WO1995012679A1

WO1995012679A1 - Anti-helminth vaccines

Info

Publication number: WO1995012679A1
Application number: PCT/GB1994/002411
Authority: WO
Inventors: Trevor Stanley Smith; Fiona Callander Smith; Danielle Mcmichael-Phillips
Original assignee: Biotechnology And Biological Sciences Research Council
Priority date: 1993-11-05
Filing date: 1994-11-03
Publication date: 1995-05-11
Also published as: ZA948729B; GB9322808D0; AU8064894A

Abstract

The present invention provides a protective helminth parasite antigen which is characterized by possessing transport protein activity and which, in native form, is a membrane-associated protein, or a functionally-equivalent variant, or antigenic fragment or precursor thereof, and its use in the stimulation of an immune response against helminth parasites in an animal. Preferably, the antigen has η-glutamyl transpeptidase activity.

Description

ANTI-HELMINTH VACCINES

This invention relates to novel helminth parasite antigens and their use in the control of disease caused by helminth parasites, particularly parasitic nematodes of the gastro-intestinal tract of mammals.

Helminth parasites, particularly nematodes, infect or infest a wide range of animals, including man, and are a widespread and significant source of disease and ill-thrift, not only in animals, but also in man. Such parasites thus represent a considerable worldwide drain on economic resources. This is particularly true in animal husbandry, where parasite infections of grazing animals, such as sheep and cattle, are often difficult and expensive to control and may result in significant economic losses.

Particular mention may be made in this regard of the blood-sucking nematode Haemonchus contortus, a parasite of ruminants, most notably sheep. Also worthy of particular mention from the economic viewpoint are the non-blood feeding nematodes Ostertagia ostertagi and Ostertagia (Teladprsagia) circumcincta (o. circumcincta has recently been reclassified as T.circumcincta , although the new name is not yet in wide usage) .

Other parasitic helminths of economic importance include the various species of the following helminth families:- Trichcstrpngylus, Nematpdirus, Pictypcaulus, Cppperia, Ascaris, Dirofiioria. Trichuris, Strpngylus,

Fasciola. Oesophaσostomum. Bunostomum and Metastrongylus. In addition to domestic livestock, pets and humans may also be infected, not infrequently with fatal results.

At present, control of helminth parasites of grazing livestock relies primarily on the use of anthelmintic drugs, combined with pasture management. Such techniques have not proved entirely satisfactory however, due to their expense and inconvenience and to a rapid increase in drug resistance. Anthelmintic drugs need to be administered frequently and appropriate pasture management is often not possible on some farms and even where it is, it can place constraints on the best use of available grazing.

To overcome these problems, attempts have been made to achieve immunological means of control. Although there has been some success in identifying certain protective antigens as potential vaccine candidates, most notably in Haemonchus. this approach has proved difficult and, other than for the cattle lungworm Dictyocaulus Viviparus, has yet to come to commercial fruition.

The most success to date has been achieved with the protein doublet H110D, an integral membrane protein isolated from the gut of H.contortus and described by Munn in WO88/00835. H110D now represents the most promising vaccine candidate to date.

Munn has also described and proposed as a vaccine, contortin, a helical polymeric extracellular protein associated with the luminal surface of H.contortus intestinal cells (Munn et al.. Parasitology £4.: 385-397, 1987) .

A third Haemonchus gut membrane protein with protective antigenic properties has also been discovered and termed H45 (Munn and Smith, WO90/11086) .

Whilst proteins such as H110D and H45 can be used as the basis for a vaccine against Haemonchus. there is nonetheless a continuing need for new and improved helminth parasite vaccines and in particular for a vaccine which may be used across a broad range of helminth genera.

