Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2182178
S WO 95/26402
PCT/GB95/00665
Vaccines against=helminthic parasites
This invention relates to novel helminth antigens
and their use in the control of disease caused by
helminth parasites, particularly parasitic nematodes of
the gastro-intestinal tract of mammals.
Helminth parasites, particularly nematodes, infect
or infest a wide range of animals, including man, and
are a widespread and significant source of disease and
ill-thrift, not only in animals, but also in man. Such
parasites thus represent a considerable worldwide drain
on economic resourdes. This is particularly true in
animal husbandry, where parasite infections of grazing
animals, such as sheep and cattle, are often difficult
and expensive to control and may result in significant
economic losses.
Particular mention may be made in this regard of
the blood-sucking nematode Eiemonrhus contortus, a
parasite of ruminants, most notably sheep. E. rontorWIR
(or other Baemonchns species including B. plarei) also
parasitises cattle. Infection with aftemounhuR leads to
a condition known as haemonchosis, which is frequently
fatal if untreated and represents one of the major
helminth infections causing problems in animal husbandry
today.
Also worthy of particular mention from the economic
viewpoint are the non-blood feeding nematodes OstertagiR
ostertagi and OstPrtagia, (9'elednrsRgis) fircrmrinrtR (S).
pirrmitrinrtµ has recently been reclassified as
T.rircnmcinnta, although the new name is not yet in wide
= usage).
Other parasitic helminths of economic importance
= include the various species of the following helminth
faualies:- Trirhostronculus., Pemarodirus, Pict-yocaulus,
conperia, Ascaris, nirnfilPri, Trichuris., Zrronsylus,
Fascio1R, OpsophagostomuM, Bunostomum Metastrongylus,
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WO 95/26402 2 I 8 2 1 78 PCT/GB95/00665
11,
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Neca r or , Ancylostoma, and schistosomes.
At present, control of helminth parasites of
grazing livestock relies primarily on the use of
anthelmintic drugs, combined with pasture management.
Such techniques have not proved entirely satisfactory
however, due to their expense and inconvenience and to a
rapid increase in drug resistance. Antihelmintic drugs
need to be administered frequently and appropriate
pasture management is often not possible on some farms
and even where it is, it can place constraints on the
best use of available grazing.
To overcome these problems, attempts have been made
to achieve immunological means of control. Although
there has been some success in identifying certain
protective antigens as potential vaccine candidates,
most notably in gaemonchuq, this approach has proved
difficult and, other than for the cattle lungworm
pintyocaulus rixiiiaras., has yet to come to commercial
fruition.
EP-A-0434909 describes a 35kd cysteine proteinase
which forms part of a high molecular weight
fibrinogenolytic protein complex present in glycerol
extracts of Eaemonchus contortus. Although proposed as
a potential vaccine candidate, protective immune
responses elicited by this antigen can be seen to be
inconsistent and statistically insignificant.
The most success to date has been achieved with the
protein doublet H110D, an integral membrane protein
isolated from the gut of R.contortus and described by
Munn in W088/00835. H110D now represents the most
promising vaccine candidate to date.
Munn has also described and proposed as a vaccine,
contortin, a helical polymeric extracellular protein
associated with the luminal surface of F contort-31F
intestinal cells (Munn Pr a1., Parasitology 24.: 385-397,
1987).
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A further Haemonchus gut membrane protein with
protective antigenic properties has also been discovered
and termed H45 (Munn and Smith, W090/11086).
W094/02169 describes, inter alia, the antigen termed
H-gal-GP, also an integral gut membrane protein of
Haemonchus. This antigen is a galactose-containing
glycoprotein complex and has been shown to confer
protective immunity in animals against Haemonchus.
As far as other helminth genera, particularly non-
blood feeding helminth species, are concerned, there are
fewer reports in the literature of successful
immunisation. In the case of 0.circumcincta, McGillivery
et al. have reported the identification of a 31kd
protective antigen, isolated from 3rd stage larvae using
sera from sheep which developed immunity after infection
(McGillivery et al., 1990, Int. J. Parasitol., 20: 87-93,
and W091/01150). However, only partial protection of
sheep against challenge infection could be demonstrated.
Whilst proteins such as H110D, H45 and H-gal-GP can
be used as the basis for a vaccine against Haemonchus,
there is nonetheless a continuing need for new and
improved helminth parasite vaccines and in particular for
a vaccine which may be used across a broad range of
helminth genera. There is especially a need for a
vaccine which may be used against non-blood feeding
helminths such as Ostertagia.
In accordance with one aspect of the present
invention there is provided a protective helminth
parasite antigen obtainable from adult helminths of the
genera Haemonchus or Ostertagia and characterised by:
(i) in native form being an integral membrane protein;
(ii) having a native localisation in the parasite gut;
(iii) being capable of binding to a thiol affinity
medium; (iv) being recognised by sera from animal hosts
immunized with thiol SepharoseTM binding proteins of an
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adult helminth parasite, containing antibodies capable of
one or both of inhibiting parasite growth or development;
and (v) being encoded by a nucleic acid molecule which
(a) encodes a protein which has at least 70% identity to
a protein encoded by the nucleotide sequence shown in any
one of Figures 16 to 21 or a complementary sequence
thereof, or (b) which hybridises under non-stringent
binding conditions (6 x SSC / 50% formamide at room
temperature) and washing under conditions of higher
stringency of 2 x SSC and 65 C with a complement of the
nucleotide sequence shown in any one of Figures 16 to 21;
or a variant thereof which is characterised by features
(iii) and (iv) above, is capable of raising host
protective antibodies, and is encoded by a nucleic acid
molecule which encodes a protein which has at least 70%
identity to a protein encoded by the nucleotide sequence
shown in any one of Figures 16 to 21 or a complementary
sequence thereof; or a precursor thereof which contains
said antigen.
In accordance with another aspect of the present
invention there is provided a protective helminth
parasite antigen that is a protease obtainable from adult
helminths of the genera Haemonchus or Ostertagia by
subjecting a crude extract of a helminth parasite to
extraction using a non-ionic detergent capable of
extracting integral membrane proteins followed by
chromatography on a thiol affinity medium and elution of
bound antigens, and characterised by: (i) in native form
being an integral membrane protein; (ii) having a native
localisation in the parasite gut; (iii) being capable of
binding to a thiol affinity medium; and (iv) being
recognised by sera from animal hosts immunised with thiol
Sepharose binding proteins of an adult helminth parasite,
containing antibodies capable of one or both of
inhibiting parasite growth or development.
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The present invention accordingly seeks to provide
novel antigens for use as helminth parasite vaccines and
in particular as protective immunogens in the control of
diseases caused by helminth parasites.
More specifically, the present invention is based on
the finding that extracts of helminth parasites
containing integral membrane proteins having thiol-
binding activity are capable of conferring protective
immunity against the parasites in animals. Such
proteins, when liberated from the membranes in which
ak 02382178 2006-12-12
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they are bound, for example by the use of detergents,
are novel and of use in the manufacture of vaccines
against helminth infections.
According to one aspect, the present invention thus
provides a protective helminth parasite antigen
obtainable from adult helminths by chromatography of a
Triton X_100TM extract of whole parasites on a thiol
affinity medium, and characterised by
(i) in native form being an integral membrane
protein;
(ii) having a native localisation in the parasite
gut;
(iii) being capable of binding to a thiol affinity
medium; and
(iv) being recognised by sera from immunised animal
hosts, containing antibodies capable of
inhibiting parasite growth and/or development,
or a functionally-equivalent variant, or antigenic
fragment or precursor thereof.
A further aspect of the invention provides such
protective antigens, and functionally-equivalent
variants, antigenic fragments or precursors thereof, for
use in stimulating an immune response against helminth
parasites in a human or non-human, preferably mammalian,
especially preferably ruminant, animal.
A precursor for the antigen in question may be a
larger protein which is processed, eg. by proteolysis,
to yield the antigen per se. Such precursors may take
the form of zymogens ie. inactive precursors of enzymes,
activated by proteolytic cleavage, for example analogous
to the pepsin/pepsinogen system or the well known
zymogens involved in the blood clotting cascade.
The novel antigens of the invention are not
recognised by sera from naturally immune animals. In
other words, they are not normally, in native form,
accessible to the immune system of the infected host and
are thus "hidden", "concealed" or "cryptic" antigens.
2 1 8 2173
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PCT/GB95/00665
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The term "protective antigens" or "protective
antigenic activity" as used herein defines those
antigens and their fragments or precursors, capable of
generating a host-protective, ie. immunogenic, immune
response, that is a response by the host which leads to
generation of immune effector molecules, antibodies or
cells which damage, inhibit or kill the parasite and
thereby "protect" the host from clinical or sub-clinical
disease and loss of productivity. Such a protective
immune response may commonly be manifested by the
generation of antibodies which are able to inhibit the
metabolic function of the parasite, leading to stunting,
lack of egg production and/or death.
As mentioned above, included within the scope of
the invention are functionally-equivalent variants of
the novel antigens and their fragments and precursors.
"Functionally-equivalent" is used herein to define
proteins related to or derived from the native protein,
where the amino acid sequence has been modified by
single or multiple amino acid substitution, addition
and/or deletion and also sequences where the amino acids
have been chemically modified, including by
deglycosylation or glycosylation, but which nonetheless
retain protective antigenic activity eg. are capable of
raising host protective antibodies and/or functional
immunity against the parasites. Within the meaning of
"addition" variants are included amino and/or carboxy
terminal fusion proteins or polypeptides, comprising an
additional protein or polypeptide fused to the antigen
sequence. Such functionally-equivalent variants
mentioned above include natural biological variations
= (eg. allelic variants or geographical variations within
a species) and derivatives prepared using known
techniques. For example, functionally-equivalent
=
proteins may be prepared either by chemical peptide
synthesis or in recombinant form using the known
techniques of site-directed mutagenesis including
WO 95/26402 21 8 2 1 7 8 PCT/GB95/00665
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deletion, random mutagenesis, or enzymatic cleavage
and/or ligation of nucleic acids. Functionally-
equivalent variants according to the invention also
include analogues in different parasite genera or
species.
