US20080145379A1

US20080145379A1 - Recombinant DNA molecules encoding aminopeptidase enzymes and their use in the preparation of vaccines against helminth infections

Info

Publication number: US20080145379A1
Application number: US11/591,569
Authority: US
Inventors: Margaret Graham; Trevor Stanley Smith; Edward Albert Munn; David Patrick Knox; Joanna Jane Oliver; Susan Elizabeth Newton
Original assignee: Agricultural and Food Research Council
Current assignee: Agricultural and Food Research Council
Priority date: 1992-05-08
Filing date: 2006-11-02
Publication date: 2008-06-19
Also published as: NO944245L; EP0640133B1; ZA933216B; DE69329195D1; US20030078398A1; NO944245D0; FI945221A; BG62700B1; AU4077593A; GB9209993D0; RU94046017A; JPH07508879A; CN1053699C; EP0640133A1; RU2140986C1; DE69329195T2; CZ274494A3; ATE195346T1; NZ252221A; US6534638B1

Abstract

The present invention provides nucleic acid molecules containing nucleotide sequences encoding helminth aminopeptidase enzymes, and antigenic fragments and functionally-equivalent variants thereof, their use in the preparation of vaccines, for use against helminth parasites, and synthetic polypeptides encoded by them.

Description

This application is a divisional of U.S. Ser. No. 09/129,366, filed Aug. 5, 1998 which is a continuation of U.S. Ser. No. 08/335,844, now U.S. Pat. No. 6,066,503, issued May 23, 2000 which was a filing under 35 USC § 371 of PCT/GB93/00943, filed May 7, 1993 which claimed priority from GB 9209993.6, filed May 8, 1992.
The present invention relates to the preparation of protective antigens by recombinant DNA technology for use as anthelmintic agents and as protective immunogens in the control of diseases caused by helminth parasites.
Helminth parasites are responsible for a wide range of diseases and infestations of domestic animals which, leading as they do to loss of production and even animal mortality, are of considerable economic importance. Thus for example, the blood feeding nematode Haemonchus infects the lining of the gastrointestinal tract of ruminants, causing anaemia and weight loss and if untreated frequently leads to death. Animals infected with the related non-blood feeding nematode Ostertagia similarly fail to thrive and may die if untreated. Other genera of helminths of economic importance include Trichostrongylus and Nematodirus which cause enteritis in various animals, and trematodes.
Problems are also caused by nematodes such as hookworms (eg. Necator, Ancylostoma Uncinaria and Bunostomum ssp) and flukes (eg. Fasciola, Paramphistomum and Dicrocoelium) and their relatives which in addition to ruminants and domestic pets, also infect humans, frequently with fatal results.
Control of helminth parasites presently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques have a number of drawbacks however—frequent administration of drugs and pasture management are often not practical, and drug-resistant helminth strains are becoming increasingly widespread.
There is therefore a need in this field for an effective anti-helminth vaccine and many efforts have been concentrated in this area in recent years. However, as yet there are no commercially available molecular or sub-unit vaccines for the major helminth species, particularly for the gastrointestinal nematodes of ruminants, such as Haemonchus and Ostertagia.
Most promising results to date have been obtained with novel proteins isolated from Haemonchus, which have potential as protective antigens not only against Haemonchus but also against a range of other helminths. In particular the protein doublet H11OD, found at the luminal surface of the intestine of H. contortus has been shown to confer protective immunity against haemonchosis in sheep.
H11OD from H. contortus has an approximate molecular weight of 110 kilodaltons (kd) under reducing and non-reducing conditions, as defined by SDS-PAGE, and is described in W088/00835 and W090/11086. The term “H11OD” as used herein refers to the protein doublet H110D as defined in W088/00835 and W090/11096. Corresponding proteins have also recently been shown in other helminth species, eg. Necator americanus.
A number of methods for the purification of H11OD have been described in W088/00835 which suffice for the characterisation of the protein, and may be scaled up to permit production of the protein in experimentally and commercially useful quantities. There is however a need for an improved and convenient source from which to prepare not only H11OD but also related antigenic proteins, especially for a process based on recombinant DNA technology and expression of the proteins in suitably transformed prokaryotic or eukaryotic organisms.
The present invention seeks to provide such an improved procedure. Sequence determination of cDNAs for H11OD from Haemonchus contortus has been performed and the predicted amino acid sequences have been found to display homology with a family of integral membrane aminopeptidases (systematic name: α-amino acyl peptide hydrolase (microsomal)).
The mammalian integral membrane aminopeptidases are located in several tissues, eg. on the microvillar brush border of intestines, and kidney. Their role in the kidney is unclear, but in the intestine their function is to cleave the small peptides which are the final products of digestion (for reviews, see Kenny & Maroux, 1982; Kenny & Turner, 1987; Noren et al, 1986; Semenza, 1986).
In one aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences which encode helminth aminopeptidase enzymes or antigenic portions thereof substantially corresponding to all or a portion of the nucleotide sequences as shown in FIG. 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15) or sequences coding for helminth aminopeptidase enzymes which are substantially homologous with or which hybridise with any of said sequences.
A nucleic acid according to the invention may thus be single or double stranded DNA, cDNA or RNA.
Variations in the aminopeptidase-encoding nucleotide sequences may occur between different strains of helminth within a species, between different stages of a helminth life cycle (e.g. between larval and adult stages), between similar strains of different geographical origin, and also within the same helminth. Such variations are included within the scope of this invention.
“Substantially homologous” as used herein includes those sequences having a sequence identity of approximately 50% or more, eg. 60% or more, and also functionally-equivalent allelic variants and related sequences modified by single or multiple base substitution, addition and/or deletion. By “functionally equivalent” is meant nucleic acid sequences which encode polypeptides having aminopeptidase activities which are similarly immunoreactive ie. which raise host protective antibodies against helminths.
Nucleic acid molecules which hybridise with the sequences shown in FIG. 2, 3, 4 or 5 (composed of SEQ ID NOS: 1 to 15) or any substantially homologous or functionally equivalent sequences as defined above are also included within the scope of the invention. “Hybridisation” as used herein defines those sequences binding under non-stringent conditions (6×SSC/50% formamide at room temperature) and washed under conditions of low stringency (2×SSC, room temperature, more preferably 2×SCC, 42° C.) or conditions of higher stringency eg. 2×SSC, 65° C. (where SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.2).
Methods for producing such derivative related sequences, for example by site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids are well known in the art, as are methods for determining whether the thus-modified nucleic acid has significant homology to the subject sequence, for example by hybridisation.
Provision of a nucleic acid molecule according to the invention thus enables recombinant aminopeptidase enzymes, or immunogenic fragments thereof, to be obtained in quantities heretofore unavailable, thereby permitting the development of anti-helminth vaccines.
In another aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences encoding one or more polypeptides capable of raising protective antibodies against helminth parasites, which sequences incorporate one or more antigenic determinant-encoding regions from the aminopeptidase-encoding sequences as shown in FIG. 2, 3, 4 or 5 (composed from SEQ ID NOS: 1 to 15).
The present invention also extends to synthetic polypeptides comprising one or more amino acid sequences constituting an aminopeptidase enzyme or antigenic portions thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in FIG. 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15), or a functionally-equivalent variant thereof other than a synthetic polypeptide corresponding to the protein doublet H110D, or a synthetic polypeptide corresponding to any of the individual polypeptide sequences disclosed in WO90/11086.
Alternatively viewed, the invention also provides synthetic polypeptides comprising an amino acid sequence constituting an aminopeptidase enzyme or an antigenic portion thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in FIG. 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15) or a functionally-equivalent variant thereof, substantially free from other Haemonchus contortus components.
The invention further extends to vaccine compositions for stimulating immune responses against helminth parasites in a human or non-human animal, comprising at least one synthetic polypeptide as defined above, together with a pharmaceutically acceptable carrier.
W090/11086 discloses a number of polypeptide or partial polypeptide sequences obtained by proteolytic digestion or chemical cleavage of the protein doublet H110D as follows:

(a)	Met Gly Tyr Pro Val Val Lys Val Glu Glu Phe

(b)	Met Gly Phe Pro Val Leu Thr Val Glu Ser

(c)	Met Gly/Phe Asn Phe Lys Ile Glu/Val Thr/Glu
	Ala Gly

(d)	Met Lys Pro/Glu Thr/Val Lys Asp/Ala Thr/Lys
	Leu - Ile Thr

(e)	Met Leu Ala Leu Asp Tyr His Ser - Phe Val

(f)	Met Leu Ala Glu/Tyr Asp Gln/Ala Glu Asp Val

(g)	Met Gly Phe Pro Leu Val Thr Val Glu Ala Phe
	Tyr

(h)	Met Lys Thr Pro Glu Phe Ala Val/Leu Gln Ala
	Phe/Thr Ala Thr Ser/Gly Phe Pro

(i)	Lys His/Tyr Asn/Val Ser Pro Ala Ala Glu Asn/
	Leu Leu Asn/Gly

(j)	Lys - Thr Ser Val Ala Glu Ala Phe Asn

(k)	Lys Ala Ala Glu Val Ala Glu Ala Phe Asp - Ile
	- - - Lys Gly

(l)	Lys Ala Val Glu Val/Pro Ala Glu Ala Phe Asp
	Asp Ile Thr? Tyr - - Gly Pro Ser

(m)	Lys - Glu Glu Thr Glu Ile Phe Asn Met

(n)	Lys - - - Pro Phe Asn/Asp Ile Glu Ala
	Leu

(o)	Asp Gln Ala Phe Ser Thr Asp Ala Lys

(p)	Met Gly Tyr Pro Val Val Lys Val Glu Glu Phe
	Ala Thr Ala Leu

(q)	Met Gly Phe Pro Val Leu Thr Val Glu Ser -
	Tyr? - Thr

(r)	Met Glu/Phe Asn Phe Leu Ile Glu/Val Thr/Glu
	Ala Gly - Ile Thr

(s)	Met Gly Phe Leu Val Thr Val Glu Ala Phe Tyr -
	Thr Ser

(t)	Met Lys Thr Pro Glu Phe Ala Val/Leu Gln Ala
	Phe/Thr Ala Thr Ser/Gly Phe Pro

(u)	Met Lys Pro/Glu Thr/Val Leu Asp/Ala Thr/Lys
	Leu - Ile Thr - Gly

(v)	Met Leu Ala Leu Asp Tyr His Ser - Phe Val
	Gly?

