF MELBOURNE, a body corporate organised and existing under the laws of the State of Victoria, of Grattan Street, Parkville, Victoria 3052, Australia.

TITLE OF INVENTION: Antigenic polypeptides of T. ovis and vaccines containing such polypeptides.

NUMBER OF SEQUENCES: 3 CORRESPONDENCE ADDRESS: ADDRESSEE: A J PARK SON STREET: HUDDART PARKER BUILDING, POST OFFICE SQUARE CITY: P 0 BOX 949, WELLINGTON COUNTRY: NEW ZEALAND COMPUTER READABLE FORM: MEDIUM TYPE: 3.5,DS,HD FLOPPY DISC COMPUTER: IBM PC COMPATIBLE OPERATING SYSTEM: MS-DOS SOFTWARE: WORD PERFECT 5.1 FOR WINDOWS CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 7 APRIL 1994

CLASSIFICATION:

ATTORNEY/AGENT INFORMATION: NAME: BENNETT, MICHAEL R.

-V H I I t'~C~ WO 94/22913 1PCT/NZ94/0029 22 TELECOMMUNICATION INFORMATION: TELEPHONE: (64 4) 473 8278 TELEFAX: (64 4) 472 3358 472 3351 INFORMATION FOR SEQUENCE ID NO. 1: SEQUENCE CHARACTERISTICS: LENGTH: 601 BASE PAIRS TYPE: NUCLEIC ACID STRANDEDNESS: SINGLE TOPOLOGY: LINEAR MOLECULE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO. 1: 1 GAATTC CCC GCC TTG TCC GAG AGA Ala Leu Ser Glu Arg 34 TT GGC GAA AGT ACA CTT TCA GGA Phe Gly Glu Ser Thr Leu Ser Gly 67 CTG AGG AGA TAC ATG CAT TGG AGT Leu Arg Arg Tyr Met His Trp Ser 100 CAC AAT GCC CTA CTG CTA AGT TGG His Asn Ala Leu Leu Leu Ser Trp 133 AAA TTA GCC GAA AGG GGC GTC AAG Lys Leu Ala Glu Arg Gly Val Lys 166 GTG TTC GCA AGT CCA GTC TCC AAA Val Phe Ala Ser Pro Val Ser Lys 199 ATC GTG CAT AAA AGC GCG AGC GTC Ile Val His Lys Ser Ala Ser Val 232 CGT ATC CTT CTT CGG GGA TTG CTG Arg Ile Leu Leu Arg Gly Leu Leu 265 GAA TAC GTG CTG ACC ACA CAA GCG Glu Tyr Val Leu Thr Thr Gin Ala 298 TTC GGT CCC ATT TTT GTT TAC ACA Phe Gly Pro Ile Phe Val Tyr Thr TAC GTT Tyr Val GAC ACC Asp Thr CAC AAG His Lys ATT GGC Ile Gly CAT GTC His Val CCT CAC Pro His GCG AAG Ala Lys GCC AAC Ala Asn CTA GGA Leu Gly CTT CAC Leu His

