AU634754B2 - Vaccine for the preventative treatment of infection of liver fluke in ruminants - Google Patents

Description

VACCINE FOR THE PREVENTATIVE TREATMENT OF INFECTION OF LIVER FLUKE IN RUMINANTS

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 (Fascioliasis) is a major worldwide problem in the animal industry. Fascioliasis 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 wool 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. japonicum 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 S. mansoni infections worm burdens were reduced by 67% in rats and 52% in hamsters, respectively, following vaccination with a GST of M_p 28,000 (Sm28 or p28) [6]. Similarly, a GST of M_r26,000 from S. japonicum (Sj26) induced 30% protection in mice against an

SUBSTITUTE SHEET 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 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 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 fascioliasis comprising irradiated metacercariae of F. αiαantica. The use of irradiated metacercariae for vaccination of sheep aαainst F. hepatica has been reported to be unsuccessful [12].

PCT Application No. WO8801277 (Australian National University) is described in Chemical Abstract 110. No. 121367g (M J Howell). cDNAs prepared from mRNA ofTaenia ovis were cloned in E. coli and expressed as cro-lac fusion proteins. Sheep vaccinated with these proteins produce a low antibody response to T. ovis. 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 metacercariae of F. hepatica. The GST proteins are purified from adult worms of F. hepatica by affinity chromatography on glutathione-agarose. 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 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 Fasciola αiαantica 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 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 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. 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 markers is indicated (in kDa).

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Figure 2. Two dimensional SDS-PAGE analysis of I 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 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 japonicum (Sj) and mammalian Mu class GSTs. Homologous regions are boxed. Rat (Rn), mouse (Mm); bovine (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), S. japonicum (Sj26) and the mouse (Mm GST1). CT18.3: chymotryptic peptide of 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 Oterminal region of Schistosoma GSTs. Si26: S. japonicum Mr 26.000 GST; Sm26 : S. mansoni Mr 26,000 GST; T21.5b, T21.6a: F. hepatica tryptic peptides.

Figure 6. EUSA 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 ( _a ) 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: normal sheep serum; panel C: sera from sheep immunized with GST in Freund's adjuvant; panel D: sera from sheep infected with F. heoatica 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 flane 3). The position of the molecular weight markers is indicated (kDa).

Figure 8. Panel A shows the average RBC hemoglobin levels over 36 weeks of infection with F. hepatica in uninfected control sheep ( ), infected control sheep

( ) and GST-vaccinated sheep (. ). Panel B shows average RBC hemoglobin levels in sheep over 36 weeks of infection in uninfected control sheep ( ), infected control sheep ( — ), GST group 1 vaccinated sheep

( ) and GST group 2 vaccinated sheep (-..-..).

Figure 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-vaccinated 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 (-..-..).

Figure 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 igroup 1 vaccinated sheep (. ) and GST group 2 vaccinated sheep

(-..-..).

Figure 11. Panel A shows the average fecal egg counts over 36 weeks of infection with hepatica in infected control sheep (- - -) and GST-vaccinated sheep ( ).

Panel 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 ( — ).

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

Figure 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 M_r 26,000-26,500.

Figure 14. DNA sequence of the GST 1 cDNA.

Figure 15. DNA sequence of the GST 7 cDNA.

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

Figure 17. DNA sequence of the GST 47 cDNA. 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 of S. jaoonicumr 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, TO.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/vTriton 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 phenyimethylsulphonyi fluoride) ata ratio of 1 ml /worm. Occasionally washed whole worms stored at -20°C, were thawed at RT and then homogenized into TNET. These iysates 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 ϊsoenzymes were purified by affinity binding to glutathione (GSH) agarose (Sigma, St Louis, USA). Briefly, TNET Iysates 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 water and stored at -70°C. The GST content and purity were assessed by Coomassie blue and silver staining of SDS-PAGE gels. Generation of Peptides

Approximately 300g of affinity purified Fh GSTs were reduced in the presence of 1% w/v SDS, 10mM 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 carboxyamidomethyiation 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 resolubilized in the buffer of choice. In the case of the trypsin digest, the GST pellet was taken up in 200/d 1% v/v trimethyiamine (pH 8.0), and a further 7μg trypsin (Worthington,

Freehold, USA) added. Digestion occurred overnight at 37°C. The chymotrypsin digest was prepared by addition of 200μl 0.1 M NH₄HC0₃, pH 7.8, (C0₂) and 10/tg chymotrypsin (Worthington) and proteoiysis 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,/C₈ reverse phase column (Pharmacia). The elution was monitored at 214nm and peptide 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 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 471 A Protein Sequencer. Derivitized amino acids were resolved on a 25cm Zorbax PTH column (Dupont) (at 38°C) using isocratic delivery of the resolving buffer (5.529% v/v tetrahydrofuran, 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

For 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 aπd 10% glycerol, pH 6.8), and electrophoresed under reducing conditions on 13% acrylamide slab gels [13]. Relative molecular weights (M_r) 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% 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 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 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 H-,0. Following a further wash (30 minutes) in H-,0, 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 with the developer solution until the desired intensity of staining was achieved. The reaction was arrested by the addition of 2.3 M citric acid (5 ml per 100 ml of developer).