(w)	Met Leu Ala Glu/Tyr Asp Gln/Ala Glu Asp Val

(x)	Lys His/Tyr Asn/Val Ser Pro Ala Ala Glu Asn/
	Leu Leu Asn/Gly

(y)	Lys - Thr Ser Val Ala Glu Ala Phe Asn

(z)	Lys Ala Ala Glu Val Ala Glu Ala Phe Asp -
	Ile - - - Lys Gly

(aa)	Lys Ala Val Glu Val/Pro Ala Glu Ala Phe Asp
	Asp Ile Thr? Tyr - - Gly Pro Ser

(bb)	Lys - Glu Gln Thr Glu Ile Phe Asn Met

(cc)	Lys - - - Pro Phe Asn/Asp Ile Glu Ala
	Leu

(dd)	Asp Gln Ala Phe Ser Thr Asp Ala Lys

Uncertainties are shown either by the form Phe/Gly, where the first three letter code represents the most likely correct amino acid based on the strength of the signal, or by a question mark; a sign “-” means an unknown residue.
The specific individual polypeptide sequences which are disclosed in WO09/11086 are disclaimed.
The term “polypeptide” as used herein includes both full length protein, and shorter peptide sequences.
“Functionally equivalent” as used above in relation to the polypeptide amino acid sequences defines polypeptides related to or derived from the above-mentioned polypeptide sequences where the amino acid sequence has been modified by single or multiple amino acid substitution, addition or deletion, and also sequences where the amino acids have been chemically modified, including by glycosylation or deglycosylation, but which nonetheless retain protective antigenic (immunogenic) activity. Such functionally-equivalent variants may occur as natural biological variations or may be prepared using known techniques, for example functionally equivalent recombinant polypeptides may be prepared using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of amino acids.
Generally, the synthetic polypeptides according to the invention represent protective antigenic sequences. The term “protective antigen” as used herein defines those antigens capable of generating a host-protective (immunogenic) immune response ie. a response by the host which leads to the generation of immune effector molecules, antibodies or cells which sterilise the fecundity of, 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.
The synthetic polypeptides according to this aspect of the invention may be prepared by expression in a host cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly described 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 according to the invention into a host cell.
The synthetic polypeptide so expressed may be a fusion polypeptide comprising a portion displaying the immunogenicity of all or a portion of an aminopeptidase enzyme and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto. For example, it may be desirable to produce a fusion protein comprising a synthetic aminopeptidase or other polypeptide according to the invention coupled to a protein such as β-galactosidase, phosphatase, glutathione-S-transferase, urease, hepatitis B core antigen (Francis et al., 1989) and the like. Most fusion proteins are formed by expression of a recombinant gene in which two coding sequences have been joined together with reading frames in phase. Alternatively, polypeptides can be linked in vitro by chemical means. All such fusion or hybrid derivatives of aminopeptidase-encoding nucleic acid molecules and their respective amino acid sequences are encompassed by the present invention. Such suitable recombinant DNA and polypeptide expression techniques are described for example in Sambrook et al., 1989. Alternatively, the synthetic polypeptides may be produced by chemical means, such as the well-known Merrifield solid phase synthesis procedure.
Further aspects of the invention include use of a nucleic acid molecule or a synthetic peptide or polypeptide as defined above, for the preparation of a vaccine composition for stimulating immune responses in a human or non-human, preferably mammalian animal against helminth parasite infections.
Alternatively viewed, the invention also provides a method of stimulating an immune response in a human or non-human, preferably mammalian, animal against a helminth parasite infection comprising administering to said animal a vaccine composition comprising one or more polypeptides encoded by a nucleotide sequence as defined above.
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 synthetic polypeptides according to the invention together, where appropriate, with one or more suitable adjuvants eg. aluminium hydroxide, saponin, QuilA, or more purified forms thereof, muramyl dipeptide, mineral oils, or Novasomes, 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 a patient. Additional components such as preservatives may be included.
An alternative vaccine formulation may comprise a virus or host cell eg. a microorganism (eq. vaccinia virus, adenovirus, Salmonella) having inserted therein a nucleic acid molecule (eq. a DNA molecule) according to this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.
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 7-28 day interval.
As mentioned above, the amino acid translation of the nucleotide sequences depicted in FIG. 2, 3, 4 or 5 show sequence homology with a family of integral membrane aminopeptidase enzymes. This was determined by searching various databases available in the Genetics Computer Group Sequence analysis software package, version 7.01, November 1991 (Devereux et al., (1984)), using translations of the sequences shown in FIG. 2, 3, 4 or 5. Two such comparisons are shown in FIG. 6.
Expression of the aminopeptidase-encoding sequences according to the invention can, as mentioned above, be achieved using a range of known techniques and expression systems, including expression in prokaryotic cells such as E. coli and in eukaryotic cells such as yeasts or the baculovirus-insect cell system or transformed mammalian cells and in transgenic animals and plants. Particularly advantageously, the nucleotide sequences may be expressed using the transgenic nematode system such as the system for the nematode Caenorhabditis described for example in Fire, (1986); Fire et al., (1989); Spieth et al., (1988); Han et al., (1990).
A further aspect of the invention provides a method for preparing a synthetic polypeptide as defined above, which comprises culturing a eukaryotic or prokaryotic cell containing a nucleic acid molecule as defined above, under conditions whereby said polypeptide is expressed, and recovering said polypeptide thus produced.
Further aspects of the invention thus include cloning and expression vectors containing nucleotide sequences according to the invention. Such expression vectors include appropriate control sequences such as for example translational (eg. start and stop codes) and transcriptional control elements (eg. promoter-operator regions, ribosomal binding sites, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.
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, as mentioned above, baculovirus and also adenovirus and vaccinia 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.
Transformed or transfected eukaryotic or prokaryotic host cells or transgenic organisms containing a nucleic acid molecule according to the invention as defined above, form a further aspect of the invention.
Eukaryotic expression systems in general, and the nematode expression system in particular, have the advantage that post-translational processing, and particularly glycosylation can occur—in the case of the transgenic nematode system, a glycosylation corresponding to that found in the native protein may be expected. This represents an important aspect of the invention, since in many cases post-translational processing is required for the recombinant protein to express optimum biological activity.
Mammalian cell expression systems, also have a number of advantages. Mammalian host cells provide good reproduction of the native form and protective epitopes of the antigen since a eukaryotic expression system will give rise to more similar glycosylation patterns, disulphide bonding and other post-translational modifications than E. coli which may produce an insoluble protein requiring refolding and having poor reproduction of the native form. In addition mammalian glycosylation is unlikely to induce an immune response which distracts from a protective anti-protein response. For protection of humans and domestic animals, it is thus preferable to use human or animal fibroblast or myeloma cell lines such as HeLa—a human cell line; BHK—baby hamster kidney cells; VERO, a monkey kidney cell line; FR3T3, Fisher rat fibroblasts; NIH3T3, a mouse fibroblast cell line; C127I, a mouse mammary tumour cell line; CV-1, African green monkey kidney fibroblasts; 3T6, mouse embryo fibroblasts; L cells, a mouse cell line; CHO, a Chinese Hamster Ovary cell line; NSO NSI, SP2 and other mouse myeloma cell lines and rat myeloma cell lines such as YB2/0 and Y3.
Vectors appropriate for different classes of mammalian cell lines are well known in the art. In general, these will comprise a promoter and/or enhancer operably connected to a nucleotide sequence encoding the antigen or fragment thereof. Suitable promoters include SV40 early or late promoter, eg. PSVL vector, cytomegalovirus (CMV) promoter, mouse metallothionein I promoter and mouse mammary tumour virus long terminal repeat. The vector preferably includes a suitable marker such as a gene for dihydrofolate reductase or glutamine synthetase. Vectors of those types are described in W086/05807, W087/04462, W089/01036 and W089/10404.
Transfection of the host cells may be effected using standard techniques, for example using calcium phosphate, DEAE dextran, polybrene, protoplast fusion, liposomes, direct microinjection, gene cannon or electroporation. The latter technique is preferred and methods of transfection of mammalian cell lines using electroporation are described by Andreason et al., 1980. In general, linear DNA is introduced more readily than circular DNA.
In the case of the protein H11OD, it has been found to have a unique and unusual glycosylation pattern, which is thought to contribute to immunoactivity since many monoclonal antibodies so far obtained to H110D from Haemonchus recognise carbohydrate epitopes which may be of importance in developing useful vaccines.
In particular the following glycosylation pattern for H110D from Haemonchus has been demonstrated:

i. about 65% of oligosaccharides are N-linked, the remainder O-linked;
ii. the major part (eg. about 48%) of the N-linked oligosaccharide is of the complex class;
iii. substantially all (eg. greater than 95%) of the oligosaccharides are uncharged;
iv. the relative molar content of the constituent monosaccharides is N-acetylgalactosamine 1.0, fucose 3.6, galactose 4.1, glucose 4.4, mannose 6.2 and N-acetylglucosamine 5.2;
v. the oligosaccharides, other than the major oligosaccharide (designated oligosaccharide D), are substantially resistant to degradation by a broad range of exo-glycosidases (eg. α-D-mannosidase, β-D-mannosidase, β-D-glucosidase, β-D-galactosidase, α-D-galactosidase, α-L-fucosidase, β-D-xylosidase, β-D-N-acetylqlucosaminidase).

