Stable forms of antigenic Taenia ovis polypeptides

This invention relates to stable forms of peptide antigens of T. ovis suitable for use in vaccines to protect ruminants against infection by cestode parasites. The antigens are preferably obtained by expression of DNA coding therefor in a recombinant host cell. Aspects of the invention include DNA encoding the antigens, vectors containing the DNA and hosts which express the antigens.

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. 
Previous investigations conducted into vaccination against T.ovis infection 
with oncosphere antigens are reviewed by Rickard, M. D. and Williams, J. 
F., Hydatidosis/Cystercercosis: Immune mechanisms and Immunisation against 
infection, Adv Parasitology 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 & 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 
.beta.-galactosidase fusion proteins were selected using antibodies in 
serum from sheep infected with T.ovis . Some fusion proteins were shown to 
correspond with native antigens (92.5 to 180kD) present in adult and 
oncosphere stages of T.ovis , but trials of the host-protective nature of 
purified fusion proteins were not reported. 
In Johnson, K. S. et al, Nature 338, 585-587 (1989), the present inventors 
have described the identification and cloning of a native polypeptide of 
T.ovis capable of generating a protective immunological response in 
ruminants against T.ovis infection. However, the recombinant polypeptide 
described in this paper has been found to be less stable than is optimal 
for the production of a commercial vaccine. 
It is accordingly an object of the present invention to provide a stable 
form of 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. 
SUMMARY OF INVENTION 
Accordingly, in one aspect the present invention may broadly be said to 
consist in a purified stable antigenic peptide comprising a fragment of a 
T.ovis polypeptide, which fragment 
(a) has a molecular weight of from about 23 kD to about 24 kD: and 
(b) is capable of generating a protective immunological response to T.ovis 
in a ruminant 
or a subfragment or variant thereof having substantially equivalent 
stability and immunological activity thereto. 
Preferably, the antigenic polypeptide fragment has the amino acid sequence 
set out in FIG. 1. 
Conveniently, the protective antigenic peptide of the invention is obtained 
by expression of the DNA sequence coding therefor in a host cell or 
organism. 
In a further aspect, the invention consists in a stable antigenic peptide 
comprising a fragment of a T.ovis polypeptide which is capable of 
generating a protective immunological response to T.ovis in a ruminant, 
which fragment has the amino acid sequence encoded by the DNA sequence of 
FIG. 1 herein, or a derivative or variant thereof having substantially 
equivalent stability and immunological activity thereto. 
In still a further aspect, the invention consists in a composition of 
matter capable of generating a protective immunological response to T.ovis 
infection in a ruminant which essentially consists of: 
(a) a stable peptide comprising a polypeptide fragment having the amino 
acid sequence of FIG. 1; 
(b) a stable immunologically active subfragment of (a): 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 
equivalent stability and immunological activity thereto. 
In still a further aspect, the invention consists in a vaccine which 
includes a stable immunogenic peptide thereof as defined above in 
combination with a pharmaceutically acceptable carrier and/or adjuvant 
therefor. 
In still a further aspect, the invention may be said to consist in a method 
of protecting a ruminant against infection by a cestode parasite 
comprising administering to said ruminant an immunologically effective 
amount of: 
(a) a stable peptide as defined above: 
(b) a composition as defined above: or 
(c) a vaccine as defined above. 
In additional aspects, the invention relates to a DNA isolate which 
comprises a DNA sequence encoding the immunologically effective antigenic 
peptide of the invention: to transformed hosts capable of expressing the 
antigenic peptide encoded; and to methods of producing the antigenic 
peptide comprising culturing the said transformed hosts.

DETAILED DESCRIPTION OF THE INVENTION 
Previous investigations by the inventors have identified a 47-52 kD 
molecular weight fraction of the antigenic complement of T.ovis as a major 
antigen to which antibodies were present in immune sheep. The cloning and 
recombinant production of this native antigen in general and of a major 
fragment of the antigen in particular (termed the "45W" antigen) has been 
reported (Johnson, K. S. et al, Nature (1989), supra), and forms the 
subject of U.S. Ser. No. 07/349,723, the disclosure of which is 
specifically incorporated herein by reference. 
However, during the course of subsequent investigations into the production 
of a commercial vaccine based upon this major fragment it was found that 
although it was perfectly possible to produce a vaccine composition 
protective against T.ovis, the stability of the polypeptide fragment 
encoded by the 45W EcoRI/EcoRI DNA fragment itself was less than ideal. 
