CA2045663A1 - Vaccine for the preventative treatment of infection of liver fluke in ruminants - Google Patents
Vaccine for the preventative treatment of infection of liver fluke in ruminantsInfo
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- CA2045663A1 CA2045663A1 CA002045663A CA2045663A CA2045663A1 CA 2045663 A1 CA2045663 A1 CA 2045663A1 CA 002045663 A CA002045663 A CA 002045663A CA 2045663 A CA2045663 A CA 2045663A CA 2045663 A1 CA2045663 A1 CA 2045663A1
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- Prior art keywords
- gst
- vaccine
- antigen
- sheep
- gsts
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
- C12N9/1088—Glutathione transferase (2.5.1.18)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
A vaccine for the preventative treatment for infection of liver fluke in ruminant animals where the antigen is glutathione-S-transferase extracted from adult worms of F. hepatica. The antigen may also be synthetic equivalent molecules and equivalent molecules prepared by recombinant DNA techniques. Methods of preparation, and use are also disclosed.
Description
WO 9~/08819 PCr/AU90/0002~
, 2~4a~
VACCINE FOR THE PREVE:~TATIVE TREATME T OF
INFECTION OF LIVFR FLUKE IN hiUMlNANTS
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.
BACKGROUl~iD OF THE INVENTION
EHective control of infection with liver fluke (Fascioliasis) is a major woridwide problem in the animal industry. Fascioliasis is caused by infection with the trematode parasite Fasciola hepatica (F. heDatica~. In ,oarticular, 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 antlgen vaccine approach and the potential of identifying and subsequently inducing an immune attack on a functional mdecule 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. jaDonicum in which 2 major defined antigens, glutathione-S-transferase (GST) [6,7] and paramyosin [8] have been studied fortheirvaccination potential.
: 25 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 l9]. 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 Mr 28,000 (Sm28 or p28) [6]. Similariy, a GST of Mr26,000 from S. jaDOniCum (Sj26) induced 30% protection in mice against an SlJ B ST~ E S ~l EET ~
~ _ I
WO 90/08~19 fi~ PCl`/AU90/00027 homologous cercarial challenge l7] though vaccinating effects in mice using Sj26 alone have been inconsistent l10].
In a recent report [11 j no protective effect of F heDatica GST was detected in rats against challenge with metacercariae. The authors concluded that GSTs ~do not confer any5 protection on rats against a challenge infection (with metacercariae of F. heDatica~~ that F. heDatica GSTs are almost cerlainly not host-protective antigens in rats~ and that ~nuke GSTs seem to be out of reach of the host immune system . Thus these authors havediscounted 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. heDatica 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 lg to affinity purify juvenile-specific antigens (JSA) from Iysates of juvenile fluke. The JSA
fraction conferred 652/o protection in rats against infection with F. hepatica.
European Patent Specification 11438 (Vaccines Intemational Ltd) describes a vaccine against bovine fascioliasis comprising irradiated rnetacercariae of F. aiaantica. The use of irradiated metacercariae for vaccination of sheep against F. her~atica has been reported to be unsuccessful [12].
PCT Application No. WO8801277 (Australian National University) is described in Chemical Abstract 110 No. 1213679 (M J Howell). cDNAs prepared from mRNA of Taenia 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 OFTHE 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 numinants the vaccine comprising glutathione-S-transferase (GST) derived from adult womms of F. heDatica.
A vaccine containing GST is able to stimulate immunity in sheep to infection with metacercariae of F. her~atica. The GST proteins are purified from adult worms of F.
heDatica by affinity chromatography on glutathione-agarose.
WO 90/0881~ PCr/AU90/00027 2043~63 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 pl values.
5 Direct peptide sequencing of some of the protein components present in the GST mixture has identified two major N terminal sequences and 8 other ssquences 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.
: .
10 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.
l~ is likely that the vaccine of the present invention will be effective against other members of the Fasciola genus, such as Easciola 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 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 moleculespurified from a crude homogenate of F. heDatica adult worms by aHinity chromatography on glutathione agarose. The position of the molecular weight WO 90t08X19 PC~IAU90/()0027 6~
markers is indicated (in kDa).
Figure 2. Two dimensional SDS-PAGE analysis of 1125 labelled glutathione-binding molecules purified from a crude homogenate of F. heoatica 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-temminal sequences obtained for GSTs of F. heDatica (Fh) to the N-termini of GSTs of other helminths (Schistocephalus 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 ~. Comparison of the sequence of tryptic and chymotryptic peptides of the GSTs of F. heDatica to homologous regions in GSTs of S. mansoni (Sm26), S.
japonicum (Sj26) and the mouse (Mm GST1). CT18.3: chymotrvptic peptide of F. hepatica; TO.7a, TO.7b, T16.3a, T16.3b, T16.2a, T16.2b: tr,vptic peptides of F. heDatica. 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. jaDonicum Mr 26,000 GST;
Sm26: S. mansoni Mr 26,000 GST; T21 .5b, T21.6a: F. heDatica tr,vptic peptides.
:
20 Figure 6. ELISA analysis of native F. heDatica GST probed with antisera from sheep immunized with GST in Freund's adjuvant (-), infected with F. hepatica for 12 wks (-), infected with F. hepatica for 6 wks ( ~ ) and normal sheep serum (r), Figure 7. Westem blot analysis of native F. hepatlca GST probed with antisera from dfflerent sheep. Panel A: an amido black stain of the native protein; panel B:
nommal sheep serum; Panel C: sera from sheep immunized with GST in Freund's adjuvant; panel D: sera from sheep infected with F. heDatica for 6 weeks; panel E: sera from sheep infected with F. heDatica 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 ofthe molecular weight markers is indicated (kDa).
30 Figure 8. Panel A shows the average RBC hemoglobin levels over 36 weeks of infection with F. hçpatica 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 . ,..-. .
:
WO 90/~8819 PCrJAU90/OOnl7 2~ 3 sheep ( ), infected control sheep (-- -), GST group 1 vaccinated sheep (.. ) and GST group 2 vaccinatr,~d sheep (-.. -.. ).
Figure 9. Panel A shows the average asparlate aminotransferase serum levels over 36 weeks of infection w~h F. her~atica 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 (-..-..).
10 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 (- -15 -), GST group 1 vaccinated sheep (.. ) and GST group 2 vaccinated sheep ( ) ., Figure 11. Panel A shows the average fecai egg counts over 36 weeks of infection with F
hepatica In infected control sheep (---) and aST-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 (- - -3.
, , Figure 12. Final worm burdens in sheep infected with F. heDatica and sacrificed over a period of 13 weeks (weeks 44 - a~7).
Figure 13. Westem blot analysis of F. heDatica 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 identlfied are of Mr 26,000-26,500.
Figure 14. DNA sequence of the GST 1 cDNA.
Figure 15. DNA sequence of the GST 7 cDNA.
30 Figure 16. DNA sequence of the GST 42 cDNA. Dashes indicate unassigned sequence.
Figure 17. DNA sequence of the GST 47 cDNA.
.,. -:, Figu!e 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. jawnicum; Fh26a, Fh26b: N-terminal amino acid sequences of GSTs of F. heoatica; GST1,7,42,47,50: amino acid sequences predicted from the cloned GST cDNAs of F. heDatica. T.05, TO.7b/0.6, T21.5: tryptic peptides of F. heoatica: CT18.3:chymotr,vptic peptide of F. hepatica. The sequences have been aligned to maximise the homology. Dashes indicate unassigned residues.
