Parasite Immunology, 2010, 32, 414–419
Attempts to immunize sheep against Haemonchus contortus using
a cocktail of recombinant proteases derived from the protective
antigen, H-gal-GP
E. CACHAT, G. F. J. NEWLANDS, S. E. EKOJA, H. MCALLISTER & W. D. SMITH
Moredun Research Institute, Edinburgh, Scotland, UK
SUMMARY
The objective of this study was to determine whether an anti-
gen cocktail containing recombinantly expressed versions of
most of the protective proteases of H-gal-GP, a known pro-
tective antigen from Haemonchus contortus, would confer
any protection to lambs in a vaccine-challenge trial. Hae-
monchus contortus metalloendopeptidases, MEP1, MEP3
and MEP4, were expressed as soluble recombinant proteins
in insect cells, but attempts to express the H. contortus
aspartyl proteases, PEP1 and PEP2, by the same techniques
were not successful. Recombinant H. contortus PEP1 was
therefore expressed in Escherichia coli and refolded. Groups
of sheep were immunized thrice with either native H-gal-GP,
a cocktail of recombinantly expressed proteins (rMEP1,
rMEP3, rMEP4 and rPep1), or adjuvant only (QuilA in
PBS). All sheep were challenged with 5000 infective larvae
1 week after the final vaccination. High levels of serum anti-
bodies that recognized H-gal-GP were detected in both the
native antigen and recombinant cocktail-immunized groups
by the time of challenge, but protective immunity was only
observed in the group immunized with native H-gal-GP.
Keywords Haemonchus, H-gal-GP, vaccine, recombinant
INTRODUCTION
Haemonchus contortus is the single, most important nema-
tode parasite of sheep and goats in the world. As it prefers
warm moist climates, H. contortus is much more prevalent
in the tropics and sub-tropics than in temperate zones.
The parasite inhabits the true stomach or abomasum,
Correspondence: W.D. Smith, Moredun Research Institute,
Pentlands Science Park, Penicuik, Edinburgh, Scotland, UK
(e-mail: smitd@mri.sari.ac.uk).
Received: 20 October 2009
Accepted for publication: 18 December 2009
414
DOI: 10.1111/j.1365-3024.2010.01208.
where the late larval and adult stages feed on blood. It is
capable of causing substantial blood loss, so that the host
becomes anaemic and may die.
Existing control strategies have relied on the use of
anthelmintic drugs combined with pasture management.
However, drug-resistant strains of H. contortus are becom-
ing increasingly widespread and the need to find alterna-
tive control methods is pressing. Vaccination is one
potential solution but to date there are no commercial
vaccines for any gut nematode parasite of any host.
Various antigens have been isolated from the intestinal
cells of H. contortus and shown to be protective when
tested in vaccine trials in sheep. However, only two of
these antigens, H-gal-GP and H11, have consistently con-
ferred protection to a degree which would exceed a con-
ventional anthelmintic treatment regime (i.e. >80%
efficacy in >80% of the flock; 1) and which would there-
fore be commercially useful. Native H-gal-GP and H11
have each been shown to reduce faecal egg counts by more
than 90% in vaccinated sheep and, when used in combina-
tion, their effect in a controlled field trial was highly bene-
ficial for grazing Merino lambs; preventing deaths,
reducing anaemia and substantially reducing transmission
of infective larvae (2).
H-gal-GP is an intestinal cell brush border complex
thought to be involved in the digestion of the parasite’s
blood meal. The protective components of the complex
are a family of four metalloendopeptidases (MEPs) and
two aspartyl proteases (PEPs) (3,4). The native complex
loses some of its protective capacity when SDS dissoci-
ated, which indicates that conformational epitopes are
important and may explain why bacterially expressed
insoluble recombinants do not protect (3,4). In this paper,
soluble recombinant versions of most of the protective H-
gal-GP proteases were expressed and subsequently evalu-
ated as a candidate vaccine antigen cocktail in a sheep
protection trial.