It has been shown that there is no immunological
cross-reactivity between antigens of the invention and
proteins contained in glycerol or other water-soluble
extracts of helminth parasites. More particularly, it
has been shown that proteins present in glycerol
extracts of BasTrirorrus prepared according to EP-A-
0434909 do not cross-react with sera raised against, and
reactive with, antigens according to the present
invention. However, there is immunological cross-
reactivity between antigens of the invention of
different parasite origin.
Polyacrylamide gel electrophoresis (PAGE) studies
carried out on the antigens of the invention have shown
differing behaviour on reducing and non-reducing gels.
In particular, under reducing conditions, the antigens
resolve into a more complex set of bands. Similarities
in the band profiles under different conditions are
however apparent between antigens of different parasite
origin. 'Thus for example broad similarities may be
observed between antigens of B.contortus and OstertagiP
rinmimcincta
In the case of the nematode worm B.contortus.,
preferred antigens of the invention have the following
molecular weights as determined by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) under reducing and non-
reducing conditions:
(a) about 60kd, 97-120kd and 205kd or greater (10t gel,
non-reducing); and
=
(b) about 37-38, 51, 52, 56, 62, 70, 77, 100, 120, 160
and 175-180kd (10% gel, reducing).
2182178
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PCT/GB95/00665
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Preferred antigens of the nematode worm
0.circumninrft have the following molecular weights as
determined by SDS-PAGE under reducing and non-reducing
conditions:
(a) about 45 and 60kd (10% gel, non-reducing); and
(b) about 29, 37, 51, 52, 56, 66, 70, 77, 100, 120,
175kd (10% gel, reducing).
Substrate gel analysis, described in more detail
below, shows the following molecular weights (7.5%
gelatin substrate gel, non-reducing):
= E. rontortuF
(a) about 37-38, 52, 70 and 100kd (pH 5);
(b) about 77 and 88kd (pH 8.5).
0. circumcincta
(a) about 40, 50, 70 and >300kd (pH 5);
(b) about 70, 85 and 97kd (pH 8.5).
Lectin binding studies have shown that some, but
not all, of the antigens of the invention may be
glycosylated. In particular, for antigens from Es
pontortuR binding of concanavalin A, wheatgerm, Helix
pomatia, jacalin and peanut lectins has been observed,
but not with Dolichos bifluorous agglutinin and soybean
lectins.
Of the preferred F rontorrns, antigens mentioned
above, those having molecular weights of 52, 56, 62, 77,
120 and 175kd (determined by SDS-PAGE on a 10% gel under
reducing conditions) bound to lectin, indicating the
= presence of glycosylated structures.
In the case of 0.circumcinrta, lesser degrees of
= glycosylation are observed, with only concanavalin A
binding to a group of antigens at 55-70 kd. Thus, the
pattern of glycosylation may vary between antigens of
different parasite origin.
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A preferred feature of antigens according to the
invention is proteolytic activity. This may be
demonstrated using general proteinase substrates such as
gelatin and azocasein. However, more particular
proteinase activities may also be observed, most notably
cysteine proteinase-like, serine protease-like and
metalloproteinase-like activities. Such activities may
be demonstrated both by spectrophotometric assay of
thiol-binding detergent extracts containing antigens of
the invention and by substrate gel analysis. As will be
described in more detail below, in the case of the
latter, broadly similar activity profiles may be
observed between antigens of different parasite origin.
In contrast, antigens according to the invention show no
aminopeptidase, aspartate proteinase or neutral
endopeptidase activity.
Whilst not wishing to be bound by theory, it is
believed that blockage of enzymic activity of such
antigens by antibodies may contribute to the protective
antigenic response.
As mentioned above, and as will be described in
more detail below, antigens of the invention may be
prepared by extracting whole adult worms with a
detergent capable of extracting integral membrane
proteins, and subjecting the detergent extracts to
chromatography on .a thiol affinity medium e.g. thiol
SepharoseTM.
Extractability with strong detergents, particularly
non-ionic detergents such as Triton, indicates the
integral membrane nature of the antigens. Retention of
the antigens on thiol affinity medium indicates that the
antigens have thiol binding character ie. they are
integral membrane thiol binding proteins.
Thus, the invention also provides a process for the
preparation of the above-mentioned antigens of the
invention which comprises the steps of subjecting a
crude extract of a helminth parasite, preferably
2 1 8 2 1 7 8
=
W095/26402 PCT/GB95/00665
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Baemonchus contortus or Ostertagia, sp., to detergent
extraction using a detergent capable of solubilising
integral membrane proteins, followed by chromatography
of the detergent extract on a thiol affinity medium and
elution of the bound antigens.
The crude extract of the helminth parasite may be
prepared using conventional biochemical and surgical
techniques eg. by homogenisation of the whole or a
portion of the parasite. Thus, for example the
parasites may be subjected to homogenisation in a
suitable buffer or medium such as phosphate buffered
saline (PBS) and the insoluble material (ie. the pellet)
may be recovered by centrifugation, whereby to fonu the
required crude extract. If desired, a preliminary
detergent extraction step, employing a gentle detergent
such as Tween (which removes membrane-associated
proteins but will not solubilise integral membrane
proteins) may also be used in preparing the crude
extract to remove contaminating membrane associated
proteins.
Thiol affinity chromatography techniques are well
known in the art and are defined herein to include all
forms of chromatography using media having affinity for
free thiol groups. Such techniques includes for
example, in addition to the "classical" thiol affinity
media, also covalent chromatography eg. metal chelate
chromatography. A range of thiol affinity media are
available (for example from Pharmacia or Sigma).
Mention may be made of thiol Sepharose and thiolpropyl
Sepharose.
Protection trials using antigens prepared by Triton
extraction and Thiol Sepharose chromatography have shown
that immunity may be induced in animals against
= challenge by the helminth parasites.
Viewed from a different aspect, the invention can
also be seen to provide use of a helminth parasite
antigen as hereinbefore defined, and fragments,
2 1 8 2 1 7 8
WO 95/26402 PCT/GB95/00665 =
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precursors and functionally-equivalent variants thereof,
for the preparation of a vaccine composition for use in
stimulating an immune response against helminth
parasites in a human or non-human, animal.
The invention also provides a vaccine composition
for stimulating an immune response against helminth
parasites in a human or non-human animal comprising one
or more antigens, antigenic fragments, precursors or
functionally-equivalent variants thereof, as defined
above, together with a pharmaceutically acceptable
carrier or diluent, and a method of stimulating an
immune response against helminth parasites in a human or
non-human animal, comprising administering to said
animal a vaccine composition as defined above.
The animal preferably is mammalian and more
preferably a ruminant. Especially preferred animals are
sheep, cattle and goats.
Antigens according to the invention may be obtained
from a range of helminth parasite genera. Preferably,
however the helminths will be nematodes, especially
preferably gastro-intestinal nematodes including for
example waemonChua and Ostertagia sp. (For the
avoidance of doubt, the term "Ostert gia," as used herein
includes leladorsagia sp.). Such antigens may be used
to prepare vaccines against a range of helminth
parasites including any of those mentioned above.
Preferred are those antigens, so called "broad spectrum"
antigens, which are capable of stimulating host
protective immune responses against, in addition to the
parasite from which they were isolated, a broad range of
other parasites.
As mentioned above, one of the ways in which the
=
antigens of the invention may exert their host
protective effects is by raising inhibitory antibodies
which inhibit the growth, maintenance and/or development
of the parasite. Such antibodies and their antigen-
binding fragments (eg. F(ab)2, Fab and Fv fragments ie.
2 1 8 2 1 76
=
W095/26402 PCT/GB95/00665
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fragments of the "variable" region of the antibody,
which comprises the antigen binding site) which may be
mono- or polyclonal, form a further aspect of the
invention, as do vaccine compositions containing them
and their use in the preparation of vaccine compositions
for use in immunising hosts against parasites. Such
inhibitory antibodies may be raised hy use of idiotypic
antibodies. Anti-idiotypic antibodies may be used as
immunogens in vaccines.
In addition to the extraction and isolation
techniques mentioned above, the antigens may be prepared
by recombinant DNA technology using standard techniques,
such as those described for example by Sambrook et al.,
1989, (Molecular Cloning, a laboratory manual 2nd
Edition, Cold Spring Harbor Press).
Nucleic acid molecules comprising a nucleotide
sequence encoding the antigens of the invention thus
form further aspects of the invention.
Nucleic acid molecules according to the invention
may be single or double stranded DNA, cDNA or RNA,
preferably DNA, and include degenerate, substantially
homologous and hybridising sequences which are capable
of coding for the antigen or antigen fragment or
precursor concerned. By "substantially homologous" is
meant sequences displaying at least 601, preferably at
least 70% or 801 sequence homology. Hybridising
sequences included within the scope of the invention are
those binding under non-stringent conditions (6 x
SSC/50W formamide at room temperature) and washed under
conditions of low stringency (2 x SSC, roam temperature,
more preferably 2 x SCC, 42 C) or conditions of higher
= stringency eg. 2 x SSC, 65 C (where SSC = 0.15M NaCl,
0.015M sodium citrate, pH 7.2), as well as those which,
= but for the degeneracy of the code, would hybridise
under the above-mentioned conditions.
Derivatives of nucleotide sequences capable of
encoding antigenically active antigens or antigen
¨ - ¨
213218
WO 95/26402 7 PCT/GB95/00665 =
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variants according to the invention may be obtained by
using conventional methods well known in the art.
cDNA fragments encoding cysteine proteinases have
been obtained from T-1,2211torrus. by PCR amplification (as
described in more detail below) and have been sequenced.