Materials and Methods 1 0 Parasites Fasciola heDatica 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 saiine (PBS) and homogenized in TNi_T 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. Occasionaily washed whoie worms stored at -20C, were thaweci at RT and then homogenked into TNi_T. These Iysates were clarified by centrifugation (10,0009, 30 minutes, 4C) and stored at -20C. Adult worms of the Compton strain of F. hepatica were simiiarly 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 intemmediate snaii host LYmnaea truncatula in the laboratory and subsequently through sheep. Aduit parasites of the Compton strain were obtained fresh from the biie ducts of infected sheep, washed in PBS
at 37C and stored at -70C.
Purification ot F. he,oatica GSTs GST isoereymes were purified by affinity binding to giutathione (GSH) agarose (Sigma, St Louis, USA). Briefly, TNET Iysates of aduit worms were passed down a GSH agarosecolumn, and the matrix washed with severai voiumes of PBS, prior to elution with a GSH
containing buffer (1.5mg/mi GSH in 50mM Tris (pH 9.3) ) [7]. Fractions shown to contain protein were pooled, dialysed against PBS or distilled water and stored at -70C. The GST
content and purity were assessed by Coomassie blue and silver staining of SDS-PAGE gels.
"
, W O 90/08819 PC~r/AU90/00027 2 ~
Generation of Peptides Approximately 3009 of affinity purified Fh GSTs were reduced in the presence of 1% w/v SDS, 10mM Dl~, in 100mM Tris (pH 8.0) for 60 minu~es at 58C. On cooling to ambient temperature, iodoacetamide was added to a final concentration of 22mM and 5 carboxyamidomethy1ation proceeded for 15 minutes at RT. Protease was added to 1-2%
(w/w), and the mixlure precipitated at -20C (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 10 taken up in 200~1 1% v/v trimethylamine (pH 8.0), and a further 7~9 trypsln (Worthington, Freehold, USA) added. Digestion occurred overnight at 37C. The chymotrypsin digest was prepared by addition of 200~-1 0.1 M NH4HCO3, pH 7.8, (CO2) and 10~.9 chymotrypsin (~orthington) and proteolysis conducted at 37C for 4 hours. Digestion was arrested by storage at -20C.
15 The ensuing peptides were separated by reverse phase chromatography using an - organic/aqueous gradient delivered by an FPLC system (Pharrnacia). Complete digests were primarily resolved with a 0-92.5% v/v acetonitrile (AcN) gradient in 15-20mM
ammonium formate, pH 4.0 (CO2) applied over 46 minutes, onto a Pro PRC 5/10 C1/C8 reverse phase column (Pharmacia). The elution was monitored at 214nm and peptide20 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 trfluoracetic acid. The elution was monitored at 21 4nm. Collected peptide peaks were stored at -2ûC, and dried by vacuum centrifugation (Savant Instnuments, 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 Melboume, using an ABI Model 471 A Protein Sequencer.
Derivitized amino acids were resolved on a 25cm Zorbax PTH column (Dupont) (at 38C) 30 using isocratlc 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 1ml/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, 5û mM dithiothreitol .
WO 90/08819 ~ PCI`/AU90/00027 and 1o% glycerol, pH 6.8), and electrophoresed under reciucing conditions on 13%acrylamide slab gels 113]. Relative molecular weights (M,) 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 w ith 5% methanol and 7% acetic acid, then dried under vacuum before autoradiography. Two-ciimensional electrophoresis was performed by the method of O'Farrell [14]. For the first dimension, isoelectric focusing (IEF) was perfommeci in glass tubes using a 1:1 mixture of pH 5-7 and pH 7-9 ampholytes (Pharmacia, Uppsala, Sweden). SDS-PAGE, using 13% acryiamide slab gels, was used for the second 10 dimension. The gels were prepareci for autoradiography following electrophoresis as described above.
Silver staining ot gels On occasion, electrophoresis gels were silver stained by the method of Morrissey [15I. In brief, the gels were rinsed in H2O 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/O glutaraldehyde for 30 minutes. At this stage the gel was left overnight in a large volume of H2O. Following a further wash (30 minutes) in H2O, the gel was immersed in a fresh 0.1% AgNO3 solution for 30 minutes and then rinsed once in H2O
and twice in developer solution (3 % Na2CO3, 0.05 % formalin). The gel was then stained 20 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).
. Westem blotting Electrophoretic transfer of proteins from poiyacryiamide gels to nitrocellulose paper was performed according to the method of Bumette [16], with a transfer time of 18 hours at 15 25 voits. The nitrocellulose sheet was blocked with 5 % skim milk powder in PBS for 3 hours ; at room temperature. The antiserum was diluteci 1 in 100 in PBS, added to the nitrocellulose sheet, and incubateci for 1 hour. The sheet was washed three times in PBS containing 0.1 % Tween 20. Affinity-purified rabbit an~i-sheep immunoglobulin (Biorad) or goat anti-rabbit immunoglobulin (Kinkegaard and Perry i abs, 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 washeci 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 1 2~Li of hydrogen peroxide. The location of the transferred protein was established by staining in a solution of 0.û04 % amido black in 45 % methanol 35 /10 % acetic acid.
-; -' .
WO 90/08819 P~/AU90/~)0027 9 2~a~3 lociination oI proteins The native GSTs of F. heDatica were radioiodinated using the Bolton and Hunter procedure [17].
EUSA
5 The ELiSA was perfommed as described by Milner et al [18] with the following changes.
Pdyvinyi microtitre plates were coated overnight at 4 C with 50~L1 purHied GST (5 ~.g/ml) in 0.1 M sodium carbonate buffer (pH 9.5). Sera were diluted in ELiSA buHer (0.1 M Tris Ha, 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 50~1 of the appropriate dilution was incubated in the 10 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. Affin~y-purHied rabbit anti-sheep immunoglobulin conjugateci to horse radish peroxidase (Biorad) was diluted in ELISA buffer, 50 ILI 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 Na2HPO4 pH
15 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. heDatica. All animals were screened before use for the absence 20 of F. hepatica eggs in their feces.
A group of 10 sheep were immunked by subcutaneous injection of 100,ug of purHied GST
of F. heDatica in Freund's complete adjuvant (FCA) 16 weeks prior to infection followed by a boost with 100~.9 of purHied GST in Freund's incomplete adjuvant (IFA) 12 weeks prior to infection. The sheep were given subsequent boosts of 100~ 9 of purHied GST in PBS at 25 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, all sheep, except 3 of the 8 unimmunked sheep which were kept as uninfected controls, were infected intraruminally with 500 metacercariae (Compton Paddock Laboratories, UK) suspended in a 0.4% w/v 30 suspension of high viscosity carboxymethyi cellulose (Sigma). Sera from all sheep were coilected 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 glutamyitransferase (EC 2.3.2.2.) (GGT) [20] and red blooci cell (RBC) hemoglobin [21] on a Roche Cobas MIRA automatic WO 90/08~19 C;~ PCr/AU90/00027 analyser (Basel, Switzerland). Serum was stored frozsn at -20 C until use.
Fecal egg counts (FEC) were parfo~med by the method of Kelly et al ~z] 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 ~ecal 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.
10 Statistical sign'~icance was calculated by the Mann Whitney U statistic [23].
Construction of cDNA libraries in /~ZAP and /~gt11 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 125]. The cDNA libraries were construc~ed in phage vectorslgt1 1 and ~ZAP by CLONTECH
15 (Palo Alto, USA) using the procedure of Gubler and Hoffman [26].
