NZ232327A - Vaccine for preventative treatment of liver fluke infection in ruminants comprising as an antigen glutathione-s-transferase (gst) of f. hepatica - Google Patents

Description

212127 Priority Dal Comptate Ppt.cin . 1,1.1 . 3 ! r.i«u! Clas--:: ^V"?in^V»! n" "r** < 0.""*. 17////?] f-'.O, teMi ■»!, '-o: ...&&£ NEW ZEALAND PATENTS ACT. 1953 No.: 232327 Date: 31 January 1990 COMPLETE SPECIFICATION "Vaccine for the Preventative Treatment of Infection From Liver Fluke in Ruminants" WE, DARATECH PTY LIMITED, a company incorporated in Victoria, Australia of 6th Floor, 409 St Kilda Road, Melbourne, Victoria 3004, Australia hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- ■■ ttZTjT - la - FIELD OF THE INVENTION This invention relates to vaccines for the preventative treatment for infection of liver fluke in ruminant animals. The Invention also relates to methods for the preventative treatment for infection of liver fluke in ruminant animals.

BACKGROUND OF THE INVENTION Effective control of infection with liver fluke (Fascioiiasis) is a major worldwide problem in the animal industry. Fascioiiasis is caused by Infection with the trematode parasite Fasciola hepatica (F. hepatica^. In particular, in ruminants such as sheep and cattle, it can cause serious economic losses due to wasting, death and reduced woof and milk production [1]. Current control methods rely heavily on the use of anthelmintic chemicals but these methods are not always effective [2].

Despite considerable efforts there has been little progress towards production of a vaccine for the prevention of infection with liver fluke in sheep or cattle. There has been only one study examining the efficacy of a defined antigen against liver fluke infection in ruminants. A 12 kilodalton (kDa) polypeptide isolated from F. hepatica. has been shown to induce significant protection in calves [3,4]. This latest study highlights the utility of the defined antigen vaccine approach and the potential of identifying and subsequently inducing an Immune attack on a functional molecule which may not normally be antigenic during natural infections [5].

This approach has been applied to the search for a vaccine against the related trematodes Schistosoma mansoni and S. laponicum in which 2 major defined antigens, glutathione-S-transferase (GST) [6,7] and paramyosin [8] have been studied for their vaccination potential. The GSTs (glutathione transferase; EC 2.5.1.18) are a family of multifunctional proteins involved in the metabolism of a broad range of xenobiotics and the binding and possible transport of endogenous anionic compounds such as bilirubin and heme [9]. in reactions catalysed by these enzymes, electrophilic substrates are neutralised following conjugation with glutathione, rendering the product water soluble and facilitating excretion. In the schistosome parasite these enzymes have been suggested to play a role both in the solubilization of haematin, and in detoxifying products of lipid peroxidation [7]. In & mansoni infections worm burdens were reduced by 67% in rats and 52% in hamsters, —respectively, following vaccination with a GST of M 28,000 (Sm28 or p28) [6]. Similarly, •Uj I a GST of Mr26,000 from S. faponicum (SJ26) induced 30% protection in mice against an €32237 2Z2.3Z7 homologous cercarial challenge [7] though vaccinating effects in mice using Sj26 alone have been Inconsistent [10].

In a recent report [11], no protective effect of F. hepatica GST was detected in rats against challenge with metacercariae. The authors concluded that GSTs "do not confer any 5 protection on rats against a challenge infection (with metacercariae of F. hepatica)*. that *F. hepatica GSTs are almost certainly not host-protective antigens in rats" and that "fluke GSTs seem to be out of reach of the host immune system". Thus, these authors have discounted GSTs of fluke as potential vaccine molecules.

US Patent Specification 4743446 (National Research Development Corp) describes antigens 10 specific to the juvenile stage of F. hepatica which are prepared by raising an antiserum against the juvenile flukes, absorbing this antiserum with antigens extracted from adult flukes, separating the immunoglobulins (Ig) from the unabsorbed antiserum and using these Ig to affinity purify juvenile-specific antigens (JSA) from lysates of juvenile fluke. The JSA fraction conferred 65% protection in rats against infection with F. hepatica.

European Patent Specification 11438 (Vaccines International Ltd) describes a vaccine against bovine fascioiiasis comprising irradiated metacercariae of F. qiaantica. The use of irradiated metacercariae for vaccination of sheep against F. hepatica has been reported to be unsuccessful [12].

PCT Application No. W08801277 (Australian National University) is described In Chemical 20 Abstract 110. No. 121367g (M J Howell). cDNAs prepared from mRNA of Taenia ovls were cloned in E. coll and expressed as cro-lac fusion proteins. Sheep vaccinated with these proteins produce a low antibody response to T. ovls. These antigens are claimed to be useful for vaccination against helminth parasites such as T. ovis and F. hepatica.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a vaccine for the prevention of infection with liver fluke and which is suitable for use in ruminant animals.

In order to achieve this object the present invention provides in one form a vaccine for the preventative treatment for infection of liver fluke in ruminants, the vaccine comprising glutathione-S-transferase (GST) derived from adult worms of F. hepatica.

A vaccine containing GST is able to stimulate immunity in sheep to infection with riHtacercariae of F. hepatica. The GST proteins are purified from adult worms of E. hepatica by affinity chromatography on glutathione-agarose. —30. -3 APR 1990 V-v C , "252257 232? * 7 The GST proteins purified by glutathione-agarose chromatography comprise a mixture of proteins of similar molecular weight of about 26,000 and 26,500 Da. These proteins can ^ be fractionated by two dimensional SDS-PAGE into about 10-11 individual components with different apparent pi values.

Direct peptide sequencing of some of the protein components present in the GST mixture has identified two major N terminal sequences and 8 other sequences which are unique but show a significant level of homology to amino-acid sequences of other GST proteins from Schistosoma species, and certain mammalian species. These results show that the major proteins isolated by glutathione-agarose affinity chromatography are GSTs.

The GST used in the present invention may be extracted as described above or alternatively the parts of the molecule responsible for this vaccination effect may be synthesised as peptide molecules or by means of genetic engineering. It will be appreciated that a protective immune response can be achieved by vaccination with a peptide fragment of the GST described. Anti-idiotype antibodies corresponding to the vaccinating epitopes of the 15 GST molecule may also be used as a vaccine.