The nucleotide sequences, and corresponding predicted
amino acid sequences of such fragments, identified as
DM.1, DM.2, DM.3, DM.4, DM.4a and 1DM.5, are shown in
Figures 16 to 21 respectively. These sequences, and
their degenerate and allelic variants, and fragments
thereof, form a further aspect of the invention.
Antigens according to the invention may be prepared
in recombinant form by expression in a host cell
containing a recombinant DNA molecule which comprises a
nucleotide sequence as broadly defined above,
operatively linked to an expression control sequence, or
a recombinant DNA cloning vehicle or vector containing
such a recombinant DNA molecule. Alternatively the
polypeptides may be expressed by direct injection of a
naked DNA molecule into the host cell. Synthetic
polypeptides expressed in this manner form a further
aspect of this invention (the term "polypeptide" is used
herein to include both full-length protein and shorter
length peptide sequences).
The antigen so expressed may be a fusion
polypeptide comprising all or a portion of an antigen
according to the invention and an additional polypeptide
coded for by the DNA of the recombinant molecule fused
thereto. This may for example be p-galactosidase,
glutathione-S-transferase, hepatitis core antigen or any
of the other polypeptides commonly employed in fusion
= proteins in the art. Such fusion proteins may also
comprise the antigen in a form, eg. a pro-enzyme, which
= may be secreted ie. the antigen may be expressed
together with signal and secretion-directing sequences.
Other aspects of the invention thus include cloning
and expression vectors containing the DNA coding for an
2 1 8 2 1 7
=
W095/26402 PCT/GB95/00665
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antigen of the invention and methods for preparing
recombinant nucleic acid molecules according to the
invention, comprising inserting nucleotide sequences
encoding the antigen into vector nucleic acid, eg.
vector DNA. Such expression vectors include appropriate
control sequences such as for example translational (eg.
start and stop codons, ribosomal binding sites) and
transcriptional control elements (eg. promoter-operator
regions, termination stop sequences) linked in matching
reading frame with the nucleic acid molecules of the
invention. Optional further components of such vectors
include secretion signalling and processing sequences.
Vectors according to the invention may include
plasmids and viruses (including both bacteriophage and
eukaryotic viruses) according to techniques well known
and documented in the art, and may be expressed in a
variety of different expression systems, also well known
and documented in the art. Suitable viral vectors
include baculovirus and also adenovirus, herpes and
vaccinia/pox viruses, preferably non-permissive pox
viruses. Many other viral vectors are described in the
art.
= A variety of techniques are known and may be used
to introduce such vectors into prokaryotic or eukaryotic
cells for expression, or into germ line or somatic cells
to form transgenic animals. Suitable transformation or
transfection techniques are well described in the
literature.
The invention also includes transformed or
transfected prokaryotic or eukaryotic host cells, or
transgenic organisms containing a nucleic acid molecule
according to the invention as defined above. Such host
cells may for example include prokaryotic cells such as
F rnli, eukaryotic cells such as yeasts or the
baculovirus-insect cell system, transformed mammalian
cells and transgenic animals and plants. Particular
mention may be made of transgenic nematodes (see for
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example Fire, 1986, EMBO J., a. 2673 for a discussion of
a transgenic system for the nematode Caenorhabditis).
A further aspect of the invention provides a method
for preparing an antigen of the invention as
hereinbefore defined, which comprises culturing a host
cell containing a nucleic acid molecule encoding all or
a portion of said antigen or precursOr thereof, under
conditions whereby said antigen is expressed and
recovering said antigen thus produced.
The antigens of the invention and functionally
equivalent antigen variants may also be prepared by
chemical means, such as the well known Merrifield solid
phase synthesis procedure.
Water soluble derivatives of the novel antigens
discussed above form a further aspect of the invention.
Such soluble forms may be obtained, for example, by
proteolytic digestion.
A vaccine composition may be prepared according to
the invention by methods well known in the art of
vaccine manufacture. Traditional vaccine formulations
may comprise one or more antigens or antibodies
according to the invention together, where appropriate,
with one or more suitable adjuvants eg. aluminium
hydroxide, saponin, quil A, or more purified forms
thereof, muramyl dipeptide, mineral or vegetable oils,
Novasomes or non-ionic block co-polymers or DEAF
dextran, in the presence of one or more pharmaceutically
acceptable carriers or diluents. Suitable carriers
include liquid media such as saline solution appropriate
for use as vehicles to introduce the peptides or
polypeptides into an animal or patient. Additional
components such as preservatives may be included.
An alternative vaccine formulation may comprise a
virus or host cell eg. a microorganism (eg. vaccinia or
pox virus, adenovirus or Salmonella) which may be live,
killed or attenuated, having inserted therein a nucleic
acid molecule (eg. a DNA molecule) according to this
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invention for stimulation of an immune response directed
against polypeptides encoded by the inserted nucleic
acid molecule.
Vaccination may also take place by direct injection
of a naked DNA molecule according to the invention, for
in situ expression of the antigens.
Administration of the vaccine composition may take
place by any of the conventional routes, eg. orally or
parenterally such as by intramuscular injection,
optionally at intervals eg. two injections at a 7-35 day
interval.
The antigens may be used according to the invention
in combination with other protective .antigens obtained
from the same or different parasite species. Thus a
vaccine composition according to the invention may
comprise one or more of the antigens defined above
together with the antigens H11 D and H45 mentioned
above. Such a combined vaccine composition may contain
smaller amounts of the various antigens than an
individual vaccine preparation, containing just the
antigen in question. Combined vaccines are beneficial
where there is a likelihood that "adaptive selection" of
the parasite may occur when a single antigen vaccine is
used.
The invention will now be described in more
detail with particular reference to the isolation of
thiol binding proteins from E.ermtnrrus. and
0.circumcincta. In the following non-limiting Examples,
the drawings represent:
Figure 1 shows molecular weights of Thiol-Sepharose
binding integral membrane proteins (TSBP) from adult 11,_
ponrortus
TSBP were fractionated in 1()% polyacrylamide gel
slabs under non-reducing (Lane 2, Figure la) and
reducing conditions (Lane 2, Figure lb). Molecular
weight markers are shown in Lane 1, Figure la & b;
Figure 2 shows gelatin-substrate gel analysis of
CA 02182178 2006-12-12
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proteinases in TSBP from adult H. contortus.
TSBP, in the absence or presence of specific
proteinase inhibitors, were fractionated using 7.51;
gelatin-substrate gel analysis and zones of proteolysis
visualised as described in Example 2. The pH refers to
the incubation buffer and Lane 1, pH 5 or pH 8.5 is a
control, Lane 2 pH 5, Lane 2 and 3 - pH 8.5 were
incubated in the presence of E64 (100 M), PMSF (1.0mM) or
EDTA (1.0mM) respectively prior to CoomassieTM staining;
Figure 3 shows reactivity of biotinylated lectins
with TSBP from H. contortus.
TSBP were fractionated by 10% reducing SDS-PAGE and
blot transferred to Immobilon-P. The blot was cut into
strips and blot strips probed with 1) Dolichos bifluorus
agglutinin, 2) Soybean, 3) Wheatgerm, 4) Helix Pomatia,
5) Concanavalin A, 6) Jacalin and 7) Peanut biotinylated
lectins;
Figure 4 shows cryostat sections of adult
contortus probed with serum from sheep immunised with
TSBP.
Sections were incubated with sera from TSBP
immunised lambs (A) and sera from control lambs
immunised with adjuvant alone (B);
Figure 5 shows circulating antibody responses in
lambs immunised with TSBP.
TSBP were fractionated by 10%. reducing SDS-PAGE and
blot transferred to Immobilon-PTM. The blot was cut into
strips and half (Lanes 1 to 8) were treated with
periodate and the remainder used untreated (Lanes 9 to
16). Blot strips were probed with sera from lambs
immunised with TSBP prior to Baemonchus challenge or
with sera from challenge controls (Lanes 7, 8, 15 & 16);
Figure 6 shows cryostat sections of adult B. ,
contortus probed with fluorescein isothiocyanate
conjugated anti-ovine immunoglobulin.
Worms in panel A were retrieved from lambs which
had been immunised with TSBP prior to Baemonchus
CA 02182178 2006-12-12
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challenge. Worms in panel B were from challenge
controls;
Figure 7 shows Faecal Egg Output from lambs
immunised with TSBP (-v-), with TSBP from a saline/TweenTm
extract (-s-), or with challenge controls (-EH
following challenge with 5000 E. contortus infective L3;
Figure 8 shows reactivity of anti-TSBP antibody
with adult F. contortus antigen extracted in an
aqueous/glycerol buffer.
Western blot strips of TSBP (Lane 1), or an 11,õ_
contortus extract prepared as described by Cox et al,
1991 (Lane 2) were probed with anti-TSBP antiserum.
Figure 9 shows gelatin-substrate gel analysis of
TSBP proteinases which were either unbound (Lane 2) or
bound to Mono Q and eluted with 100mM (Lane 3), 200mM
(lanes 4 & 5), 300mM (Lane 6) or 1M (Lane 7) NaCl. Lane
6 shows the starting TSBP profile and panel A was
incubated at pH 5, panel B at pH 8.5.
Figure 10 shows molecular weights of Thiol-
Sepharose binding integral membrane proteins from adult
Ostertagia circumcincta.
TSBP were fractionated in 10t SDS-polyacrylamide
gel slabs under non-reducing (Figure 9A) and reducing
(Figure 9B) conditions. In Figures 9A & B, Lanes 1 and
2 are TSBP extracted from adult 0.circumcincta and
F. contortus respectively;
Figure 11 shows gelatin-substrate gel analysis of
proteinases in Thiol-Sepharose binding integral membrane
proteins from adult Ostertagia circumcincta.