,~
lmmunoscreening ot cDNA libraries The cDNA libraries were screened for expression of GSTs of F. heDatica 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. heDatica at a dilution of 1/600. Filters were blocked in a buffer containing 10mM Tris HCI, pH8.0, 150mM NaCl, 0.05% Tween 20, 1% gelatin. Positive plaques identified in a primary screen were picked, replated at a lower dens-~y and rescreened with the rabbit antisenum until individual positive plaques were identHied.
.
Absorption of rabbit anti-GST serum on GST1 25 Antibodies in the rabbit anti-GST senum were depleted of specificities to sequences :expressed in Ihe GST1 clone before the 1ZAP library was rescreened to identriy other GSTs of F. heDatica. Undiluted rabbit antiserum (lml) was incubated with 1ml of a sonicate of E coli expressing s-galactosidase for 16 hours at 4~C to deplete anti- E. coli specificities.
This depleted serum was diluted to 1/600 with PBS and 10ml of this sarum was incubated 30 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.
': -.
W O 90/08819 P ~ /A~90/00027 20~63 DNA Hybridization .
Plaque hybridization of radiolabelled GST1 or GST7 insert DNA to the 1ZAP library was performed as described by Maniatis et al 125]. 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 AZAP phage clones was isolated by excision in v,vo of the pBluescript phagemid under the conditions recommended byStratagene (La Jolla, USA). Phagemid DNA was extracted by the method of Bimboim and 10 Doly [27]. Double-stranded DNA sequencing of cDNA inserts was performed by the chain termination method [28].
Characterization ot proteins purified by glutathione agarose chromatography The purification of native GSTs from mammalian or Schistosoma species by glutathione-15 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. heDatica. In order to isolate GSTs of F. heDatica, adult worms were Iysed in buffer containing Triton X-- 100 and the clarified Iysate was applied to a glutathione-agarose column as described in Materials and Methods. The column was washed with PBS and the bound material eluted 20 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 25 about 10-11 components which exhibit dfflerent apparent pl 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. Altematively, each component could be the product of an individual GST gene within a clonal fluke population.
WO 90/08819 PC~/AU90/00027 ~ - 12-Amino acid sequence of native GSTs ot F. heDatica N temminal amino acid sequences of the purified F. heDatica 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]
5 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 respecthely ~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 10 other GST sequences. Peptide CT18.3 is homologous to sequences in the Schistosoma GSTs whereas the TO.7A, TO.7b and T16 series of peptides show greatest identity to mouse GST1. Two peptides, T21.5b and T21.6a, are identical and show 69% identity with the Gterrninal region of the Mr 26,000 GST of S. jaDonicum and S. mansoni.
TABLE 1 Identities in N-terminal amino acid sequence between GSTs of F. hepatica and other species.
. f~eference Other sDecies1 % identty Fh26a2 Fh26bZ
31 Ss 24 60 66 Sm 26 76 77 20 7 Sj 26 72 70 Rn GST1 56 59 . 34 Hs GST 58 62 37 Bi GST 55 60 32 Mm GST1 52 55 25 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. heDatica.
These results show that the abundant proteins of Mr 26,000 and 26,500 purified by affinity 30 ehromatography on glutathione-agarose are homologous to the GSTs of both Schistosoma and mammalian species.
-, ~ .
WO 90/08819 PCI`/AU90/00027 Antibody r~sponse o~ sheep to the purified ~ST antigen The antibody response to GST in infectecl sheep and sheep vaccinated with GST inFreund's complete adjuvant was analysed by ELISA and Westem blotting. As shown in Fig 6, GST vaccinated animals exhibited a strong antibody response to the vaccine antigen 5 whereas sheep infected with F. her~atica 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. he~atica (Fig 7).
Parameters analysed during vaccination trial To assess the progression of the liver fluke infection and to monitor the health of the 10 animals throughout the vaccination trial three parameters were analysed. The level of ~BC
hemoglobin was assayed as an indicator of anemia. Senum 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 contro! infected animals (i.e. 10 PBS vaccinated controls and 5 non-15 vaccinated controls), 1 animal died from a dog attack and 3 animals died (one at week 5and two at week 7) as a result of liver fluke infeclion. 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 20 demonstrated a decrease in RBC hemoglobin with time, dropping to below 8 g/L by week 36. The GST vaccinated sheep displayed levels consistentiy 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 triai, it was found that 4 of the animals (GST group 25 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 parenchymai damage in the trial 30 animals. The GST-vaccinated animals consistently displayed levels of senJm 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 senum levels over the initial 10 weeks with a slightly delayed maximum reached at week 6 compared to week 4 a~ 3 - 14-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 v ere analysed to monitor damage resulting from the establishment of parasites in the bile ducts. The level of GGT in the GST-vaccina~ed animals demonstrated a profile similar to that recorded for the infected control animals (Fig 1 Oa) 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 2subpopulation.
A~l 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 d-dferent (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 11uke 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 - 25 reduction in worm burden of 55 % (p < 0.001). When the GST vaccinated animals were considered as subpopulations, the GST group 1 group exhibited a mean worrn 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 dfflerent groups of animals was compared. As shown in Table 2, there was no significant effect ofvaccination on the egg output per womm although there is a tendency towards higher egg output in the GST-vaccinated animals.
, " ' ' ' -2 ~ 3 Cloning and expression ot GST genes ot _. hepatica Rabbit antiserum was raised to the purified GST fraction by subcutaneous injection of F.
heDatica 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 two5 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. heDatica 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 15 incornplete, 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 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-g6h) among the predicted polypeptide sequences which extends throughout the sequences(Table 3). This result shows that adult F. he~atica express at least f~e dfflerent mRNAs for GST. Comparison of the sequences of the F. heDatica 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. heDatica.
~,~ 4~ 6 1 6 --L~L ¦ FC~ ~iOR\,~FC/~NOR~vl ¦
! lDE~Tl~Y I Bl ~DE.~
CO~ROLI ~W6~ 1-i8 ~6 10.,5 S ~h6li li- ~59 5.oi W6,' 1-`- ~ 4.63 W686 397 1~' 4.9i W69; '~1~ 10.36 \~69l 8;5 i3 Ll~
W695 i99~ ^9 14.36 ~696 W69~ 101 y_~ l'08 ';~ 1.3 TOT.~L ~ ~VG ^ S~1 9 - 9 '~: - li3.`0 - 1.'o 1.
1 5 I G_ T I G' -; ~ ~ l î . ' ' G~9; 1,; ^0 3.65 ~65; 1100 ,~ /~9 .
W6, l 19 / 13 10.9 ¦ C-ST o G~3 ~;,5 -no ]1.SS
Gi81 169- 1;1 1'~ 9 W609 ~5i 110 6.8~
:: W6~0 1*3; 1-~ 1''.;6 W69? 169~ 13~ 9.~5 TOT~L .~G: SE~I¦ 1114 - ~i710, - ''`10.33 - 1.0;
GST 1 AVG: SEl~l50/: 1S7 5' - '611.15 1.91 GST ~ AVG - S~L1600: æ2 1'9 = 1610.66 _ 1.02 .', .
.
Data derived from 10 control aiimals represerlt an average count from weelcs ;2, i4 ar~d i6.
; Worm burde:l at ~ime o~ sacrihce.
. . .
:....... . . .. . ., . , ~ , .
:
. -.
:, ' , WO 90/08819 PCr/AU9OtO(3()27 ?