 2010 Moredun Research Institute
Journal compilation  2010 Blackwell Publishing Ltd
Volume 32, Number 6, June 2010
MATERIALS AND METHODS
Expression of recombinant MEPs
Haemonchus contortus MEP1 and MEP4 (accession num-
bers AF102130 and AF132519, respectively) were expressed
in Spodoptera frugiperda Sf9 insect cells by tebu-bio labora-
tories (Le Perray-en-Yvelines, France) under proprietary
agreement. cDNAs encoding the extracellular domain of
both MEP1 and MEP4 were cloned into expression vectors
that were transfected into Sf9 cells using Escort lipofection
agent (Sigma-Aldrich Ltd., Dorset, England). After selec-
tion under antibiotic pressure (1Æ75 lg ⁄ mL puromycin),
cells were subcultured, amplified and tested for soluble pro-
tein expression by SDS–PAGE stained with Coomassie
Blue and immunoblots probed with sheep antiserum to
H-gal-GP. Large-scale expression (5 L) was achieved in
serum-free medium IPL-41 (Invitrogen Ltd., Paisley, UK)
supplemented with 1% lipid concentrate (Invitrogen), 2%
yeastolate (Invitrogen), 10 lg ⁄ mL gentamycin (Sigma) and
Pluronic F68 (Sigma). The supernatants containing the
recombinant proteins were stored at 4C.
Haemonchus contortus MEP3 (accession number
AF080172) was expressed in Sf9 insect cells by Eurogene-
tec (Seraing, Belgium). The extracellular domain of MEP3
(bp 98–2515) was amplified by PCR, cloned into pCR2Æ1
and sequenced, showing one mutation at position 1453
(T fi A), when compared with database entry AF080172,
inducing the change of an Asn into a Lys. It was then
subcloned between the BamHI and NcoI sites of the pMel-
BacB transfer vector, chosen to target secretion with the
Mellitin signal sequence. The construction of the recombi-
nant baculovirus was achieved by co-transfection of pMel-
BacB ⁄ MEP3 with Bac-N-Blue linear virus DNA
(Invitrogen) in Sf9 cells. After selection by end-point dilu-
tion, a high-titre stock was produced (400 mL) in serum-
containing medium. Culture conditions (harvest time and
multiplicity of infection, MOI) were optimized in serum-
free medium (Insect-Xpress; Lonza Wokingham Ltd.,
Berkshire, UK) using Sf9 or Tn5 cells and monitored by
SDS–PAGE and immunoblots. Large-scale expression
(4 L) was achieved to a cell density of 1Æ8 · 106 cells ⁄ mL
and a MOI of two with Tn5 cells. The culture supernatant
was harvested by low-speed centrifugation (750·g, 10 min)
and stored at )80C.
Purification of recombinant MEPs
Insect cell culture supernatants containing recombinant
MEPs were buffer exchanged on a Sephadex G25 col-
umn (GE Healthcare, Buckinghamshire, UK) equili-
brated with 20 mM Tris-HCl, 0Æ01% w ⁄ v NaN3, pH 8Æ5,
 2010 Moredun Research Institute
Journal compilation  2010 Blackwell Publishing Ltd, Parasite Immunology, 32, 414–419
To immunize sheep against H. contortus using a recombinant cocktail
at 4C. CHAPS was added to the desalted protein to a
final concentration of 0Æ05% w ⁄ v and the protein mate-
rial was loaded on a MonoQ HR 5 ⁄ 5 anion-exchange
column (GE Healthcare) equilibrated at room tempera-
ture with running buffer (20 mM tris-HCl, 0Æ01% w ⁄ v
NaN3, 0Æ05% w ⁄ v CHAPS, pH 8Æ5). The column was
eluted with a linear gradient of 0–0Æ5 M NaCl in running
buffer at 1 mL ⁄ min over 20 mL. Fractions were col-
lected and analysed by nonreducing SDS–PAGE. Those
fractions containing proteins of the appropriate molecu-
lar weight (around 90 kDa for rMEP1, 120 kDa for
rMEP3 and 140 kDa for rMEP4) were pooled and con-
centrated using Amicon Ultra concentrators (Millipore,
Watford, UK) with a 10 kDa cut-off. The concentrated
fractions were loaded on a Superose 12 size-exclusion
column equilibrated with 10 mM Tris-HCl, 150 mM
NaCl, 0Æ01% w ⁄ v NaN3, pH 7Æ4. As before, fractions
were collected, analysed, pooled and concentrated. The
recombinant MEPs were stored at )80C.