TSBP, in the absence or presence of class-specific
proteinase inhibitors, were fractionated in gelatin-
substrate gels and, after extensive washing and
overnight incubation in buffer of appropriate pH, zones
of proteolysis visualised by Coomassie blue
counterstaining;
Figure 12 shows circulating antibody responses in
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lambs immunised with TSBP from 0.circumcincta.
TSBP were fractionated in 10% SDS-PAGE gel slabs
under reducing conditions prior to blot transfer to
Immobilon-P. The blot was cut into strips and probed
with pooled sera from the protection trial described in
Example 12 (Figure 12A) or using individual sera from
the same trial (Figure 128);
Figure 11 shows cyrostat sections of adult
Ostertagia circumcinctR probed with fluorescein
isothiocyanate conjugated anti-ovine immunoglobulin.
Panels A and B show transverse sections of worms
retrieved from TSBP immunised lambs (Panel A) and
control lambs (Panel B); and
Figure 14 shows group mean faecal egg output from
lambs immunised with TSBP from 0.circumcincta as well as
adjuvant only controls.
Figure 19,(A) shows the nucleotide sequence of
oligonucleotide PR primers used in amplification of 11.,_
rontortfls. cysteine proteinase gene fragments. The
active site cysteine and asparagine residues as well as
the peripheral conserved glycine residue are shown in
bold type. Restriction enzyme recognition sites were
added to the 5 ends of all the primers, (except 550J),
to allow rapid directional cloning of amplified
products. Figure 15(8) shows the distribution of the
primers along the gene length. Arrows indicate the
direction in which DNA amplification is initiated from
each primer.
Figure 16 shows the nucleotide and predicted amino
acid sequences of cysteine proteinase encoding cDNA
fragment DM.1;
Figure 17 shows the nucleotide and predicted amino
acid sequences of cysteine proteinase encoding cDNA
fragment DM.2;
Figure 18 shows the nucleotide and predicted amino
acid sequences of cysteine proteinase encoding cDNA
fragment DM.3;
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Figure 19 shows the nucleotide and predicted amino
acid sequences of cysteine proteinase encoding cDNA
fragment DM.4;
Figure 20 shows the nucleotide and predicted amino
acid sequences of cysteine proteinase encoding cDNA
fragment DM.4a;
Figure 21 shows the nucleotide and predicted amino
acid sequences of cysteine proteinase encoding cDNA
fragment DM.5;
Figure 22 shows the alignment of the predicted
amino acid sequences from PCR-amplified cDNA fragments
encoding cysteine proteinases isolated from adult IL
contortus with the published amino acid sequence of AC-1
of Cox et al. 1990, Mol. Biochem. Parasitol. 41, 25-34,
which encodes a dominant 35kDa cystein proteinase
isolated from a North American strain of F. contortus.
Using AC-1 as the reference sequence, only regions of
divergent amino acids are shown. Potential
glycosylation sites (N-X-T/S) are underlined. * denotes
end of sequence data with ** indicating termination
codons. Direct alignment indicated that one amino acid
had been deleted in the predicted sequence for DM.3 as
compared with the other predicted sequences. To
maintain alignment, a gap (indicated by ik) has been
included in the DM.3 sequence. The boxed region
indicates the position of primer 550J, the sequence of
which corresponds to AC-1;
Figure 23 shows an autoradiograph of a Southern
blot in which adult H. contortus genomic DNA digested
with HaeIII (Ha), HindIII (H) or EcoRI (E) was probed
with 32P dATP labelled cysteine proteinase-encoding PCR
fragments DM.1 (lane 1), DM.2 (lane 4), DM.3 (lane 2)
and DM.4 (lane 6). Dm.2a, which differs in nucleotide
sequence from DM.2 by 3t, and DM.3a, which differs from
DM.3 by lt were also used as probes and are shown in
lanes 5 and 3 respectively. lkbp DNA molecular weight
standards (Gibco BRL) are shown in lane 7;
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Figure 24 shows an autoradiograph of a Northern
blot in which adult H. contortus total RNA (T) and mRNA
(m) was probed with 32P dATP labelled cysteine
proteinase-encoding PCR fragments DM.1 (lane 1), DM.2
(lane 3), DM.3 (lane 2) and DM.4 (lane 4). RNA markers
(Gibco BRL) are shown in lane 5.
FXAMPLE 1
Matacials.._ansillathada
1) 2r.enaratian-sist_membranezlaauncLextrant_ir9.IILarialt.
parpsirps of H rdrrnrtris..
Clean adult parasites (21 days old) were harvested
from the abomasa of donor lambs which had been raised
under worm-free conditions and experimentally infected
with a pure strain of F. contortils.. The harvested
parasites were homogenised in 15g aliquots in 100m1 ice-
cold phosphate buffered saline, pH 7.4 containing
phenylmethane-sulphonylfluoride (PMSF), N-ethylmaleimide
and EDTA, all at 1mM, after which the homogenate was
centrifuged at 10,000g and 4 C for 15 minutes. The
resultant pellet was re-extracted in 80m1 of the same
buffer containing 0.11 v/v Tween 20. Following
centrifugation at 10,000g for 15 minutes at 4 C the
supernatant was removed. This step was repeated and the
pellet extracted for 2 hours at 4 C in 40m1 2% reduced
Triton X-100. This extract was centrifuged at 100,000g
for 1 hour at 4 C and the supernatant containing the
solubilised membrane proteins retained. The supernatant
was filtered (0.22gM) and then diluted by the addition
of 3 volumes 0.5M NaCl, 10mM Tris, pH 7.4 containing Ca2*
and mn2* at 100gm and 10gM respectively and immediately
applied to a column containing Thiol-Sepharose.
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2) Chromatography on Thiol-Sepharose
A 5m1 bed volume Thiol-Sepharose (Pharmacia, U.K.)
column was equilibrated in 0.52o- reduced Triton X-100
(Aldrich), 10mM Tris, 0.5M NaC1, pH 7.4 containing CaC12
(100 M) and MnC12 (10 M) respectively. The supernatant,
described above, was then applied to the Thiol-Sepharose
column at a rate of 5m1/h and the elution of
proteinaceous material was monitored by measuring the
eluate absorbance at 280nm. Unbound material was eluted
by washing the column with 10 to 20 volumes of column
equilibration buffer. When the 0D280 had returned to a
steady base-line, bound materials were eluted from the
column by washing with equilibration buffer containing
50mM dithiothreitol (DTT). The peak fractions were
pooled and DTT was removed by passage through a
Sephadex-G25TM column which was equilibrated in 10mM Tris,
0.1%- reduced Triton X-100, 0.196- sodium azide, pH 7.4.
The protein peak fractions were again pooled and the
protein content determined by the B.C.A. method (Pierce,
U.K.). The eluates were supplemented with iodoacetate
(1mM final concentration) and stored at -70 C prior to
innoculation into lambs. The proteins present in these
eluates are, henceforth, referred to as Thiol Sepharose
binding proteins (TSBP).
EXAMPLE 2
1. Molecular weight of Thiol-Sepharose binding
integral membrane proteins (TSBP) of H.contortus
Approximately 2 g TSBP, extracted from adult B.
contortus were fractionated using 109r; SDS-PAGE (Laemmli,
1977) under reducing and non-reducing conditions. Gels
were stained using a sensitive silver staining procedure
(BioRad, U.K.).
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Results:
Typical profiles obtained are shown in Figure la
(non-reduced) and Figure lb (reduced).
TSBP were fractionated under non-reducing
conditions into a prominent band at 60kDa as well as a
strongly staining zone above 205kDa (Lane 2, Figure la)
which, in some gels resolved partially as three
components. In addition, faintly staining material was
evident between 97 and 120kDa (Lane 2, Figure la).
Under reducing conditions TSBP were fractionated into a
more complex banding pattern (Lane 2, Figure lb) and the
molecular weight of these components was calculated by
reference to the marker track (Lane, 1, Figure lb) and
the outcome of this analysis is summarised in Table 1.
Table 1 The molecular weights of TSBP fractionated
using 10t SDS-PAGE and reducing conditions.
175-180, 160, 120, 100, 77, 70, 62, 56, 52, 51 and
37-38kDa
The 62 and 37-38kDa peptides were the most
prominent peptides observed (Lane 2, Figure lb).
PNAMPLF
proteinase properties of TSBP of H contortus
Proteinase activity associated with TSBP was
monitored using both a spectrophotometric assay with
azocasein as substrate to estimate total proteinase
activity as well as gelatin-substrate analysis to enable
the characterisation of individual proteinases_
Methods:-
a) Spectronhotometric assay
TSBP (2gg protein in 10g1) were mixed with 100g1
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sterile buffer (0.1M acetate, pH5) and 20A1 azocasein
(1mg/m1) in the same buffer. The reaction mixture was
supplemented with penicillin (500 iu/ml) and
streptomycin (5mg/m1), vortexed briefly and then
incubated overnight at 37 C. Undigested protein was
precipitated by the addition of 1M perchloric acid
(130A1) and incubation on ice for 30 minutes. Following
centrifugation at 11,000g for 5 minutes the 0D405 of the
resulting supernatant was determined. Blank reactions,
containing sterile water instead of TSBP, were run in
parallel. The optimal pH for enzyme activity was
determined in a series of reactions using buffers over
the pH range 3 to 10.
The effects of various proteinase inhibitors on
TSBP proteinases were monitored by incubating TSBP with
buffer supplemented with inhibitor for 30 minutes prior
to addition of azocasein and antibiotics. Inhibitors
tested and the final reaction concentrations were
phenylmethanesulphonylfluoride (PMSF, 1.0mM), L-
transepoxysuccinyl-L-leucylamido-(4-guanidino)-butane
(E64, 100AM), ethylenediaminetetraacetic-acid (EDTA,
1.0mM) and pepstatin (10AM).