It is likely that the vaccine of the present invention will be effective against other members of the Fasciola genus, such as Fasclola aiaantica which is believed to be the predominant cause of liver fluke infection in tropical zones.

Preferably the vaccine further comprises adjuvants. Any adjuvants commonly used in 20 similar vaccines may be used but non-oil based adjuvants such as of the aluminium hydroxide type are preferred.

Preferably the vaccine further comprises molecules derived from members of the Fasciola genus or other parasites. It is likely that other molecules, unrelated to GST, may also induce a protective immune response in ruminants and that a cocktail vaccine comprising 25 these other molecules together with GST may be an effective vaccine.

Whilst the vaccine of this invention has most economic value with sheep and cattle it is useful for other ruminants as well. -3 APR1W0S BRIEF DESCRIPTION OF THE DRAWINGS |Figure1. One dimensional SDS-PAGE analysis of the glutathione-binding molecules purified from a crude homogenate of F. hepatica adult worms by affinity chromatography on glutathione agarose. The position of the molecular weight n 7, ? ° 7 7 £32 3-2.7 markers is Indicated (in kDa).

Figure 2. Two dimensional SDS-PAGE analysis of I125 labelled glutathione-binding molecules purified from a crude homogenate of F. hepatica adult worms by affinity chromatography on glutathione-agarose. The anode is on the right of 5 the figure. The position of the molecular weight markers is indicated (in kDa).

Figure 3. Comparison of the N-terminal sequences obtained for GSTs of F. hepatica (Fh) to the N-termini of GSTs of other helminths /Schistoceohalus solidus (Ss), Schistosoma mansoni (Sm), Schistosoma iaponicum (Sj) and mammalian Mu class GSTs. Homologous regions are boxed. Rat (Rn), mouse (Mm); bovine 10 (Bi) and human (Hs) GSTs are also represented. The bracketed residues indicate uncertain amino acid assignments.

Figure 4. Comparison of the sequence of tryptic and chymotryptic peptides of the GSTs of F. hepatica to homologous regions in GSTs of S. mansoni (Sm26), & japonicum (SJ26) and the mouse (Mm GST1). CT18.3: chymotryptic peptide of 15 F. hepatica: T0.7a, T0.7b, T16.3a, T16.3b, T16.2a, T16.2b: tryptic peptides of F. hepatica. The bracketed residues indicate uncertain amino acid assignments.

Figure 5. Comparison of the sequence of tryptic peptides of the GSTs of F. hepatica to the C-terminal region of Schistosoma GSTs. Sj26: S. [aponlcum Mr 26,000 GST; Sm26: S. mansoni Mr 26,000 GST; T21,5b, T21.6a: F. hepatica tryptic peptides.

Figure 6. ELISA analysis of native F. hepatica GST probed with antisera from sheep immunized with GST in Freund's adjuvant (a), infected with F. hepatica for 12 wks (•), infected with F. hepatica for 6 wks (■) and normal sheep serum (*).

Figure 7. Western blot analysis of native F. hepatica GST probed with antisera from different sheep. Panel A: an amido black stain of the native protein; panel B: 25 normal sheep serum; panel C: sera from sheep immunized with GST in Freund's adjuvant; panel D: sera from sheep infected with F. hepatica for 6 weeks; panel E: sera from sheep Infected with F. hepatica for 12 weeks. Sera were used at a dilution of 1/100 (lane 1), 1/300 (lane 2) or 1/1000 (lane 3). The position of the molecular weight markers is indicated (kDa).

Figure-a« Panel A shows the average RBC hemoglobin levels over 36 weeks of infection I? with F. hepatica in uninfected control sheep ( ), infected control sheep ; j i (...) and GST-vaccinated sheep (. ). Panel B shows average RBC hemoglobin levels in sheep over 36 weeks of infection in uninfected control 232227 sheep { ), infected control sheep (- - -), GST group 1 vaccinated sheep ( ) and GST group 2 vaccinated sheep Rgure 9. Panel A shows the average aspartate aminotransferase serum levels over 36 weeks of infection with F. hepatica in serum from uninfected control sheep ( ), infected control sheep (- - -) and GST-vaccfnated sheep (. ).

Panel B shows average aspartate aminotransferase serum levels in sheep over 36 weeks of infection In serum from uninfected control sheep ( ), infected control sheep (- - -), GST group 1 vaccinated sheep (. ) and GST group 2 vaccinated sheep Rgure 10. Panel A shows the average L - gamma glutamyltransferase levels over 36 weeks of infection with F. hepatica in uninfected control sheep ( ), infected control sheep (- - -) and GST-vaccinated sheep ( ). Panel B shows average L - gamma glutamyltransferase serum levels in sheep over 36 weeks of infection in serum from uninfected control sheep ( ), infected control sheep (- - -), GST group 1 vaccinated sheep (. ) and GST group 2 vaccinated sheep Rgure 11. Panel A shows the average fecal egg counts over 36 weeks of infection with F. hepatica in infected control sheep (—) and GST-vaccinated sheep (. ).

Pane! B shows average fecal egg counts levels in sheep over 36 weeks of infection in Infected control sheep ( ), GST group 1 vaccinated sheep (. ) and GST group 2 vaccinated sheep (- - -).

Rgure 12. Hnal worm burdens in sheep infected with F. hepatica and sacrificed over a period of 13 weeks (weeks 44 - 57).

Rgure 13. Western blot analysis of F. hepatica GST probed with rabbit antiserum to the native GST fraction. The GST was fractionated into 10-11 components by two dimensional SDS-PAGE. The anode is on the right of the figure. The bands identified are of Mr 26,000-26,500.

Rgure 14. DNA sequence of the GST 1 cDNA.

Rgure 15. DNA sequence of the GST 7 cDNA.

Rgure 16. DNA sequence of the GST 42 cDNA. Dashes indicate unassigned sequence.

Figure 17. DNA sequence of the GST 47 cDNA. 222. 327 Figure 18. DNA sequence of the GST 50 cDNA.

Figure 19. Comparison of the amino acid sequences of cloned GST sequences and GST peptides of F. hepatica. Sm26: Mr 26,000 GST of S. mansoni: Sj26: Mr 26,000 GST ofS. japonicum; Fh26a, Fh26b: N-terminal amino acid sequences of GSTs of F. hepatica: GST1,7,42,47,50: amino acid sequences predicted from the cloned GST cDNAs of F. hepatica. T.05, T0.7b/0.6, T21.5: tryptic peptides of F. hepatica: CT18.3:chymotryptic peptide of F. hepatica. The sequences have been aligned to maximise the homology. Dashes indicate unassigned residues.