Expression, purification and refolding of recombinant
Pepsin
Haemonchus contortus pepsin PEP1 (accession number
Z72490) was expressed in E. coli. The cDNA encoding the
mature enzyme, bp 175–1287, was cloned into the pET-22b
expression vector and transformed into BL21-CodonPlus
(DE3)-RIPL competent cells (Stratagene Europe, Leicester,
UK). Recombinant clones were selected and checked for
PEP1 expression, following Stratagene’s protocol for iso-
propyl b-D-1-thiogalactopyranoside (IPTG) induction
(1 mM IPTG at 37C for 3 h). Levels of expression were
monitored by SDS–PAGE stained with Coomassie Blue and
immunoblots probed with sheep antiserum to H-Gal-GP.
Large-scale expression (3 L) was performed in luria broth
(LB)-shaken medium supplemented with 50 lg ⁄ mL chl-
oramphenicol and 50 lg ⁄ mL ampicillin at 37C and shaken
at 200 rpm. Cells were harvested 3Æ5 h after IPTG induction
by centrifugation at 5000·g and pellets were pooled. Bacte-
rial inclusion bodies containing the recombinant protein
were extracted from cell pellets using the BugBuster Protein
Extraction Reagent (Merck Chemicals Ltd., Nottingham,
UK) following the manufacturer’s instructions.
rPEP1 was extracted from purified inclusion bodies
by a base extraction method. The pellets of purified
inclusion bodies were resuspended in lysis buffer (20 mM
Tris, 5 mM EDTA, 5 mM b-mercaptoethanol, pH 8Æ0)
using a hand homogenizer. Suspensions were stirred on
ice and 0Æ2 M NaOH was added dropwise to increase
the pH from 8Æ0 to 11Æ7. The solution was stirred on
ice for 30 min and the pH was re-adjusted to 8Æ0 by
dropwise addition of 0Æ2 M HCl. b-mercaptoethanol was
415
E. Cachat et al.
added to a final concentration of 5 mM and the solution
was stirred on ice for a further 30 min. The supernatant
was collected after centrifugation at 86 500·g, 4C for
25 min and dialysed against 50 mM Tris, pH 8Æ0 at 4C.
The refolded rPEP1 was filter-sterilized through a
0Æ22 lm filter and stored at 4C. To determine the con-
formational state of rPEP1, it was analysed by chroma-
tography on a Superose 12 size-exclusion column (GE
Healthcare).
MEP and PEP activity assays
The endopeptidase activity of recombinant MEPs was
measured in a two-stage assay utilizing the fluorogenic
substrate Glu-Ala-Ala-Phe-4-MNA (Sigma), as described
previously (5).
The aspartyl protease activity of rPEP1 was measured by
its ability to hydrolyse the peptide substrate H-Pro-Thr-
Glu-Phe-Phe(NO2)-Arg-Leu-OH (Bachem), as described
previously (6).
Preparation of native H-gal-GP
This was the peanut lectin-binding fraction of detergent-
extracted membranes of adult H. contortus prepared as
described previously (7).
Sheep and design of vaccine trial
Twenty-one castrated Blackface · Leicester male lambs,
aged 9 months and weighing between 35 and 52 kg at the
time of the first vaccination, were allocated to three groups
of seven balanced for weight. These lambs had been reared
indoors from birth at the Moredun Research Institute
under conditions designed to exclude nematode parasites
and fed a maintenance diet of concentrates and hay.
Each group of sheep was immunized thrice, 3 weeks
apart, with either 100 lg native H-gal-GP, 200 lg
recombinant cocktail (23 lg rMEP1, 63 lg rMEP3,
20 lg rMEP4 and 100 lg rPEP1) or with PBS buffer.
QuilA adjuvant (5 mg; Brenntag, Denmark) was mixed
with each dose of immunogen, which was injected intra-
muscularly as a 2 mL dose (1 mL being administered to
each back leg). Protein concentration of recombinant
and native protein solutions were determined by Pierce
BCA assay (Fisher Scientific, Loughborough) performed
as per the manufacturer’s protocol.
Blood samples were obtained for serology on most
weeks of the trial. All sheep were challenged with 5000 H.
contortus larvae 1 week after the final immunization. Fae-
cal egg counts were made three times per week from day
18 or day 19 after challenge. All sheep were sacrificed
416
Journal compilation  2010 Blackwell Publishing Ltd, Parasite Immunology, 32, 414–419
Parasite Immunology
5 weeks post-challenge and the abomasa were collected for
worm counts.