2~45~3 T~ble 3 I~e~ ies in amino ~c;d sequenc: be~we^3 GSTs o~ F he~a~ic~ ~nd Schistosom~ pr~ic;ed ~om ~he DN.'~ sequ~:lces of tbe c'oned Gâ 15.t ~GST1 ¦GS~7 ~GaT~2 ¦GS~7 ~GS~SO ¦ Sm2S lSj26 ¦ GST~ 1 ~ 100 5i Ga~ 7 ~ 68 ¦ 100 ¦
C-~ 21 68 1 84 1- --GS'" 4, ¦ 7~ ¦ 76 1 84 ~ 100 ¦
i CS~ 501 6" 1 87 j 9~ ~ 90 ~ 1 ~S-~2~ 1 52 1 5~ 57 1 48 1 100 0 I Sj25 1 56 1 56 1 52 1 59 1 48 1 80 1 100 1 l The C70 ide~ betwe^n each paiiwLse com~arison o~ the predic:e~ am~oa~:d se~uencos ot ~e GST cDNA ciones.
DISCUSSION
) The GSTs of adult worms of F. heDatica comprise two major components of approximate 15 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. heDatica identified two major N-terminal sequences. In addition, peptides derived from intemal or ~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
20 heDatica. The isolation and sequencing of near identical peptides indicates the high degree of heterogeneity in the F. heoatica GST fraction and implies the expression of multiple GST
genes in this parasite.
The isolation of cDNA sequences encoding GSTs of F. heDatica was achieved by immunoscreening of cDNA expression libraries using rabbit antisera to the native GSTs.
25 Each of the five cDNA sequences cloned encodes a different primary amino acid sequence WO 90/08819 PCr/AU90/00027 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 showidentity or high simiiarity with the peptide sequences obtained from the native fluke GSTs showing that these cDNAs encode the GSTs expressed in the adult womm. The finding 5 that multiple GST sequences are expressed in a population of adult worrns implies either the presence of multiple GST genes within the F. heDatica genome or that multiple polymorphic variants of one or a few GST alleles exist within a genetically heterogeneous worm population.
-i The vaccination potential of trematode GST has been demonstrated in S. mansoni and S
10 jaDonicum 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. heDatica in the rat, immunity was not induced following subcutaneous vaccination with 250~,9 of GST in Freund's adjuvant [11]. The relevance of the rat model in fascioliasis has been questioned '~
15 [39] and it was therefore our aim in the present study to investigate the vaccination potential of F. heDatica 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 pattem consistent with 20 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 /0 reduction (p c 0.001) in woml 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 reduc~ion 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 ~hus 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. ja~onicum in laboratoryanimals.
The nature of the protective immune response directed against the parasite remains uncertain. A strong humoral response to GST of F. heoatica has been induced in all the vaccinated animals but the members of GST group 1 do not exhibit a dfflerentially higher antibody titre relative to the GST group 2 animals. It is therefore uncertain if a humoral - 35 response and/or a T-cell response is necessary to induce the protective effect observed.
In addition, the animals usad in this trial were outbred merino wethers which will exhibit . .
,9 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 5 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
heoatica has facilitated induction of a response to a novel parasite antigen leading to the death of the parasite.
WO 90/08819 ~, O a~ $ ~ 6 3 PCI /AU90/00027 REFERENCES
1. Haroun, E.M., and G.V. Hillyer. 1986. Vet. Parasitol. 20:63.
2. Dawes, B., and D.L Hughes. 1964. Adv Parasito! 2 97.
, 2~4a~
VACCINE FOR THE PREVE:~TATIVE TREATME T OF
INFECTION OF LIVFR FLUKE IN hiUMlNANTS
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.
BACKGROUl~iD OF THE INVENTION
EHective control of infection with liver fluke (Fascioliasis) is a major woridwide problem in the animal industry. Fascioliasis is caused by infection with the trematode parasite Fasciola hepatica (F. heDatica~. In ,oarticular, 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 antlgen vaccine approach and the potential of identifying and subsequently inducing an immune attack on a functional mdecule 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. jaDonicum in which 2 major defined antigens, glutathione-S-transferase (GST) [6,7] and paramyosin [8] have been studied fortheirvaccination potential.
: 25 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 l9]. 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 Mr 28,000 (Sm28 or p28) [6]. Similariy, a GST of Mr26,000 from S. jaDOniCum (Sj26) induced 30% protection in mice against an SlJ B ST~ E S ~l EET ~
~ _ I
WO 90/08~19 fi~ PCl`/AU90/00027 homologous cercarial challenge l7] though vaccinating effects in mice using Sj26 alone have been inconsistent l10].
In a recent report [11 j no protective effect of F heDatica GST was detected in rats against challenge with metacercariae. The authors concluded that GSTs ~do not confer any5 protection on rats against a challenge infection (with metacercariae of F. heDatica~~ that F. heDatica GSTs are almost cerlainly not host-protective antigens in rats~ and that ~nuke GSTs seem to be out of reach of the host immune system . Thus these authors havediscounted 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. heDatica 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 lg to affinity purify juvenile-specific antigens (JSA) from Iysates of juvenile fluke. The JSA
fraction conferred 652/o protection in rats against infection with F. hepatica.
European Patent Specification 11438 (Vaccines Intemational Ltd) describes a vaccine against bovine fascioliasis comprising irradiated rnetacercariae of F. aiaantica. The use of irradiated metacercariae for vaccination of sheep against F. her~atica has been reported to be unsuccessful [12].
PCT Application No. WO8801277 (Australian National University) is described in Chemical Abstract 110 No. 1213679 (M J Howell). cDNAs prepared from mRNA of Taenia 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 OFTHE 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 numinants the vaccine comprising glutathione-S-transferase (GST) derived from adult womms of F. heDatica.
A vaccine containing GST is able to stimulate immunity in sheep to infection with metacercariae of F. her~atica. The GST proteins are purified from adult worms of F.
heDatica by affinity chromatography on glutathione-agarose.
WO 90/0881~ PCr/AU90/00027 2043~63 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 pl values.
5 Direct peptide sequencing of some of the protein components present in the GST mixture has identified two major N terminal sequences and 8 other ssquences 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.
: .
10 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.
l~ is likely that the vaccine of the present invention will be effective against other members of the Fasciola genus, such as Easciola 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 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 moleculespurified from a crude homogenate of F. heDatica adult worms by aHinity chromatography on glutathione agarose. The position of the molecular weight WO 90t08X19 PC~IAU90/()0027 6~
markers is indicated (in kDa).
Figure 2. Two dimensional SDS-PAGE analysis of 1125 labelled glutathione-binding molecules purified from a crude homogenate of F. heoatica 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-temminal sequences obtained for GSTs of F. heDatica (Fh) to the N-termini of GSTs of other helminths (Schistocephalus 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 ~. Comparison of the sequence of tryptic and chymotryptic peptides of the GSTs of F. heDatica to homologous regions in GSTs of S. mansoni (Sm26), S.
japonicum (Sj26) and the mouse (Mm GST1). CT18.3: chymotrvptic peptide of F. hepatica; TO.7a, TO.7b, T16.3a, T16.3b, T16.2a, T16.2b: tr,vptic peptides of F. heDatica. 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. jaDonicum Mr 26,000 GST;
Sm26: S. mansoni Mr 26,000 GST; T21 .5b, T21.6a: F. heDatica tr,vptic peptides.
