AU6583794A - Antigenic polypeptides of (t. ovis) and vaccines containing such polypeptides - Google Patents

Description

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 Parasitoloσv 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 β-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 18 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 ammo acid sequence which comprises the ammo 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 18 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) a polypeptide having the ammo acid sequence of Figure 5; (b) a peptide fragment of the polypeptide (a) having substantially equivalent protective immunological activity thereto; or

(c) a variant of (a) or (b) 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.

In still a further aspect, the invention provides a DNA molecule which is selected from the group consisting of:

(a) a nucleotide sequence encoding the antigenic polypeptide defined above; (b) a nucleotide sequence encoding a peptide fragment of the antigeni polypeptide of (a) , which f agment has substantially equivalent protective immunological activity to the polypeptide of (a) ; and

(c) a nucleotide sequence encoding a variant of the polypeptide of (a) or a variant of the peptide fragment of (b) 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 (a) or the peptide fragment (b) .

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, 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 m which:

Figure 1 is a silver stain of the 18 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 18 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-18 recognise the native T. ovis oncosphere 18 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 18 kDa antigen; Lane 5: Sheep antibody to GST-18 Fusion protein.

Figure 4 shows the results obtained from analysis of GST-18 fusion protein by SDS-PAGE. Lane 1: Pharmacia Low Molecular Weight markers; Lane 5: GST-18 Fusion protein.

Figure 5 represents the nucleotide sequence of T. ovis 18 cDNA and the predicted ammo acid sequence of the polypeptide encoded. 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 a 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 18 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 derivative 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:

(a) Ala Ser Thr Pro Gly;

(b) Asn Asp Glu Gin;

(c) His Arg Lys;

((dd)) MMeett LLeeuu HHee _ VVaall;; and

((ee)) PPhhee TTyyrr TTrrpp.. 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. 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 recombmantly 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 (4) 1563- 1575 (1988)), E. coli K12 strain 294 (ATCC 31446), E. coli B, E. coli 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 marcesanε or the attenuated strain Bacille Calmette-Guerin (BCG) of Mycobacteriu 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 β-lactamase

(penicillinase) and lactose promoter systems (Chang et al . , Nature 275 615 (1978); Itakura et al . , Science 198 1056 (1977); Goeddel et al . , Nature 281 544 (1979)) and a tryptophan (trp) promoter system (Goeddel et al . , Nucleic Acids Res 8.4057 (1980); EP0 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 trpl 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 trpl 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 Reσ 1_ 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, 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 for 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 viral 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 Hindlll site toward the Bgll 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 (e.g. 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 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 390 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 nucleotide sequence of Figure 5) .

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. 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 eguimolar amounts of the desired components, appropriately tailored for correct matching, and treatment with an appropriate ligase (eg T₄ 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 lmmunologically appropriate adjuvant or carrier.

Examples of appropriate adjuvants known to those skilled in the art are saponins (or derivative or related material), muramyldipeptide, trehalose dimycollate, Freund's complete adjuvant, Freund's incomplete adjuvant, other water in oil emulsions, double emulsions, dextran, diethylaminoethyl- dextran, potassium alum, aluminium phosphate, aluminium hydroxide, bentonite, zymosan, polyelectrolytes, retinol, calcium phosphate, protamine, sarcosme, glycerol, sorbitol, propylene glycol, fixed oils and synthetic esters of higher fatty acids. Saponins 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 lmmunogenic 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. The amount of the vaccine administered to the host 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 T. 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. 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 of 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 identification 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 Example 3.