=
b) pelatin-substrste gel analysis.
TSBP were fractionated by non-reducing SDS-PAGE in -
7.5% gel slabs containing 0.1% gelatin. The electrode
buffer (Laemmli, 1977) was chilled on ice prior to use.
After electrophoresis, SDS was eluted from the gel by
extensive washing with 2.5W Triton X-100 for 30 minutes.
Gel slabs were incubated overnight at 37 C in 0.1M
acetate buffer pH 5, 2mM with respect to DTT or 0.1M
= Tris, pH 8.5. For some experiments TSBP were incubated
with proteinase inhibitors prior to electrophoresis and
= inhibitors were also included in the incubation buffer
at the final concentrations indicated above.
Proteinases were visualised by Coomassie blue counter-
staining.
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2..eallita: -
Ana lys es indicated that passage of membrane bound
extracts of adult E. contorrus, over Thiol Sepharose
resulted in an 8 or 24 fold enrichment of proteinase
activity against azocasein respectively as determined at
pH 5. At pH 8.5 enrichment could not be quantified
because activity in the start material was low.
TSBP proteinase activity at pH 5 was eliminated by
the class specific cysteine proteinase inhibitor, E64
and relatively unaffected by the serine, PMSF (28%);
metallo, EDTA (115k) or aspartate, Pepstatin (0)
proteinase inhibitors where (n) represents the percent
activity lost in comparison to a control preparation in
the absence of the inhibitor. At pH 8.5 proteolysis was
weak and was markedly reduced in the presence of PMSF or
EDTA (52 and 43% respectively).
At pH 5 (Lane 1, pH 5, Figure 2), two prominent
zones of proteolysis at 37-38 and 52kDa were visualised
by gelatin-substrate gel analysis as well as faint bands
around 70 and 100kDa. These activities were abolished
= by E64 (Lane 2, pH5, Figure 2). At pH 8.5, proteinase
activity resolved as two sharp bands at 70 and 88kDA
(Lane 2, pH 8.5, Figure 2) the latter activity being
completely inhibited by both PMSF and EDTA (Lanes 2 & 3 .
respectively, pH 8.5, Figure 2) while the former
activity was markedly reduced in the presence of either
of these compounds.
These results indicated that TSBP contained several
cysteine proteinases active at acidic pH and
serine/metallo proteinases active at alkaline pH.
=
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FXAMPLE 4
jomrtin binding properties of TSBP glyrans from
R.contortus.
Methods:-
TSBP (25gg in 5g1) were fractionated using 10% SDS-
PAGE and reducing conditions. Fractionated proteins
were blot transferred onto Immobilon P nylon membranes
(Millipore) and the blot subsequently blocked overnight
in 2% globin free bovine serum albumin (BSA, Sigma)
dissolved in TEST (50mM Tris base, 150mM NaCl, 0.05%
Tween 20, pH 7.5). Blots were extensively washed in
TBST and then cut into strips. Individual blot strips
were incubated with a 10 gg/ml final concentration of
the following biotinylated lectins (Vector Laboratories,
U.K.) in 2% BSA in TEST for 1 hour. Lectins tested were
Dolichos bifluorus Agglutinin, Soybean, Peanut, Wheat
germ, Helix Pomatia, Concanavalin A and
Jacalin. Following incubation with the lectins, the
blot strips were extensively washed prior to incubation
(lh) with streptavidin horse radish peroxidase which had
been diluted 1:500 in BSA/TSBT. After further thorough
washing in TSBT the blot strips were incubated in
diaminobenzidine tetra-hydrochloride (30mg/50m1 TSBT
containing 50g1 30% hydrogen peroxide) substrate
solution.
Results:-
ronranavaliu A (Lane 5, Figure 3) bound to the
broadest range of glycoproteins, MWts 175-180, 120, 62,
56 and 52kDa, while Wheat germ, Helix Pomatia, Jacalin
and Peanut (Lanes 3, 4, 6 and 7 respectively, Figure 3)
all bound to a single band at 175-180kDa. Dolichos
(Lane 1, Figure 3) bound to a 77kDa glycoprotein giving
a weak signal. These data indicated that some, but not
all, components of TSBP were glycosylated.
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Exampl
ligacialisatian_o_L_ThaEi.ma-cantartual
Methods:
Cryostat sections of adult g. contortus were
incubated with sera from sheep which had been immunised
with TSBP in Freund's complete adjuvant or from control
sheep immunised with adjuvant alone. After extensive
washing the parasite sections were incubated with
fluorescein isothiocyanate-conjugated horse antibodies
with specificity for sheep immunoglobulin. After
further extensive washing the sections were viewed using
a UV fluorescence microscope and photographed.
Results.--
Sections incubated with anti-TSBP sera showed
pronounced fluorescence at the intestinal brush border
membrane as well as a limited region of the subcutis
(Panel A, Figure 4). No specific staining was evident
in sections which were incubated with sera from control
sheep (Panel B, Figure 4). These results suggested that
TSBP were mainly localised in the intestinal brush
border membrane and, to some extent, in the subcuticular
region.
EXAMPLE 6
Antibody responses of sheep immunised with TSB? of
Ii.onntortus
Methods:
The antibody responses of lambs immunised with TSBP
= prior to challenge with E. contortus (see Example 7)
were evaluated by western blotting. TSBP were run on
10t SDS-PAGE under reducing conditions and then blot
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transferred onto Immobilon P. The blots were cut in
half and one half was treated with sodium periodate to
block carbohydrate epitopes while the other was used
untreated. The blot sections were then cut into strips
and probed with sera (diluted 1/200 in TBST) from
individual experimental lambs. Antigen recognition was
defined by using a periodate-treated and untreated blot
strip for each individual lamb serum.
In addition, cryostat sections were prepared from
adult JL contortps, which had been recovered from sheep
immunised either with TSBP in Freund's complete adjuvant
or with adjuvant alone. The sections were incubated
with fluorescein isothiocyanate conjugated antibodies
specific for sheep immunoglobulin and, after thorough
washing, viewed under a UV fluorescence microscope and
photographed.
Results.
Following :immunisation with TSBP, sera from
vaccinated sheep recognised a variety of antigens on
non-periodate treated blots with particularly strong
signals evident at 35-40 and 60-62kDa (Lanes 9 to 14,
Figure 5). In addition, a group of antigens in the 50-
55 kDa region was recognised to a varying degree as well
as an antigen at 120kDa. Both control lamb sera (Lanes
15 & 16, Figure 5) reacted with a 70kDa antigen.
Periodate treatment markedly reduced the variety of
antigens recognised by immunised lamb sera (Compare
Lanes 1 to 6 (+ periodate) with 9 to 14 (no periodate),
Figure 5). A prominent antigen at about 60-62k1Ja was
recognised by all sera from immunised lambs as well as a
group of antigens around 50-55k0a Lanes 1 to 6, +
periodate, Figure 5). Reactivity with the 35-40kDa
antigen was abolished by periodate treatment. Control
=
lamb sera again recognised an antigen at 70kDa (Lanes 7
& 8, periodate, Figure 5).
Sheep immunoglobulin was detected on the luminal
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surface of the gut of parasites retrieved from sheep
immunised with TSBP (Arrowed, Panel A, Figure 6) but not
in parasites retrieved from challenge controls (Panel B,
Figure 6) indicating that an element of the protective
response stimulated by immunisation with TSBP was
directed at components on the gut surface of the
parasite.
wtx AMPLE 7
Protection Trials: H.contortus
Sheep: Greyface/Suf folk cross lambs 3 to 4 months of
age at the start of the experiment were allocated into
two groups balanced for sex and weight. The lambs had
been reared indoors from birth in conditions designed to
exclude accidental infection with nematode parasites.
Infective larvae were from a strain which had been
maintained at the Moredun Institute, Edinburgh by
repeated passage of jasemonrhus contortus through worm-
free donor lambs.
Group No. of Antigen Dose/lamb* Challenge
lambs
1 6 TSBP 3 x 200gg 5000 L3 2wks
2 6 Adjuvant - after final
alone immunisation
* Total protein injected at each immunisation. Lambs
were killed 30 days after challenge.
TSBP were emulsified in Freund's complete adjvant
and control preparations were prepared in the same way
except that phosphate buffered saline was substituted
for antigen. Two ml of "vaccine" were injected
intramuscularily into each hind leg. Lambs were
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immunised on three occasions at 3 weekly intervals and
antibody responses were adsessed by Western blotting
immediately prior to challenge. Blood samples for
assessment of antibody titres were taken before and at
regular intervals during the experiment by jugular
venepuncture. The establishment of challenge infection
was assessed by determining faecal agg counts from 15
days post-infection until the lambs were killed 30 days
post-infection. Worms were retrieved from the abomasa
as described below and the total worm number was
estimated.
Parasitological techniques.-
Faecal egg counts were performed using a modified
McMaster technique and expressed as eggs per gram fresh
faeces. Worm counts were performed on aliquots of
gastric washings and mucosal digests. Counted worms
were sexed and classified by their stage of development.
Results,¨
a) Antibody responses,
The circulating antibody responses of lambs
immunised with TSBP are described in Example 5 above.
b) Faecal eqg counts
Group mean faecal egg outputs are plotted in Figure
7 and individual counts are shown in Table 2.