Such oligosaccharides and glycoproteins containing them form a further aspect of this invention.
Oligosaccharide D of the Haemonchus H110D glycoprotein is of the N-linked type and has a novel structure consisting of two fucose residues attached by an α-1,3 linkage and an α-1,2 linkage to a mannose (N-acetylglucosamine) 2 core.
Another aspect of the invention thus provides an oligosaccharide having the structure:
and more particularly the structure:
especially when linked to a protein, eg. a recombinant protein such as a helminth aminopeptidase-protein or an antigenic fragment thereof, or when used to generate anti-idiotypic antigens for immunisation especially of very young animals.
Animal glycoproteins generally have fucose α-1,6 linkages and the fucose α-1,3 linkage of the oligosaccharide of the present invention is an unusual feature.
This invention will now be described in more detail with particular reference to the protein H110D from Haemonchus contortus. However, by a variety of techniques such as histochemistry and DNA hybridisation, H110D equivalents have been observed in other parasite species. It is believed that the H110D protein is a multigene complex and that in addition, the nucleotide sequences encoding it, may exhibit sequence variations between different strains and different life cycle stages of the helminth. Moreover there may exist multiple enzyme forms (isoenzymes) which may be differentially expressed at different stages, or in different strains. In this study DNA sequences, and thus the predicted amino acid sequences, have been determined from cDNA clones and PCR products obtained from mRNA corresponding to the H110D gene by recombinant DNA technology from different sources, and at different parasitic stages of H. contortus life cycle.
Sequencing of cDNA and PCR products has enabled us to identify three closely related H110D sequences which are here designated H11-1 (SEQ ID NO: 19), H11-2 (SEQ ID NO: 20) and H11-3 (SEQ ID NO: 21). H11-1 comprises three contiguous and overlapping sequences, cDNA clone AustB1 (SEQ ID NO: 6), PCR product A-648 (SEQ ID NO: 9) and at the 3′ end PCR product 014-178 (SEQ ID NO: 12); H11-2 comprises the PCR products A-650 and 2.5 kb (SEQ ID NOS: 10 and 7 respectively); H11-3 comprises the PCR products 3.5 kb and A-649 (SEQ ID NOS: 8 and 11 respectively). The specific relationships between the individual sequenced cDNA and PCR product clones and H11-1, -2 and -3 are summarised in FIG. 1 and shown in detail in FIGS. 3, 4 and 5.
Differences and variations in the sequences obtained from the cDNA clones and PCR products have been observed, as can be seen in particular from FIGS. 2, 3, 4 and 5 (composed of SEQ ID NOS: 1 to 15 and 19 to 21) and as summarised in Table 1.

TABLE 1

Homologies of the deduced amino acid sequences obtained by
translation of the nucleotide sequences shown in FIG. 2.

	% Similarity	% Identity

H11-1:H11-2	77	63
H11-1:H11-3	79	65
H11-2:H11-3	82	69

The differences can be attributed to different mRNAs (of the multigene family). In addition, the variations may be due, at least in part, to different variants of the H110D-encoding sequence or mRNA present at different stages of the life cycle or in strains differing in geographical origin.

Table 2 additionally shows levels of identity and similarity between the corresponding predicted amino acid sequences and two published mammalian aminopeptidase sequences.

TABLE 2

Homologies of the H110D amino acid sequences with rat aminopeptidase
M (ApM) and mouse aminopeptidase A (ApA).

	% Similarity	% Identity

H11-1:ApM	55	32
H11-1:ApA	55	31
H11-2:ApM	52	31
H11-2:ApA	54	31
H11-3:ApM	53	32
H11-3:ApA	52	30

FIG. 1 shows a map of the H. contortus H110D cDNA and PCR product clones sequenced and their relationships and relative positions along the H110D mRNA;

FIG. 2 shows the H110D nucleotide sequences designated H11-3. (SEQ ID NO: 21, derived from cloned PCR products SEQ ID NOS: 8 and 11 and cDNA clone M1AUS, SEQ ID NO: 5), H11-2 (SEQ ID NO: 20, derived from cloned PCR products SEQ ID NOS: 7 and 10) and H11-1 (SEQ ID NO: 19, derived from cloned PCR products SEQ ID NOS: 9 and 12 and cDNA clone AustB1, SEQ ID NO: 6);

FIG. 3 shows the sequence H11-3 (SEQ ID NO: 21) (shown in FIG. 2) with alignment of the cDNA clones M1 and M1AUS (SEQ ID NOS: 1 and 5);

FIG. 4 shows the sequence H11-2 (SEQ ID NO: 20, shown in FIG. 2) and the alignment of the cDNA clone B2 (SEQ ID NO: 4);

FIG. 5 shows the sequence designated H11-1 (SEQ ID NO: 19) and alignment of the cDNA B1A and Aust B1 (SEQ ID NOS: 2 and 6 respectively);

FIG. 6 shows a) the predicted amino acid sequences (SEQ ID NOS: 22, 23 and 24) derived from the DNA sequences H11-1, H11-2 and H11-3 shown in FIG. 2; bi) and ii) show the predicted amino acid sequence of H11-3 compared with the published amino acid sequences of rat microsomal aminopeptidase M (Watt et al., 1989) and mouse microsomal aminopeptidase A (Wu et al., 1990) respectively; identities are enclosed in boxes, dashes indicate spaces introduced to maximise the level of homology between the compared sequences. The conventional single letter code for amino acids is used. The horizontal line above the sequence indicates the position of the transmembrane region and the asterisks show the position of the zinc-binding motif. Levels of similarity are shown in Tables 1 and 2;

FIG. 7 Shows the alignments of amino acid sequences (designated Pep A, Pep B, Pep C, Pep D and Pep E) obtained from CNBr and Lys-C fragments of H110D as previously described (International patent application WO90/11086 and as listed earlier, polypeptide sequences (a), (b), (e), (k) and (aa), respectively) and three new sequences (SEQ ID NOS: 16, 17 and 18) obtained from H110D following digestion by elastase or thermolysin with the translations of a) H11-1, b) H11-2 and c) H11-3.

In a further-aspect the invention also provides nucleic acid molecules comprising one or more nucleotide sequences which substantially correspond to or which are substantially complementary to one or more sequences selected from the sequences of clones M1, B1A, B1A-3′, B2, M1AUS, AustB1, 014-015 (2.5PCR), 014-872 (3.5PCR clone 2), A-648 (5′ end of B1), A-650-(5′ end of 2.5PCR), A-649 (5′ end of 3.5PCR), 014-178 (3′ end of AustB1 clone 2), 014-178 (3′ end of AustB1 clones 3 & 6), 014-872 (3.5PCR clone 10) and 014-872 (3.5PCR clone 19), H11-1, H11-2 and H11-3, SEQ ID NOS: 1 to 15 and 19 to 21 respectively as shown in FIGS. 2, 3, 4, and 5 or sequences which are substantially homologous with or which hybridise with any of the said sequences.

As mentioned above, comparison of the sequences of various of the clones mentioned above, against computer databases of known sequences, reveals substantial homology with the family of microsomal aminopeptidase enzymes (EC 3.4.11.-). Enzymological activity and inhibitor studies performed with the H110D protein and sub-fractions thereof confirm that the protein is in fact microsomal aminopeptidase (α-amino acyl peptide hydrolase (microsomal)). Such studies have further shown that both aminopeptidase A-like and aminopeptidase M-like activities are exhibited, and that each of the components of the H110D doublet individually exhibit enzyme activity.

Studies with proteolytic digestion of H110D have also been carried out. Using the enzyme elastase, it was found that H110D may be partially cleaved, forming two fractions, a detergent-soluble fraction (which remained with the membrane) and a water-soluble fraction (which is designated H11S). H11S occurs in the form of a protein dimer which may be reduced to two components. Interestingly, it was found that only aminopeptidase M-like activity is associated with the water-soluble H11S fraction, whereas aminopeptidase A-like activity is only associated with the detergent-soluble fraction.

The following Example provides a description of the studies leading to determination of the sequences shown in FIGS. 1 to 7, with reference to the following additional Figures in which:

FIG. 8 shows Western blots of integral membrane proteins present in a detergent extract of Haemonchus contortus adults probed with affinity purified antibodies eluted from potential H110D clones; a) antigens in a detergent extract of Haemonchus recognised by antiserum to the extract; b) antibodies eluted from a strip such as that shown in a) re-tested against a blot of the detergent extract confirm the success of the elution step; c) antibodies as in b) which bind to clone M1 expressed protein strongly recognise a region at 110 kd (and a relatively sharp band at about 205 kd; d) there is no antibody binding when a non-recombinant is used to adsorb the serum;

FIG. 9 shows a Northern blot of mRNA purified from 11, 15 and 23 day-old Haemonchus contortus probed with a) cDNA clone M1 (SEQ ID NO: 1); b) cDNA clone M1 AUS (SEQ ID NO: 5); c) cDNA clone B1A (SEQ ID NOS: 2 and 3); d) cDNA clone AustB1 (SEQ ID NO: 6); e) cloned PCR product 014-872 (3.5-2, SEQ ID NO: 8); and f) cloned PCR product 014-015 (SEQ ID NO: 7). The

numbers

11, 15 and 23 indicate the age of the Haemonchus from which the mRNA was obtained;

FIG. 10 shows Southern blots of Haemonchus contortus genomic DNA probed with cDNA clones M1AUS (SEQ ID NO: 5), B1A (SEQ ID NOS: 2 and 3) and AustB1 (SEQ ID NO: 6) and PCR products 014-872 (3.5-2, SEQ ID NO: 8) and 014-015 (SEQ ID NO: 7); a) blots were washed at a moderate stringency, b) blots were washed at a high stringency; for each probe, track 1 contained a HindIII digest of λDNA as marker or was left blank, tracks 2 and 3 contained EcoRI and HindIII digests respectively of Haemonchus genomic DNA;

FIG. 11 shows Western blots of recombinant GST-M1 and GST-B1A fusion proteins probed with affinity purified antibodies to electrophoretically purified H110D (H110DE);

FIG. 12 shows Western blots of ConA H110D antigen probed with antisera to ConA H110D and to recombinant GST-M1 and GST-B1A fusion proteins;

FIG. 13 shows a) the results of analysis of H110D protein and aminopeptidase enzyme activities in fractions obtained by ion exchange chromatography of ConA H110D on a MonoQ column;

b) SDS-PAGE of the fractions shown in FIG. 13 a);

FIG. 14 shows a) the pH values at which fractions were obtained in a free-flow isoelectric focussing experiment;

b) SDS-PAGE under reducing conditions of the fractions from 14a) in which the lower band of the H110D doublet is found in Fraction 6 and the upper band in Fraction 16, with varying amounts of each in the intervening fractions;

c) Western blots of the fractions shown in 14b) probed with i) monoclonal antibodies designated TS 3/19.7 and ii) affinity purified polyclonal anti-M1 antibodies; control antibodies gave no detectable reaction;

FIG. 15 shows a) the pH values at which fractions were obtained in another free-flow isoelectric focussing experiment; b) SDS-PAGE under reducing conditions of fractions from 15a) used in enzyme assays, in which the lower band of the H110D doublet is found in Fractions 4-6 and the upper band in Fractions 16-18 with varying amounts of each band in the intervening fractions; c) microsomal aminopeptidase specific activities of fractions shown in 15b);