GAA

Glu

TCG

Ser

GGC

Gly

AGC

Ser

TCA

Ser

CAC

His

GGA

Gly

ACA

Thr

CGC

Arg

GCC

Ala 66 99 132 165 198 231 264 297 330

II

WO 94/22913 WO 94/22913 CUIfNZ941 00029 23 331 AAA ACA TGG CAG ACT GAT GCC AAT CAC CCT CAA 363 Lys Thr Trp Gin Thr Asp Ala Asn HIS Pro Gin 364 CAA TCC TAACTTCTGG GATTCTATTG CGCACATCCC AGCAGTTCTC 409 Gin Ser 410 GCTCCAATCC TCTCGTCTTC ACGGAGCAGA GGATGCATCG GCAGCAGTTT 459 460 TTTPGTGCCC ACTTCTACAC AACGCAAATC ATCACTTGCA TAACTGTCAA 509 510 ATGAAAGGAA CAGGTGTTGT CTCAGTTGGC TTTAAAAAAA AAAAAAAAAA 559 560 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGGAATTC 601 WO 94122913 WO 94174fl3IICTINZ94/00029 24 INFORMATION FOR SEQUENCE ID NO. 2: SEQUENCE CHARACTERISTICS: LENGTH: 360 BASE PAIRS TYPE: NUCLEIC ACID STRANDEDNESS: SINGLE TOPOLOGY: LINEAR MOLECULE TYPE: cDNA SEQUENCE DESCRIPTION: SEQ ID NO. 2: 1 GCC TTG TCC GAG AGA Ala Leu Ser Glu Arg TTT GGC GAA AGT ACA CTT TCA GGA Phe Gly Glu Ser Thr Leu Ser Gly 58 CTG AGG AGA TAC ATG CAT TOG AGT Leu Arg Arg Tyr Met His Trp Ser 91 CAC AAT GCC CTA CTG CTA AGT TG His Asn Ala Leu Leu Leu Ser Trp 124 AAA TTA GCC GAA AGO OGC GTC MAG Lys Leu Ala Oiu Arg Oly Val Lys 157 GTG TTC GCA AGT CCA OTC TCC AMA Val Phe Ala Ser Pro Val. Ser Lys 190 ATC GTG CAT AAA AGC OCG AGC GrC Ile Val His Lys Ser Ala Her Val 223 COT ATC CTT CTT COG GGA TTG CTG Arg Ile Leu Leu Arg Gly Leu Leu 256 GMA TAC OTG CTG ACC ACA CAA OCO Oiu Tyr Val Leu Thr Thr Gin Ala 289 TTC GGT CCC ATT TTT OTT TAC ACA Phe Gly Pro Ile Phe Val Tyr Thr 322 AAA ACA TOG CAG ACT OAT 0CC MAT Lys Thr Trp Gin Thr Asti Ala Asn TAC, GTT GMA 24 Tyr Val Glu GAC ACC TCO3 Asp Thr Ser CAC MAG GGC His Lys Gly ATT GOC AGC Ile Gly Ser CAT GTC TCA His Val Her CCT CAC CAC Pro His His GCG MAG GGA Ala Lys Gly GCC MAC ACA Ala Asn Thr CTA GGA CGC Leu Gly Arq OTT CAC 0CC Leu His Ala CAC CCT CMA His Pro Gin 57 123 1 56 169 222 255 288 321 354 355 CMA TCC 360 Gin Ser

'N

WO 94/22913 PCT/NZ94/00029 25 INFORMATION FOR SEQUENCE TD NO. 3: SEQUENCE CHARACTERISTICS: LENGTH: 120 AMINO ACIDS TYPE: AMINO ACID TOPOLOGY: LINEAR MOLECULE TYPE: PROTEIN SEQUENCE DESCRIPTION: SEQ ID NO. 3: 1 Ala Leu Ser Glu Arg Tyr Val Glu 8 9 Phe Gly Glu Ser Thr Leu Ser Gly Asp Thr Ser .9 Leu Arg Arg Tyr Met His Trp Ser His Lys Gly 31 His Asn Ala Leu Leu Leu Ser Trp Ile Gly Ser 41 42 Lys Leu Ala Glu Arg Gly Val Lys His Val Ser 52 53 Val Phe Ala Ser Pro Val Ser Lys Pro His His 63 64 Ile Val His Lys Ser Ala Ser Val Ala Lys Gly 74 Arg Ile Leu Leu Arg Gly Leu Leu Ala Asn Thr 86 Glu Tyr Val Leu Thr Thr Gin Ala Leu Gly Arg 96 97 Phe Gly Pro Ile Phe Val Tyr Thr Leu His Ala 107 108 Lys Thr Trp G2n Thr Asp Ala Asn His Pro Gin 118 119 Gin Ser 120 Ib_

Claims (27)

1. A purified antigenic polypeptide having an amino acid sequence which comprises the amino acid sequence of SEQ ID NO. 3 and which is capable of generating a protective immunological response to T. ovis infection in a susceptible host, or a peptide fragment or variant of said polypeptide having substantially equivalent host-protective immunological activity thereto.

2. A polypeptide according to claim 1 having a molecular weight of about 16 kDa calculated by SDS-PAGE.

3. A polypeptide according to claim 1 including the amino acid sequence of SEQ ID NO. 3.

4. A polypeptide according to claim 1 consisting of the amino acid sequence of SEQ ID NO. 3.

5. A polypeptide or peptide fragment or variant thereof as claimed in any one of claims 1 to 4 which is the product of expression of a nucleotide sequence coding therefor in a host cell or organism.