The present invention accordingly seeks to provide novel antigens for use as helminth parasite vaccines and in particular as protective immunogens in the control of diseases caused by helminth parasites. More specifically, the present invention is based on the finding that extracts of H.contortus containing a membrane-associated protein having γ-glutamyl- transpeptidase (γGTP) activity are capable of conferring protective immunity in animals against Haemonchus. yGTP functions as a transporter of amino acids across biological membranes. It has also been observed that membrane-bound transporters of other biological molecules also have useful protective antigenic activity. Such proteins, when liberated from the membranes with which they are associated, for example by the use of detergents, are novel and of use in the manufacture of vaccines against helminth infections.

According to one aspect, the present invention thus provides a protective helminth parasite antigen which is characterised by possessing transport protein activity and which, in native form, is a membrane-associated protein, or a functionally-equivalent variant, or antigenic fragment or precursor thereof.

In a preferred aspect the antigen has γ-glutamyl- transpeptidase activity.

A further aspect of the invention provides such protective antigens, and functionaly-equivalent variants, antigenic fragments or precursors thereof, for use in stimulating an immune response against helminth parasites in a human or non-human, preferably mammalian, especially preferably ruminant, animal.

The term "transport protein activity" is used herein to describe protein which are involved in the active transport across biological membranes of biomoleculeε such as amino acids, peptides, carbohydrates, cofactors, nucleotides or ions. Many specific transport proteins are known for the transport of amino acids and sugar, for example.

A precursor for the antigen in question may be a larger protein which is processed, eg. by proteolysis, to yield the antigen per se. Such precursors may take the form of zymogens ie. inactive precursors of enzymes, activated by proteolytic cleavage, for example analogous to the pepsin/pepsinogen system or the well known zymogens involved in the blood clotting cascade.

The novel antigens of the invention are not recognised by sera from naturally immune animals. In other words, they are not normally, in native form, accessible to the immune system of the infected host and are thus "hidden", "concealed" or "cryptic" antigens.

The term "protective antigens" or "protective antigenic activity" as used herein defines those antigens and their fragments or precursors, capable of generating a host-protective, ie. immunogenic, immune response, that is a response by the host which leads to generation of immune effector molecules, antibodies or cells which damage, inhibit or kill the parasite and thereby "protect" the host from clinical or sub-clinical disease and loss of productivity. Such a protective immune response may commonly be manifested by the generation of antibodies which are able to inhibit the metabolic function of the parasite, leading to stunting, lack of egg production and/or death.

As mentioned above, included within the scope of the invention are functionally-equivalent variants of the novel antigens and their fragments and precursors. "Functionally-equivalent" is used herein to define proteins related to or derived from the native protein, where the amino acid sequence has been modified by single or multiple amino acid substitution, addition and/or deletion and also sequences where the amino acids have been chemically modified, including by deglycosylation or glycosylation, but which nonetheless retain protective antigenic activity eg. are capable of raising host protective antibodies and/or functional immunity against the parasites. Within the meaning of "addition" variants are included amino and/or carboxy terminal fusion proteins or pclypeptides, comprising an additional protein or polypeptide fused to the γ-GTP antigen sequence. Such functionally-equivalent variants mentioned above include natural biological variations (eg. allelic variants or geographical variations within a species) and derivatives prepared using known techniques. For example, functionally-equivalent proteins may be prepared either by chemical peptide synthesis or in recombinant form using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids. Functionally-equivalent variants according to the invention also include analogues in different parasite genera or species.

As mentioned above, γ-GTP enzymes function to transport glutamic acid and other amino acids across cell membranes. Other specific transport proteins exist for the transport across membranes of other nutrients, including other amino acids, carbohydrates e.g. sugars and ions. Such proteins are involved for example in the nutrient assimilation process in the gut of mammals.

Whilst, not wishing to be bound by theory, it is believed that blockage of such proteins by antibodies may contribute to the protective antigenic response.