FIG. 16 shows protection of sheep by vaccination with separated upper (U), lower (L), recombined (U+L) and intermediate doublet (D) bands from H110D; a) parasite egg output, expressed as eggs per gram faeces, b) worm burden at post-mortem, relative to controls;

FIG. 17 shows protection of sheep by vaccination with a water-soluble fragment (H11S) obtained from H110D by digestion with elastase and H11A, the residual detergent-soluble H110D. a) parasite egg output, expressed as eggs per gram faeces; b) worm burden at post-mortem, relative to controls (C);

FIG. 18 shows examples of the relationship between inhibition of Ai), Bi) aminopeptidase M-like and Aii), Bii) aminopeptidase A-like activities of H110D by antisera of individual sheep vaccinated with H110D with levels of protection measured by Ai, ii) % reduction of worm burden at post-mortem and Bi, ii) % reduction reduction of faecal egg count; □ anti-H110D, ▪ anti-horse ferritin control;

FIG. 19 shows the histochemical localisation of aminopeptidase enzyme activities in adult Haemonchus contortus the light micrographs of cryo-sections of adult female Haemonchus contortus show aminopeptidase activity (red reaction product appears as dark band (arrowed) in these black and white photographs) associated only with the microvilli (mv) of the intestine (i). None of the other tissues (eg. cuticle (c), hypodermis (h), genital tract (gt), wall muscle (wm)) show activity. In a) the substrate was L-leucine 4-methoxy-β-naphthylamide, in b) the substrate was L-glutamic acid α-(4-methoxy-β-naphthylamide);

FIG. 20 shows a map of the 3.5 PCR product (clone 2) (SEQ ID NO: 8) sub-cloned into the baculovirus expression vector pBlueBacII”;

FIG. 21 shows a Western blot of extracts from baculovirus-infected insect SpodoPtera frugiperda (Sf)₉cells probed with anti-H110DN antibodies. Two cloned plaques, P3A and P4A expressed the full-length immuno-positive H110D (arrowed), the controls did not.

EXAMPLE

Methods

Construction of U.K. λgt11 Library
mRNA Isolation
Adult Haemonchus contortus (0.5 gm) of UK origin snap-frozen in liquid nitrogen were ground in liquid nitrogen using a pre-chilled mortar and pestle. The RNA was extracted from the grindate with 10 volumes of 4 M guanidine hydrochloride in 25 mM sodium citrate containing 0.5% w/v sarkosyl and 0.7% w/v 2-mercaptoethanol, followed by extraction with phenol and chloroform using the method of Chomczynski & Sacchi (1987). Messenger RNA (mRNA) was prepared from this by affinity chromatography-on oligo dT cellulose (twice) as described in Maniatis et al (1982) and the quality was assessed by in vitro translation using a rabbit reticulocyte lysate kit and ³⁵S-methionine from Amersham International plc, according to the manufacturer's instructions. Polypeptides up to 120 kd were detected.

Complementary DNA Preparation

First strand complementary DNA (cDNA) was synthesized from 1 μg mRNA using random priming and avian reverse transcriptase and the second strand was synthesized using a replacement reaction with RNase H and E. coli DNA Polymerase I followed by repair of 3′ overhangs using T4 DNA Polymerase, according to the method of Gubler & Hoffman (1983). The yield of double-stranded (ds) cDNA was approximately 400 ng from 1 μg mRNA. The ds cDNA was examined by electrophoresis in a 1% agarose gel followed by autoradiography. The ds cDNA was in the size range 0.2-9.4 kilobases-(Kb), with the majority being in the range 0.5-2.3 Kb.
Cloning of cDNA in λgt11
Non-size selected cDNA was used to construct a library in λgt11 using the Amersham cDNA cloning system (kit no. RPN 1280, Amersham International plc) and in vitro packaging extracts (kit no. N334, Amersham International plc) as described in the manufacturer's instructions, and EcoRI linker oligonucleotides (5′GGAATTCC). The resulting library was plated on E. coli strain Y1090 in the presence of isopropylthio-β-D-galactoside (IPTG) and 5-bromo, 4-chloro, 3-indolyl β-D-galactoside (X-gal), under which conditions recombinant λgt11 appear as clear (“white”) plaques and wild-type non-recombinant λgt11 as blue plaques. The library contained 90% white plaques and the cloning efficiency was calculated to be 4×10⁷plaque forming units (pfu)/μg cDNA and a library titre of 2×10⁶plaque forming units per ml. Analysis of the DNA from 20 recombinants picked at random revealed an average insert size of 0.51 Kb. However this mean was distorted by one clone with an insert of 3.5 Kb. The majority of the inserts were >300 base pairs (bp). This unamplified λgt11 library derived from UK worm mRNA was then immunoscreened.

Preparation of Antibody Probes

Antiserum to Integral Membrane Proteins

Intestines were dissected from adult Haemonchus contortus (of UK origin) and homogenised in ice-cold phosphate buffered saline (PBS), pH 7.4, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM phenylmethylsulphonyl fluoride (PMSF). The homogenate was centrifuged for 10 minutes using a microfuge and the pellet resuspended in the same buffer containing 0.1% v/v Tween 20 (Tween is a Trade mark). After re-centrifugation, the pellet was resuspended in the same buffer containing 2% v/v Triton X-100 and extracted for two hours at 4° C. This extract was centrifuged as above, to obtain a supernatant containing integral membrane proteins (IMP).
A sheep was hyperimmunised with IMP in Freund's Complete Adjuvant (FCA) by intramuscular injection of 50, 50, 120 and 130 μg of IMP given on weeks 0, 7, 11 and 15. Six weeks after the final injection, serum was harvested, and designated serum EE-068.

Preparation of Integral Membrane Proteins by Detergent Extraction of Haemonchus Contortus

An extract was prepared by homogenizing worms in 5-10 volumes of PBS containing 1 mM EDTA and 1 mM PMSF. The suspension was centrifuged at 10,000×g for 20 minutes at 4° C. and the pellet washed in the same buffer containing 0.1% v/v Tween 20 then. extracted with 5 volumes 2% v/v Triton X-100 as described above. The supernatant was re-centrifuged at 100,000×g for 1 hour, and the resulting supernatant, which was enriched in H110D but contained other IMP, was used in Western blotting experiments and for the preparation of non-denatured H110D (see below).

Preparation of H110D and Affinity Purified Anti-H110DN

The extract enriched for H110D, was subjected to affinity chromatography on ConA-agarose followed by ion exchange chromatography on MonoQ (as described in WO88/00835 and WO90/11086). The purified H110D was injected intramuscularly into lambs in FCA. Three doses of 100 μg were given at 3 week intervals. Serum collected from the lambs 4 weeks after the final injection was affinity purified by absorption to a column containing purified H110D which had been coupled to cyanogen bromide activated Sepharose (Pharmacia). Coupling of H110D to the Sepharose, binding of antiserum and elution of anti-H110D antibodies were according to the instructions supplied by Pharmacia. These affinity purified antibodies are designated anti-H110DN. The “N” distinguishes these antibodies from those raised to denatured, electrophoretically purified H110D, which are designated anti-H110DE.

Western Blotting

Western blotting was carried out using standard procedures (Johnstone et al., 1982).

Isolation and Characterisation of Clones

Immunoscreening of the U.K. λgt11 Library
The method used to immunoscreen the library was essentially as described by Bowtell et al (1986). Prior to use, the serum (EE-068) was depleted of anti-E. coli antibodies by absorption with lysates and whole cells of E. coli Y1090. The library was plated on E. coli Y1090 cells at a density of 10³pfu per 90 mm diameter plate. Plates were overlaid with nitrocellulose filters impregnated with IPTG and incubated overnight. The filters were washed with TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.05% v/v Tween 20) and then blocked with 5% v/v horse serum in TBST for 2 hours. Serum EE-068 diluted 1 in 200 in TBST containing 5% horse serum was added and the filters incubated for 4 hours with gentle rocking. The filters were again washed in TBST, then incubated with horseradish peroxidase (HRP)-conjugated horse anti-sheep IgG diluted 1 in 500 in TBST containing 5% v/v horse serum for 2 hours. (Anti-serum to sheep IgG was raised in a horse, the anti-sheep IgG purified by affinity chromatography on a sheep IgG Sepharose column, and the antibodies conjugated to HRP by the method of Nakane & Kawaoi, 1974.) Filters were further washed in TBST and positive plaques detected using 0.6 mg/ ml 3,3′-diaminobenzidine (DAB) and 0.1% v/v hydrogen peroxide. Twenty-five putative positives were picked and were rescreened with affinity purified anti-H110DN as described above. Following this secondary screen recombinants were still positive, with the clone designated as M1 giving the strongest signal.

Affinity Purification of Antibody on Recombinant Phage

Confluent plates were prepared on E. coli Y1090 lawns by plating 10³pfu of each of the antibody-positive λclones or non-recombinant λgt11 negative control phage. The lawns were incubated for 4 hours at 42° C. then overlaid with filters impregnated with IPTG and further incubated overnight at 37° C. The filters were removed from the plates and washed in TBST prior to being blocked with 5% v/v horse serum for 1 hour. The filters were then incubated with a 1 in 100 dilution of antiserum EE-068 for 6 hours, before being thoroughly rinsed with TBST. Bound antibodies were eluted from the filters by two applications of 2 ml of elution buffer (5 mM glycine, 500 mM NaCl, 0.2. Tween 20, pH 2.3) for 2 to 3 minutes each, neutralised by addition of 200 μl of 1 M tris-HCl, pH 7.4, diluted 1 in 200 and used to immunoscreen a Western blot of an H110D-enriched extract.