Materials and Methods Parasites Fasciola hepatica adult worms used for purification of GSTs were collected from the livers of sheep slaughtered and processed at local abattoirs in Melbourne. The parasites were transported on ice, washed twice in phosphate buffered saline (PBS) and homogenized in TNET buffer (0.5% v/v Triton X-100 (Triton X-100 is a non-ionic detergent supplied by Rohm & Haas), 10mM EDTA, 0.15M NaCI, in 50mM Tris (pH 7.8) supplemented with 2mM phenylmethylsulphonyl fluoride) at a ratio of 1 ml/worm. Occasionally washed whole worms stored at -20°C, were thawed at RT and then homogenized into TNET. These lysates were clarified by centrifugation (10,000g, 30 minutes, 4°C) and stored at -20°C. Adult worms of the Compton strain of F. hepatica were similarly obtained from livers of sheep infected with metacercariae obtained from Compton Paddock Laboratories, U.K. This isolate had been maintained in the laboratory by passage through the intermediate snail host Lvmnaea truncatula in the laboratory and subsequently through sheep. Adult parasites of the Compton strain were obtained fresh from the bile ducts of infected sheep, washed in PBS at 37°C and stored at -70°C.

Purification of F. hepatica GSTs GST isoenzymes were purified by affinity binding to glutathione (GSH) agarose (Sigma, St Louis, USA). Briefly, TNET lysates of adult worms were passed down a GSH agarose column, and the matrix washed with several volumes of PBS, prior to elution with a GSH containing buffer (1.5mg/ml GSH in 50mM Tris (pH 9.3)) [7]. Fractions shown to contain protein were pooled, dialysed against PBS or distilled wati&indjstored at -70°C. The GST content and purity were assessed by Coomassie blue and silver staining of 5DS=PAG&qels.

Generation of Peptides Approximately 300g of affinity purified Fh GSTs were reduced in the presence of 1% w/v SDS, tOmM DTT, in 100mM Tris (pH 8.0) for 60 minutes at 58°C. On cooling to ambient temperature, iodoacetamide was added to a final concentration of 22mM and 5 carboxyamidomethylation proceeded for 15 minutes at RT. Protease was added to 1-2% (w/w), and the mixture precipitated at -20°C (18 hours) in 10 volumes of acetone (Aristar, BDH). The pelleted material was washed with 2 changes of acid-acetone (0.005% v/v HCI in acetone), 2 changes of acid-ethanol and once in ethanol. The pellet was air dried and resoiubilized In the buffer of choice. In the case of the trypsin digest, the GST pellet was 10 taken up in 200<d 1% v/v trimethylamine (pH 8.0), and a further 7pg trypsin (Worthington, Freehold, USA) added. Digestion occurred overnight at 37°C. The chymotrypsin digest was prepared by addition of 200/J 0.1M NH4HC03, pH 7.8, (C02) and 10/tg chymotrypsin (Worthington) and proteolysis conducted at 37°C for 4 hours. Digestion was arrested by storage at -20°C.

The ensuing peptides were separated by reverse phase chromatography using an organic/aqueous gradient delivered by an FPLC system (Pharmacia). Complete digests were primarily resolved with a 0-92.5% v/v acetonitrile (AcN) gradient in 15-20mM ammonium formate, pH 4.0 (C02) applied over 46 minutes, onto a Pro PRC 5/10 C^Cg reverse phase column (Pharmacia). The elution was monitored at 214nm and peptide 20 peaks collected via a timed loop. The void volume and peptide peaks suspect of heterogeneous content were refractionated on a Pep PRC 5/5 C2/C18 reverse phase column (Pharmacia) most often using a 0-60% v/v AcN gradient in 0.1% v/v trifluoracetic acid. The elution was monitored at 214nm. Collected peptide peaks were stored at -20°C, and dried by vacuum centrifugation (Savant Instruments, Hicksville, USA) prior to amino 25 acid analysis.

Amino acid sequencing N-terminal and peptide sequencing was conducted at the Department of Veterinary Preclinical Sciences, University of Melbourne, using an ABI Model 471A Protein Sequencer. Derivitized amino acids were resolved on a 25cm Zorbax PTH column (Dupont) (at 38°C) 30 using isocratic delivery of the resolving buffer (5.529% v/vtetrahydrofuran, 30.17% v/v AcN, 60.5mM sodium acetate (pH 3.8), 0.00907mM sodium acetate (pH 4.6) at 1 ml/minute.

SDS-PAGE l^pr one-dimensional SDS polyacrylamide gel electrophoresis (SDS-PAGE), samples were resuspended in sample buffer (62.5 mM Tris-HCI containing 3% SDS, 50 mM dithiothreitol •g-5225'7 23z -izy and 10% glycerol, pH 6.8), and electrophoresed under reducing conditions on 13% acrylamide slab gels [13]. Relative molecular weights (Mr) were calculated with reference to protein molecular weight standards (Biorad, Richmond, USA). Following electrophoresis, gels were stained and fixed in 0.05% w/v Coomassie blue R250 in 50% methanol and 10% 5 acetic acid for 20 minutes, destained with 5% methanol and 7% acetic acid, then dried under vacuum before autoradiography. Two-dimensional electrophoresis was performed by the method of O'Farrell [14]. For the first dimension, isoelectric focusing (IEF) was performed in glass tubes using a 1:1 mixture of pH 5-7 and pH 7-9 ampholytes (Pharmacia, Uppsala, Sweden). SDS-PAGE, using 13% acrylamide slab gels, was used for the second 10 dimension. The gels were prepared for autoradiography following electrophoresis as described above.