:
20 Figure 6. ELISA analysis of native F. heDatica GST probed with antisera from sheep immunized with GST in Freund's adjuvant (-), infected with F. hepatica for 12 wks (-), infected with F. hepatica for 6 wks ( ~ ) and normal sheep serum (r), Figure 7. Westem blot analysis of native F. hepatlca GST probed with antisera from dfflerent sheep. Panel A: an amido black stain of the native protein; panel B:
nommal sheep serum; Panel C: sera from sheep immunized with GST in Freund's adjuvant; panel D: sera from sheep infected with F. heDatica for 6 weeks; panel E: sera from sheep infected with F. heDatica 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 ofthe molecular weight markers is indicated (kDa).
30 Figure 8. Panel A shows the average RBC hemoglobin levels over 36 weeks of infection with F. hçpatica 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 . ,..-. .
:
WO 90/~8819 PCrJAU90/OOnl7 2~ 3 sheep ( ), infected control sheep (-- -), GST group 1 vaccinated sheep (.. ) and GST group 2 vaccinatr,~d sheep (-.. -.. ).
Figure 9. Panel A shows the average asparlate aminotransferase serum levels over 36 weeks of infection w~h F. her~atica 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 (-..-..).
10 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 (- -15 -), GST group 1 vaccinated sheep (.. ) and GST group 2 vaccinated sheep ( ) ., Figure 11. Panel A shows the average fecai egg counts over 36 weeks of infection with F
hepatica In infected control sheep (---) and aST-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 (- - -3.
, , Figure 12. Final worm burdens in sheep infected with F. heDatica and sacrificed over a period of 13 weeks (weeks 44 - a~7).
Figure 13. Westem blot analysis of F. heDatica 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 identlfied are of Mr 26,000-26,500.
Figure 14. DNA sequence of the GST 1 cDNA.
Figure 15. DNA sequence of the GST 7 cDNA.
30 Figure 16. DNA sequence of the GST 42 cDNA. Dashes indicate unassigned sequence.
Figure 17. DNA sequence of the GST 47 cDNA.
.,. -:, Figu!e 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. jawnicum; Fh26a, Fh26b: N-terminal amino acid sequences of GSTs of F. heoatica; GST1,7,42,47,50: amino acid sequences predicted from the cloned GST cDNAs of F. heDatica. T.05, TO.7b/0.6, T21.5: tryptic peptides of F. heoatica: CT18.3:chymotr,vptic peptide of F. hepatica. The sequences have been aligned to maximise the homology. Dashes indicate unassigned residues.
Materials and Methods 1 0 Parasites Fasciola heDatica 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 saiine (PBS) and homogenized in TNi_T 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. Occasionaily washed whoie worms stored at -20C, were thaweci at RT and then homogenked into TNi_T. These Iysates were clarified by centrifugation (10,0009, 30 minutes, 4C) and stored at -20C. Adult worms of the Compton strain of F. hepatica were simiiarly 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 intemmediate snaii host LYmnaea truncatula in the laboratory and subsequently through sheep. Aduit parasites of the Compton strain were obtained fresh from the biie ducts of infected sheep, washed in PBS
at 37C and stored at -70C.
Purification ot F. he,oatica GSTs GST isoereymes were purified by affinity binding to giutathione (GSH) agarose (Sigma, St Louis, USA). Briefly, TNET Iysates of aduit worms were passed down a GSH agarosecolumn, and the matrix washed with severai voiumes of PBS, prior to elution with a GSH
containing buffer (1.5mg/mi GSH in 50mM Tris (pH 9.3) ) [7]. Fractions shown to contain protein were pooled, dialysed against PBS or distilled water and stored at -70C. The GST
content and purity were assessed by Coomassie blue and silver staining of SDS-PAGE gels.
"
, W O 90/08819 PC~r/AU90/00027 2 ~
Generation of Peptides Approximately 3009 of affinity purified Fh GSTs were reduced in the presence of 1% w/v SDS, 10mM Dl~, in 100mM Tris (pH 8.0) for 60 minu~es at 58C. On cooling to ambient temperature, iodoacetamide was added to a final concentration of 22mM and 5 carboxyamidomethy1ation proceeded for 15 minutes at RT. Protease was added to 1-2%
(w/w), and the mixlure precipitated at -20C (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 10 taken up in 200~1 1% v/v trimethylamine (pH 8.0), and a further 7~9 trypsln (Worthington, Freehold, USA) added. Digestion occurred overnight at 37C. The chymotrypsin digest was prepared by addition of 200~-1 0.1 M NH4HCO3, pH 7.8, (CO2) and 10~.9 chymotrypsin (~orthington) and proteolysis conducted at 37C for 4 hours. Digestion was arrested by storage at -20C.
15 The ensuing peptides were separated by reverse phase chromatography using an - organic/aqueous gradient delivered by an FPLC system (Pharrnacia). Complete digests were primarily resolved with a 0-92.5% v/v acetonitrile (AcN) gradient in 15-20mM
ammonium formate, pH 4.0 (CO2) applied over 46 minutes, onto a Pro PRC 5/10 C1/C8 reverse phase column (Pharmacia). The elution was monitored at 214nm and peptide20 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 trfluoracetic acid. The elution was monitored at 21 4nm. Collected peptide peaks were stored at -2ûC, and dried by vacuum centrifugation (Savant Instnuments, 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 Melboume, using an ABI Model 471 A Protein Sequencer.
Derivitized amino acids were resolved on a 25cm Zorbax PTH column (Dupont) (at 38C) 30 using isocratlc 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 1ml/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, 5û mM dithiothreitol .
WO 90/08819 ~ PCI`/AU90/00027 and 1o% glycerol, pH 6.8), and electrophoresed under reciucing conditions on 13%acrylamide slab gels 113]. Relative molecular weights (M,) 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 w ith 5% methanol and 7% acetic acid, then dried under vacuum before autoradiography. Two-ciimensional electrophoresis was performed by the method of O'Farrell [14]. For the first dimension, isoelectric focusing (IEF) was perfommeci in glass tubes using a 1:1 mixture of pH 5-7 and pH 7-9 ampholytes (Pharmacia, Uppsala, Sweden). SDS-PAGE, using 13% acryiamide slab gels, was used for the second 10 dimension. The gels were prepareci for autoradiography following electrophoresis as described above.
Silver staining ot gels On occasion, electrophoresis gels were silver stained by the method of Morrissey [15I. In brief, the gels were rinsed in H2O 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/O glutaraldehyde for 30 minutes. At this stage the gel was left overnight in a large volume of H2O. Following a further wash (30 minutes) in H2O, the gel was immersed in a fresh 0.1% AgNO3 solution for 30 minutes and then rinsed once in H2O
and twice in developer solution (3 % Na2CO3, 0.05 % formalin). The gel was then stained 20 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).
. Westem blotting Electrophoretic transfer of proteins from poiyacryiamide gels to nitrocellulose paper was performed according to the method of Bumette [16], with a transfer time of 18 hours at 15 25 voits. The nitrocellulose sheet was blocked with 5 % skim milk powder in PBS for 3 hours ; at room temperature. The antiserum was diluteci 1 in 100 in PBS, added to the nitrocellulose sheet, and incubateci for 1 hour. The sheet was washed three times in PBS containing 0.1 % Tween 20. Affinity-purified rabbit an~i-sheep immunoglobulin (Biorad) or goat anti-rabbit immunoglobulin (Kinkegaard and Perry i abs, 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 washeci 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 1 2~Li of hydrogen peroxide. The location of the transferred protein was established by staining in a solution of 0.û04 % amido black in 45 % methanol 35 /10 % acetic acid.