DNA Sequencing of the M1 Clone

Lambda DNA was isolated from the M1 clone according to the methods described in Maniatis et al (1982). The 2.38 Kb KpnI-SstI fragment containing the 300 bp M1 fragment was isolated by gel electrophoresis, purified using a GENECLEAN kit (Stratagene) (GENECLEAN is a registered trade mark of BI0101) and subcloned into pBluescriptII SK⁺ (Stratagene). The EcoRI fragment was purified using the same methods and re-subcloned into the same vector.
The nucleotide sequence of the M1 insert was determined using a T7 Sequencing kit (Pharmacia, U.K.), using both the M13 forward and reverse primers.
Preparation of Australian λgt11 and λZap cDNA Libraries
mRNA Isolation
5 gm adult Haemonchus contortus (Australian McMaster susceptible strain) snap-frozen in liquid nitrogen were ground in liquid nitrogen and the RNA extracted using hot phenol by the method of Cordingley et al. (1983). Yield of total RNA was 10.35 mg. 1.3 mg of this RNA was used to prepare mRNA by affinity chromatography on oligo dT cellulose (2 sequential purifications) using the method described by Maniatis et al. (1982). Yield of mRNA was 21.6 μg. Quality of mRNA was assessed by in vitro translation in rabbit reticulocyte lysate in the presence of ³⁵S-methionine (Amersham) according to the supplier's instructions. The translation products obtained had clearly distinguished bands including bands >200 kd in size as demonstrated by electrophoresis on SDS-polyacrylamide gels followed by fluorography.
cDNA Synthesis and Library Preparation
1 μg mRNA was used to make cDNA by priming with oligo dT or random primers, using a cDNA synthesis kit from Amersham International plc following the manufacturer's instructions. Yield was 115 ng double stranded (ds) cDNA. The quality of the cDNA was examined by electrophoresis of the ³²P-labelled DNA on an alkaline agarose gel as described by the Amersham cDNA kit instructions. Size of the cDNA (by comparison with λ-HindIII markers, New England Biolabs) was from 150 bp to >10 Kb, with most of the products being in the size range 0.6-5 Kb. The oligo dT-primed and random-primed ds cDNAs were pooled and ligated to excess EcoRI 8-mer linkers (5′GGAATTCC3′ New England Biolabs, Catalogue No. 1018) which had been labelled with γ-³²P-ATP and T4 polynucleotide kinase. The linkered cDNA was digested with EcoRI and excess linkers were removed by Sepharose 4B (Pharmacia) chromatography according to the methods described by Maniatis et al. (1982). Fractions from the column were pooled in two lots, one containing cDNA larger than 2 Kb and one of cDNA less than 2 Kb. Each pool was then ligated separately to 1 μg EcoRI cut, phosphatased λZapII arms (Stratagene) and packaged separately using Gigapack Gold (Stratagene, registered trademark). The larger sized cDNA yielded 1.3×10⁵recombinants and the smaller cDNA 1.4×10⁵recombinants; these were pooled to yield a library of 2.7×10⁵. The λZap library was amplified by plating on XL1-Blue cells (Stratagene) at 2×10⁴pfu per 135 mm plate. The titre of the amplified library was 7×10⁷pfu/ml.
A further 2 μg mRNA was used to make cDNA as described above, but using only oligo dT as primer. The yield of ds cDNA was 740 ng. This cDNA was treated with EcoRI methylase as described in Maniatis et al (1982) prior to addition of EcoRI linkers, and in this case 12-mer linkers (5′CCGGAATTCCGG3′ New England Biolabs, Catalogue No. 1019) were used. Following digestion of the linkered cDNA with EcoRI, all fractions from a Sepharose 4B column which contained cDNA were pooled, and ligated to 2 μg EcoRI cut, phosphatased λgt11 arms (Stratagene). The ligation mix was split in two and packaged with two lots of Gigapack Gold (Stratagene); these were pooled to yield a λgt11 library of 7×10⁶pfu. The library was amplified by plating on ST9 cells at 5×10⁵pfu per 135 mm plate. The titre of the amplified λgt11 library was 4.5×10¹¹pfu/ml.
Screening of the Australian λgt11 Library with Antisera to H110D
Antisera were raised by injecting sheep with H110D protein (of UK origin) which had been electro-eluted from polyacrylamide after electrophoresis in SDS according to the following method: ConA H110D prepared as described in WO 88/00835 and WO 90/11086 was electrophoresed on SDS polyacrylamide gels (Laemmli 1970) to obtain electro-eluted H110D. After electrophoresis, the area of the polyacrylamide gel containing H110D was cut out, placed in an electroeluter (Atto) and elution carried out for 3 hours at 10 watts. The electroeluted H110D (designated H110DE) was concentrated on a Centriprep 10 (Amicon) and buffer exchanged on a PD10 column (Pharmacia) into 50 mM ammonium bicarbonate/0.07% SDS, mixed with adjuvants and then injected into sheep. Immunoglobulins from the sera were precipitated with ammonium sulphate (Johnstone and Thorpe, 1982). The precipitated antibodies were resuspended at 60 mg/ml in phosphate buffered saline, dialysed against phosphate buffered saline and diluted 1:10 in Tris buffered saline (TBS) containing 5% w/v low fat milk powder. 10 mg of ConA H110D was made to 0.5% SDS, heated to 100° C. for 3 minutes and dried onto a nitrocellulose filter. Following washes with TBS containing 0.2% v/v Tween 20 and 0.5% Triton X-100 (TBSTT) the filter was incubated for 1 to 2 hours at room temperature with the antibodies to H110DE. After washing the filter for 2 hours with TBSTT, the bound antibodies were eluted with 3 ml of 0.1M glycine, 0.15M NaCl pH 2.6 for 2 minutes and immediately adjusted to neutral pH by the addition of 75 μl of 1.5 M Tris pH 8.0. These affinity purified antibodies, designated anti-H110DE, were used to screen 5×10⁵pfu of the Australian λgt11 cDNA library as described above.
5×10⁵recombinants from the λgt11 library derived from Australian Haemonchus contortus were immunoscreened and three positives picked. Following further screening two of these recombinants were still positive and were designated B1A and B2.

Sequencing of B1A and B2 Clones

The two clones were digested with EcoRI, yielding a single insert of approximately 500 bp for B1A and three fragments, B2A (about 400 bp), B2B (about 100 bp) and B2C (about 100 bp), for B2. These were subcloned into pBluescript SK⁺ (Stratagene) and sequenced using a Sequenase 2.0 kit (United States Biochemicals).
Expression of clones M1 and B1a
The M1 (SEQ ID NO: 1) and B1A (SEQ ID NOS: 2 and 3) inserts were expressed in E. coli, using a pGEX vector (Smith and Johnson 1988). This vector expresses proteins at the C-terminus of Schistosoma japonicum glutathione-S-transferase (GST). The M1 and B1A EcoRI inserts were ligated to EcoRI-cut, phosphatased pGEX1 and transformed into E. coli strain JM101 according to the methods described in Maniatis et al. 1982. Eight progeny were picked from each transformation and 2 ml cultures were grown for 6 hours at 37° C. IPTG was added to induce fusion protein synthesis, and the incubation continued overnight. Cells were harvested by centrifugation, disrupted by boiling in sample buffer (Laemmli, 1974), and the extracts analysed by SDS-PAGE and by Western blotting using affinity purified sheep antibodies specific for the SDS-denatured H110D doublet (anti-H110DE—see above). Bound antibodies were detected using alkaline-phosphatase conjugated rabbit anti-sheep IgG alkaline phosphatase conjugate (Jackson Immunoresearch) followed by colour development with 5-bromo,4-chloro,3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT). Cultures of immunopositive clones were grown and induced as above and disrupted by sonication. The sonicates were separated into soluble and insoluble fractions by centrifugation (Sorvall RC-2B centrifuge, HS4 rotor, 7000 rpm, 30 minutes, 4° C.). The insoluble pellets were resuspended in 8 M urea by sonication, and samples of fractions examined by SDS-PAGE. The fusion proteins were found to be in the insoluble inclusion body fraction. Each of these preparations was used to vaccinate 2 sheep three times at 150 μg fusion protein per dose in Freunds adjuvants. Positive control sheep were immunised with native ConA H110D protein, and negative control sheep were immunised with solubilised protein from E. coli containing the pGEX vector without an Haemonchus insert. Sera from vaccinated sheep were analysed by Western blotting against H110D.
Screening of the Australian λZap Library by DNA Hybridisation with M1 and B1A Inserts
M1 and B1A plasmid DNAs (cloned in pBluescript) were digested with EcoRI and the inserts isolated by electrophoresis in TBE (tris-borate-EDTA; 89 mM tris-borate, 89 mM boric acid, 2 mM EDTA pH approximately 8.3) buffer in 1% agarose gel, followed by purification using a GENECLEAN kit. The isolation and purification were repeated to avoid contamination of the probe with plasmid DNA sequences which would hybridise to λZAP sequences, causing unacceptable levels of background. The purified insert DNAs were labelled with α-³²P-dCTP using a Nick Translation kit from Promega Biotech according to the manufacturer's instructions. Labelled DNA was separated from unincorporated label by spin column chromatography (Maniatis et al., 1982). Eight 135 mm plates of the λZAP library were plated at 10⁵pfu/plate, and plaque lifts performed onto nitrocellulose filters (Maniatis et al., 1982). Following baking in a vacuum oven for two hours at 80° C., filters were prehybridised for two hours, then hybridised at 42° C. overnight (as described below in the Southern Blot analysis section). Four filters were screened with the M1 probe and four with the B1A probe. Filters were washed twice in 2×SSC containing 0.5% SDS, once in 1×SSC containing 0.5% SDS and once in 0.5×SSC containing 0.5% SDS, all at 50° C., and autoradiographed. Potential positive plaques were picked, and re-screened with the probes. High titre phage stocks were prepared from confirmed positives (designated M1AUS for the M1-hybridising clone and AustB1 for the B1A-hybridising clone) and the clones rescued into pBLUESCRIPT according to the λZAP manufacturer's instruction manual (Stratagene), using BB4 as the host E. coli strain. Plasmid DNA minipreps of the resultant progeny were prepared by alkaline lysis (Maniatis et al., 1982) and digested with EcoRI. Digests were analysed by agarose gel electrophoresis.

Sequencing of the M1AUS Insert

DNA sequencing was carried out on purified pBLUESCRIPT plasmid DNA using the United States Biochemicals version 2.0 Sequenase kit, according to the manufacturer's instructions. For the first sequencing reactions primers from the ends of the vector sequence were used to prime the reactions. The sequencing data obtained from these reactions was used to design a second pair of primers and from the data generated with these second primers a third pair were designed. In this way the DNA was sequenced by ‘walking along’ from both the 5′ and 3′ ends.

Sequencing of the AustB1 Insert

This was carried out using Sequenase 2.0 T7 polymerase (USB Biochemicals) as described for the sequencing of the M1AUS insert.