Silver staining of gels ? On occasion, electrophoresis gels were silver stained by the method of Morrissey [15]. In brief, the gels were rinsed in H20 and soaked in 50 % methanol / 10 % acetic acid fixative 15 for 30 minutes. After a 5 minute immersion in 5 % methanol / 7% acetic acid solution, the gel was treated with 10% glutaraldehyde for 30 minutes. At this stage the gel was left overnight in a large volume of H20. Following a further wash (30 minutes) in HzO, the gel was Immersed in a fresh 0.1 % AgN03 solution for 30 minutes and then rinsed once in H20 and twice in developer solution (3 % Na2C03,0.05 % formalin). The gel was then stained 20 with the developer solution until the desired Intensity of staining was achieved. The reaction i was arrested by the addition of 2.3 M citric acid (5 ml per 100 ml of developer). ) Western blotting ) Bectrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose paper was performed according to the method of Bumette [16], with a transfer time of 18 hours at 15 25 volts. The nitrocellulose sheet was blocked with 5 % skim milk powder in PBS for 3 hours at room temperature. The antiserum was diluted 1 in 100 in PBS, added to the nitrocellulose sheet, and incubated for 1 hour. The sheet was washed three times in PBS containing 0.1 %Tween 20. Affinity-purified rabbit anti-sheep immunoglobulin (Biorad) or goat anti-rabbit immunoglobulin (KInkegaard and Perry Labs, Gaithersburg, USA) was diluted 1 in 300 in 30 PBS and added to the sheet and incubated at room temperature for 1 hour. The sheet was washed 3 times in PBS / 0.1 % Tween 20 (Tween 20 is a non-ionic detergent) and developed using 4 ml of a 3 mg/ml solution of 4-chloro-1 -napthol (Sigma) in cold methanol mixed with 20 ml PBS containing 12jtl of hydrogen peroxide. The location of the transferred Rljptein was established by staining In a solution of 0.004 % amido black in 45 % methanol /10 % acetic acid. '3 APR (990 i v -tytrtTl lodination of proteins The native GSTs of F. hepatica were radioiodinated using the Bolton and Hunter procedure [17].

EUSA The EUSA was performed as described by Milner et al [18] with the following changes.

Polyvinyl microtitre plates were coated overnight at 4*C with 50/J purified GST (5 /tg/ml) in 0.1 M sodium carbonate buffer (pH 9.5). Sera were diluted in ELISA buffer (0.1 M Tris HCI, 0.5 M NaCI, 2 mM EDTA, 0.05 % Tween 20,0.05 mM thiomersal, pH 8.0) supplemented with 0.2 % bovine serum albumin, and 50/d of the appropriate dilution was incubated in the microtiter plate for 1 hour at 37*C. The wells were washed 3 times between incubations with PBS containing 0.1% v/v Tween 20. Affinity-purified rabbit anti-sheep immunoglobulin conjugated to horse radish peroxidase (Biorad) was diluted in EUSA buffer, 50 id was added to each well and incubated for 1 hour at 37*C. The substrate, 1 mM 2,2-Azinobis (3-ethylbenzthIazoie sulphonic acid) (ABTS) in 0.062 M citric acid / 0.076 M Na2HPOA pH 4.0,0.03 % hydrogen peroxide, was added to each well. After 1 hour, the optical density at 414 nm was measured with an automated Titertek Multiskan spectrophotometer.

Vaccination protocol Merino-cross wethers were obtained from a farm in Deniliquin, New South Wales, with no history of infection with F. hepatica. All animals were screened before use for the absence of F. hepatica eggs In their feces.

A group of 10 sheep were Immunized by subcutaneous injection of 100>ig of purified GST of F. hepatica In Freund's complete adjuvant (FCA) 16 weeks prior to infection followed by a boost with 100/ig of purified GST in Freund's incomplete adjuvant (IFA) 12 weeks prior to infection. The sheep were given subsequent boosts of 100»ig of purified GST in PBS at approximate 4 week intervals throughout the 52 weeks of the trial. A group of 10 control sheep were treated identically, with PBS substituted for the GST antigen. A group of 8 sheep were not immunized. On the day of challenge, all sheep, except 3 of the 8 unimmunized sheep which were kept as uninfected controls, were infected intraruminally with 500 metacercariae (Compton Paddock Laboratories, UK) suspended in a 0.4% w/v suspension of high viscosity carboxymethyl cellulose (Sigma). Sera from all sheep were collected immediately prior to immunization and every 2-4 weeks thereafter for 52 weeks.

Serum taken at each time interval was assayed for the liver enzymes aspartate "aminotransferase (EC 2.6.1.1.) (AST) [19] and L-gamma glutamyltransferase (EC 2.3.2.2.) (GGT) [20] and red blood cell (RBC) hemoglobin [21] on a Roche Cobas MIRA automatic -<?. J l <c j r a *2322*7 analyser (Basel, Switzerland). Serum was stored frozen at -20*C until use.

Fecal egg counts (FEC) were performed by the method of Kelly et al [22] with the following changes. One gram of feces was suspended in 9ml of water and passed through a sieve into a tapered urine flask to remove coarse fecal material. The eggs were allowed to settle 5 for 6 minutes and most of the supernatent removed. This procedure was repeated once and yielded about 10 ml of sediment containing F. hepatica eggs. Several drops of 0.1 % new methylene blue were added to the final sediment to a volume of 10 ml and poured into a square lined petri dish. The number of eggs/g feces were counted under a dissecting microscope.

Statistical significance was calculated by the Mann Whitney U statistic [23]. Construction of cDNA libraries in AzAP and AgtH Total RNA was extracted from adult worms of the Compton strain of F. hepatica by the method of Chirgwin et al [24]. Poly(A)+ RNA was selected by oligo dT chromatography [25]. The cDNA libraries were constructed in phage vectors Agt1 1 and feAP by CLONTECH 15 (Palo Alto, USA) using the procedure of Gubler and Hoffman [26].

Immunoscreening of cDNA libraries \ •> j The cDNA libraries were screened for expression of GSTs of F. hepatica using the Protoblot method as described In the Protoblot Technical Manual purchased from PROMEGA (Madison, USA). The library was screened with a rabbit antiserum raised to the purified 20 GSTs of F. hepatica at a dilution of 1/600. Filters were blocked in a buffer containing 10mM Tris HCI, pH8.0, 150mM NaCI, 0.05% Tween 20, 1% gelatin. Positive plaques identified in a primary screen were picked, replated at a lower density and rescreened with the rabbit antiserum until individual positive plaques were identified.