If required, monoclonal antibodies can be prepared in accordance with the procedure of Kohler and Milste (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 Immunoassavs in Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier, Amsterdam, New York, Oxford, 117-121 (1990). Alternatively, the production of antigen binding fragments (Fv, ScFv, Fab etc) can be achieved by recombinant means (Hodgson J, "Making Monoclonols 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 al . , 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 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 10mM tris-HCl buffer pH 7.5 containing 1% dithiothreitol (DTT), 5 mM MgCl₂ 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 CuCl₂ (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 18 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 fraction 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 1mL 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 1 mL 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. 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 conclusively that Fraction C also contains highly effective immunogens even though they are not immuno-dominant 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 3mm preparative SDS PAGE gel containing a 12-18% polyacrylamide gradient. After electrophoresis for 17h the proteins were visualised by the copper staining method described in Example 1. The region of the gel between 30 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) . 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 2mL dose, 1mL was injected subcutaneously over the rib area and 1mL 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 antige (s). Western Blots of sera from groups immunised with Fractions C2 and C3 revealed that the sheep had made antibodies to antigens at 18,16, and 12 kDa (Fraction C3) and 24,20 and 18 kDa (Fraction C2) (Fig 3). Both the protei ιn stain (Fig 2) and antibody reaction (Fig 3) indicated that the 18 kDa protein was common to both fractions C2 and C3. This protein was therefore 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 18 kDa antigen of the 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 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 in Example 1. The 20-14 kDa region was located by reference to Pharmacia Low Molecular Weight Marker proteins run on the same gel. Strips containing the 18 kDa protein and the regions immediately adjacent to the 18 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 20 mL 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 18 kDa protein. (Figure 3, lane 4).

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

Example 4

The T.ovis oncosphere λgt11 cDNA library (Johnson et al . , Nature 338 585-587 (1989)) was screened using a 1/50 dilution of rabbit anti-18 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-18 kDa serum by low pH elution from nitrocellulose filters impregnated with λgt11 - expressed fusion protein. These antibodies were used m plaque arrays to determine which clones were related (sibling analysis) and in immunoblots to confirm that antibody to the recombinant protein recognised the 18 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 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 λgt11 into the GST fusion protein expression system, the vector pGEX-1T was constructed to allow in- frame 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 with EcoRI and BamHI 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 EcoRI and BamHI and the small linker fragment purified by electrophoresis on a 20% polyacrylamide gel in tris-Borate-EDTA buffer. The linker band was visualised with ethidium bromide staining, cut out and DNA eluted into d.H₂0 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-IT plasmid DNA was cut with EcoRI . treated with phosphatase and ligated to the insert cDNA's derived from the λgt11 clone described above. Ligations were transformed into E.coli JM101 and clones expressing the GST- 18 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-18 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 14,205 Da based on the predicted amino acid sequence.

Example 5 Two vaccination trials were undertaken to investigate the immunogenicity of the fusion protein GST-18.

Trial A

Five Romney sheep were immunised by subcutaneous injection on three occasions four weeks apart, with 50μg of GST-18 affinity purified fusion protein and 1mg Saponm adjuvant.

Three weeks after the third injection, these sheep plus eight 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 numbers of cysts.

Trial B

Ten Romney lambs were immunised by subcutaneous injection on two occasions four weeks apart, with 100 μg GST-18 affinity purified fusion protein and 10 mg 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 and B are shown in Table 3.

Table 3

Cyst counts at post mortem of sheep immunised with GST-18.

Group Cyst No. in individual Mean % Significance sheep Protection *

Trial A

Control 0 1 1 3 8 10 23.0

56 105

GST-18 0 0 1 1 3 1.0 96 NSD

Trial B

Control 13 16 22 27 35 35 39.1 37 48 50 52 56

GST-18 0 0 0 0 0 0 0 0.1 99 P<0.01 0 0 1

*Mann-Whitney Test

NSD, not significantly different. (P>0.05)

The results show that the fusion protein GST-18 is a potent immunogen.

Serum samples from sheep immunised with GST-18 were analysed by immunoblotting against native oncosphere antigens. (Fig.3) Antibodies to the recombinant GST-18 recognised the 18 kDa native antigen indicating that the recombinant protein shares one or more epitopes with the native 18 kDa protein.