Polymerase Chain Reactions

Preparation of cDNA
mRNA (1 μg) from 11 day old post-infection U.K. H. contortus, prepared as described for adult UK worms, was mixed with T17 adaptor-primer (5′GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT 3′) in diethyl pyrocarbonate (DEPC)-treated water, then heated to 65° C. for 5 minutes and immediately placed on ice. Methylmercury hydroxide was added to a final concentration of 28.6 mM and the mixture incubated at room temperature for 3 minutes. 2-mercaptoethanol was added to a final concentration of 14.2 mM and the mixture was placed on ice. To synthesize cDNA, RNAse Guard (Pharmacia) was added to 1 unit/μl, Reverse Transcriptase buffer (Life Sciences) to 1 times concentration, dATP, dGTP, dCTP and dTTP each to 1 mM, and AMV Reverse Transcriptase (Life Sciences) to 2 units/μl (all given as final concentrations). The reaction was incubated at 41° C. for 75 minutes, then extracted with phenol and chloroform and purified by spun column chromatography (Maniatis et al, 1982). The purified reaction mix was diluted 2.5-fold and stored at 4° C.
PCR Amplification of the cDNA Using M1AUS-Specific Primers
PCR reactions were carried out using a Programmable Thermal cycler (M.J. Research Inc.). The reaction mix contained 1 μl out of the 250 μl diluted cDNA prepared as described above, 25 μmol of the first strand T17-adaptor-primer, 25 μmol of second strand amplification primer (either that based on positions 865-884 (5′ACGGGTGTTCGGTTTCCGTAT 3′) or that based on positions 30-49 (5′GCTGAATCTAACTCCAATCC 3′) of the M1AUS sequence (SEQ ID NO: 5)), 1× Taq buffer (Northumbria Biologicals Ltd) and 0.5 mM each of dATP, dTTP, dGTP and dCTP, in a 100 μl reaction volume and covered with 40 μl mineral oil to prevent evaporation. This mix was then heated in the thermal cycler to 95° C. for 2 minutes then held at 72° C. Whilst at 72° C. 2 units of Taq Polymerase (Northumbria Biologicals Ltd) was added and mixed gently with the other reactants. The following program was then carried out in the thermal cycler:
Step 1 Anneal at 50° C. for 5 minutes
Step 2 Extend at 72° C. for 40 minutes
Step 3 Denature at 94° C. for 40 seconds
Step 4 Anneal at 50° C. for 2 minutes
Step 5 Extend at 72° C. for 3 minutes
Step 6 39 cycles of steps 3 to 5
Step 7 Final extension at 72° C. for 15 minutes

Step 8 Hold at 4° C.

These conditions were established from Frohman et al., (1988).
Cloning of the PCR products
The PCR products from the above reactions were separated by electrophoresis in an agarose gel. Bands of DNA of approximately 2.5 and 3.5 kb were electroeluted onto glass fibre (Whatman), phenol extracted and purified by G50 chromatography (Pharmacia) (Sambrook et al., 1989). The purified DNA was ligated into pT7Blue T-vector (Novagene)-following the manufacturer's instructions.

Sequencing of the 2.5 kb and 3.5 kb PCR Products

DNA sequencing was carried out with a Sequenase 2.0 kit (US Biochemicals) using the “oligonucleotide walking” technique described in the section on sequencing of M1AUS.

Polymerase Chain Reactions for the 5′ Ends

Preparation of First Strand cDNA
1 μg of mRNA from 11 day post-infection UK Haemonchus contortus prepared as described for adult worms was mixed with a constant primer (5′AAIGAAAGCGGATGGCTTGAIGC 3′) designed from a conserved region in AustB1 and the 2.5 kbPCR and 3.5 kbPCR products (SEQ ID NOS: 6, 7 and 8 respectively). The mixture was heated to 65° C. for 5 min., placed on ice and methyl mercury hydroxide added to a final concentration of 28.6 mM. The mixture was incubated at room temperature for 5 min., then 2-mercaptoethanol added to a final concentration of 142 mM and the mixture placed on ice. First strand DNA was prepared using reagents from the 5′ RACE system (Gibco/BRL) at a final concentration of 20 mM Tris/HCl pH 8.4, 50 mM KCl, 2.5 mM MgCl₂, 100 μg/ml BSA, 0.5 mM of dATP, dCTP, dGTP, dTTP. 200 Units of Superscript Reverse Transcriptase were added and the reaction was incubated at 42° C. for 30 min. and then heated at 55° C. for 5 min. RNAse H was added to a final concentration of 100 Unit/ml and the reaction incubated at 55° C. for 10 min. and then placed on ice. The cDNA was purified through a Glassmax spin column (Gibco/BRL) and stored at −20° C.
C-Tailing of the cDNA
⅕ of the first strand cDNA was heated at 70° C. for 5 min then chilled on ice for 1 min. Reagents from the 5′RACE system (Gibco/BRL) were added to a final concentration of 10 mM Tris/HCl pH 8.4, 25 mM KCl, 1.25 mM MgCl, 50 ug/ml BSA, 0.2 mM dCTP. 500 Units/ml Terminal transferase were added and the reaction incubated at 37° C. for 10 min, then heated at 70° c. for 15 min and stored on ice.
PCR Amplification using AustB1, 2.5 kbPCR and 3.5 kbPCR Specific Primers
The PCR reactions were carried out in a programmable Thermal Cycler (M.J. Research Inc.). For the 3′ end one of 3 primers was used.
1. A primer specific for the 2.5 kb PCR product based on positions 374 to 394 (5′TGTTGTGGCTAATTTCGTCCA 3′).
2. A primer specific to the 3.5 kb product based on positions 1210 to 1229 (5′CATCTTIAGTTATCTGACCAG 3′).
3. A primer specific for the cDNA clone AustB1 based on positions 357 to 377 (5′ GACCATCGCTGATGAAGTCGG 3′). For the 5′ end of the reactions a common ‘Anchor primer’ (5′CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG3′) was used. Each reaction mixture contained 4 μl of the 50 μl of C-tailed cDNA, 25 pMol of the appropriate 2 primers, 1× Taq polymerase buffer (Boehringer/Mannheim) and 0.5 mM each of dATP, dCTP, dGTP and dTTP to a final volume of 100 μl. This mix was covered with 50 μl of mineral oil and heated to 95° C. in the cycler for 2 min. The reaction mix was held at 80° C. whilst 0.6 units of Taq Polymerase were added and then put through the following programme:

1. Anneal at 50° C. for 5 min.

2. Extend at 72° C. for 10 min.

3. Denature at 94° C. for 45 sec.

4. Anneal at 50° C. for 1 min.

5. Extend at 72° C. for 2.5 min.

6. 39 cycles of 3 to 5.

7. Extend at 72° C. for 15 min.

8. Hold at 4° C.

Cloning of the 5′ PCR Products

The PCR products were separated by electrophoresis on an agarose gel and bands of the expected size, circa 1.3 kb, were cut out, the DNA purified using a GENECLEAN kit and ligated into PT7Blue T-Vector (Novagene) according to the manufacturer's instructions.

Polymerase Chain Reaction for the Production of the 3′ End of AUSTB1

The first strand cDNA used was that described for the production of cDNA for use with M1AUS primers. A specific primer from 1414 to 1435 (5′TCTTGAAGAAATGAAAAAGCTT 3′) in AustB1 (SEQ ID NO: 6) was used with the T17 Adaptor primer used for the M1AUS PCR and the reactions carried out in a thermal cycler (M.J. Research Inc). The reaction mixture consisted of 25 pMol of each primer, 2 μl of cDNA, 1× Taq Polymerase buffer (Boehringer Mannheim), 0.5 mM dATP, dCTP, dGTP and dTTP in 100 μl. These were covered with 50 μl mineral oil and heated to 95° C. for 2 min. 0.6 μl of Taq polymerase was added and the same programme cycle carried out as for the 5′ PCR described above.

Cloning and Sequencing of the 3′ Products.

The products of the PCR were separated by electrophoresis in an agarose gel and a band of the expected size, 1.3 kb, cut out and the DNA purified using a GENECLEAN kit and ligated into PCR-Script (Stratagene) according to the manufacturer's instructions.

Sequencing of the Cloned PCR Products

The DNA from the PCR clones was sequenced using a Sequenase 2.0 kit (United States Biochemical) as instructed by the manufacturer. Oligonucleotide primers were used to “walk along” the DNA of the clones from both the 5′ and 3′ ends.

Analysis of all DNA Sequences

Sequences were analysed using the GCG (Genetics Computer Group) Sequence Analysis Software Package, Devereux et al., 1984.

Northern and Southern Blot Analyses

Preparation of Northern Blots

Northern blots were performed in formaldehyde gels essentially as described in Maniatis et al. (1982). mRNA samples (from 11-, 15- and 23-day old adult H. contortus) were treated with 17.5% v/v formaldehyde and 50% v/v formamide in MOPS buffer (20 mM 3-(N-morpholino)propanesulphonic acid, pH 7.0, 8 mM sodium acetate, 1 mM EDTA) at 65° C. for 15 minutes, and cooled on ice. Gels were electrophoresed in MOPS buffer, and blotted onto Duralon membranes by capillary transfer as described in Sambrook et al., (1989).

Preparation of Southern Blots

Two gm of adult Haemonchus contortus which had been snap-frozen in liquid nitrogen were ground to a fine powder n liquid nitrogen. The powder was added slowly to 25 ml of lysis buffer (0.05 M Tris-HCl, pH 8, 0.1 M EDTA, 1% w/v Sarkosyl, 0.05 mg/ml proteinase K (Boehringer Mannheim)) and incubated for two hours at 65° C. The suspension was then extracted twice with one volume of phenol plus chloroform, twice with two volumes of chloroform, and ethanol. precipitated. The precipitated genomic DNA was resuspended in 20 ml of Tris, EDTA buffer (TE, pH 8) overnight at 4° C. on a rocking table, then dialysed against two changes of one litre of TE. RNA was removed by incubating with DNase-free RNase A Type 1 (Sigma) at a final concentration of 20 μg/ml, at 37° C. for one hour, followed by one extraction with phenol-chloroform, one extraction with chloroform, and ethanol precipitation, as above. The precipitated genomic DNA pellet was washed twice with 70% v/v ethanol, and resuspended in one ml TE, as above.
Genomic DNA was digested with EcoRI or HindIII (25 μg of DNA in each digest) overnight at 37° C., then electrophoresed at 5 μg per track on a 1%. w/v agarose gel in Tris-acetate buffer. The gel was Southern blotted by capillary transfer as described in Maniatis et al., (1982) onto Hybond-N membrane (Amersham International). DNA was fixed onto the membrane using ultraviolet light, according to the manufacturer's recommendations.