Absorption of rabbit anti-GST serum on GSTI Antibodies In the rabbit anti-GST serum were depleted of specificities to sequences expressed in the GST1 clone before the AzAP library was rescreened to identify other GSTs of F. hepatica. Undiluted rabbit antiserum (1ml) was incubated with 1 ml of a sonicate of E. coli expressing B-galactosidase for 16 hours at 4°C to deplete anti- E. coll specificities. This depleted serum was diluted to 1/600 with PBS and 10ml of this serum was incubated ' 35~" ion a filter to which an induced confluent lawn of clone GST1 had been absorbed. After 1 [hour at room temperature, the serum was removed and used to screen the AZAP library. 'n One positive plaque was obtained (termed GST 7) which was rescreened to purity.

APR woa DNA Hybridization Plaque hybridization of radiolabeled GST1 or GST7 insert DNA to the AzAP library was performed as described by Maniatis et al [25]. Radiolabeled probes were prepared as described by using the BRL (Gaithersburg, USA) nick translation kit as recommended by the supplier.

Isolation and sequencing of cDNA inserts Phagemid DNA containing cDNA inserts from positive AzAP phage clones was isolated by excision in vivo of the pBluescript phagemid under the conditions recommended by Stratagene (La Jolla, USA). Phagemid DNA was extracted by the method of Birnboim and Doly [27]. Double-stranded DNA sequencing of cDNA inserts was performed by the chain termination method [28].

RESULTS OF EXAMPLE 1 Characterization of proteins purified by glutathione agarose chromatography The purification of native GSTs from mammalian or Schistosoma species by glutathione-agarose chromatography has been previously described [7,29]. Howell et ai [11] have recently used this approach to identify multiple GSTs in adult worms of F. hepatica. In order to isolate GSTs of F. hepatica. adult worms were lysed in buffer containing Triton X-100 and the clarified lysate was applied to a glutathione-agarose column as described in Materials and Methods. The column was washed with PBS and the bound material eluted with a glutathione buffer. The GST bound to the column was analysed by SDS-PAGE in one or two dimensions to determine the protein heterogeneity of the sample. We routinely found that the GST fraction comprised two major components of approximate Mr 26,000 and 26,500 by one dimensional SDS-PAGE (Fig 1). Similar results were obtained by Howell et ai (1988). When analysed in two dimensional gels, the GST fraction fractionated into about 10-11 components which exhibit different apparent pi values (Fig 2). We believe, without limiting the scope of the invention, the GST fraction comprises protein extracted from a population of individual adult worms isolated from several infected sheep livers. Since each sheep could be infected with several strains of F. hepatica which may exhibit sequence polymorphisms within GST isoenzymes, the multiple protein components observed within our GST fraction could represent allelic variants of one or a few GST isoenzymes within the polymorphic fluke population studied. Alternatively, each component coijild be the product of an Individual GST gene within a clonal fluke population. 23JL3Z7 Amino acid sequence of native GSTs of F. hepatica N terminal amino acid sequences of the purified F. hepatica GSTs revealed two different but related sequences. Comparison of these sequences (Fh26a, Fh26b) with the corresponding regions of Schistosoma [7,30,31] and known mammalian GSTs [31,37] showed very high levels of homology (Fig. 3). Conservation of several key regions of sequence resulted in Identities of 52-76% and 55-77% for Fh26a and Fh26b respectively (Table 1).

The amino acid sequence of several tryptic and chymotryptic peptides isolated from the digests of the GST fraction are shown in Figures 4 and 5 together with alignments with other GST sequences. Peptide CT18.3 is homologous to sequences in the Schistosoma GSTs whereas the T0.7A, T0.7b and T16 series of peptides show greatest identity to mouse GST1. Two peptides, T21.5b and T2l.6a, are identical and show 69% identity with the C-terminal region of the Mr 26,000 GST of S. iaoonicum and S. mansoni.

TABLE 1 Identities in N-terminal amino acid sequence between GSTs of F. hepatica and other species.

Reference Other species1 % identity Fh26a2 Fh26b' 31 Ss 24 60 66 Sm26 76 77 7 S] 26 72 70 Rn GST1 56 59 34 Hs GST 58 62 37 Bi GST 55 60 32 Mm GST1 52 55 36 Rn GST2 56 59 33 Mm GST2 52 55 1. GSTs of species listed in Fig 3 2. N-terminal sequences of GSTs of F. hepatica.

These results show that the abundant proteins of Mr 26,000 and 26,500 purified by affinity chromatography on glutathione-agarose are homologous to the GSTs of both Schistosoma and mammalian species. 23ZZ2-7 Antibody response of sheep to the purified GST antigen The antibody response to GST in infected sheep and sheep vaccinated with GST in Freund's complete adjuvant was analysed by EUSA and Western blotting. As shown in Fig 6, GST vaccinated animals exhibited a strong antibody response to the vaccine antigen whereas sheep infected with F. hepatica for 6 or 12 weeks exhibited a very poor response. Similarly, by Western blotting of purified GST, only sera from GST vaccinated sheep detected the native GSTs of F. hepatica (Fig 7).

Parameters analysed during vaccination trial To assess the progression of the liver fluke infection and to monitor the health of the animals throughout the vaccination trial three parameters were analysed. The level of RBC hemoglobin was assayed as an indicator of anemia. Serum was assayed for the presence of the liver enzymes, AST and GGT as indicators of liver damage. Fecal samples were collected for egg counts as an indicator of the establishment of adult parasites. During the trial, of the 15 control infected animals (i.e. 10 PBS vaccinated controls and 5 non-vaccinated controls), 1 animal died from a dog attack and 3 animals died (one at week 5 and two at week 7) as a result of liver fluke infection. The results for these 3 animals have been included in the group analysis of the 14 infected control animals shown in Figs 8-11.

The RBC hemoglobin levels in the uninfected control animals remained consistently high around a mean of 12 g/L over the period of the trial. The infected control animals demonstrated a decrease in RBC hemoglobin with time, dropping to below 8 g/L by week 36. The GST vaccinated sheep displayed levels consistently orientated around the median between the uninfected and the infected control animals (Fig 8a). When the GST vaccinated animals were analysed further as two sub-populations (Fig 8b), based solely upon relative RBC haemoglobin levels through the trial, it was found that 4 of the animals (GST group 1) displayed consistently higher levels of RBC hemoglobin than the infected controls, while the remaining 5 animals (GST group 2) demonstrated a decrease with time, consistent with the infected controls. These results suggest that a subpopulatlon of the GST vaccinated animals (GST group 1) did not exhibit the anemia characteristic of liver fluke infection.