Parasitology procedures
Infective larvae were from a culture of H. contortus which
has been maintained at the Moredun Research Institute
for several years.
Faecal samples were collected at 18 or 19, 21, 23, 26,
28, 30 and 33 days post-challenge. The methods for faecal
egg counting, enumeration of worm burdens and for
obtaining clean adult parasites for protein preparations
have been described before (8).
ELISAs
Determination of anti-H-gal-GP antibody levels was per-
formed by ELISA. Briefly, Microlon 96K microtitre plates
(Greiner-Bio-One, Frickenhausen, Germany) coated with
50 lL of a 5 lg ⁄ mL H-gal-GP solution in carbonate buf-
fer (50 mM carbonate, pH 9Æ6) overnight at 4C. Wells
were blocked for 2 h at room temperature with 10% (w ⁄ v)
Infasoy (Cow & Gate Ltd., Trowbridge, Wiltshire, UK) in
TNTT (10 mM Tris-HCl, 0Æ5 M NaCl, 0Æ05% Tween 20,
0Æ01% w ⁄ v thiomersal, pH 7Æ4). Serum from each animal
was diluted 1 : 800 in TNTT; 50 lL was added per well
and the plate was incubated for 2 h at room temperature.
Mouse monoclonal anti-goat ⁄ sheep IgG-horse radish per-
oxidase (HRPO) conjugate (50 lL; Sigma), diluted
1 : 10 000 in TNTT, was added and incubated for 1 h at
room temperature. Sigma-Fast OPD substrate was added
(50 lL) and incubated at room temperature in the dark
for 7 min. The reaction was stopped by the addition of
25 lL 2Æ5 M sulphuric acid. Absorbance values were read
at 490 nm. All titrations were performed in triplicate.
SDS–polyacrylamide gel electrophoresis and western blot
analysis
SDS–PAGE was performed using commercially available
pre-cast 4–12% bis-tris gradient gels (Invitrogen), with a 3-
(N-morpholino)propanesulfonic acid (MOPS) buffer sys-
tem. Samples were prepared and run under reducing or
nonreducing conditions, according to the manufacturer’s
protocol and stained with Coomassie blue.
For western blot analysis, proteins were transferred from
SDS–PAGE gels onto Immobilon P, polyvinylidene fluor-
ide (PVDF) membrane (Millipore) using a semi-dry blot-
ting apparatus (Sigma). Blots were probed with
appropriately diluted primary antisera in TNTT and sheep
immunoglobulin was subsequently detected with monoclo-
nal anti-sheep ⁄ goat IgG-HRPO conjugate (Sigma). HRPO
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Volume 32, Number 6, June 2010 To immunize sheep against H. contortus using a recombinant cocktail

activity was revealed with 3,3¢-diaminobenzidine (Sigma-
Fast DAB tablet set; Sigma) as substrate.
Statistical analysis
Arithmetic means are shown throughout with their stan-
dard errors. Statistical differences were calculated by ANO-
VA on untransformed data.
RESULTS
was expressed in E. coli, purified from inclusion bodies
and refolded by base extraction. Its apparent molecular
weight was around 40 kDa, in agreement with its
previously observed molecular weight (4). None of the
recombinants were enzymatically active when compared
with native H-gal-GP, but they all reacted with sheep anti-
serum to H-gal-GP. When native H-gal-GP was probed in
an immunoblot, with antiserum raised in sheep immunized
with the recombinant cocktail, all the major protein bands
were recognized by that antiserum (Figure 1).

SDS–PAGE analysis of the antigen preparations

V1 V2 V3 Ch
The protein profile of native H-gal-GP closely resembled 1·1

Mean OD 490 ± SE
that of previously published preparations (6) and is shown
0·9
with the various recombinant antigen preparations in
Figure 1. Under nonreducing conditions, H-Gal-GP is 0·7
separated into different protein zones. The zone around 0·5
230 kDa includes MEP1, MEP2 and MEP4. MEP3
0·3
appears as a dimer around 190 kDa and the zone around
40 kDa corresponds to the pepsins PEP1 and PEP2. 0·1
Recombinant MEPs were expressed as soluble proteins
–0·1
in insect cells and purified by chromatography. Their 0 1 2 3 4 5 6 7 8 9 10
apparent molecular weights are around 92, 110 and Weeks
145 kDa for rMEP1, rMEP3 and rMEP4, respectively.