INDUSTRIAL APPLICATION

In accordance with the present invention there is provided a polypeptide antigen of T. 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 and/or its fragments stimulates almost complete immunity against challenge infection with T. ovis 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. SEQUENCE LISTING

(1 ) GENERAL INFORMATION

(1) 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.

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

(3) NUMBER OF SEQUENCES: 3

(4) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: A J PARK & SON

(B) STREET: HUDDART PARKER BUILDING, POST OFFICE SQUARE

(C) CITY: P O BOX 949, WELLINGTON

(D) COUNTRY: NEW ZEALAND

(5) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5,DS,HD FLOPPY DISC

(B) COMPUTER: IBM PC COMPATIBLE

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 5.1 FOR WINDOWS

(6) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 7 APRIL 1994

(C) CLASSIFICATION:

(7) ATTORNEY/AGENT INFORMATION:

(A) NAME: BENNETT, MICHAEL R. (8) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (64 4) 473 8278

(B) TELEFAX: (64 4) 472 3358

472 3351

(2) INFORMATION FOR SEQUENCE ID NO. 1: (1 ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 574 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 1:

GAATTC CGT CGG TTT GGT CTC ATC TTG TTG GTG 33 Arg Phe Gly Leu He Leu Leu Val 4 GCC GTC GTT TTG GCG AGC GGT GGC CGA AAT CCC 66

Ala Val Val Leu Ala Ser Gly Gly Arg Asn Pro 7 GGC AAA CGT AGT ATC GTG CCA TAC ATT CGC TGC 99

Gly Lys Arg Ser He Val Pro Tyr He Arg Cys 00 TTC GCC CTT AGA AAT GAA CGA ATC GCG GTG GTT 132

Phe Ala Leu Arg Asn Glu Arg He Ala Val Val 33 TGG GAT ACT AAA GAT ATG GCT GGC TAT GAC GTG 165

Trp Asp Thr Lys Asp Met Ala Gly Tyr Asp Val 66 AAG AAG ATC GAA GTG ACA GTA GAA AAG GCA ATA 198 Lys Lys He Glu Val Thr Val Glu Lys Ala He 99 GAT CCA CAC AAG ACC TGG AAT ACA ACA GTC AGC 231 Asp Pro His Lys Thr Trp Asn Thr Thr Val Ser 32 GTG GAC AAT GGA AAA GTC ATT ATG AGC GCG TTG 264 Val Asp Asn Gly Lys Val He Met Ser Gly Leu 65 AAG GCC AAC ACA ATT TAC AGA GTG GGC GTA AAC 297

Lys Ala Asn Thr He Tyr Arg Val Gly Val Asn 98 GGC TAT CGA AAC GAT TTC ATG GTG TTT GGT TCG 330

Gly Tyr Arg Asn Asp Phe Met Val Phe Gly Ser 331 GAG CGT TTC GTG ACA ACA CTT TCG AAA AAG AAG 3

Glu Arg Phe Val Thr Thr Leu Ser Lys Lys Lys

364 ACC AAA AGC AGG AAG GCC CGA GGA TTG TAGATGCTTG 4

Thr Lys Ser Arg Lys Ala Arg Gly Leu

401 CATGTCCGTA GTGCCTGCGT CAAACCTAAC GTTTGCGCAA AGCGGGCAAT 4

451 GCATTTTGTC GTCAATCCTT CGCAAGCAAG CAAATATAAT GTGACCTTTC 5

501 ATGTTAACGA ATAATTCTTC GATTACAAAT ACAGGGTGTT GGTAATGCTA 55

551 AAAAAAAAAA AAAAAAAGGAATTC 57

(3) INFORMATION FOR SEQUENCE ID NO. 2:

(1 ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 381 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: cDNA

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 2:

