Note: Descriptions are shown in the official language in which they were submitted.
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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
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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.
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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
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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
. ,..-. .
:
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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.
.,. -:,
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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.
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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.
-; -
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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
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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.
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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].
RESULTS OF EXAMPLE 1
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.
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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.
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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
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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.
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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.
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TABLE 2
-
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.
. . .
:....... . . .. . ., . , ~ , .
:
. -.
:, '
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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
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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
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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.
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