H-gal-GP Recombinant Adjuvant only
Their predicted molecular weights, however, are 85Æ6, 87Æ9
Figure 2 Serum anti-H-gal-GP antibody responses. Serum
and 103Æ3 kDa (5). A likely explanation for these discrep-
anti-H-gal-GP ELISA responses (group mean OD € SE) from
ancies is the presence of potential N-linked glycosylation sheep immunized with H-gal-GP ( ), recombinant cocktail (¤) or
sites (and O-linked glycosylation sites, in the case of adjuvant only (d). The times of the three immunizations and
MEP4) along the protein backbones. Recombinant PEP1 subsequent challenge are indicated by arrows.

(a) (b) (c)

M 1 2 3 4 1 2 3 4 M 1 2 3 4 5 6 M
kDa kDa
250 -
130 -
250 -
95 -
130 -
95 - 72 -
72 -
55 - 55 -
43 -
36 - 34 -
28 -
26 -
17 - 17 -

Figure 1 SDS–PAGE and immunoblot profiles of the recombinant cocktail components and native H-gal-GP. (Panel A) Coomassie blue
stained SDS–PAGE gel profiles of recombinant proteins; M = Molecular weight standards; Lanes 1 to 3 – rMEP1, rMEP3 and rMEP4,
respectively, under nonreducing conditions; Lane 4 – rPEP1 under reducing conditions. (Panel B) Immunoblot profiles of the same
recombinant proteins probed with sheep anti-H-gal-GP; Lanes 1 to 3 – rMEP1, rMEP3 and rMEP4, respectively; Lane 4 – rPEP1. (Panel
C) Native H-gal-GP under reducing or nonreducing conditions; M = Molecular weight standards; Lanes 1 and 2 – Coomassie blue stained
SDS–PAGE gel of H-gal-GP run under reducing or nonreducing conditions, respectively; Lanes 3 and 5 – immunoblot of H-gal-GP under
reducing conditions, probed with sheep anti-H-gal-GP or sheep anti-recombinant cocktail, respectively; Lanes 4 and 6 – immunoblot of
H-gal-GP under nonreducing conditions, probed with sheep anti-H-gal-GP or sheep anti-recombinant cocktail, respectively.

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E. Cachat et al.
Serology
Group mean serum anti-H-gal-GP antibody responses are
illustrated in Figure 2. OD values close to zero were
detected in all sheep at the start of the trial and remained
low in the adjuvant-only control group, except on Weeks 7
and 8 when values reached about 0Æ1.
By the time of the second immunization, the sheep
immunized with H-gal-GP showed a rise in OD, which
increased sharply by Week 4, 7 days after the first vaccine
boost. The mean OD values of this antigen group
remained above 0Æ9 from Week 5 to the end of the trial.
During the first 4 weeks of the trial, the responses of
the sheep immunized with the recombinant cocktail or the
native H-gal-GP antigen were very similar. The mean OD
values of the recombinant group remained around 0Æ7
from Week 4 to Week 6, when the second vaccine boost
was administrated, causing a slight rise to 0Æ8 a week after
the boost. By Week 10, the mean OD fell back to 0Æ7 and
then dropped to 0Æ5 by the end of the trial.
Faecal egg counts and worm burdens
From Day 21, mean worm egg counts were substantially
reduced in the group immunized with native H-gal-GP
compared with the controls which received adjuvant only
(Figure 3). No significant difference between the counts of
the recombinant and control groups was detected.
Over the course of the trial, the group immunized with
H-gal-GP shed 88Æ5% fewer eggs than the controls
(P < 0Æ01), but the group immunized with the recombinant
proteins shed only 2Æ5% fewer, a difference which was not
statistically significant. Fewer worms were recovered from
5000
4000
Mean epg (±SE)
3000
2000
1000
0 since earlier trials suggested that the combination of metal-
15 20 25 30 35
Days after challenge
H-gal-GP Recombinant Adjuvant only
Figure 3 Faecal egg counts of immunized and control sheep after
challenge. Group mean € SE faecal egg counts (eggs per g),
from sheep immunized with H-gal-GP ( ), recombinant cocktail
(¤) or adjuvant only (d) and subsequently challenged with 5000
Haemonchus contortus larvae.