A variety of γ-GTP enzymes are known and described in mammals, although not much is yet known concerning equivalent enzymes in helminths. γ-GTP-like activity may be assayed using γ-glutamic acid p- nitroanilide/glycylglycine as substrate, or by other techniques known in the art e.g. histochemistry using y- glutamyl-4-methoxy-2-naphthylamide as substrate in the presence of glycylglycine and diazotized 4'-amino-2', 5' -diethoxybenzanilide (Fast blue BBN) on cryostat sections of frozen tissue by the method of Rutenburg et al. 1969 J. Histochem Cytochem 17, 517.

In the case of the nematode worm Haemonchus contortus. the yGTP antigen of the invention exhibits an apparent molecular weight (Mr) of about 58,000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non- reducing conditions.

The γ-GTP antigens of the present invention are associated with the membranes of the helminth parasites. In particular, studies using various detergents have shown that γ-GTP-like activity may be localised in an integral membrane fraction, that is a fraction extractable with ionic detergents such as Triton X-100 or polyoxyethylene 9-lauryl ether (Thesit) , as well as in a fraction which is more loosely associated with cellular membranes, and which may be extracted with gentler detergents eg. polyoxyethylene sorbitan monolaurate (Tween 20) , (Triton, Thesit and Tween are Trade Marks) .

In the case of the helminth Haemonchus contortus it has been found that up to 74% of γ-GTP-like activity may be localised in a Thesit detergent soluble extract, and that about 16% may be localised in a Tween detergent soluble extract.

Thus, antigens according to the invention may be obtained by extraction of helminth parasites using appropriate detergents.

A further aspect of the invention thus provides a method for preparing the above-mentioned antigens of the invention which comprises the step of subjecting a crude extract of a helminth parasite to detergent extraction, eg. using Tween 20 or a like detergent, and recovering the solubilised proteins.

The detergent extract may then be subjected to further purification using conventional procedures eg. centrifugation, selective precipitation, electrophoresis, chromatography and the like. Fractions containing the antigen of the invention may be identified by assaying for γ-GTP activity using known techniques.

Alternatively, the detergent extraction may take place using a stronger detergent, such as Thesit or Triton X-100, which is capable of solubilising integral membrane proteins. In the case of Haemonchus. this is less preferred however, as such detergents also extract the integral membrane antigens H110D and H45 mentioned above.

Antigens obtainable by such methods form a further aspect of the invention.

The crude extract of the helminth parasite may be prepared using conventional biochemical and surgical techniques eg. by homogenisation of the whole or a portion of the parasite. Thus, for example the parasites may be subjected to homogenisation in a suitable buffer or medium such as phosphate buffered saline (PBS) and the insoluble material (ie. the pellet) may be recovered by centrifugation, whereby to form the required crude extract.

Thus, a suitable purification protocol for the antigens of the invention might comprise (i) homogenising adult parasite worms in PBS, recovering the PBS-insoluble material (eg. by centrifugation) , optionally repeating the PBS-washing step one or more times, (ii) extracting the PBS-insoluble material (ie. the pellet) with Tween 20 (eg. in PBS) , (iii) recovering the solubilised fraction containing the Tween-soluble proteins (ie. the supernatant) followed by (iv) ion exchange chromatography eg. on a Mono Q column, and/or gel filtration eg. using Superose.

Protection trials using antigens prepared either by Tween or by Thesit extraction have shown that immunity may be induced in sheep against challenge by H.contortus. It has been observed in this regard that a greater effect is achieved against male worms than female worms. This is opposite to the effect observed with the antigen H110D, and means for example that the antigens of the invention have utility in augmenting the effects of H110D eg. in a combined vaccine. Viewed from a different aspect, the invention can also be seen to provide use of a helminth parasite antigen as hereinbefore defined, and fragments, precursors and functionally-equivalent variants thereof, for the preparation of a vaccine composition for use in stimulating an immune response against helminth parasites in a human or non-human, animal.