CGG TTT GGT CTC ATC TTG TTG GTG 24

Arg Phe Gly Leu He Leu Leu Val 5 GCC GTC GTT TTG GCG AGC GGT GGC CGA AAT CCC 57

Ala Val Val Leu Ala Ser Gly Gly Arg Asn Pro 8 GGC AAA CGT AGT ATC GTG CCA TAC ATT CGC TGC 90

Gly Lys Arg Ser He Val Pro Tyr He Arg Cys 1 TTC GCC CTT AGA AAT GAA CGA ATC GCG GTG GTT 123

Phe Ala Leu Arg Asn Glu Arg He Ala Val Val 24 TGG GAT ACT AAA GAT ATG GCT GGC TAT GAC GTG 156

Trp Asp Thr Lys Asp Met Ala Gly Tyr Asp Val 57 AAG AAG ATC GAA GTG ACA GTA GAA AAG GCA ATA 189

Lys Lys He Glu Val Thr Val Glu Lys Ala He 90 GAT CCA CAC AAG ACC TGG AAT ACA ACA GTC AGC 222

Asp Pro His Lys Thr Trp Asn Thr Thr Val Ser 23 GTG GAC AAT GGA AAA GTC ATT ATG AGC GCG TTG 255

Val Asp Asn Gly Lys Val He Met Ser Gly Leu 56 AAG GCC AAC ACA ATT TAC AGA GTG GGC GTA AAC 288

Lys Ala Asn Thr He Tyr Arg Val Gly Val Asn 89 GGC TAT CGA AAC GAT TTC ATG GTG TTT GGT TCG 321

Gly Tyr Arg Asn Asp Phe Met Val Phe Gly Ser 22 GAG CGT TTC GTG ACA ACA CTT TCG AAA AAG AAG 354

Glu Arg Phe Val Thr Thr Leu Ser Lys Lys Lys 55 ACC AAA AGC AGG AAG GCC CGA GGA TTG 381

Thr Lys Ser Arg Lys Ala Arg Gly Leu (4) INFORMATION FOR SEQUENCE ID NO. 3:

(1 ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 127 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) TOPOLOGY: LINEAR

(2) MOLECULE TYPE: PROTEIN

(3) SEQUENCE DESCRIPTION: SEQ ID NO. 3:

Arg Phe Gly Leu He Leu Leu Val 8

Ala Val Val Leu Ala Ser Gly Gly Arg Asn Pro 19

0 Gly Lys Arg Ser He Val Pro Tyr He Arg Cys 30

1 Phe Ala Leu Arg Asn Glu Arg He Ala Val Val 41

2 Trp Asp Thr Lys Asp Met Ala Gly Tyr Asp Val 52

3 Lys Lys He Glu Val Thr Val Glu Lys Ala He 63

4 Asp Pro His Lys Thr Trp Asn Thr Thr Val Ser 74

5 Val Asp Asn Gly Lys Val He Met Ser Gly Leu 85

6 Lys Ala Asn Thr He Tyr Arg Val Gly Val Asn 96

Gly Tyr Arg Asn Asp Phe Met Val Phe Gly Ser 107

8 Glu Arg Phe Val Thr Thr Leu Ser Lys Lys Lys 118

9 Thr Lys Ser Arg Lys Ala Arg Gly Leu 127

Claims

CLAIMS :

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 18 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 i 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:

(a) the polypeptide of claim 4;

(b) a peptide fragment of (a) having equivalent protective immunological activity thereto; and

(c) a variant of (a) or (b) 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) a nucleotide sequence encoding the antigenic polypeptide of claim 1 ; (b) a nucleotide sequence encoding a peptide fragment of the antigenic polypeptide of (a) , which fragment has substantially equivalent protective immunological activity to the polypeptide of (a) ; and (c) a nucleotide sequence encoding a variant of the polypeptide of (a) or a variant of the peptide of (b) 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 (a) or the peptide fragment of (b) .

10. 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 variant 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 cultur g 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 n 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.

24. An optionally-labelled DNA molecule comprising part or all of the nucleotide sequence of 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.