AST serum levels were analysed to assess the level of liver parenchymal damage in the trial animals. The GST-vaccinated animals consistently displayed levels of serum AST similar to the infected control animals (Fig 9a). When the GST-vaccinated animals were assessed as •Ssub-populations (Fig 9b), the GST group 1 animals displayed lower serum levels over the inmal 10 weeks with a slightly delayed maximum reached at week 6 compared to week 4 -tttzrr 23ZZZ7 in the infected control animals. The animals in GST group 2 did not display any differences in AST serum levels from the infected control animals.

GGT levels in serum are an indicator of damage to the liver and specifically the bile ducts and were analysed to monitor damage resulting from the establishment of parasites in the bile ducts. The level of GGT in the GST-vaccinated animals demonstrated a profile similar to that recorded for the infected control animals (Pig 10a) with a rise in the levels of enzyme in serum detectable by week 2, peak values by week 12 and a slow decrease after this time. No comparable release of GGT into serum was detected in the uninfected control animals. When the GST-vacclnated animals were analysed as sub-populations (Fig 10b), GST group 1 displayed lower GGT levels over the initial 12 weeks and with maximal levels not attained until week 14. GGT levels In the GST group 2 again coincided with the infected controls. This suggests that the GST group 1 subpopulation of animals have a decreased and delayed onset of liver damage compared with the controls and the GST group 2 subpopulation.

All infected animals within the trial displayed large variations in their FEC. The mean FEC of the GST-vaccinated animals are lower than the Infected control animals but these values are not significantly different (Fig 11a). Analysis of the two GST sub-populations indicates that the GST group 1 has a lower mean FEC relative to the infected control animals, while the FEC of GST group 2 are consistent with those of the infected control animals (Fig 11 b).

Total fluke counts The sheep were slaughtered over a period of 13 weeks (weeks 44 - wk 57), post infection, and the worm burdens within each liver were ascertained (Rgure 12 and Table 2). The 10 infected controls sacrificed to date, contained an average of 241 parasites in comparison to the GST-vaccinated animals with a mean of 107 parasites representing an overall reduction in worm burden of 55 % (p < 0.001). When the GST vaccinated animals were considered as subpopuiations, the GST group 1 group exhibited a mean worm count of 54, representing a reduction of 77% (p < 0.001), whereas the GST group 2 group exhibited a mean worm count of 149, representing a reduction of 38% (p < 0.025). Moreover, one third of the GST-vaccinated animals exhibited worm burdens of less than 15 % of the mean burden in the control animals.

As an indicator of average worm fecundity, the average FEC/worm in the different groups of animals was compared. As shown in Table 2, there was no significant effect of m although there is a tendency towards higher egg £■32257" J?3Z 3*7 Cloning and expression of GST genes of F. hepatica Rabbit antiserum was raised to the purified GST fraction by subcutaneous injection of F. hepatica GST In Freund's adjuvant. This antiserum identifies various GST species of Mr 26,000 and 26,500 on Western blots of the purified GST fraction separated by two 5 dimensional SDS-PAGE (Fig 13). This antiserum was used to isolate cDNA sequences of F. hepatica encoding GST by immunoscreening of a gt11 or ZAP cDNA library synthesised from poly(A)+ RNA isolated from adult F. hepatica worms.

Two cDNA clones (termed GST1 and GST7) were identified. The cDNA sequence of GST7 was used to isolate other homologous cDNA sequences in the library by DNA-DNA 10 hybridization which identified 3 other cDNA sequences (termed GST42, GST47 and GST50). The DNA sequence of these five cloned cDNAs was determined by the chain termination method of Sanger et al [28]. The DNA sequence of clones GST1, 7, 42, 47 and 50 are shown in Figs 14-18. Clones GST1, 7, 42 and 47 contain a polyA tail Indicating that we have cloned the 3' end of these mRNAs. Whilst the DNA sequence of GST 47 is 15 incomplete, the majority of the sequence is presented in Rgure 17. As this is in a region of high homology to GSTs 1,7,42 and 50 the incompleteness does not effect the working of the invention.

The amino acid sequences predicted by each of the cDNA sequences is shown in Fig 19 together with an alignment with the Mr26,000 GST sequences of Schistosoma [7,30]. Each 20 cDNA sequence predicts a single open reading frame. The GST 1 amino acid sequence begins 22 amino acids from the N terminus of GST peptides (Fh26b) and shows a degree of homology with this sequence. The GST 7 amino acid sequence begins 7 amino acids from the N terminus of GST (Fh26a) and is identical to this sequence. The GST47 amino acid sequence begins 6 amino acids from the N terminus of GST (Fh 26a,b) and shows 25 high homology with these sequences. The GST42 and GST50 sequences are much shorter. Comparison of these 5 cloned cDNA sequences shows a high level of identity (65-96%) among the predicted polypeptide sequences which extends throughout the sequences (Table 3). This result shows that adult F. hepatica express at least five different mRNAs for GST. Comparison of the sequences of the F. hepatica GSTs, predicted from the cDNAs, 30 with the Schistosoma GST sequences shows a high level of homology (48-59%) confirming that these cloned cDNAs encode the GSTs of F. hepatica. -£-52257-*£2 3,27 TABLE 2 D ANIMAL FEC2 WORM3 FEOWORM IDENTITY BURDEN CONTROL7 W654 2538 236 .75 W673 1172 209 .61 W674 1357 290 4.68 W686 897 182 4.93 W693 2715 250 .86 W694 835 184 4.54 W695 3997 269 14.86 W696 2242 302 7.42 W698 1012 240 4.22 Y73 1208 250 4.83 TOTAL AVG t SEM 1797 ± 329 241 ± 13 8 JO = 1.16 GST 1 G255 558 32 17.44 G293 173 8.65 W653 1100 145 7.59 W671 197 18 .94 GST 2 G258 2375 200 11.88 G381 1697 131 12.95 W669 753 110 6.85 W670 1483 120 1136 W692 1692 183 9.25 TOTAL AVG ± SEM 1114 : 237 107 = 22 .88 ± 1.03 GST 1 AVG tSEM 507 ± 187 54 = 26 11.15 ± 1.91 GST 2 AVG i SEM 1600 i 232 149 = 16 .66 ± 1.02 1 Data derived from 10 control animals " - FEC represent an average count from weeks 32, 34 and 36. 3 Worm burden at time of sacrifice. 2323>*7 Table 3 Identities in amino add sequence between GSTs of F. hepatica and Schistosoma predicted from the DNA sequences of the cloned GSTs/ GST1 GST7 GST42 GST47 GST 50 Sm26 Sj26 GST 1 100 GST 7 68 100 GST 42 68 84 100 GST 47 76 76 84 . 100 GST 50 65 87 96 90 100 Sin26 52 -55 55 57 48 100 Sj26 56 56 52 '59 . 48 80 100 1. The % identity between each pairwise comparison of the predicted aminoacid sequences of the GST cDNA clones.