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Table 2
Days post infection
Group Sheei 15. 12 aa 22. 2.1 21 2.1
1179 0 6 36 225 567 749 666
TSBP 1805
0 12 27 63 324 504 531
Immunised 1812 3 6 116 477 81 9 15
1151 3 9 3 6 60 141 153
1836 0 6 21 33 39 414 90
1178 3 0 0 129 252 405 279
Mean 1 7 34 156 221 370 289
1058 9 639 3393 2412 3753 2637 2133
1140 0 1170 1584 2592 4500 3249 2556
Challenge 499 0 756 2799 2700 5535 3861 2619
Controls 1106 3 729 2313 4590 6309 4302 4185
1175 0 441
2646 2781 6822 4464 4824
1035 6 27 1917 4446 6255 5220 6993
Mean 3 627 2442 3254 5529 3956 3885
From day 17, the mean faecal egg count of lambs
immunised with TSBP was always significantly lower
(p<0.01; Two Sample T-test) than those of the controls.
c) Final worm burdens
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Ta-ble
grroup Sheep Total Worms Males. Females
1179 2729 1563 1166
TSBP 1805 2600 1655
945
Immunised 1812 136 74 62
1151 1592 1061 531
1836 1381 887 494
1178 1528 960 568
Mean 1661 1033 628
1058 3912 1955 1987
1140 3072 1657 1415
Challenge 499 3158 1614 1544
controls 1106 3810 1864 1946
1175 3468 1814 1654
1035 3401 1635 1766
Mean 3470 1757 1714
Final worm burdens are summarised in Table 3.
Lambs immunised with TSBP had significantly reduced
(53%, p<0.01) final worm burdens compared to the
controls, with the female parasites being markedly more -
susceptible. No immature stages of the parasite were
observed.
sxAMPLE 8
Zvidence that Thiol Sepharose binding proteins from H.
= Pontortns differs from the cysteine proteinase of EP-A-
0434909
Sera from lambs immunised with TSBP (Figure 5) did
not recognise any periodate-treated antigens at 35kDa
while lambs immunised with the AC1 containing complex
=
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(EP-A-0434909) strongly recognised a 35kDa antigen. In
addition, these sera also recognised recombinant AC1
expressed in a non-glycosylated form in a bacterial
expression vector confirming that protein epitopes of
the native protein were antigenic. Here (Figure 5), no
proteins in the size range 29 to 40kDa were recognised
by anti-TSBP sera on periodate treated Western blots.
Finally, E. contortvs, were extracted in an aqueous
glycerol buffer as described in EP-A-0434909 and the
proteins solubilised by this procedure probed with anti-
TSBP serum (Figure 8) on periodate treated Western
blots. Anti-TSBP serum did not recognise any components
present in the aqueous/glycerol extract (Lane 2, Figure
8) indicating that TSBP are quite distinct from the AC1
fibrinogenase complex.
In a separate group pooled saline (Si) and Tween 20
(52) extraction supernatants (see Example 1) were
applied to Thiol-Sepharose and the resultant bound
proteins used, to immunise a group of lambs prior to
challenge with E. contortus. The results shown in
Figure 7 and in Tables 4 and 5 demonstrated that these
components were not protective.
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Table 4.
Individual faecal egg counts for lambs immunised with
Sl/S2 TSBP
Days after challenge
Group Sheep 15 17 20 22 24 27 29
1074 0 945 1098 1557 2592
1737 1800
1069 6 1269 1503 2403 3501
1593 1017
1833 9 1242 4716 5130 6588
3303 4158
31/52 1813 6 1296 1692 1647 3465 3447 3267
TSBP 1022 6 693 2196 1404 2250 2520 2871
1182 0 828 1665 2700 4626 4491 3681
Mean 4 1046 2145 2474 3837 2849 2799
1058 9 639 3393 2412 3753 2637 2133
1140 0 1170 1584 2592 4500 3249 2556
499 0 756 2799 2700 5535 3861 2619
controls 1106 3
729 2313 4590 6309 4302 4185
1175 0 441 2646 2781 6822 4464 4824
1035 6 27 1917 4446 6255 5220 6993
Mean 3 627 2442 3254 5529 3956 3885
Table 9.
Summary of the contrasting effects of immunisation of
lambs with 51/52 or Triton X-100 extracted (53) TSBP
from H. contortus on worm burdens following challenge
with the same parasite
Group Mean Worm t protection I
Male (SE)
Count (SE)
51/52 3202 (328) 7.7 55.5
(2.2)
TSBP
53 1661 (385) 53.1 61.5
(2.0)
TSBP
Control 3470 (138) 50.7
(0.8)
2 8 2 1 78
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FxAMpLE 9
Evidence that cvsteine orotéase activity within the
phini sepharnse binding proteins of H contortus is
associated with the protective caDacity cf these
oroteins
Haemonchus TSBP were separated by ion exchange
chromatography on a column containing MonoQ medium.
This column was equilibrated in 10mM Tris-HCl, pH 7.4
containing 0.1% reduced Triton X-100. TSBP diluted in
this buffer was applied to the column and after the
unbound fraction had been collected, bound material was
sequentially eluted with the following concentrations of
NaC1 in the same buffer:- 100mM, 200mM, 300mM and 1M.
Analysis by conventional and gelatin substrate SDS-PAGE
revealed that the predominant polypeptide of about 60kd
was mainly eluted with 200mM NaCl, whereas the protease
activity was mostly eluted before (100mM NaCl) or after
this (300mM and 1M NaCl). The fractions were pooled as
follows:-
1) the fraction which did not bind to the column
2) those fractions which were enriched for the
60kd protein, and
3) those fractions which were enriched for
protease activity.
The protease profiles are shown in Figure 9. The
protective capacity of these fractions were compared
with unfractionated TSBP in a sheel trial using the
techniques described in Example 7.
Thirty five 3-4 month old Greyface x Suffolk worm-
free lambs were allocated to 5 equal groups balanced for
sex and weight. Group 1 was immunised with TSBP, Group
2 was immunised with the fraction which did not bind to
MonoQ, Group 3 was immunised with the fraction enriched
for protease activity, Group 4 was immunised with the
fraction enriched for the 60kd protein, whereas Group 5
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received adjuvant and phosphate buffered saline only.
The dose of antigen used for group 1 was 200 jig protein
per injection. Groups 2, 3 and 4 each received an
amount equivalent to 200 yg of TSBP separated into the
= respective fractions.
The antigens were given as three injections at 3
weekly intervals. All immunogens were emulsified in an
equal volume of Freund's complete adjuvant for the first
injection but an equal volume of incomplete Freund's
adjuvant was used for the booster doses. The first
vaccine dose was administered as 4 subcutaneous
injections each of 0.5 ml, 2 on each flank, whereas the
boosters were given intramuscularly as two 2m1
=
injections, one into each back leg.
As in Example 7, blood samples were obtained for
serology. Two weeks after the final immunisation all
sheep were challenged with 5,000 infective H.contortils,
larvae each. Faecal egg counts were determined 3 times
a week from 14 days after challenge until the sheep were
killed for worm counts 34 days after infection.
The results are summarised in Table 6. One sheep
in Group 2 died of causes unrelated to the experiment
before it was challenged.
The egg count of the control sheep averaged over
the experiment ranged from 2057 to 3791 eggs per gm.
There was evidence that Group 1 sheep were partially
protected as their egg counts were significantly lower
(p<0.02 Students t test), with 4 of the 7 animals
scoring less than 1000 epg. Mean total worm counts of
Group 1 were also lower than those of the controls,
although this differenve was not statistically
significant. However, the proportion of male worms was
significantly higher (p<0.02 Students t test) in Group 1
compared to the controls.
When the same comparisons were made between either
Group 2 or 4 and the controls, no significant
differences were found, but Group 3 sheep shed
2 1 8 2 1 7 8
WO 95/26402 PCT/GB95/00665
=
- 36 -
significantly fewer eggs and contained a significantly
higher proportion of male worms, (p<0.02 Students t
test), a result identical to Group 1.
It was concluded that the protective component
within the TSBP used to immunise Group 1 was associated
with the protease enriched fraction which served as the
antigen for Group 3.
Table 6
Worm Total Mean egg count
Sex ratio Worm averaged
over
Group Sheep No. (t male) Count experiment
1. Thiol binding 1426 70.8 1326
2897
1295 73.7 177 67
1481 50.5 2994 1652
1413 72.7 338 354
1507 53.0 3220 2990
1500 56.8 341 728
1390 74.3 1698 648
Mean 64.5 1442 1334
2. MonoQ-unbound 1509 52.8 3238
2125
1414 51.0 3452 2847
1379 47.9 3114 3090
1346 48.5 2922 3785
1543 53.5 2695 2859
1380 55.2 3480 2803
1367 died
Mean 51.5 3150 2918
SWO 95/26402 218217E
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3. Protease rich 1436 61.7 2195
1854
1480 74.1 950
952
' 1273 50.0 2820
2372
1374 80.6 523
383
1396 57.6 1631
1446
1418 80.2 977
557
1508 52.9 2828
1361
Mean 65.3 1703
1275
= 4. 60kd rich 1523 68.2
1301 2532
1486 56.6 2375
3676
1337 57.1 2490
2790
No Tag 63.0 3696
2478
1487 49.4 3183
3475
1358 49.8 2390
4111
1296 43.8 547
1058
Mean 55.4 2283
2874
S. Control ' 1282 48.1 3253
3807
1514 38.2 1056
2190
1375 45.8 2229
2084
. .. 1548 48.5 2866
2057
1405 50.7 2820
3207
1236 51.9 2867
3097
1421 53.4 2618
3791
Mean 48.1 2530
2890
FIX4MPLE 10
Materials and Methods
1.
Preparation of Thiol-Sepharose Binding Protein
pyrrart from adult Ostertagia rirrnmrinrrft
A detergent extract of clean adult parasites
obtained from the local abbatoir was prepared
following exactly the procedures described in step (1)
,
WO 95/26402 21 8 2 1 78
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of Example 1..
2. Chromatography on Thini-
sepharnne
The supernatant from step (1) above was applied
to a Thiol-Sepharose column and eluted as described in
Example 1. Treatment and storage of the eluates was
as described in Example 1.