The invention also provides a vaccine composition for stimulating an immune response against helminth parasites in a human or non-human animal comprising one or more antigens, antigenic fragments, precursors or functionally-equivalent variants thereof, as defined above, together with a pharmaceutically acceptable carrier or diluent, and a method of stimulating an immune response against helminth parasites in a human or non-human animal, comprising administering to said animal a vaccine composition as defined above.

The animal preferably is mammalian and more preferably a ruminant.

Antigens according to the invention may be obtained from a range of helminth parasite genera. Preferably, however the helminths will be nematodes, especially preferably gastro-intestinal nematodes including for example HaemPIlChUS, Ostertagia and Trichostronσylus. (For the avoidance of doubt, the term "Ostertagia" as used herein includes Teladorsaσia sp.) Such antigens may be used to prepare vaccines against a range of helminth parasites including any of those mentioned above. Preferred are those antigens, so called "broad spectrum" antigens, which are capable of stimulating host protective immune responses against, in addition to the parasite from which they were isolated, a broad range of other parasites.

As mentioned above, one of the ways in which the antigens of the invention may exert their host protective effects is by raising inhibitory antibodies which inhibit the growth, maintenance and/or development of the parasite. Such antibodies and their antigen- binding fragments (eg. F(ab)₂, Fab and Fv fragments ie. fragments of the "variable" region of the antibody, which comprises the antigen binding site) which may be mono- or polyclonal, form a further aspect of the invention, as do vaccine compositions containing them and their use in the preparation of vaccine compositions for use in immunising hosts against parasites. Such inhibitory antibodies may be raised by use of idiotypic antibodies. Anti-idiotypic antibodies may be used as immunogens in vaccines.

In addition to the extraction and isolation techniques mentioned above, the antigens may be prepared by recombinant DNA technology using standard techniques, such as those described for example by Sambrook et al. , 1989, (Molecular Cloning, a laboratory manual 2nd Edition, Cold Spring Harbor Press) .

Nucleic acid molecules comprising a nucleotide sequence encoding the antigens of the invention thus form further aspects of the invention.

Nucleic acid molecules according to the invention may be single or double stranded DNA, cDNA or RNA, preferably DNA, and include degenerate, substantially homologous and hybridising sequences which are capable of coding for the antigen or antigen fragment or precursor concerned. By "substantially homologous" is meant sequences displaying at least 60%, preferably at least 70% or 80% sequence homology. Hybridising sequences included with the scope of the invention are those binding under non-stringent conditions (6 x SSC/50% formamide at room temperature) and washed under conditions of low stringency (2 x SSC, room temperature, more preferably 2 x SCC, 42^DC) or conditions of higher stringency eg. 2 x SSC, 65°C (where SSC = 0.15M NaCl, 0.015M sodium citrate, pH 7.2), as well as those which, but for the degenerary of the code, would hybridise under the above-mentioned conditions. Derivatives of nucleotide sequences capable of encoding antigenically active antigens or antigen variants according to the invention may be obtained by using conventional methods well known in the art.

Antigens according to the invention may be prepared in recombinant form by expression in a host cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly defined above, operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. Synthetic polypeptides expressed in this manner form a further aspect of this invention (the term "polypeptide" is used herein to include both full-length protein and shorter length peptide sequences) .

The antigen so expressed may be a fusion polypeptide comprising all or a portion of an antigen according to the invention and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto. This may for example be β-galactosidase, glutathione-S-transferase, hepatitis core antigen or any of the other polypeptides commonly employed in fusion proteins in the art.

Other aspects of the invention thus include cloning and expression vectors containing the DNA coding for an antigen of the invention and methods for preparing recombinant nucleic acid molecules according to the invention, comprising inserting nucleotide sequences encoding the antigen into vector nucleic acid, eg. vector DNA. Such expression vectors include appropriate control sequences such as for example translational (eg. start and stop codons, ribosomal binding sites) and transcriptional control elements (eg. promoter-operator regions, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.

Vectors according to the invention may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art. Suitable viral vectors include baculovirus and also adenovirus and vaccinia viruses. Many other viral vectors are described in the art.

A variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression, or into germ line or somatic cells to form transgenic animals. Suitable transformation or transfection techniques are well described in the literature.

The invention also includes transformed or transfected prokaryotic or eukaryotic host cells, or transgenic organisms containing a nucleic acid molecule according to the invention as defined above. Such host cells may for example include prokaryotic cells such as E.coli. eukaryotic cells such as yeasts or the baculovirus-insect cell system, transformed mammalian cells and transgenic animals and plants. Particular mention may be made of transgenic nematodes (see for example Fire, 1986, EMBO J., __.. 2673 for a discussion of a transgenic system for the nematode Caenorhabditis) .

A further aspect of the invention provides a method for preparing an antigen of the invention as hereinbefore defined, which comprises culturing a host cell containing a nucleic acid molecule encoding all or a portion of said antigen, under conditions whereby said antigen is expressed and recovering said antigen thus produced.

The antigens of the invention and functionally equivalent antigen variants may also be prepared by chemical means, such as the well known Merrifield solid phase synthesis procedure.

Water soluble derivatives of the novel antigens discussed above form a further aspect of the invention. Such soluble forms may be obtained by proteolytic digestion, for example using the enzyme elastase. Generally speaking enzymic digestion of the antigens yields two fractions, a detergent soluble fraction (which remains with the membrane) and a water-soluble fraction.

A vaccine composition may be prepared according to the invention by methods well known in the art of vaecine manufacture. Traditional vaccine formulations may comprise one or more antigens or antibodies according to the invention together, where appropriate, with one or more suitable adjuvants eg. aluminium hydroxide, saponin, quil A, or more purified forms thereof, uramyl dipeptide, mineral or vegetable oils, Novasomes or non-ionic block co-polymers or DEAE dextran, in the presence of one or more pharmaceutically acceptable carriers or diluents. Suitable carriers include liquid media such as saline solution appropriate for use as vehicles to introduce the peptides or polypeptides into an animal or patient. Additional components such as preservatives may be included.

An alternative vaccine formulation may comprise a virus or host cell eg. a microorganism (eg. vaccinia virus, adenovirus or Salmonella) which may be live, killed or attenuated, having inserted therein a nucleic acid molecule (eg. a DNA molecule) according to this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.

Administration of the vaccine composition may take place by any of the conventional routes, eg. orally or parenterally such as by intramuscular injection, optionally at intervals eg. two injections at a 7-35 day interval.

The antigens may be used according to the invention in combination with other protective antigens obtained from the same or different parasite species. Thus a vaccine composition according to the invention may comprise one or more of the antigens defined above together with the antigens HllOD and H45 mentioned above and/or glutathione s-transferase. Such a combined vaccine composition may contain smaller amounts of the various antigens than an individual vaccine preparation, containing just the antigen in question. Combined vaccines are beneficial where there is a likelihood that "adaptive selection" of the parasite may occur when a single antigen vaccine is used.

Animals which may benefit from the present invention may be any human or non-human animal, but companion animals, particularly dogs and cats and domestic animals, especially ruminants are preferred. Particular mention may be made of sheep, cattle, pigs, deer and goats.

The invention will now be described further in the following non-limiting Example in which:

Figure 1 shows H. contortus Tween extracted γGTP enriched fractions run on a 6-16% reducing SDS- polyacrylamide gel

1. Fractions purified by ion exchange (Mono Q) and

2. Gel filtration (Superose 6) and selected for high γGTP activity. Contaminants A &B present - MWs of 130,000 and 90,000.