DISCUSSION The GSTs of adult worms of F. hepatica comprise two major components of approximate Mr 26,000 and 26,500 which can be further fractionated into 10-11 components by two dimensional SDS-PAGE. Direct sequencing of the GST fraction of F. hepatica identified two major N-terminal sequences. In addition, peptides derived from internal or C-terminal regions of GSTs were Identified by homology with other known GSTs. From these data, it is evident that the glutathione binding molecules purified do represent the GSTs of F. hepatica. The isolation and sequencing of near identical peptides indicates the high degree of heterogeneity in the F. hepatica GST fraction and implies the expression of multiple GST genes in this parasite.

The isolation of cDNA sequences encoding GSTs of F. hepatica was achieved by ~Trffi^pnoscreening of cDNA expression libraries using rabbit antisera to the native GSTs. Each of the five cDNA sequences cloned encodes a different primary amino acid sequence -g-32257 J22>2 2>Z7 which shows up to 59% homology with other cloned GST sequences Including GSTs from Schistosoma and mammalian species. Regions of the cloned GST sequences also show identity or high similarity with the peptide sequences obtained from the native fluke GSTs showing that these cDNAs encode the GSTs expressed in the adult worm. The finding 5 that multiple GST sequences are expressed in a population of adult worms implies either the presence of multiple GST genes within the F. hepatica genome or that multiple polymorphic variants of one or a few GST alleles exist within a genetically heterogeneous worm population.

The vaccination potential of trematode GST has been demonstrated in S. mansoni and S. [aponicum since immunization with native and recombinant forms of Sm28 and Sj26 was able to induce significant levels of protection against homologous experimental infections in the rat and mouse models [7,38]. However in a study with GST of F. hepatica in the rat, immunity was not induced following subcutaneous vaccination with 250jig of GST in Freund's adjuvant [11]. The relevance of the rat model in fascioiiasis has been questioned [39] and it was therefore our aim in the present study to investigate the vaccination potential of F. hepatica GST in the sheep, a natural and highly susceptible host of this parasite.

The health of the animals and the progression of the infection was monitored by the assay of several biochemical parameters in erythrocytes and serum. A subpopulation of the GST vaccinated animals (GST group 1) displayed a clear biochemical pattern consistent with 20 both a reduced worm burden as well as a delay in the establishment of these worms in the bite ducts. The subsequent finding of a 77 % reduction (p < 0.001) in worm burden in these animals was complementary to the biochemical findings. A statistically significant reduction y (p < 0.025) in worm burden of 38 % was also demonstrated in the GST group 2. An overall reduction in worm burden of 55 % (p < 0.001) was demonstrated in the vaccinated group 25 as a whole. The fecundity of parasites in the GST-vaccinated animals does not appear to have been affected following establishment in the bile ducts as evidenced by the FEC/worm ratio which is slightly higher in the vaccinated animals relative to the infected controls. We ~n. have thus been able to demonstrate a highly significant level of protection by vaccination y with GST in sheep, equivalent to or exceeding, the protection demonstrated with S. mansoni and S. |aponicum in laboratory animals.

The nature of the protective immune response directed against the parasite remains uncertain. A strong humoral response to GST of F. hepatica has been induced in all the vaccinated animals but the members of GST group 1 do not exhibit a differentially higher antibody titre relative to the GST group 2 animals, it is therefore uncertain if a humoral response and/or a T-cell response is necessary to induce the protective effect observed, in addition, the animals used in this trial were outbred merino wethers which will exhibit -3 APR 1990, -052237 genetically-based qualitative and quantitative variability in their immune response to GST.

Without limiting the scope of this invention we believe the evidence presented here suggests that the parasites in the vaccinated sheep have been eliminated or retarded by vaccination prior to establishment in the bile ducts. The target of the immune attack could be GST in the metacercariae and /or in the newly excysted juvenile resulting in the subsequent damage and elimination of the parasite.lt is also possible that immune attack on the GST of & hepatica has facilitated induction of a response to a novel parasite antigen leading to the death of the parasite.

"•Us*. a -3-g223?- o?32327 - 20 ' REFERENCES I. Haroun, E.M., and G.V. Hillyer. 1986. Vet. Parasitol. 20:63. 2. Dawes, B., and D.L Hughes. 1964. Adv. Parasitol. 2:97. 3. Hillyer, G.V., E.M. Haroun, and M. Soler De Galanes. 1987. Am. J. Trop. Med. Hvq. 37:363. 4. Hillyer. G.V.. M.I. Garcia Rosa. H. Alicea. and A. Hernandez. 1988. Am. J. Trop. Med. Hvo.38:103.

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J. Immunol. 138:3448. 7. Smith, D.B., K.M. Davern, P.G. Board, W.U. Tiu, E.G. Garcia, and G.F. Mitchell. 1986. Proc. Natl. Acad. Scl. USA 83:8703. 8. Pearce, E.J., S.L James, S. Hieny, D.E. Lanar and A. Sher. 1988. Proc. Natl. Acad.Sci. U.S.A. 85:5678. 9. Mannervik, B. 1985. Adv. Enzvmol. 57:357.

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II. Howell, M.J., P.G. Board, and J.C. Boray. 1988. J. Parasitol. 74:715. 12. Campbell, N J., P. Gregg, J.D. Kelly and J.K. Dineen. 1978. Vet. Parasitol. 4:143. 13. Laemmli, U.K. 1970. Nature 227:680. 14. O'Farrell, P.H. 1975. J Biol. Chem. 250:4007.

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Bumette, W.N. 1981. Anal. Blochem. 112:195.

^^Pf?l99oi -252237 222.32.~7 17. Bolton, A.E. and W.M. Hunter. 1973. J. Biochem 133:529. 18. Milner, A.R., K.B. Jackson, K. Woodruff, and I.J. Smart. 1985. J. Clin. Micro. 22:539. 19. International Federation of Clinical Chemistry, Expert Panel on Enzymes. 1977. Clin. Chem. 23:887.