Western blotting

Electrophoretic 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 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 immunogiobulin (Biorad) or goat anti-rabbit immunoglobulin (Kinkegaard and Perry Labs, Gaithersburg, USA) was diluted 1 in 300 in 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 12μl of hydrogen peroxide. The location of the transferred protein was established by staining in a solution of 0.004 % amido black in 45 % methanol / 10 % acetic acid. lodination of proteins

The native GSTs of F. hepatica were radioiodinated using the Boiton 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 50μl purified GST (5 μg/ml) in 0.1 M sodium carbonate buffer (pH 9.5). Sera were diluted in EUSA buffer (0.1 M Tris HO, 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 50μl 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 μl was added to each well and incubated for 1 hour at 37° C. The substrate, 1 mM 2,2-Azinobis (3-ethylbenzthiazole sulphonic acid) (ABTS) in 0.062 M citric acid / 0.076 M Na₂HP0₄ 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 Deniiiquin, 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 10Qμg of purified GST of F. hepatica in Freund's complete adjuvant (FCA) 16 weeks prior to infection followed by a boost with 100μg of purified GST in Freund's incomplete adjuvant (IFA) 12 weeks prior to infection. The sheep were given subsequent boosts of 100μg of purified GST in PBS at approximatly 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, ail 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 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 for 6 minutes and most of the supematent 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 Agt11

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 gtl 1 aπdfcAP by CLONTECH (Palo Alto, USA) using the procedure of Gubler and Hoffman [26].

Immunoscreening of cDNA libraries

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 GSTs of F. hepatica at a dilution of 1/600. Filters were blocked in a buffer containing

10mM Tris HCI, pHδ.O, 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 GST1

Antibodies in the rabbit anti-GST serum were depleted of specificities to sequences expressed in the GST1 clone before the λZAP library was rescreened to identify other GSTs of F. hepatica. Undiluted rabbit antiserum (1ml) was incubated with 1ml of a sonicate of E. coli expressing β-galactosidase for 16 hours at 4°C to deplete anti- E. coli specificities. This depleted serum was diluted to 1/600 with PBS and 10m! of this serum was incubated on 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/ZAP library. One positive plaque was obtained (termed GST 7) which was rescreened to purity. DNA Hybridization

Plaque hybridization of radiolabelled GST1 or GST7 insert DNA to the ZAP library was performed as described by Maniatis et al [25]. Radiolabelled 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 λZAP 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 al [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 al (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 could be the product of an individual GST gene within a clonal fluke population. 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 GSTl. Two peptides, T21.5b and T21.6a, are identical and show 69% identity with the Oterminal region of the M_r 26,000 GST of S. japonicum and S. mansoni.

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

Reference Other species¹

31 Ss 24

30 Sm 26

7 Sj 26

35 Rn GST1

34 Hs GST

37 Bi GST

32 Mm GST1

36 Rn GST2

33 Mm GST2

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. 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 ELISA 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 subpopulation 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 2 sub-populations (Fig 9b), the GST group 1 animals displayed lower serum levels over the initial 10 weeks with a slightly delayed maximum reached at week 6 compared to week 4 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 (Fig 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-vaccinated 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 11b).

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 (Figure 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 vaccination on the egg output per worm although there is a tendency towards higher egg output in the GST-vaccinated animals. 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 M_p 26,000 and 26,500 on Western blots of the purified GST fraction separated by two 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 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 incomplete, the majority of the sequence is presented in Figure 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 M_r26,000 GST sequences of Schistosoma [7,30]. Each 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 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, with the Schistosoma GST sequences shows a high level of homology (48-59%) confirming that these cloned cDNAs encode the GSTs of F. hepatica. TABLE 2

Table 3 Identities in amino acid sequence between GSTs of F. hepatica and Schistosoma predicted from the DNA sequences of the cloned GSTs/

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 M 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-termiπal 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 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 immunoscreening 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 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 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 japonicum 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 250μg of GST in Freund's adjuvant [11]. The relevance of the rat model in fascioliasis 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 both a reduced worm burden as well as a delay in the establishment of these worms in the bile 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 (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 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 have thus been able to demonstrate a highly significant level of protection by vaccination with GST in sheep, equivalent to or exceeding, the protection demonstrated with S. mansoni and S. japonicum 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 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 F. hepatica has facilitated induction of a response to a novel parasite antigen leading to the death of the parasite.