Preparation of Probes

pBLUESCRIPT plasmids containing the M1AUS, B1A or AustB1 inserts were digested with EcoRl. pT7Blue plasmids containing 3.5 kbPCR product inserts were digested with BamHl and those containing 2.5 kbPCR product inserts were digested with BamHl and Xbal. Digests were electrophoresed, the inserts recovered and radioactively labelled with α-³²P-dCTP by nick translation as described above under screening of the λZAP library.

Hybridization Conditions

For Southern blots
The membranes were cut into strips and pre-hybridised in hybridisation buffer as described earlier, for 3 hours at 28° C. Genomic DNA Southern blot strips were hybridised to each of the above probes overnight at 28° C., washed twice at room temperature (24° C.) then twice at 42° C., in 2×SSC containing 0.1% w/v SDS (moderate stringency) and autoradiographed. Following development of the autoradiographs, strips were re-washed at a high stringency (0.1×SSC, 0.1% w/v SDS at 65° C.) and re-autoradiographed.

For Northern Blots

For Probes M1, M1AUS and B1A (SEQ ID NOS: 1, 5 and 2 Respectively)

The Northern blot of mRNA from 11, 15 and 23 day-old Haemonchus contortus was probed first with the M1 insert. The filter was prehybridised for 2 hours at 42° C. in 2×SSC (where 20×SSC=3 M NaCl, 0.3 M sodium citrate, pH 7.2) containing 5×Denhardt's (0.1% w/v Ficoll 400 (Pharmacia), 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin Fraction V (Sigma Chemical Corp)), 0.5% SDS (sodium dodecylsulphate), 10% dextran sulphate, 0.1 mg/ml salmon testes DNA and 50% de-ionised formamide. The hybridisation to the probe was performed in the same buffer overnight at 42° C. The filters were washed twice for 30 minutes in 2×SSC containing 0.5% SDS and 50% formamide, twice for 30 minutes in 2×SSC containing 0.5% SDS and twice for 30 minutes in 2×SSC. The first wash was at 42° C. and all remaining washes were at 37° C. After autoradiography the blot was stripped by washing in boiling 0.1% SDS and re-autoradiographed to ensure removal of the probe. The same blot was then probed with the M1AUS insert, washed and autoradiographed. The blot was again stripped and checked and when clear was then probed with the B1A insert. After stripping again the blot was then probed as described below.

For Probes AustB1, 2.5 kb and 3.5 kb PCR Products (SEQ ID NOs: 6, 7 and 8)

The Northern blot was hybridised with the AustB1 insert using the conditions of moderate stringency as described for Southern blot hybridisation. After autoradiography the blot was stripped with boiling 0.1% SDS according to the membrane manufacturer's instructions (Amersham), then probed with the 2.5 kb PCR insert (clone 2), stripped again and probed with the 3.5 kb PCR (clone 2) insert.

Digestion of H110D and Assays of Enzyme Activity

Preparation of H110D

Native H110D (H110DN) was prepared according to the methods described in WO88/00835 and WO90/11086.

Preparation of an Elastase Fragment (H11S) of H110D

Adult Haemonchus were homogenized in 10 volumes of ice cold PBS/0.02% sodium azide and then centrifuged for 20 minutes at 13000 rpm. The pellet was resuspended in 10 volumes of PBS/azide, rehomogenised and the centrifugation repeated. Following resuspension in 50 mM MOPS buffer pH 7.4 (the volume for suspension is 1 ml for each 0.17 g of worms) and pre-warming at 37° C. for 30 minutes, the pellet material was digested with elastase (800 μl/20 ml of suspension; 1 mg/ml fresh stock solution made up in 1 mM HCl/2 mM Ca²⁺) for one hour. The digestion was stopped by the addition of 3,4 dichloroisocoumarin (300 μl of stock 10 mM in DMSO/200 ml of digest). The mixture was centrifuged at 13000 rpm for 20 minutes and the pelleted material retained. The supernatant was ultracentrifuged at 100000 g for 1 hour 20 minutes. The resultant supernatant liquid was applied to a ConA column and the binding fractions obtained. For analysis, this fraction was run on an SDS-polyacrylamide gel and electrophoretically transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore), lightly stained with Coomassie blue, the 105 kd band excised and analysed in a gas phase amino acid sequenator. For vaccination studies, the ConA binding fractions were further purified by concentrating and applying to a Superose 12 (Pharmacia) gel filtration column and collecting those fractions containing aminopeptidase activity.

Thermolysin Digestion of H110D

The H110D doublet was purified by electroelution from a preparative scale 8% SDS-polyacrylamide gel to give H110DE, as described in WO90/11086 and by electroelution of a dimeric form, running at just over 200 kd (which yielded the characteristic doublet at 110 kd when re-run on SDS-PAGE) to give H110DE. Solutions of H110DE were concentrated to 200 g, calcium chloride was added to 5 mM and the mixture warmed to 37° C. A freshly prepared solution of 1 mg/ml Thermolysin (in 1 mM HCl, 2 mM CaCl₂) was added in a ratio of 0.1 μg Thermolysin per μg H110DE. The mixture was incubated at 37° C. for 120 minutes and the reaction then stopped by addition of 5 μl of 0.5M EDTA.
The protein fragments were separated by 15% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore). Following staining with Coomassie blue the most intense discrete bands were excised and analysed in a gas phase amino acid sequenator.

Preparation of an H110D Fraction (H11A) Enriched for Aminopeptidase A Activity

The pelleted material obtained after elastase treatment by centrifugation at 17000 g for 20 min. (see above) was resuspended in PBS at 4° C. and repelleted by centrifugation then resuspended in 1% Tween in PBS/azide and left (with stirring) for 1 hour. The suspension was centrifuged at 17.000 g for 20 minutes and the supernatant removed. The pellet was repeatedly extracted with 1% Thesit in PBS/azide. The supernatants after centrifugation at 17,000 g for 20 minutes were combined and ultracentrifuged for 1 hour 20 minutes at 100,000 g. The supernatant was applied to a ConA affinity column (Affigel, Biorad) and the bound material eluted and further fractionated by ion exchange chromatography on a MonoQ column.

Assays of Enzyme Activities

α-amino acylpeptide hydrolase (microsomal) aminopeptidase activities in H110D preparations were characterised by assays, in solution, using L-leucine, methionine, phenylalanine, α-glutamic acid and lysine p-nitroanilides (pNA). All the amino acid p-nitroanilide substrates (except α-glutamic acid-) were obtained from Sigma, Poole, Dorset UK, α-glutamic acid was from Fluka, Dorset, UK. Single hydrophobic (leucine- or phenyalanine-) and charged (α-glutamic acid-) amino acid pNA known to be substrates for mammalian aminopeptidase-M (ApM) and -A (ApA) respectively, were chosen to measure the effect of enzyme inhibitors and serum inhibition on H110D aminopeptidase activities.

(a) Microplate Assay

Micro-ELISA plate (Dynatech Immulon 1, Virginia, USA) wells were each filled with 250 μl of either 50 mM HEPES or MOPS pH 7 plus 1-10 μl of the fraction to be assayed. The plates were then pre-incubated at 37° C. for 10 minutes prior to the addition of 10 μl of 25 mM amino acid p-nitroanilide substrate per well. The time zero optical density (OD) at 405 nm was then measured using an ELISA plate reader and the plates were then incubated at 37° C. for 15-30 minutes. The final OD reading was then taken as before and the OD change per minute per milligram of protein calculated.

(b) Inhibitor Sensitivity

The method used for the enzyme assay was as described in (a) above except that the inhibitors were added to the 250 μl of buffer plus 10 μl of 1 mg/ml ConA H110D and pre-incubated for 10 minutes at 37° C. prior to addition of the substrates (leucine-pNA or α-glutamic acid-pNA). The percentage inhibition was calculated as follows
x−y/x×100=percentage inhibition.
Where x=the ΔOD/min of the enzyme with no inhibitor added and y=ΔOD/min of the enzyme plus inhibitor. Nine compounds with differing enzyme class inhibition were tested individually: Amastatin, Bestatin (metalloprotease, aminopeptidase), 1,10 phenanthroline, EDTA (metalloprotease), phosphoramidon (metalloprotease, thermolysin, collagenase), Aprotinin (serine protease), Pepstatin (aspartic protease), PMSF (serine and cysteine protease) and E64 (cysteine protease). Amastatin, bestatin, 1,10 phenanthroline, PMSF and pepstatin were obtained from Sigma, Dorset, UK. All the other inhibitors were obtained from Boehringer-Mannheim. Each inhibitor was used at two concentrations equal to or greater than the maximum concentrations recommended by Boehringer-Mannheim.

(c) Assay of Antiserum Inhibition of Enzyme

The assay was as described in (a) above except that two micro-ELISA plates were set up. In one plate 10 μl of ConA H110D (1 mg/ml) plus 10 μl of antiserum from each sheep per well were pre-incubated at 37° C. for 15 minutes. The second plate, with 250 μl of HEPES/bicarbonate buffer plus 10 μl of either phenylalanine-pNA or α-glutamic acid-pNA substrate per well was also pre-incubated at 37° C. for 15 minutes. The ConA H110D-serum mixtures were then added to the buffer-substrate mixtures in the second plate with a multi-pipette. The ΔOD/min was determined. For the purposes of the correlations, percentage inhibition was calculated using the formula in (b) above here x represents the mean ΔOD/min for the wells which contained enzyme plus serum from negative control sheep (vaccinated with horse spleen ferritin) and y=the ΔOD/min for the wells which contained enzyme plus sera from individual sheep vaccinated with H110D. For the purposes of the correlations (FIG. 18), the percentage protection was calculated using the formula in (b) above where x=either the average faecal egg output per gram or the average worm burden of the controls, and y=either the faecal output per gram during the experiment or the worm burden of the individual sheep vaccinated with H110D.

Localisation of Enzyme Activity by Histochemistry

Aminopeptidase activity was demonstrated on 10 μm cryostat sections of adult Haemonchus contortus using L-leucine 4-methoxy-β-naphthylamide and L-glutamic α-4-methoxy-β-naphthylamide as substrates by the methods of Nachlas et al. (1957), Nakane et al. (1974) and Lojda et al. (1980).