-; -' .
WO 90/08819 P~/AU90/~)0027 9 2~a~3 lociination oI proteins The native GSTs of F. heDatica were radioiodinated using the Bolton and Hunter procedure [17].
EUSA
5 The ELiSA was perfommed as described by Milner et al [18] with the following changes.
Pdyvinyi microtitre plates were coated overnight at 4 C with 50~L1 purHied GST (5 ~.g/ml) in 0.1 M sodium carbonate buffer (pH 9.5). Sera were diluted in ELiSA buHer (0.1 M Tris Ha, 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 50~1 of the appropriate dilution was incubated in the 10 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. Affin~y-purHied rabbit anti-sheep immunoglobulin conjugateci to horse radish peroxidase (Biorad) was diluted in ELISA buffer, 50 ILI 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 Na2HPO4 pH
15 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. heDatica. All animals were screened before use for the absence 20 of F. hepatica eggs in their feces.
A group of 10 sheep were immunked by subcutaneous injection of 100,ug of purHied GST
of F. heDatica in Freund's complete adjuvant (FCA) 16 weeks prior to infection followed by a boost with 100~.9 of purHied GST in Freund's incomplete adjuvant (IFA) 12 weeks prior to infection. The sheep were given subsequent boosts of 100~ 9 of purHied GST in PBS at 25 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, all sheep, except 3 of the 8 unimmunked sheep which were kept as uninfected controls, were infected intraruminally with 500 metacercariae (Compton Paddock Laboratories, UK) suspended in a 0.4% w/v 30 suspension of high viscosity carboxymethyi cellulose (Sigma). Sera from all sheep were coilected 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 glutamyitransferase (EC 2.3.2.2.) (GGT) [20] and red blooci cell (RBC) hemoglobin [21] on a Roche Cobas MIRA automatic WO 90/08~19 C;~ PCr/AU90/00027 analyser (Basel, Switzerland). Serum was stored frozsn at -20 C until use.
Fecal egg counts (FEC) were parfo~med by the method of Kelly et al ~z] 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 ~ecal 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.
10 Statistical sign'~icance was calculated by the Mann Whitney U statistic [23].
Construction of cDNA libraries in /~ZAP and /~gt11 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 125]. The cDNA libraries were construc~ed in phage vectorslgt1 1 and ~ZAP by CLONTECH
15 (Palo Alto, USA) using the procedure of Gubler and Hoffman [26].
,~
lmmunoscreening ot cDNA libraries The cDNA libraries were screened for expression of GSTs of F. heDatica 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. heDatica at a dilution of 1/600. Filters were blocked in a buffer containing 10mM Tris HCI, pH8.0, 150mM NaCl, 0.05% Tween 20, 1% gelatin. Positive plaques identified in a primary screen were picked, replated at a lower dens-~y and rescreened with the rabbit antisenum until individual positive plaques were identHied.
.
Absorption of rabbit anti-GST serum on GST1 25 Antibodies in the rabbit anti-GST senum were depleted of specificities to sequences :expressed in Ihe GST1 clone before the 1ZAP library was rescreened to identriy other GSTs of F. heDatica. Undiluted rabbit antiserum (lml) was incubated with 1ml of a sonicate of E coli expressing s-galactosidase for 16 hours at 4~C to deplete anti- E. coli specificities.
This depleted serum was diluted to 1/600 with PBS and 10ml of this sarum was incubated 30 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.
': -.
W O 90/08819 P ~ /A~90/00027 20~63 DNA Hybridization .
Plaque hybridization of radiolabelled GST1 or GST7 insert DNA to the 1ZAP library was performed as described by Maniatis et al 125]. 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 AZAP phage clones was isolated by excision in v,vo of the pBluescript phagemid under the conditions recommended byStratagene (La Jolla, USA). Phagemid DNA was extracted by the method of Bimboim and 10 Doly [27]. Double-stranded DNA sequencing of cDNA inserts was performed by the chain termination method [28].
Characterization ot proteins purified by glutathione agarose chromatography The purification of native GSTs from mammalian or Schistosoma species by glutathione-15 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. heDatica. In order to isolate GSTs of F. heDatica, adult worms were Iysed in buffer containing Triton X-- 100 and the clarified Iysate was applied to a glutathione-agarose column as described in Materials and Methods. The column was washed with PBS and the bound material eluted 20 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 25 about 10-11 components which exhibit dfflerent apparent pl 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. Altematively, each component could be the product of an individual GST gene within a clonal fluke population.
WO 90/08819 PC~/AU90/00027 ~ - 12-Amino acid sequence of native GSTs ot F. heDatica N temminal amino acid sequences of the purified F. heDatica 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]
5 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 respecthely ~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 10 other GST sequences. Peptide CT18.3 is homologous to sequences in the Schistosoma GSTs whereas the TO.7A, TO.7b and T16 series of peptides show greatest identity to mouse GST1. Two peptides, T21.5b and T21.6a, are identical and show 69% identity with the Gterrninal region of the Mr 26,000 GST of S. jaDonicum and S. mansoni.
TABLE 1 Identities in N-terminal amino acid sequence between GSTs of F. hepatica and other species.
. f~eference Other sDecies1 % identty Fh26a2 Fh26bZ
31 Ss 24 60 66 Sm 26 76 77 20 7 Sj 26 72 70 Rn GST1 56 59 . 34 Hs GST 58 62 37 Bi GST 55 60 32 Mm GST1 52 55 25 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. heDatica.
These results show that the abundant proteins of Mr 26,000 and 26,500 purified by affinity 30 ehromatography on glutathione-agarose are homologous to the GSTs of both Schistosoma and mammalian species.
-, ~ .
WO 90/08819 PCI`/AU90/00027 Antibody r~sponse o~ sheep to the purified ~ST antigen The antibody response to GST in infectecl sheep and sheep vaccinated with GST inFreund's complete adjuvant was analysed by ELISA and Westem blotting. As shown in Fig 6, GST vaccinated animals exhibited a strong antibody response to the vaccine antigen 5 whereas sheep infected with F. her~atica 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. he~atica (Fig 7).
Parameters analysed during vaccination trial To assess the progression of the liver fluke infection and to monitor the health of the 10 animals throughout the vaccination trial three parameters were analysed. The level of ~BC
hemoglobin was assayed as an indicator of anemia. Senum 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 contro! infected animals (i.e. 10 PBS vaccinated controls and 5 non-15 vaccinated controls), 1 animal died from a dog attack and 3 animals died (one at week 5and two at week 7) as a result of liver fluke infeclion. 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 20 demonstrated a decrease in RBC hemoglobin with time, dropping to below 8 g/L by week 36. The GST vaccinated sheep displayed levels consistentiy 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 triai, it was found that 4 of the animals (GST group 25 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 parenchymai damage in the trial 30 animals. The GST-vaccinated animals consistently displayed levels of senJm 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 senum levels over the initial 10 weeks with a slightly delayed maximum reached at week 6 compared to week 4 a~ 3 - 14-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 v ere analysed to monitor damage resulting from the establishment of parasites in the bile ducts. The level of GGT in the GST-vaccina~ed animals demonstrated a profile similar to that recorded for the infected control animals (Fig 1 Oa) 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 2subpopulation.