In further investigations, the inventors have now located a subfragment of 
the 45W antigen which exhibits enhanced stability while also having 
equivalent if not superior immunological activity as compared to the 45W 
antigen. It is to this more stable subfragment that the present invention 
is primarily directed. 
The present invention therefore has as its first aspect an antigenic 
peptide comprising a fragment of a T.ovis polypeptide which is capable of 
generating a protective immunological response to T.ovis . The molecular 
weight of this fragment has been calculated as about 23,841 Da from the 
DNA sequence determined for the molecule. 
In a preferred form of this aspect of the invention, the immunologically 
active peptide includes a polypeptide fragment having the amino acid 
sequence of FIG. 1. It will however be appreciated that modifications can 
be made to the native sequence of the polypeptide fragment whilst at least 
substantially retaining both its stability and biological activity. Such 
modifications to the native amino acid sequence to result in the 
insertion, substitution or deletion of one or more amino acids are 
specifically within the scope of this invention. 
The antigenic polypeptide fragments of the invention can be prepared in a 
variety of ways. For example, they can be obtained by isolation from a 
natural source, by synthesis using any suitable known technique (such as 
by the stepwise solid phase approach described by Merrifield (1963) J. 
Amer Chem. Soc 85 2149-2156) or, as is preferred, through employing 
recombinant DNA techniques. 
The variants of the polypeptide fragments can similarly be made by any of 
those techniques known in the art. For example, variants can be prepared 
by site-specific mutagenesis of the DNA encoding the native amino acid 
sequence. 
Site-specific mutagenesis allows the production of variants through the use 
of specific oligonucleotide sequences which encode the DNA sequence of the 
desired mutation, as well as a sufficient number of adjacent nucleotides, 
to provide a primer sequence of sufficient size and sequence complexity to 
form a stable duplex on both sides of the deletion junction being 
traversed. Typically, a primer of about 20 to 25 nucleotides in length is 
preferred, with about 5 to 10 residues on both sides of the junction of 
the sequence being altered. In general, the technique of site-specific 
mutagenesis is well known in the art as exemplified by publications such 
as Adelman et al., DNA 2, 183 (1983). 
In a further aspect, the invention accordingly relates to the recombinant 
production of the stable antigenic 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 antigen expressed. 
An initial step in the method of recombinant production of 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 peptide 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 No 4 p 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 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 .beta.-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); 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 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 Reg. 7: 
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 
utilization. An 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 in recent years (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 terminator 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 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 (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 peptide encoded can be produced often in the form of a fusion 
protein by culturing the host cells. The fusion protein is then recovered 
and purified as necessary. 
Where the protein is produced in a soluble form, 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, where the peptide of 
the invention is produced as a fusion protein, the carrier portion of the 
fusion protein can prove useful in this regard. 
However, it has been the applicants' experience that only a minor 
proportion of the protein is expressed in a soluble form, with the major 
proportion being expressed in a non-soluble form contained with inclusion 
bodies within the transformed host. In such cases, a solubilisation 
technique can be employed to extract the product from the inclusion bodies 
and to render it soluble for subsequent processing and use. 
Once again, any of those conventional techniques known in the art for this 
purpose can be employed. For a general review of these techniques, 
reference can be made to the following publications, Marston, F. A. O., 
"The purification of eukaryotic polypeptides synthesized in Escherichia 
coli"; Biochem J 240 1-12 (1986); and Sharma S. K., "On the recovery of of 
Genetically Engineered Proteins from Escherichia coli", Separation Science 
and Technology 21 (8), pp 701-726 (1986). Of such techniques, urea 
solubilisation as described in Example 4 is preferred. 
Following recovery of the antigenic peptide it is purified as desired. The 
purification procedure adopted will of course depend upon the degree of 
purity required for the use to which the 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 encodes the nucleotide 
sequence of FIG. 1. This DNA sequence 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. 
However, as indicated above, the invention also contemplates variants of 
the peptide 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 equimolar amounts 
of the desired components, appropriately tailored for correct matching, 
and treatment with an appropriate ligase (eg T.sub.4 DNA ligase). 