Fzampry, 11
fklezular_waislatst_MBE_Lnanatertagiststraumainctas
Materials and Methods
The procedures described in Example 2, were
followed exactly, except that the protein applied to
the gel was less than 1 gg.
Results.:
Typical profiles obtained are shown in Figure
10A (non-reduced) and Figure 10B (reduced).
TSBP were fractionated into a prominent 60kDa
band (Lane 1, Figure 10A) and faintly defined banding
in the 45kDa region. The profile obtained was
generally similar to a Raemonchus TSBP preparation
(Lane 2, Figure 10A).
Under reducing conditions (Lane 1, Figure 10B)
Ostertagia TSBP resolved into a complex set of bands
which had broad similarities to the gaemonchus profile
(Lane 2, Figure 108). Indeed, this similarity was
confirmed by the observations that antisera to TSBP of
either parasite origin recognised many components in
heterologous TSBP after periodate treatment.
= WO 95/26402
21 321 73 PCT/GB95/00665
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FXAMPT,R, 1
proteinase Properties of TSBP of 0 ri rrnmr-i TirtR
= Proteinase activity associated with TSBP was
monitored using gelatin-substrate gel analysis as
described in Example 2 (paragraph (b)) to enable the
characterisation of individual proteinases.
Results
At pH 5, two faint zones of proteolysis at
approximately 40 and 50kDa as well as very faint
activity at 70kDa and unresolved material at the
stack/separating gel interface were observed (pH 5,
Figure 11). Although indistinct, all these
proteinases appeared to be completely inhibited by
the cysteine proteinase specific inhibitor, E64.
At pH 8.5 proteolysis was observed at 70, 85 and
97kDa and activity at 70 and 85kDa resolved,
indistinctly, into doublets (pH 8.5, Lane C, Figure
11). The 97kDa protease was inhibited by PMSF and the
chelator, 1,10 Phe. The 85 kDa doublet was inhibited
by PMSF only.
Together these data indicated that TSBP from
circumcinote comprised a mixture of cysteine
proteinases active at acidic pH as well as
serine/metallo proteinases active at alkaline pH.
MAMIE-11
Lent-in binding properties of TSBP glycans from 0
fircurrinrtA
Methods
The procedures described in Example 4 were
followed exactly, except that approximately 10 to 15mg
of protein were fractionated prior to blot transfer
2182178
WO 95/26402 PCT/GB95/00665 =
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and cutting into strips.
9esults.
Of the lectins tested only Concanavalin A bound to
TSBP, albeit faintly, in the size range 55 to 70kDa.
=
EXAMPLE 14
Antibody resn,nses of sheep immunised with TSBP
Methods
The procedures described in Example 6 were
followed. The blot strips were periodate treated.
Results
Immune lamb sera strongly recognised a group of
five antigens between 55 and 70kDa (Figure 12A) and
showed weak reactivity with antigens at about 29, 38
and 120kDa (arrowed in Figure). In addition, similar
blots strips were probed with sera from the 4
individual lambs in the TSBP immunised group
(Protection trial - see Example 15). All sera
recognised the antigens in the size range 55 to 70kDa
although the strength of recognition was variable.
Sheep immunoglobulin was detected on the luminal
surface of the gut of parasites retrieved from sheep
immunised with TSBP (arrowed, Panel A, Figure 13) but
not in parasites from control lambs (Panel B, Figure
13) indicating that an element of the protective
response was directed at components on the gut
surface.
= WO 95/26402 21 8 2 1 78
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FXAMPTtE 15
Protection of Jambs against Ost-rtagiasis following
immunisation with Tsclp from 0 cirn1minrta.
= Sheep: Greyface/Suffolk cross lambs (7 months old)
were allocated to two groups balanced for sex and
weight which had been reared indoors in conditions
designed to exclude accidental infection with nematode
parasites.
Parasites: Infective larvae were from a strain which
had been maintained at Moredun by repeated passage of
OstertagiA circumcincta through worm-free donor lambs.
Group No. Lambs Antigen Dose/lamb* challenge
1 4 TSBP 20ug 5000L3
2 wks after
2 8 Adjuvant final
alone immunisation
Total protein injected at each immunisation. Lambs
were killed 35 days after challenge.
TSBP were emulsified with an equal volume of
Freund's Complete Adjuvant and control preparations were
made in the same way except phosphate buffered saline
was substituted for antigen. Two ml. of vaccine were
injected intramuscularly into each hind leg. Lambs were
immunised on three occasions at 3 weekly intervals and
antibody titre evaluated by Western blotting immediately
prior to challenge. Faecal egg counts were monitored
from 15d post-infection until the end of the experiment
at 35d post-infection. Worms were retrieved from the
abomasa as described below and the total worm
burden/lamb estimated. Blood samples for the assessment
of antibody titres were taken before and at regular
intervals during the experiment by jugular venepuncture.
2 1 8 2 1 7 8
W095/26402 PCT/GB95/00665 411
- 42 -
Parasitologica, rechniqu-p
Faecal egg counts were performed using the modified
McMaster technique and expressed as eggs per gram fresh
faeces. Worm counts were made on aliquots of both
= washings and mucosal digests. Counted worms were sexed
and classified by their stage of development.
Statistic-al analyses
Differences between the groups were analysed using
the Student's T-test and analysis of variance.
Results:
Faecal egg cnunts.
The group mean faecal egg outputs during the
infection period for control and immunised lambs are
plotted in Figure 14 and individual lamb egg outputs are
shown in Table 7.
Mean faecal egg output from lambs immunised with
TSBP was significantly (p<0.05) lower than that from
control lambs at all sampling points except days 28 and
35. Individual responses to vaccination varied with
lambs 57 and 60 (Lanes 1 and 4, Figure 12B) generally
having the lowest egg outputs throughout the sampling
period.
Worm burdens.
Individual and group mean worm burdens are shown in
Table 7. All worms found were mature adults. The
number of worms retrieved from TSBP immunised lambs was
significantly lower (p<0.05) than from the challenge
controls. The response varied between individuals and
worm burden reflected the egg output data described
above. No difference in the sex ratio of worms
recovered from immunised or control lambs was noted.
=
W095/26402 2182178 PCT/GB95/00665
- 43 -
Table 7 - Individual faecal egg outputs final worm burdens
Days aftpr rhallengp
. araap. ,Sheep 14 18 21 25 28 32 35 Worm
burden
49 0 342 459 72 495 387 450 3830
Challenge 50 6 198 450 297 720 432 558 4979
controls 51 14 261 585 297 603 927 603 3292
52 11 432 441 252 252 693 612 3993
53 7 279 405 207 378 486 NS 3806
54 9 396 297 369 1134 495 459 4189
55 3 27 189 27 72 162 198 2088
56 2Z 111 411 saa zu 511 fili 4568
kleaaa 12 2.1i AlA ZEZ Ail 511 121 1111
57 0 0 1 6 27 9 12 232
S3 Thiol- 58 2 117 165 60 189 279 270
2823
sepharose 59 3 81 288 90 9 234 450 2047
binding 60 __6. 522. _2.E. _11 21.6.
Means 1 IQ 11.4. Al lam 141 agia 115.
FxAmPLE 16
Materia] and Methods
ppnomir DNA preparation
Using a pestle and mortar (pre-chilled to -70 C),
0.5g (wet wt) of adult E. rontortus worms frozen in
liquid nitrogen were powdered and solubilised in 5 ml
extraction buffer (50 mM Tris.HC1, pH 7.5, 100 mM sodium
chloride, 1 mM EDTA, 1% (w/v) SDS and 200 jig/ml
proteinase K). The solution was mixed and incubated at
55 C for 15 minutes and then for 1 hour at 37 C. DNase-
free RNase (Pharmacia) was added to a final
concentration of 10 jig/ml and the solution incubated at
37 C for a further 15 minutes. An equal volume of
ak 02182178 2006-12-12
- 44 -
phenol:chloroform (6:4) was added and the aqueous and
organic phases separated by centrifugation at 10,000g
for 15 minutes at 4 C. The aqueous phase was further
extracted with an equal volume of chloroform:isoamyl
alcohol (49:1). DNA was precipitated at -20 C by the
addition of 0.1 vol of 3M sodium acetate, pH 4.5, and 2
vol of ethanol and, following centrifugation at 10,000g
for 15 minutes at 4 C, the resulting DNA pellet was
dissolved in 1 ml T.E buffer (10 mM Tris.HC1, 1 mM EDTA,
pH 7.5).
RNA extraction
Powdered adult worms, (0.5g wet wt), were
solubilised in 5 ml RNA extraction buffer (4M guanidine
isothiocyanate, 25 mM sodium citrate, 0.5 5 (w/v)
sarcosyl and 0.7- 6 (v/v) 2-mercaptoethanol) in a sterile
30 ml corexTM centrifuge tube. After the addition of 5 ml
phenol (equilibrated with T.E.) and 1 ml chloroform the
solution was shaken vigorously for 5 minutes and
incubated on ice for 10 minutes. Aqueous and organic
phases were separated by centrifugation at 10,000g for
30 minutes at 4 C. The aqueous phase was retained and,
following addition of 5 ml isopropanol, stored at -20 C
for 1 hour to precipitate the RNA. PNA was pelleted by
centrifugation at 10,000g for 30 minutes at 4 C,
dissolved in 0.5 ml RNA extraction buffer and
reprecipitated in an equal volume of isopropanol at
-20 C for 1 hour. The precipitate was pelleted by
centrifugation, washed extensively with 705k ethanol and
dissolved in 0.5 ml distilled H20. Messenger RNA (mRNA)
was isolated by chromatography on oligo(dT)-cellulose
(one passage) as described in Maniatis [Maniatis, T.,
Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning.