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Parasite Immunology
the group immunized with H-gal-GP (779 € 200) com-
pared with the controls (2813 € 238; P < 0Æ001) or the
recombinant group (2784 € 370; P < 0Æ001), but there was
no significant difference between the worm burdens of the
control and recombinant groups.
DISCUSSION
Previous work showed that bacterially expressed insoluble
recombinant versions of H. contortus MEP1 and MEP3
were completely ineffective against a H. contortus chal-
lenge (3). Interestingly although, Williamson et al. (9)
showed that Ac-MEP-1, a homologue from Ancylostoma
caninum, could be expressed as a soluble and enzymati-
cally active protein in baculovirus-infected insect cells, sug-
gesting that the resulting molecule was close to its native
conformation. This finding indicated that proteases from
H-gal-GP, expressed through eukaryotic systems, might
confer better protection against H. contortus. As a result,
the extracellular domains of MEP1, MEP3 and MEP4
were expressed in the insect cell-baculovirus system and
were included in the recombinant antigen cocktail. Impor-
tantly, although the MEPs were expressed as soluble rec-
ombinants, they were not enzymatically active.
MEP2 was not included in the recombinant cocktail, as
attempts to express it in insect cells were not successful.
Besides, even though it is 55% identical to MEP1 (3),
MEP2 might not be functionally active as it does not pos-
sess a complete zinc binding domain (5).
Neither denatured native PEP1 nor insoluble E.
coli-expressed PEP1 conferred any protection when tested
earlier in sheep vaccine trials (4). However, a PEP1 homo-
logue from Necator americanus (Na-APR-2) has been
expressed as a soluble protein in insect cells (10) and Ac-
APR-1, an aspartic protease from Ancylostoma caninum,
was expressed in yeast as a soluble, catalytically active and
protective recombinant (11). We have attempted to repeat
these experiments with PEP1 and PEP2, but despite using
permutations of different constructs (prepro-, pro-enzyme,
codon-optimized and different secretion signals) and show-
ing that they were consistently expressed, neither was ever
secreted into either the yeast or insect cell culture medium
as a soluble recombinant (data not shown). Nevertheless,
lo- and aspartyl protease fractions was more protective
than either alone (3), it was decided to include a recombi-
nant pepsin in the vaccine cocktail, even though it had to
be solubilized ⁄ refolded from inclusion bodies. PEP1 was
chosen as it is fivefold more abundant than PEP2 (4).
Compared with the adjuvant-only controls, both the
native antigen and recombinant cocktail-immunized
groups showed strong circulating antibody responses, as
 2010 Moredun Research Institute
Volume 32, Number 6, June 2010
measured by both ELISA and immunoblot, following the
second and third vaccinations and these were near their
peak at the time of challenge. Despite this, however, only
the group immunized with native antigen was protected.
As mentioned earlier, failures of insoluble recombinant
versions of individual H-gal-GP proteases to induce
protective immunity have also been reported (3,4). In fact,
inducing reliable protective immunity with any recombi-
nant antigen from H. contortus has so far proved elusive,
a subject of considerable debate (12,13).
In the case of H-gal-GP, the failure of a recombinant
cocktail of most of its protective components to confer
protection may be explained from its structural form,
which has recently been elucidated by electron microscopy.
Further details of the shape of the complex will be pub-
lished elsewhere, but intriguingly it is about the size of the
smallest of viruses and contains an internal chamber with
three openings (14). This complex quaternary structure
must have many conformational epitopes which are not
present in a mixture of its individual components. Presum-
ably, some or many of these are vital for protective immu-
nity. If so, it will be extremely tricky, using current
recombinant DNA technologies, to co-express the various
components of H-gal-GP for them to assemble correctly
into the quaternary structure of the complex.
One possible way in which this problem may be
addressed is to identify peptides that mimic protective
conformational epitopes in a phage display library. The
antisera produced in this study may prove to be very use-
ful in differentiating between epitopes that represent
potentially protective native structures and those that rep-
resent structures found in the nonprotective recombinant
protein fraction.
ACKNOWLEDGEMENTS
The authors would like to thank Stephen Smith for techni-
cal assistance and Philip Skuce for his preliminary work
on recombinant expression. This work was funded by Pfiz-
er Animal Health and by the Scottish Government Rural
and Environment Research and Analysis Directorate.
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