In addition to the protective stable antigens of the invention and the 
method of producing these, a further and most important aspect of the 
present invention relates to the use of the protective antigen as the 
active agent in a ruminant vaccine against T.ovis infection. In this 
aspect, the protective antigen of the invention can be administered either 
alone or in the form of a composition comprising the protective antigen of 
the invention as the active ingredient together with a pharmaceutically 
acceptable diluent carrier or adjuvant. 
Examples of suitable adjuvants known to those skilled in the art are 
saponins (or derivative or related material), muramyl dipeptide, trehalose 
dimycollate, Freunds'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, sarcosine, glycerol, sorbitol, propylene glycol, 
fixed oils and synthetic esters of higher fatty acids. Saponins have been 
found to be particularly effective adjuvants. 
The protective antigen of the invention may also be treated in any 
conventional way to still further enhance its stability or to conserve or 
potentiate its immunogenic efficacy. For example, the antigen may be 
treated with a suitable inhibitor, modifier, crosslinker or denaturant in 
such a way as to enhance its immunogenicity. 
In addition, the protective antigen can be administered in combination with 
other therapeutic agents such as anthelmintics, for example levamisole, or 
other vaccines. 
The vaccine can be administered to the ruminant 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 ruminant to be treated will 
depend on the type, size and body weight of the ruminant as well as on the 
immunogenicity of the vaccine preparation. Conveniently, the vaccine is 
formulated such that relatively small dosages of vaccine (1 to 5 ml) are 
sufficient to protect the ruminant. 
The immunogenicity of the antigenic peptide of the invention and its 
effectiveness as the active agent of a ruminant vaccine will be 
appreciated from the immunogenicity trials detailed in the Examples 4 and 
5. 
Specific non-limiting examples of the invention will now be described. 
EXAMPLE 1 
Identification and Initial Cloning of the Stable Antigen of the Invention 
cDNA encoding the 45W antigen is contained in E. coli 45W which has been 
deposited on Aug. 27, 1987 at the American Type Culture Collection, 12301 
Parklawn Drive, Rockville, Md. 20852, USA under accession number 67505. 
The details of derivation of E. coli 45W are fully set out in U.S. Ser. 
No. 07/349,723. 
The entire 45W complementary cDNA was cloned into the Eco RI site of the 
vector pUC18 (Yanisch-Perron Gene 33, 103-119 (1985)) prior to cloning 
into the plasmid pGEX-2T (Smith D. B. and Johnson K. S. Gene 67, 31-40 
(1988)). This plasmid pUC18-45W was digested with the restriction 
endonucleases Bam HI and Xho II. The DNA was electrophoresed on agarose 
gels and the Bam HI/Xho II fragment containing 45W DNA was purified. This 
fragment was then ligated with pGEX-2T DNA digested with Bam HI and 
transformed into the E. coli strain JM101 (Yanisch-Perron et al., 1985 
supra). 
The appropriate clones were selected on the basis of their DNA insert size 
and molecular weight of their affinity purified GST fusion proteins (Smith 
and Johnson Gene (1988) supra). The approximate molecular weight of the 
T.ovis portion of the pGEX-2T-45W (Bam HI/Xho II) fusion protein is 
23,841Da, calculated from the DNA sequence of the molecule. This fusion 
protein when electrophoresed on a 13% SDS gel and stained with Coomassie 
blue was found to be stable. 
The T.ovis portion of the 45W DNA sequence contained in the clone 
pGEX-2T-45W (Bam HI/Xho II) is set out in FIG. 1. The predicted amino acid 
sequence for this T.ovis antigen fragment is also set out in this Figure. 
It will however be appreciated that the non-GST portion of cloned fusion 
protein GST 45W(B/X) includes a small number of additional amino acids in 
addition to those encoding the fragment of the T.ovis polypeptide. These 
additional amino acids are included as a consequence of the cloning and 
expression of the T.ovis fragment as described above. 
The non-GST portion of the fusion protein is hereinafter called "45W(B/X)". 
EXAMPLE 2 
Demonstration of Antigenicity of the GST-45W(B/X) Fusion Protein 
Western blot analysis was performed with the fusion proteins prepared as 
described in U.S. Ser. No. 07/349,723 from pGEX-2T-45W (Bam HI/Xho II), 
pSj10.DELTA.ABam7Stop7-45S and pGEX-2T-45W. These fusion proteins were 
electrophoresed on SDS-polyacrylamide gels, transferred to nitrocellulose 
and probed with the 45W specific monoclonal antibody 3F10F6 prior to 
incubation with .sup.125 I-labeled protein A. 