3. Molecular weight markers;

Figure 2 shows the results of an γ-GTP sheep trial; average percentage change in packed cell volume (PCVs) . Abscissa shows days post infection; ordinate shows average percentage change in PCV; D Control group _Δ GTP group;

Figure 3 shows the results of an γ-GTP sheep trial; average faecal egg output. Abscissa shows days post infection; ordinate shows average number of eggs per gram of faeces; D Control group Δ GTP group;

Figure 4 shows the results of an γ-GTP sheep trial; average plasma anti-γGTPEF-1 levels per sheep measured by ELISA. Abscissa days post infection; ordinate shows absorbance at 405 nm; D Control group Δ GTP group;

Figure 5 shows the results of an γ-GTP sheep trial; average worm numbers ■ Male worms □ Female worms. Percentage reductions in male/female numbers with respect to controls are shown vertically above bars. Overall reductions are displayed horizontally.

Figure 6 shows light micrographs of a) transverse cryosection of adult Haemonchus contortus and b) oblique cryosection of Cooperia oncophora stained for γGTP activity by the method of Regenburg et al. The red reaction product is seen as the dark band (arrowed) associated with the microvilli at the luminal surface of the intestine of each parasite. (Reaction product is also found in some regions of the genital tract of Haemonchus (not illustrated) .)

Figure 7 shows a flow diagram of the purification procedures of Example 1 hereinafter.

Example 1

Materials anfl methcds

(a) Production of a γGTP enriched fraction (γGTPEF 1)

Adult Haemonchus contortus were homogenized in ice- cold phosphate buffered saline (pbs; 0.15M, pH7.2) containing 0.02% sodium azide in a Potter-Elvehjeim homogenizer. The suspension was then spun at 17,000g and the pellet resuspended in pbs azide. The washing process was repeated except that the pellet was resuspended in ice cold pbs containing 0.02% sodium azide and 1% Tween 20. Following a final spin at 17,000g the supernatant was removed and concentrated by polyethylene glycol 20,000 dialysis to a volume of 50mls or less and dialysed into MonoQ (Pharmacia) buffer A (20mM Tris/HCl pH 7.2, containing 0.1% Tween 20) . The samples were applied to a MonoQ HR5/5 (FPLC) column in 10-30ml batches and bound protein was separated with a 0-40% gradient of MonoQ buffer B (buffer A plus 1M sodium chloride) over 40mls at a flow rate of 0.7mls/min. Fractions containing high concentrations of polypeptides at 55-58kDa (judged by reducing SDS-PAGE) were pooled and concentrated to l-2mls using a Centriprep 10 concentrator (Amicon, USA) . This volume was applied directly to a Superose 6 HR16/50 FPLC column (Pharmacia) and elution was carried out with lOOmls of pbs at a flow rate of 0.25ml/min. Fractions were analysed and pooled selectively according to the presence of 1) 55-58kDa components on SDS-PAGE and 2) High γGTP enzyme activity (see below for assay details) . Following concentration to l-3mls and the addition of sodium azide to 0.02% protein concentrations were measured using the Pierce BCA assay.

(b) Production of a further v-GTP enriched fraction (γ- GTP 2 )

Adult Haemonchus contortus were obtained and extracted as described by Munn, Smith, Graham, Tavernor & Greenwood (1993), Int Journal for Parasitology 23, 261-269. Briefly, adult Haemonchus stored at -20°C in approximately their own weight of PBS containing 0.02% NaN₃ (PBS/a) were thawed and then homogenized in ice-cold PBS/a. The homogenate was centrifuged and the pellets were re-extracted with PBS/a. The pellets were then extracted with 1% Tween 20 followed by 1% Thesit (polyoxyethylene 9 lauryl ether, Boehringer Mannheim GmbH) in PBS/a. The Thesit extracts were ultracentrifuged and the supernatant liquids were pooled and concentrated. This provided the starting material for the fractionation scheme summarised in Fig. 7. The buffer was exchanged using a PD10 column (Pharmacia, U.K.) into 5mM-sodium acetate pH 5.2, lOOmM-NaCl, lmM- CaCl₂, lmM-MnCl₂, 0.02% NaN₃, containing 0.1% Thesit (Con A buffer) and the solution applied to a Con A-cross linked agarose column. Glycosylated proteins binding to the Con A were eluted with 500mM-methyl α-D- glucopyranoside in Con A buffer. Portions of this fraction were buffer exchanged into 20mM-Tris/HCl pH 7.2 containing 0.1% Thesit and applied to a Mono Q anion exchange column (Pharmacia) . Proteins not binding to the column did not contain γGTP activity. Bound proteins were eluted from the Mono Q with lM-NaCl in 20mM-Tris/HCl, pH7.2, 0.1% Thesit, first as a 0-25% gradient and then 100%. Fractions coinciding with the u.v. absorption peaks were run on SDS-PAGE and assayed for γGTP activity.