A~l 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 d-dferent (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 11uke 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 - 25 reduction in worm burden of 55 % (p < 0.001). When the GST vaccinated animals were considered as subpopulations, the GST group 1 group exhibited a mean worrn 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 dfflerent groups of animals was compared. As shown in Table 2, there was no significant effect ofvaccination on the egg output per womm although there is a tendency towards higher egg output in the GST-vaccinated animals.
, " ' ' ' -2 ~ 3 Cloning and expression ot GST genes ot _. hepatica Rabbit antiserum was raised to the purified GST fraction by subcutaneous injection of F.
heDatica 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 two5 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. heDatica 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 15 incornplete, 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 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-g6h) among the predicted polypeptide sequences which extends throughout the sequences(Table 3). This result shows that adult F. he~atica express at least f~e dfflerent mRNAs for GST. Comparison of the sequences of the F. heDatica 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. heDatica.
~,~ 4~ 6 1 6 --L~L ¦ FC~ ~iOR\,~FC/~NOR~vl ¦
! lDE~Tl~Y I Bl ~DE.~
CO~ROLI ~W6~ 1-i8 ~6 10.,5 S ~h6li li- ~59 5.oi W6,' 1-`- ~ 4.63 W686 397 1~' 4.9i W69; '~1~ 10.36 \~69l 8;5 i3 Ll~
W695 i99~ ^9 14.36 ~696 W69~ 101 y_~ l'08 ';~ 1.3 TOT.~L ~ ~VG ^ S~1 9 - 9 '~: - li3.`0 - 1.'o 1.
1 5 I G_ T I G' -; ~ ~ l î . ' ' G~9; 1,; ^0 3.65 ~65; 1100 ,~ /~9 .
W6, l 19 / 13 10.9 ¦ C-ST o G~3 ~;,5 -no ]1.SS
Gi81 169- 1;1 1'~ 9 W609 ~5i 110 6.8~
:: W6~0 1*3; 1-~ 1''.;6 W69? 169~ 13~ 9.~5 TOT~L .~G: SE~I¦ 1114 - ~i710, - ''`10.33 - 1.0;
GST 1 AVG: SEl~l50/: 1S7 5' - '611.15 1.91 GST ~ AVG - S~L1600: æ2 1'9 = 1610.66 _ 1.02 .', .
.
Data derived from 10 control aiimals represerlt an average count from weelcs ;2, i4 ar~d i6.
; Worm burde:l at ~ime o~ sacrihce.
. . .
:....... . . .. . ., . , ~ , .
:
. -.
:, ' , WO 90/08819 PCr/AU9OtO(3()27 ?
2~45~3 T~ble 3 I~e~ ies in amino ~c;d sequenc: be~we^3 GSTs o~ F he~a~ic~ ~nd Schistosom~ pr~ic;ed ~om ~he DN.'~ sequ~:lces of tbe c'oned Gâ 15.t ~GST1 ¦GS~7 ~GaT~2 ¦GS~7 ~GS~SO ¦ Sm2S lSj26 ¦ GST~ 1 ~ 100 5i Ga~ 7 ~ 68 ¦ 100 ¦
C-~ 21 68 1 84 1- --GS'" 4, ¦ 7~ ¦ 76 1 84 ~ 100 ¦
i CS~ 501 6" 1 87 j 9~ ~ 90 ~ 1 ~S-~2~ 1 52 1 5~ 57 1 48 1 100 0 I Sj25 1 56 1 56 1 52 1 59 1 48 1 80 1 100 1 l The C70 ide~ betwe^n each paiiwLse com~arison o~ the predic:e~ am~oa~:d se~uencos ot ~e GST cDNA ciones.
DISCUSSION
) The GSTs of adult worms of F. heDatica comprise two major components of approximate 15 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. heDatica identified two major N-terminal sequences. In addition, peptides derived from intemal or ~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
20 heDatica. The isolation and sequencing of near identical peptides indicates the high degree of heterogeneity in the F. heoatica GST fraction and implies the expression of multiple GST
genes in this parasite.
The isolation of cDNA sequences encoding GSTs of F. heDatica was achieved by immunoscreening of cDNA expression libraries using rabbit antisera to the native GSTs.
25 Each of the five cDNA sequences cloned encodes a different primary amino acid sequence WO 90/08819 PCr/AU90/00027 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 showidentity or high simiiarity with the peptide sequences obtained from the native fluke GSTs showing that these cDNAs encode the GSTs expressed in the adult womm. The finding 5 that multiple GST sequences are expressed in a population of adult worrns implies either the presence of multiple GST genes within the F. heDatica genome or that multiple polymorphic variants of one or a few GST alleles exist within a genetically heterogeneous worm population.
-i The vaccination potential of trematode GST has been demonstrated in S. mansoni and S
10 jaDonicum 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. heDatica in the rat, immunity was not induced following subcutaneous vaccination with 250~,9 of GST in Freund's adjuvant [11]. The relevance of the rat model in fascioliasis has been questioned '~
15 [39] and it was therefore our aim in the present study to investigate the vaccination potential of F. heDatica 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 pattem consistent with 20 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 /0 reduction (p c 0.001) in woml 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 reduc~ion 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 ~hus 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. ja~onicum in laboratoryanimals.
The nature of the protective immune response directed against the parasite remains uncertain. A strong humoral response to GST of F. heoatica has been induced in all the vaccinated animals but the members of GST group 1 do not exhibit a dfflerentially higher antibody titre relative to the GST group 2 animals. It is therefore uncertain if a humoral - 35 response and/or a T-cell response is necessary to induce the protective effect observed.
In addition, the animals usad in this trial were outbred merino wethers which will exhibit . .
,9 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 5 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
heoatica has facilitated induction of a response to a novel parasite antigen leading to the death of the parasite.
WO 90/08819 ~, O a~ $ ~ 6 3 PCI /AU90/00027 REFERENCES
1. Haroun, E.M., and G.V. Hillyer. 1986. Vet. Parasitol. 20:63.
2. Dawes, B., and D.L Hughes. 1964. Adv Parasito! 2 97.
3. Hillyer, G.V., E.M. Haroun, snd M. Soler De Galanes. 1987. Am. J Trop. Med.
Hyq. 37:363.
Hyq. 37:363.
4. Hillyet, G.V., M.l. Garcia Rosa, H. Alicea, and A. Hernandez. 1988. Am. J Trop.
Med. Hyg 38:103.
Med. Hyg 38:103.
5. Mitchell, G.F. 1987 Parasit. Tociav 3-106 6. Balloul, J.M., J.M. Grzych, R.J. Pierce, and A. Capron. 1987.
J. Immunol. 138:3448.
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. Sci. USA 83:8703.
` 20 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.
` 20 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. 57:3s7.
10. M~chell, G.F., E.G. Garcia, K.M. Davern, W.U. 17u, and D.B. Smith. 1988. Trans.
- Royal Soc. TroD. Med. Hva. 82:885.
- Royal Soc. TroD. Med. Hva. 82:885.
11. 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.
., .
~'' ,:
WO 90/08819 PCl/AU90/00027 -21- 2~43~3 17 Bolton, A.E. and W.M. Hunter. 1973. J Biochem 133 529 18. Milner, A.R., K.B. Jackson, K. Woodrutt, and l.J. Smart. 1985 J Clin. Micro 22:539.
., .
~'' ,:
WO 90/08819 PCl/AU90/00027 -21- 2~43~3 17 Bolton, A.E. and W.M. Hunter. 1973. J Biochem 133 529 18. Milner, A.R., K.B. Jackson, K. Woodrutt, and l.J. Smart. 1985 J Clin. Micro 22:539.