Antibody 3F10F6 was prepared as follows: 
BALB/c mice were immunised with GST 45W fusion protein and hybridomas were 
prepared according to the method of Fazekas de St. Groth and Scheidegger 
(J. Immunological Methods 35 1-21, Production of Monoclonal Antibodies; 
Strategy and tactics (1980)). Monoclonal antibody 3F10F6 was selected by 
ELISA analysis of hybridoma supernatants against GST 45W, and was purified 
by the method of Reik et al (1987, J. Immunological Methods 100, 123-130). 
The results are shown in FIG. 2. As can be seen by reference to this 
Figure, a positive reaction was obtained with antibody 3F10F6 in relation 
to the antigen expressed by host cells transformed with pGEX-2T-45W (Bam 
HI/Xho II). This indicated that the stable peptide was likely to be 
immunogenic. 
EXAMPLE 3 
Preparation of Fusion Protein for Immunogenicity Trials 
An equivalent procedure to that set out in Example 1 was adopted to prepare 
fusion protein for the immunogenicity trials. The host cell chosen to 
express the fusion protein was in this case E. coli DH5 transformed as 
follows. 
The entire 45W cDNA was cloned into the Eco RI site of the vector pUC 18 
(Yanisch-Perron, Gene 33, 103-119, 1985) prior to cloning into the plasmid 
pGEX-2T (Smith and Johnson, Gene (1988) supra). This plasmid pUC 18 was 
digested with the restriction endonucleases Bam HI and Xho II. The DNA was 
electrophoresed on agarose gels and the Bam HI/Xho II (658 bp) fragments 
containining 45W DNA were purified. These fragments were then ligated with 
pGEX-2T DNA digested with Bam HI and transformed into the E. coli strain 
DH5. 
An overnight 100 ml culture of transformed E. coli DH5 was added to a 
shaker flask containing 800 ml SOB medium containing 0.1 mg/ml ampicillin. 
After 30 minutes, fusion protein expression was induced by adding IPTG to 
0.021 mg/ml and culturing continued for a further 6 hours. The bacteria 
were pelleted by centrifugation at 5000.times.g for 30 minutes and the 
cell pellet (net weight=4.7 g) was resuspended in 22 ml PBS pH7.4. 
Lysozyme and triton-X-loo were added to 0.25 mg/ml and 0.5% final 
concentration respectively and the suspension was mixed for 10 minutes at 
ambient temperature. The bacteria were disrupted by sonication and the 
lysate was centrifuged at 4500.times.6 for 40 minutes. Soluble fusion 
protein GST-45W(B/X) was purified from the supernatant by affinity 
chromatography as follows. 
The supernatant was mixed at room temperature with 50 ml washed 
glutathione-agarose beads (sulphur linkage, Sigma) for 30 minutes. After 
absorption, the unbound supernatant was eluted and the beads washed on a 
sintered glass filter with 5.times.200 ml volumes of PBS. Fusion protein 
was eluted in 2.times.25 ml volumes (5 minutes each volume) of 50 mM 
Tris-HCl pH 8.0 containing 5 mM reduced glutathione (Sigma) freshly 
prepared. 
To solubilise the non-soluble fusion protein, the pellet was resuspended in 
15 ml of 50mM borate buffer pH 9.0 containing 2 mM EDTA and 1 mM DTT. A 5 
ml aliquot was removed and stored frozen at -18.degree. C. The remaining 
10 ml suspension was made up to 25 ml by addition of borate buffer and dry 
urea to a final concentration of 7.0M and was mixed for 2 days at ambient 
temperature. Triton X-100 was added to 0.4% final concentration and the 
solution was dialysed initially against borate buffer as above followed by 
PBS pH7.2 over 3 days. The dialysed protein solution was centrifuged at 
35000.times.g for 30 minutes at 4.degree. C. and the clear supernatant 
retained. 
Part of the urea solubilised pellet was kept for vaccination trials and 
part was further processed by affinity chromatography on 
glutathione-agarose as described previously. Amount of fusion protein 
recovered at each step is shown in Table 1. The proportion of fusion 
protein GST 45W(B/X) relative to E. coli protein was calculated by 
densitometry of Coomassie Blue stained SDS-PAGE gels (Andrews A. T., 1986, 
Electrophoresis, Oxford University Press). 