A laboratory manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, New York, p197-198, p368-369).
411 WO 95/26402 21 E 2178
PCT/GB95/00665
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cDNA synthesis
aDNA was synthesised according to the
manufacturer's instructions using the Amersham "cDNA
synthesis system plus" kit (Cat. No. RPN 1256). First-
strand synthesis was primed with random hexanucleotide
= primers.
Oligonucleotide primer ronstruction
Oligonucleotide primers (Fig 15) directed to the
consensus sequences flanking the active-site cysteine
(51 sense, 508G) and asparagine (3' antisense, 303H)
residues, and a peripheral conserved glycine residue (3'
antisense, 509G) within the canonical cysteine
proteinase molecule, were based on previously published
sequences [Sakanari, J.A., Staunton, C.E., Eakin, A.E.,
Craik, C.S. and McKerrow, J.H. (1989), Proc. Natl. Acad. .
Sci. USA 86, 4863-4867; and Eakin, A.E., Bouvier, J.,
Sakanari, J.A.. Craik, C.S. and McKerrow, J.H. (1990),
Mol. Biochem. Parasito. 39, 1-8]. In addition, a PolyT
primer (3' antisense, Poly T) was constructed to exploit
the structure of mRNA, thereby. allowing amplification of
the 3' terminus of gene sequences [Froham, M.A., Dush,
M.K. and Martin, G.R. (1988), Proc. Natl. Acad. Sci.
USA. 85, 8998-9002]. Primer 550J (3' antisense)
corresponds to a region of the AC-1 gene [Cox, G.N.,
Pratt, D., Hageman, R. and Roisvenue, R.J. (1990), Mol.
Biochem. Parasitol. 41, 25-34] which showed minimal
homology with sequences Dm.2 and DM.4 reported here.
Primer 699N corresponded to the 5' region of the AC-1
sequence. In order to minimise the degeneracy of the
primers and to increase their ability to form stable
hybrids, inosine (I) was incorporated in positions where
any one of the 4 bases could be present in the triplet
codon. Restriction enzyme recognition sites were added
to the 5' ends of primers, (except 550J), to allow rapid
and easy cloning of amplification products in a known
orientation. Oligonucleotides were synthesised by the
CA 02182178 2006-12-12
- 46 -
swell DNA Service (University of Edinburgh, Scotland).
Polymerase chain reaction
Reaction conditions were based on those described
by Eakin et al (supra) with 200 ng of genomic DNA or
cDNA being used in a total reaction volume of 50 1.
Primers were annealed at 25 C and DNA amplified in 30
cycles.
Cloning and sequencing of PCR products
Amplified products were separated on 1.0% agarose
gels containing 0.5 g/ml ethidium bromide. DNA was
visualised by UV illumination and amplified fragments of
the predicted sizes excised and extracted from the gel
using the GenecleanTM (Stratagene) method. Following
restriction with EcoRI and HindIII/XhoI, fragments were
cloned into either the BluescribeTM or BluescriptTM plasmid
vectors (Stratagene). Plasmid DNA was isolated using
the alkaline lysis method (Maniatis, supra) and
sequenced with both M13 forward and reverse primers
using the PharmaciaTm T7 sequencing kit (Catalogue No. 27-
1682-01).
Southern blot analysis
Genomic DNA (2 g) was digested with 12 units of
either EcoRI, HindIII or HaeIII for 5 hours at 37 C and
the digestion products separated on a 0.8% (w/v) agarose
gel. DNA was blotted onto HybondTM membrane (Amersham)
under standard conditions [Southern, E.M. (1975), J.
Mol. Biol. 98, 503-517]. Hybridisations were performed
at 42 C in 2xSSC (1xSSC: 150 mM sodium chloride, 15 mM
sodium citrate, pH 7.0), 0.5% (w/v) SDS, 5x Denhardt's
solution [5x: 1% (w/v) ficoliTM, 1% (w/v)
polyvinylpyrrolidone, 1% (w/v) BSA (Pharmacia)], 0.1
mg/ml salmon sperm, 50% (v/v) formamide using 32PadATP-
labelled PCR amplification products as probes.
Membranes were washed in 1xSSC/0.1% (w/v) SDS for 10
= WO 95/26402 21 821 78
PCT/6B95/00665
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minutes at room temperature with one change of buffer
followed by 2 X 15 minute washes in 0.1x5SC/0.1%.- (w/v)
= SDS at 42 C. Membranes were autoradiographed for 48-72
hours at -70 C.
= Northern blot analysis
Adult-worm total RNA (4 mg) and mRNA (2 g) were
fractionated on a 1% (w/v) denaturing formaldehyde gel
as described [Fourney, R.M., Miyakashi, J., Day III,
= R.S. and Patterson, M.C. (1989), Bethesda Research
= Laboratory Focus 10(1), 5-7] and blotted onto Hybond
membrane (Amersham). Hybridisations were carried out as
described above.
RESULTS
Amplification and cloning of PCR products
The size and designation of the PCR products
amplified from an adult E. confortns cDNA preparation
are shown in Table 8. The 5, sense primer was the same
in all reactions but was combined with different 3'
antisense primers. PCR products were cloned into either
the EcoRI/HindIII sites of the plasmid vector Bluescribe
[Stratagene (DM.1, DM.2 and DM.2a)] or the EcoRI/SmaI or
EcoRI/XhoI sites of the plasmid vector Bluescript SIC*
[Stratagene (DM.3, DM.3a and DM.4 respectively)]. DM.2a
was derived from the same PCR and cloning reaction as
DM.2 but represents a different recombinant. DM.3a was
derived using the same primer pairings as for DM.3 (i.e.
508G/550J) but in a higher stringency PCR reaction where
the primer-annealing temperature was raised to 55 C.
Table 8
Size and designation of PCR products amplified from
a cDNA preparation of adult N rnnforms in separate
reactions and using different 5'/3' primer pairings.
2 1 8 2 1 7 8
W095/26402 PCl/GB95/00665 =
- 48 -
Primer Amplified product size Sequence
pairing (5'/3') (excluding primers)
designation
=
508G/509G 114bp DM.1
508G/303H 552bp DM.2/DM.2a
508G/550J 255bp DM.3/DM.3a
508G/PolyT 711bp DM.4/DM.4a
699N/303H 742bp DM.5
Serptence analysis of PrP _products.
Nucleotide and amino acid sequence analyses are
shown in Figures 16 to 21 and in Table 9. The predicted
amino acid sequences of the cysteine proteinase-encoding
PCR fragments DM.1, DM.2, DM.3 and DM.4 were aligned
with each other and with the published sequence of AC-1
(Cox, supra) as shown in Figure 22. The amino acid
sequences could be directly aligned with each other,
except for the deletion of one amino acid (Fig. 22) in
the predicted sequence for DM.3. In adeition, the
position of the termination codon in DM.4 (Fig. 22)
indicated that it was five amino acids shorter than the
AC-1 sequence. It should also be noted that DM.4, the
product of PR using the 5' cysteine and 3' poly T
primer pairing, contained 42bp of the non-coding 3' end
of the gene which are not shown. Using AC-1 as the
reference sequence, many amino acid differences were
observed and were distributed along the gene length.
Analysis of nucleotide sequence homology (Table 9)
showed that the sequences could be divided into two
groups with DM.1 and DM.2 sharing 75% nucleotide
homology, as did DM.3 and DM.4. The latter sequences
were more similar to that of AC-1 having 70% and 67%
= W095/26402 2 1 2 1 73
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nucleotide sequence homology respectively, than were
DM.]. and DM.2, both of which shared 56% homology with
AC-1. The position and number of potential
glycosylation sites were also found to differ between
AC-1 pna the other sequences. Of the sequences isolated
from the UK strain of adult E. contortne only DM.2 and
DM.3 were found to possess single glycosylation sites,
the positions of which corresponded directly to
different glycosylation sites in AC-1. The nucleotide
sequences of DM.2a and DM.3a were found to share 97% and
99% homology with DM.2 and DM.3 respectively.
Table 9
=
Percent nucleotide sequence homology between the coding
regions of cloned PCR products amplified from a cDNA
preparation of adult B. contnrtrs. Sequences were also
compared to the published nucleotide sequence of the
cysteine proteinase-encoding cDNA fragment AC-1 Cox
(supra) isolated from an American strain of Jia,_
contortus.
2182178
WO 95/26402 PCT/GB95/00665 =
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DM . 1 DM . 2 DM . 2a DM . 3 DM . 3 a DM
. 4 AC-1
DM. 1 74% 75% 521 52% 54% 5 6
=
114bp)
DM.2 74% 97% 561 56% 55* 56*
(552bp)
DM.2a 75% 97* 56* 56% 55* 56%
(552bp)
DM.3 52* 56* 56* 99* 75% 70*
(255bp)
DM.3a 52% 56% 56% 991 74% 70%
(255bp)
DM.4 54* 551 55* 75* 74% 67*
(669bp)
AC-1 56% 56% 56% 701 70* 67%
Southern blot analysis
Cysteine proteinase-encoding fragments DM.1, DM.2,
DM.2a, DM.3, DM.3a and DM.4 were used as probes in
Southern blot hybridisations of adult IT contortus
genomic DNA which had been digested with HaeIlI, HindIII
or EcoRI. The results are shown in Fig. 23. Each of
the 4 fragments DM.1, DM.2, DM.3 and DM.4 gave different
hybridisation profiles, whilst DM.2a and DM.3a gave
profiles indistinguishable from DM.2 and DM.3
respectively.
Northern blot anaiysis
The size of the mRNA transcripts encoding the gene
fragments DM.1, DM.2, DM.3 and DM.4, amplified by PCR,
was determined by Northern blot analysis (Fig. 24). All
4 fragments hybridised at 1.3kbp.