6. A polypeptide or peptide fragment or variant thereof as claimed in claim 5 which is expressed in the host cell as a fusion protein.

7. A polypeptide or peptide as claimed in claim 6 which is expressed as a fusion protein with the enzyme glutathione s-transferase (E.C 2.5.18).

8. A composition of matter capable of generating a protective immunological response against T. ovis infection in a susceptible host which essentially consists of a component selected from the group consisting of: the polypeptide of claim 1; a peptide fragment of having equivalent protective immunological activity thereto; and a variant of or which has been modified by the insertion. substitution or deletion of one or more amino acids and which has at least substantially equivalent protective immunological activity thereto.

9. A DNA molecule which is selected from the group consisting of: a nucleotide sequence encoding the antigenic polypeptide of claim 1; a nucleotide sequence encoding a peptide fragment of the antigenic polypeptide of which fragment has substantially equivalent protective immunological activity to the polypeptide of and r~ WO 94/22913 I'CT/NZ94/00029 27 a nucleotide sequence encoding a variant of the polvpeptide of (a) or a variant of the peptide of in which the amino acid sequence of the polypeptide or peptide fragment has been modified by the insertion, substitution or deletion of one or more amino acids, which variant has at least substantially equivalent protective immunological activity to the polypeptide of or the peptide fragment of A DNA molecule comprising the nucleotide sequence of SEQ ID NO. 1.

11. A DNA molecule comprising the nucleotide sequence of SEQ ID NO. 2.

12. A DNA molecule as claimed in any one of claims 9 to 11 which has been isolated from a natural source.

13. A DNA molecule as claimed in any one of claims 9 to 11 which is cDNA.

14. A recombinant expression vector which contains a DNA molecule as claimed in any one of claims 9 to 13.

15. A host cell transformed with a vector as claimed in claim 14 and capable of expressing the polypeptide or peptide fragment or va::iant thereof which is encoded.

16. A host cell as claimed in claim 15 which is a prokaryote.

17. A host cell as claimed in claim 16 wherein the prokaryote host is E. coli.

18. A host cell as claimed in claim 15 which is a eukaryote.

19. A method of producing an antigenic polypeptide or a peptide fragment or variant thereof, which comprises culturing a host cell as claimed in any one of claims 15 to 18 and recovering the expressed product.

20. An antigenic polypeptide, peptide fragment or variant produced by the method of claim 19.

21. A vaccine comprising an immunologically-effective amount of a polypeptide, peptide fragment or variant as claimed in any one of claims 1 to 7 and 20 in combination with an immunologically appropriate adjuvant, carrier or diluent therefor.

22. A recombinant viral vaccine which includes nucleic acid encoding an antigenic polypeptide or peptide fragment or variant thereof as claimed in claim 1 and which is capable of expressing said encoded polypeptide or peptide fragment or variant in vivo in a host susceptible to infection by a cestode parasite.

23. A purified antibody or binding fragment thereof specific for an antigenic polypeptide, peptide fragment or variant as claimed in claim 1. -pll I C I WO 94/22913 IPCT/NZ94/00029 28

24. An optionall '-labelled DNA molecule comprising part or all of the nucleotide sequence o. SEQ ID NO. 2 suitable for use as a probe for identifying nucleic acid coding for a protective antigen of an Echinococcus or Taeniid parasite other than T. ovis.

25. The use of an antibody or binding fragment thereof as claimed in claim 23 to identify a protective antigen of an Echinococcus or Taeniid parasite other than T. ovis.

26. The use of a DNA molecule as claimed in claim 24 to identify DNA encoding a protective antigen of an Echinococcus or Taeniid parasite other than T. ovis.

27. The use of claim 25 or claim 26 wherein the parasite other than T. ovis is E. multilocularis, E. granulosus, E. vogelii, T. saginata, T. solium, T. multiceps or T. hydatigena.

28. A method of identifying a protective antigen of an Echinococcus or Taeniid parasite other than T. ovis which comprises the step of identifying a gene of said parasite having a nucleotide sequence which is at least substantially homologous to part or all of the nucleotide sequence of SEQ ID NO. 2. r -r ~srl~