(c) Measurement of γGTP enzyme activity usinσ a microplate assay

Micro-ELISA plate (Dynatech Immulon 1, Virginia, USA) wells were each filled with 100μl of Tris/HCl pH 9.0 plus l-10μl of the fraction to be assayed. The plates were then pre-incubated at 37°C for 15 minutes prior to the addition of lOOμl of 2mM γglutamic acid p- nitroanilide/1.9mM glycyl-glycine per well. The time zero optical density (OD) at 405nm was then measured using an ELISA plate reader and the plates were then incubated at 37°C for 15-30 minutes. The final OD reading was then taken as before and the OD change per minute per milligram of protein calculated.

(d) vGTP vaccine trial

Six sheep of 3.75-5.5 months of age were assigned to each group and were each given 3 injections of γGTPEF-1. PCVs, faecal egg counts, collection of plasma, ELISAs, post mortem procedure, worm collection and weight/numbers calculation were all carried out according to standard operating procedures.

Results

The result of the purification of the γGTPEF-1 is shown in Fig 1, the major protein band being present at approximately 58 kDa although other minor bands were visible at 90 and 130 kDa.

Changes in PCVs of the groups over the duration of the experiment are shown as percentages of pre-infection values in Fig 2. The most substantial fall in PCV was seen in the control group whilst levels in the γGTPEF-1 group were less affected.

Average faecal egg counts for both groups at time points throughout the experiment are shown in 3. The γGTP group showed a considerable (>50%) reduction in egg output with respect to controls from 21 DPI.

ELISA measurements of anti-γGTPEF-1 antibody levels are shown in Fig 4. The γGTP group did contain specific antibodies against the material injected, but the plateau levels of these antibodies were never higher than 0.88 on average.

The mean number of worms at slaughter and the percentage reductions in numbers with respect to controls, are shown in Fig 5. The γGTP group showed a greater reduction in male (32%) than female (26.5%) numbers, the overall reduction being 29%.

The results show that γGTPEF-l is a good vaccine candidate.

Example 2

As shown in Figure 6, it may be demonstrated by appropriate histochemical procedures that γGTP activity is present on the intestinal microvilli of both Haemonchus contortus and CPPPeria PUCPPftPra at which site it would be accessible to ingested host antibodies. The formation of the reaction product is inhibited by the presence of the specific inhibitors of γGTP activity, ImM phenylmethylsulphonyl fluoride (pmsf) and 3M maleic acid.

Example 3

By free-flow isoelectric focussing, γGTP activity from the Thesit extracts of Haemonchus contortus of Example 1(b) has a pi in the range 4 to 8 with the peak of activity at 5.6. Antibodies from sheep injected with fractions including this range of pis, obtained by isoelectric focussing, inhibit the in vitro activity, assayed as described above, of γGTP by 70% whereas antibodies from control sheep (injected with horse spleen ferritin) inhibit the γGTP activity by only 17%.

The γGTP activity can not be released from PBS- extracted membranes from Haemonchus by the proteolytic enzymes papain, elastase, trypsin, chymotrypsin or subtlisin used under conditions which release many other enzyme activities from the membranes.

Claims

1. A protective helminth parasite antigen which is characterised by possessing transport protein activity and which, in native form, is a membrane-associated protein, or a functionally-equivalent variant, or antigenic fragment or precursor thereof.