TABLE 1 
______________________________________ 
Amount of GST 45W (B/X) recovered during processing 
Total % Fusion Fusion 
Protein Protein Protein 
______________________________________ 
soluble affinity purified 
8.5 mg 80 6.8 mg 
urea solubilised pellet 
42.5 mg 37 15.7 mg 
urea solubilised, affinity 
3.0 mg 71 2.1 mg 
purified 
______________________________________ 
EXAMPLE 4 
Vaccination Trials With Fusion Protein Extracts 
In an initial experiment 5 Romney sheep aged 10 months were immunised with 
2 subcutaneous injections of 5 ug of GST 45W(B/X) plus 1 mg saponin in 1 
ml saline, four weeks apart. A booster inoculation of 10 ug fusion protein 
plus saponin was given three weeks later, and the sheep were infected 4 
weeks later with 1200 T.ovis eggs. Three control sheep were given 
injections of GST carrier protein in the same manner. 
In a second experiment groups of sheep were immunised as follows: 
______________________________________ 
Total dose 
Group Antigen of fusion protein* Adjuvant 
______________________________________ 
1 Saline control -- saponin, 1 mg 
2 Affinity purified 
58 ug saponin, 1 mg 
3 Urea extract 132 ug saponin, 1 mg 
4 Urea extract + affinity 
90 ug saponin, 1 mg 
purified 
______________________________________ 
*given in three injections 
Four weeks after the final immunisation all sheep were infected with 2400 
T.ovis eggs. 
Six weeks after infection sheep were humanely slaughtered and carcases 
examined for cysts. 
Results are shown in Table 2. 
TABLE 2 
______________________________________ 
Protection data from vaccination trials 
% 
Reduc- 
Group Antigen Number of cysts* 
Mean tion 
______________________________________ 
Trial A 
1 GST 15 28 63 35.3 -- 
2 GST 45W 0 0 0 0 20 4.0 89 
(B/X) 
affinity purified 
Trial B 
1 Saline 3 12 14 15 16 17 40 
16.7 -- 
2 GST 45W 0 0 0 0 1 0.2 99 
(B/X) 
affinity purified 
3 Urea extract 
0 0 0 1 0.25 98 
4 Urea extract + 
0 0 0 2 0.5 97 
affinity purified 
______________________________________ 
*Trial A: numbers of cysts in whole carcase 
Trial B: numbers of cysts in masseters, heart, diaphragm and external 
carcase. 
It can therefore be seen that immunisation with the fusion protein 
GST-45W(B/X) resulted in a highly significant reduction in cyst numbers 
compared with the controls used. 
EXAMPLE 5 
Antibody Response of Sheep Immunised With GST 45W(B/X) 
Sera from sheep immunised with the extracts described above were analysed 
by immunoblotting and ELISA. 
Immunoblotting was performed as described (Johnson et al Nature (1989) 
supra)) and showed that sheep immunised with the fusion protein GST 
45W(B/X) made antibodies which recognised oncosphere antigens with 
relative mobilities of 47-52000 as described for antibody responses to the 
parent antigen GST 45W (Johnson et al, Nature 1989, supra). These results 
are shown in FIG. 3. 
The GST portion of GST 45W(B/X) was removed by thrombin cleavage and the 
45W(B/X) protein obtained free from GST by depletion on 
glutathione-agarose as described (Smith and Johnson Gene 1988, supra). The 
45W(B;X) protein was used as antigen to coat microtitre plates for a 
standard ELISA as described in "Practice and Theory of Enzyme 
Immunoassays", P. Tijssen, Laboratory Techniques in Biochemistry and 
Molecular Biology; Volume 15, Elsevier Science Publishers (1985). 
Sheep antibody was detected with peroxidase-conjugated rabbit anti-sheep 
lgG (Cappel, Cooper Biomedical Inc.). 
The results are shown in FIG. 4. As can be seen from this Figure, 
immunisation of sheep with affinity purified fusion protein or urea 
extracted fusion protein stimulated strong antibody responses in all 
groups of sheep. 
In accordance with the present invention there is provided a stable peptide 
comprising a fragment of a T.ovis polypeptide which is effective in 
generating a protective immunological response against T.ovis infection in 
ruminants. It has been established that vaccination with this peptide 
stimulates almost complete immunity against challenge infection with 
T.ovis eggs. Insofar as the applicants are aware, it is the highest level 
of protection thus far achieved in a natural host-parasite system by 
injection of a single antigen. 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.