19 International Federation o~ 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. Zijlstra. 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 :: 15 23. Rohlt, F.R., and R.R. Sokal. 1969 Stalistical 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 clonina. Cold Sprina Harbor Laboratorv.
26 Gubler, U. and B.J. Hoffman. 1983 Gene 25:263 27. Birnboim, H.C. and J. Doly, 1979. Nuc.Acids.Res. _:1513.
28. Sanger, F., S. Nicklen and A.R. Coulson. 1977 Proc. Natl Acad Sci USA
74:5463.
- 25 29. Simons, P.C. and D. L VanderJagt. 1977. Anal Biochem. 82:334 30. Henkel, K.J., K.M. DaYem, M.D. Wright, A. J. Ramos and G.F. Mitchell. 1990.
Mol. Biochem. Parasitol. (in Dress) 31. Brophy, P.M., A. Papadopoulos, M. Touraki, B. Coles, W. Korting and J. Barrett.
1989. Mol Biochem Parasitol. 36 187 WO 90~08819 ~ PCr/AUgO/00027 32. Pear~on, W.R., J.J. Windle, J.F. Morrow, A.M. Benson and P. Talalay. 1983. J 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 1. 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. Warholm and H. Jornvall, 1985. Proc Natl Acad. Sci. USA 82:7202.
10 38. Balloul, J.M., P. Sondermeyer, D. Dreyer, M. Capron, J.M. Grzych, R.J. Pierce, D. Carvailo, J.P. Lecocq, and A. Capron. 1987. Nature 326:149.
s 39. Hughes, D.L 1987. In lmmune res~onses in parasitic infections: immunoloav immunopatholoav. and immunoDroDhylaxis. Volume ll: Trematodes and cestodes.
E.J.L Soulsby, ed. CRC Press, Florida, USA, P.91.
.... . . .
, : :
~ ' ' .
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 clonina. Cold Sprina Harbor Laboratorv.
26 Gubler, U. and B.J. Hoffman. 1983 Gene 25:263 27. Birnboim, H.C. and J. Doly, 1979. Nuc.Acids.Res. _:1513.
28. Sanger, F., S. Nicklen and A.R. Coulson. 1977 Proc. Natl Acad Sci USA
74:5463.
- 25 29. Simons, P.C. and D. L VanderJagt. 1977. Anal Biochem. 82:334 30. Henkel, K.J., K.M. DaYem, M.D. Wright, A. J. Ramos and G.F. Mitchell. 1990.
Mol. Biochem. Parasitol. (in Dress) 31. Brophy, P.M., A. Papadopoulos, M. Touraki, B. Coles, W. Korting and J. Barrett.
1989. Mol Biochem Parasitol. 36 187 WO 90~08819 ~ PCr/AUgO/00027 32. Pear~on, W.R., J.J. Windle, J.F. Morrow, A.M. Benson and P. Talalay. 1983. J 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 1. 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. Warholm and H. Jornvall, 1985. Proc Natl Acad. Sci. USA 82:7202.
10 38. Balloul, J.M., P. Sondermeyer, D. Dreyer, M. Capron, J.M. Grzych, R.J. Pierce, D. Carvailo, J.P. Lecocq, and A. Capron. 1987. Nature 326:149.
s 39. Hughes, D.L 1987. In lmmune res~onses in parasitic infections: immunoloav immunopatholoav. and immunoDroDhylaxis. Volume ll: Trematodes and cestodes.
E.J.L Soulsby, ed. CRC Press, Florida, USA, P.91.
.... . . .
, : :
~ ' ' .
Claims (11)
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.
(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 nucleotidesequence 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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPJ2481 | 1989-01-31 | ||
| AUPJ248189 | 1989-01-31 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2045663A1 true CA2045663A1 (en) | 1990-08-01 |
Family
ID=3773671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002045663A Abandoned CA2045663A1 (en) | 1989-01-31 | 1990-01-31 | Vaccine for the preventative treatment of infection of liver fluke in ruminants |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0456662A4 (en) |
| JP (1) | JPH04507235A (en) |
| AU (1) | AU634754B2 (en) |
| BR (1) | BR9007070A (en) |
| CA (1) | CA2045663A1 (en) |
| NO (1) | NO912942L (en) |
| NZ (1) | NZ232327A (en) |
| WO (1) | WO1990008819A1 (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2657883A1 (en) * | 1990-02-07 | 1991-08-09 | Transgene Sa | |
| GB9018238D0 (en) * | 1990-08-20 | 1990-10-03 | Trudgett Alan G | Liver fluke diagnostic system |
| ES2199947T3 (en) * | 1992-10-21 | 2004-03-01 | John Pius Dalton | VACCINE CONTAINING A TIOLPROTEASE. |
| BR9404004A (en) * | 1993-02-05 | 1999-05-25 | Daratech Pty Ltd | Isolated polypeptide antigen fragment derived or analogous nucleic acid molecule method to reduce the spread of a helminthic parasite and vaccine composition |
| EP0721466A1 (en) * | 1993-09-28 | 1996-07-17 | The University Of Melbourne | Protective antigens against parasites |
| CN1061990C (en) * | 1994-06-08 | 2001-02-14 | 北京医科大学 | Schistosome vaccine peptide No.1 |
| CN1068334C (en) * | 1994-06-08 | 2001-07-11 | 北京医科大学 | Schistosome vaccine peptide No.2 |
| CN1061991C (en) * | 1994-06-08 | 2001-02-14 | 北京医科大学 | Schistosome vaccine peptide NO.3 |
| GB9612214D0 (en) * | 1996-06-11 | 1996-08-14 | Mallinckrodt Veterinary Inc | Vaccine |
| CN105903007A (en) * | 2016-06-15 | 2016-08-31 | 青海大学 | Design and preparation method and application of novel fasciola hepatica multi-epitope vaccine |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8432401D0 (en) * | 1984-12-21 | 1985-02-06 | Bennett C E | Liver fluke antigens |
| NZ224663A (en) * | 1987-05-28 | 1990-11-27 | Amrad Corp Ltd | Fusion proteins containing glutathione-s-transferase and expression of foreign polypeptides using glutathione-s-transferase encoding vectors |
-
1990
- 1990-01-31 JP JP2502498A patent/JPH04507235A/en active Pending
- 1990-01-31 EP EP19900902123 patent/EP0456662A4/en not_active Ceased
- 1990-01-31 CA CA002045663A patent/CA2045663A1/en not_active Abandoned
- 1990-01-31 WO PCT/AU1990/000027 patent/WO1990008819A1/en not_active Application Discontinuation
- 1990-01-31 BR BR909007070A patent/BR9007070A/en not_active Application Discontinuation
- 1990-01-31 NZ NZ232327A patent/NZ232327A/en unknown
- 1990-01-31 AU AU50283/90A patent/AU634754B2/en not_active Ceased
-
1991
- 1991-07-29 NO NO91912942A patent/NO912942L/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| NZ232327A (en) | 1993-02-25 |
| BR9007070A (en) | 1991-11-12 |
| WO1990008819A1 (en) | 1990-08-09 |
| AU5028390A (en) | 1990-08-24 |
| NO912942L (en) | 1991-09-26 |
| AU634754B2 (en) | 1993-03-04 |
| EP0456662A4 (en) | 1991-12-11 |
| NO912942D0 (en) | 1991-07-29 |
| EP0456662A1 (en) | 1991-11-21 |
| JPH04507235A (en) | 1992-12-17 |
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| FZDE | Discontinued |