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Blochem. 14:421. 21. van Kampen, E.J., and W.G. Zijlstra. 1961. Clin. Chim. Acta. fr538. 22. Kelly, J.D., R.A.F. Chevis, and H.V. Whitlock. 1975. New Zealand Vet. J. 23:81. 23. Rohlf, F.R., and R.R. Sokal. 1969. Statistical tables. Freeman,W.H., San Francisco, p.241. 24. Chirgwin, J.M., A.E. Przybyla., R. J. MacDonald., and W.J. Rutter., 1979.

Biochemistry 18:5294.

. Maniatis, T., E.F. Fritsch and J. Sambrook. 1982. Molecular cloning. Cold Spring Harbor Laboratory. 26. Gubler, U. and B.J. Hoffman. 1983. Gene 25:263. 27. Birnboim, H.C. and J. Doly, 1979. Nuc.Acids.Res. 7:1513. 28. Sanger, F., S. Nicklen and A.R. Coulson. 1977. Proc. Natl. Acad. Sci. USA 74:5463. 29. Simons, P.C. and D. L Vander Jagt. 1977. Anal. Biochem. 82:334.

. Henkel, K.J., K.M. Davern, M.D. Wright, A. J. Ramos and G.F. Mitchell. 1990. Mol. Blochem. Parasitol. fin press). ophy, P.M., A. Papadopoulos, M. Touraki, B. Coles, W. Korting and J. Barrett. '989. Mol. Blochem. Parasitol. 36:187. o £32 3X7 32. Pearson, W.R., J.J. Windle, J.F. Morrow, A.M. Benson and P. Talalay. 1983. ^ Biol. Chem 258:2052. 33. Pearson, W.R., J. Relnhart, S.C. Sisk, K.S. Anderson and P.N. Adler. 1988. J. Biol. Chem. 263:13324. 34. Alin, P., B. Mannervik and H. Jornvall. 1985. FEBS Lett. 182:319.

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Claims (21)

WHAT WE CLAIM IS:

1. A vaccine for the preventative treatment of liver fluke infection in ruminants comprising as an antigen glutathione-S-transferase (GST) of F hepatica. or a synthetic polypeptide or recombinant molecule substantially the same as the said

2. A vaccine as claimed in Claim 1 wherein the vaccine is substantially free of any glutathione contamination.

3. A vaccine according to either Claim 1 or Claim 2 wherein the antigen is isolated from adult F hepatica and further characterised by: (i) being extractable by affinity chromatography on glutathione-agarose; and (ii) having a relative molecular mass of approximately 26,000 and 26,500

4. A vaccine according to either Claim 1 or Claim 2 where the antigen has a peptide sequence homology with glutathione-S-transferases (GSTs) of Schistosoma and mammalian species and having an N terminal amino acid sequence substantially as set out in Figure 3 or 4 or 5 or having at least a 52% similarity thereto.

5. A vaccine wherein the antigen according to any one of Claims 1 to 4 is an antigenic fragment thereof. GST. daltons. - 232327 .24-

6. A vaccine according to either Claim 1 or Claim 2 where the antigen primary structure substantially includes the amino acid sequences set out in Figure 19 or an antigenic fragment thereof.

7. A vaccine for the preventative treatment of liver fluke in sheep and other ruminants comprising an antigen according to any one of Claims 1-6 or an Ns antigenic fragment thereof and a pharmaceutically acceptable carrier or diluent

8. A vaccine according to any one of Claims 1 to 7 further comprising an adjuvant

9. A vaccine for the preventative treatment of liver fluke in ruminants comprising as the antigen a recombinant DNA molecule comprising all or a portion of a nucleotide sequence which is capable of being expressed as a polypeptide having the antigenicity of an antigen according to any one of Claims 1 to 6, or an antigenic fragment thereof, or a recombinant cloning vehicle or vector, or a host ^ cell comprising a said recombinant DNA molecule. 5

10. A vaccine according to Claim 9 wherein said nucleotide sequence is as substantially as set out in any one of Figures 14, IS, 16,17 or 18 or encoding an amino acid sequence substantially as set out for GST1, GST7, GST47 or GST42 in Figure 19.

11. A vaccine for the preventative treatment of liver fluke in ruminants comprising a synthetic polypeptide prepared by expression of all or a portion of a nucleotide sequence according to Claim 9 or Qaim 10. » .25. 23 2 3 2

12. Anti-idiotype antibodies corresponding to at least one antigenic determinant of the antigen according to any one of Claims 1-6 as an antigen expressed from recombinant DNA molecule defined in Claims 9 or 10.

13. A method of preventative treatment of liver fluke in ruminants comprising administering a vaccine comprising as an antigen glutathione-S-transferase (GST) of F hepatica. or a synthetic polypeptide or recombinant polypeptide substantially the same as the said GST.

14. A method as claimed in Qaim 13 wherein the vaccine is substantially free of any glutathione contamination.

15. A method of increasing the resistance of ruminants to liver fluke infection comprising at least one administration of a vaccine comprising as an antigen glutathione-S-transferase (GST) of F hepatica. or a synthetic polypeptide or recombinant polypeptide substantially the same as the said GST.

16. An isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in Figure 14 or having at least 52% simiarity thereto and which encode a glutathione-S-transferase or an antigenic fragment thereof.

17. A nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in Figure 15 or having at least 52% similarity thereto and which encode a glutathione-S-transferase or an antigenic fragment thereof. -. ^ ov * «c>;" 10 DEC1992 =;^ -26- 23 2 3 2 7;

18. A nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in Figure 16 or having at least 52% similarity thereto and which encode a glutathione-S-transferase or an antigenic fragment thereof.;

19. A nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in Figure 17 or having at least 52% similarity thereto and which encode a glutathione-S-transferase or an antigenic fragment thereof.;

20. A nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in Figure 18 or having at least 52% similarity thereto and which encode a glutathione-S-transferase or an antigenic fragment thereof.;

21. A recombinant polypeptide having an amino acid sequence substantially as set forth in Figure 19 in relation to one or more of GST1, GST7, GST47 or GST42 or having at least 52% homology thereto.;DATED THIS DAY OF F'Fte-;AJ';AGENTB® THTAPPbCANT;v;-2 • o;\ tODEC|992*