REFERENCES

I. Haroun, E.M., and G.V. Hiilyer. 1986. Vet. Parasitol. 20:63.

2. Dawes, B., and D.L. Hughes. 1964. Adv. Parasitol. 2:97.

3. Hiilyer, G.V., E.M. Haroun, and M. Soler De Galanes. 1987. Am. J. Trop. Med. Hvα. 37:363.

4. Hiilyer, G.V., M.l. Garcia Rosa, H. Alicea, and A. Hernandez. 1988. Am. J. Trop.

Med. Hvα.38:103.

5. Mitchell, G.F. 1987. Parasit. Today 3:106.

6. Balloul, J.M., J.M. Grzych, R.J. Pierce, and A. Capron. 1987.

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. Nat!. Acad. Sci. USA 83:8703.

8. Pearce, E.J., S.L James, S. Hieny, D.E. Lanar and A. Sher. 1988. Proc. Nat!. Acad.Sci. U.S.A. 85:5678.

9. Mannervik, B. 1985. Adv. Enzvmol. 57:357.

10. Mitchell, G.F., E.G. Garcia, K.M. Davern, W.U. Tiu, and D.B. Smith. 1988. Trans. Royal Soc. Trop. Med. Hvα. 82:885.

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.

15. Morrissey, J.H. 1981. Anal. Biochem. 117:307.

16. Burnette, W.N. 1981. Anal. Biochem. 112:195. 17. Boiton, 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.

20. Persijn, J.P., and w. van der Silk. 1976. J. Clin. Chem. Clin. Biochem. 14:421.

21. van Kampen. E.J.. and W.G. Ziilstra. 1961. Clin. Chim. Acta. 6:538.

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.

25. Maniatis, T., E.F. Fritsch and J. Sambrook. 1982. Molecular cloning. Cold Soring 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. I_ Vander Jagt. 1977. Anal. Biochem. 82:334.

30. Henkel, K.J., K.M. Davern, M.D. Wright, A. J. Ramos and G.F. Mitchell. 1990. Mol. Biochem. Parasitol. (in press).

31. Brophy, P.M., A. Papadopoulos, M. Touraki, B. Coles, W. Korting and J. Barrett.

1989. Mol. Biochem. Parasitol. 36:187. 32. Pearson, W.R., J.J. Windle, J.F. Morrow, A.M. Benson and P. Taialay. 1983. _*__, Biol. Chem 258:2052.

33. Pearson, W.R., J. Reinhart, 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.

35. Abramovitz, M. and I. Listowsky. 1987. J. Biol. Chem. 262:770.

36. Lai, H.C. J., G. Grove and C.P.O. Tu. 1986. Nuc. Acids Res. 14:6101.

37 Mannervik, B., P. Alin, C, Guthenberg, H. Jensson, M.K. Tahir, M. Warhoim and H. Jornvall, 1985. Proc. Natl. Acad. Sci. USA. 82:7202.

38. Balloul, J.M., P. Soπdermeyer, D. Dreyer, M. Capron, J.M. Grzych, R.J. Pierce,

D. Carvallo, J.P. Lecocq, and A. Capron. 1987. Nature 326:149.

39. Hughes, D.L. 1987. In Immune responses in parasitic infections: immunology, immunopatholoαv. and immunoprophylaxis. Volume II: Trematodes and cestodes. EJ.L Soulsby, ed. CRC Press, Florida, USA, P.91.

Claims (11)

Claims «- 2 3 -

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 DNA molecule substantially the same as the said GST.

2. A vaccine according to Claim 1 wherein the antigen is isolated from adult F. hepatica and further characterised by:

(i) being extractable by affinity chromatography on glutathione-agarose

(ii) having a relative molecular mass of approximately 26,000 and 26,500, daltons.

3. A vaccine according to Claim 1 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 as set out in Figure 3 or a related sequence and containing as part of its primary structure the amino acid sequences set out in Figures 4 and 5 or closely related sequences.

4. A vaccine wherein the antigen or set of related antigens according to any one of Claims 1 to 3 which is an antigenic fragment thereof.

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

6. A vaccine for the preventative treatment of liver fluke in sheep and other ruminants comprising an antigen according to any one of Claims 1-5 or an antigenic fragment thereof and a pharmaceutically acceptable carrier or diluent.

7. A vaccine according to any one of Claims 1 to 6 further comprising an adjuvant.

8. 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 5, or an antigenic fragment thereof, or a recombinant cloning vehicle or vector, or a host cell comprising a said recombinant DNA molecule.

9. A vaccine according to Claim 8 wherein said nucleotide sequence is as set out in any one of Figures 14, 15, 16, 17, 18, and 19 or a related sequence.

10. 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 8 or Claim 9.

11. Anti-idiotype antibodies corresponding to at least one antigenic determinant of the antigens according to any one of Claims 1-5 as an antigen expressed from recombinant DNA molecule defined in Claims 8 or 9.