2. An antigen as claimed in claim 1, and functionally- equivalent variants, antigenic fragments or precursors thereof, for use in stimulating an immune response against helminth parasites in a human or non-human animal.

3. An antigen as claimed in claim 1 or claim 2 having γ-glutamyl transpeptidase activity, or a functionally equivalent variant, antigenic fragment or precursor thereof.

4. An antigen as claimed in any one of claims 1 to 3, which is isolatable from helminths of the genera Haemonchus. Os ertaσia or Trichostronσylns. or a functionally equivalent variant, antigenic fragment or precursor thereof.

5. An antigen as claimed in any one of claims 1 to 4, or a functionally equivalent variant, antigenic fragment or precursor thereof, which antigen is obtainable from Haemonchus contortus and has an apparent molecular weight (Mr) of about 58,000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis

(SDS-PAGE) under reducing and non-reducing conditions.

6. An antigen as claimed in any one of claims 1 to 5, which is a membrane-associated protein extractable with the detergent polyoxyethylene sorbitan monolaurate, or a functionally equivalent variant, antigenic fragment or precursor thereof.

7. An antigen as claimed in any one of claims 1 to 5, which is an integral membrane protein or a functionally equivalent variant, antigenic fragment or precursor thereof.

8. A method for preparing an antigen, or a functionally equivalent variant, antigenic fragment or precursor thereof as defined in any one of claims 1 to 7, said method comprising the step of subjecting a crude extract of a helminth parasite to detergent extraction, and recovering the solubilised proteins.

9. A method as claimed in claim 8, wherein the detergent is a non-ionic detergent.

10. A method as claimed in claim 8, wherein the detergent is polyoxyethylene sorbitan monolaurate.

11. A method as claimed in any one of claims 8 to 10, further comprising one or more additional chromatographic purification steps.

12. A method as claimed in any one of claims 8, 10 and 11 comprising

(i) homogenising adult parasite worms in phosphate buffered saline (PBS) , recovering the PBS-insoluble material, optionally repeating the PBS-washing step one or more times,

(ii) extracting the PBS-insoluble material with Tween 20,

(iii) recovering the solubilised fraction containing the Tween-soluble proteins followed by

(iv) ion exchange chromatography, and/or gel filtration.

13. Use of a helminth parasite antigen as claimed in any one of claims 1 to 7, or a fragment, precursor or functionally-equivalent variant thereof, for the preparation of a vaccine composition for use in stimulating an immune response against helminth parasites in a human or non-human animal.

14. A vaccine composition for stimulating an immune response against helminth parasites in a human or non- human animal comprising one or more antigens, antigenic fragments, precursors or functionally-equivalent variants thereof, as claimed in any one of claims 1 to 7, together with a pharmaceutically acceptable carrier or diluent.

15. A method of stimulating an immune response against helminth parasites in a human or non-human animal, comprising administering to said animal a vaccine composition as claimed in claim 14.

16. An antibody, or antigen-binding fragment thereof, which is capable of selectively binding either to an antigen, antigenic fragment, precursor or functionally- equivalent variant thereof as defined in any one of claims 1 to 7, or to the idiotype of a said antigen- binding antibody.

17. A nucleic acid molecule comprising a nucleotide sequence encoding an antigen, functionally equivalent variant, antigenic fragment or precursor thereof, as claimed in any one of claims 1 to 7.

18. An expression or cloning vector comprising a nucleic acid molecule as defined in claim 17.

19. A host cell or transgenic organism containing a nucleic acid molecule as defined in claim 17..

20. A method for preparing an antigen, antigenic fragment, precursor, or functionally equivalent variant thereof, as defined in any one of claims 1 to 7, which comprises culturing a host cell containing a nucleic acid molecule encoding all or a portion of said antigen, under conditions whereby said antigen is expressed and recovering said antigen thus produced.