Expression of Recombinant H110D in the Eukaryotic Baculovirus-Insect Cell System

Construction of Expression Plasmids

The 3.5K PCR fragments described above, were generated by amplification between an oligo dT adaptor (which contained a SalI site) and oligo 872, which represents base no.'s 30-49 in the M1AUS sequence, and were cloned into the pT7Blue vector. Clone pT73.5-2 is oriented with the vector polylinker BamHI site at its 5′ end and the HindIII, XbaI and SalI sites at the 3′ end. The sequence at the 5′ end is:

	Bam HI
	5′ GG ATC CGA

	*!-----oligo 872-----------!--3.5K-----
	TTG CTG AAT CTA ACT CCA ATC C .......3′
	Leu Asn Leu Thr Pro Ile .........

The asterisk indicates the 3′ dT/dA overhang used for cloning in the pT7 Blue Vector.

The 3.5 PCR clone 2 was digested with HindIII (single site in the vector polylinker sequence at the 3′ end of the 3.5K gene) and the ends filled in with deoxynucleotides using DNA polymerase I (Klenow fragment), according to Maniatis et al (1982). A BamHI linker (5′CGGATCCG 3′, New England Biolabs Catalog no. 1021) was ligated to the blunt ends, and clones with an extra BamHI site, allowing a full-length H110D gene sequence to be excised as a BamHI fragment, selected. The BamHI fragment was isolated by electrophoresis on a 0.6% w/v agarose gel in tris-acetate buffer, followed by purification using GENECLEAN (BIO101); this procedure was carried out twice. The purified fragment was then ligated to BamHI-cut, phosphatased pBlueBac II (Invitrogen Corp.), and clones carrying the fragment in the correct orientation (ie. with the 5′ end of 3.5 PCR clone 2 placed under control of the baculovirus polyhedrin promoter) determined by digestion with NheI and XbaI. The resultant plasmid was partially digested with BamHI and the ends filled in as described above. An NcoI linker containing an ATG (5′CCCATGGG 3′; New England Biolabs Catalog no. 1040) was added and the mixture ligated. Clones which had the linker ligated at the 5′ end BamHI site of 3.5 PCR clone 2 rather than the 3′ site, were determined by digestion with NcoI. The resultant plasmid, designated pBB3.5-2(N), is depicted diagrammatically in FIG. 19.
This construction results in the insertion of an in-frame ATG at the 5′ end of the 3.5 PCR clone 2 insert, to initiate translation. The sequence surrounding this initiating ATG is:

BamHI--NcoI link-BamHI----*!-----oligo 872-----------!--
5′GGATCCCC ATG GGG ATC CGA TTG CTG AAT CTA ACT CCA ATC C..
Met Gly Ile Arg Leu Leu Asn Leu Thr Pro Ile ...

The expressed protein will be missing amino acids 2-9 of the corresponding H110D sequence, and will have 3 amino acids of linker sequence immediately following the ATG.

Generation of Recombinant Baculovirus Containing H110D Sequences

The plasmid pBB3.5-2(N) was transfected into Spodoptera frugiperda (Sf9) cells (obtainable from Invitrogen Corp), using linear Autographica californica nuclear polyhedrosis virus (ACNPV) DNA and cationic liposomes (Invitrogen Corp. transfection module), according to the manufacturer's instructions-. Cells were cultured in TC-100 medium (SIGMA) supplemented with foetal calf serum (CSL Ltd; heat-inactivated at 56° C. for 45 minutes) and antibiotics (penicillin/streptomycin, gentamycin; CSL Ltd). A control transfection, using a pBB3.5-2 (N) plasmid with the ATG inserted at the 3′ end of the 3.5 PCR clone 2 sequence, was also carried out. Recombinant plaques were selected on the basis that the pBlueBac II vector also encodes E. coli β-galactosidase (β-gal), by including X-gal in the agarose overlay at the recommended level. A selection of blue plaques were picked and subjected to two further rounds of plaque purification, after which time infected monolayers showed no evidence of contaminating wild-type virus (which would be evidenced by the presence of nuclear polyhedra). Purified viruses were designated 3.5-2-P2A, -P3A and -P4A, and were amplified by two sequential infections of Sf9 cells before use. A plaque purified from the control transfection was designated 3.5-2-rev.
Assessment of H110D Expression in Insect Cells Infected with Recombinant Baculovirus
Monolayers of Sf9 cells (1×10⁶cells in 25 cm²bottles) were infected with the 3.5-2 viruses, with wild-type (wt) virus, with a control virus expressing β-gal, or were not infected. After four days growth at 26° C., monolayers were detached by gentle shaking, the cells recovered by centrifugation (2000 rpm, 10 minutes), and the cell pellets disrupted by three cycles of freeze-thawing. The lysates were resuspended in 500 μl PBS, and 25 μl aliquots assayed for ApM activity by the micro-well assay.
15 μl aliquots (3×10⁴cell equivalents) of the above lysates were electrophoresed on denaturing 7.5% SDS-polyacrylamide gels. One gel was then stained with Coomassie blue to assess levels of expression. The other gel was Western blotted, and the blot probed with anti-H110DN (as described earlier).

Results

Analysis of Immunopositive Clones

Analysis of Antibodies Affinity Purified on Clone M1

Affinity-purified antibodies specific for each of the 5 antibody-positive clones were prepared and used to probe a Western blot of H110D-enriched extract. As shown in FIG. 8, all 5 clones appeared to recognise the H110D doublet. However, the reaction with clone M1 gave the strongest signal (FIG. 8 d) compared to the λgt11 negative control blot (FIG. 8 e). This clone was therefore investigated further.

Northern Blot Analysis With Clone M1

Northern blot analysis of Haemonchus contortus mRNA probed with the M1 insert is shown in FIG. 9. A single mRNA band was recognised, at approximately 3.5 kb. This is of sufficient size to code for a protein of about 110 kd.

Sequence Analysis of Clone M1

Analysis of restriction digests of the DNA with EcoRl showed the M1 insert to be approximately 300 bp. The DNA sequence or the M1 fragment was determined (SEQ ID NO: 1, and is shown in FIG. 3. The fragment comprises 295 bp with an open reading frame starting at base number 3.
Northern Blot Analysis with Clone B1A
Northern blot analysis of Haemonchus contortus mRNA probed with the B1A insert is shown in FIG. 9 c. As for M1, a single mRNA band was recognised, at approximately 3.5 kb.

Sequencing of Clones B1A and B2

Clones of B1A were sequenced (SEQ ID NOS: 2 and 3) and the full sequence (SEQ ID NO: 2) is shown aligned to H11-1 (SEQ ID NO: 19) and AustB1 (SEQ ID NO: 6) in FIG. 5. The insert is 484 bp and has a full ORF from the first base. The 3 fragments of B2 resulting from digestion with EcoRl were sequenced and the complete sequence for B2 (SEQ ID NO: 4) is 581 bp. It is shown aligned with H11-2 in FIG. 4. The sequence has an ORF from position 3 to 213 bp, the stop codon and untranslated region matching that of the 2.5 kb PCR product sequence (SEQ ID NO: 7).
Expression of M1 and B1A in E. coli
When subcloned into a GST expression vector, clones were obtained which expressed fusion proteins of 38-40 kd for M1 and of 45 kd for B1A. These agree with the predicted sizes for these inserts, allowing for the molecular weight of glutathione-S-transferase. Both fusion proteins reacted very strongly on Western blots with affinity-purified antibodies to H110DE (FIG. 11). The fusion proteins were expressed as insoluble inclusion bodies.
Antibody Responses in Sheep Vaccinated with M1-GST and B1A-GST Fusion Proteins
Antisera from sheep injected with the fusion proteins were tested by Western blotting against H110D preparations. Both GST-M1 and GST-B1A raised antibodies which specifically recognised the H110D doublet (FIG. 12). Sera from negative control sheep did not recognise the H110D doublet.
Isolation and Characterisation of Clones Selected by Hybridisation with M1 or B1A Insert DNA
The confirmed positive clone hybridising to the M1 probe was designated M1AUS (SEQ ID NO: 5), and the clone hybridising to B1A was designated AustB1 (SEQ ID NO: 6). Restriction digestion of purified plasmid DNAs with EcoRI indicated an insert size of about 900 bp for M1AUS and of about 1.6 Kb for AustB1. As shown in FIG. 9 b) and 9d), on Northern blots, M1AUS and AustB1 hybridised to the same-sized mRNA (about 3.5 kb) as did M1 and B1A.

Sequence Analysis of M1AUS

Full sequencing of the M1AUS fragment was carried out using synthetic oligonucleotides to “walk” along the DNA from either end. Analysis of the sequence obtained revealed that the M1AUS insert was 948 bp, as shown in FIG. 3. The sequence (SEQ ID NO: 5) begins with an ATG (which codes for methionine) and has an open reading frame (ORF) over the whole of its length. The sequence is 19 base pairs longer than the M1 sequence at the 5′ end, and 634 bp longer at the 3′ end. The sequence common to the two clones (bases 20 to 314) were identical except for two nucleotide differences in a third codon position. Comparison of all possible reading frames to various databases showed that the reading frame starting with the ATG at base number one shared homology with the members of a family of microsomal aminopeptidases.
Sequence of AustB1
Full sequencing of the AustB1 fragment was carried out using synthetic oligonucleotides to “walk” along the DNA from either end. The DNA sequence (SEQ ID NO: 6) is shown in FIG. 5. The clone is 1689 bp long and has an ORF from residue 2. This sequence forms part of H11-1 as shown in FIG. 1. The amino acid translation of this sequence showed the zinc binding site motif characteristic of aminopeptidases.
PCR Amplification of the cDNA of the H110D mRNAs PCR using M1 AUS Primers
cDNA was synthesized from Haemonchus contortus mRNA using as primer oligo-dT containing an adaptor sequence to facilitate subsequent cloning and manipulation of the DNA. This cDNA was then used to amplify the M1AUS sequence by PCR, using as the 5′ end primer a synthetic oligonucleotide based on positions 865-885. A PCR fragment of about 2.5 Kb was amplified. This is approximately the expected size of the fragment, based on the known size of the mRNA and on mammalian aminopeptidase cDNA sequences.
A second set of PCR reactions was performed using a primer near the 5′ end of M1AUS (bases 30-49). Four bands were detected on an agarose gel. The largest of these, at 3.5 kb, corresponds to the predicted size for the PCR product.
Cloning and Sequencing of 2.5 kb and 3.5 kb PCR Products from M1AUS Primers
The 2.5 kb and 3.5 kb PCR products were cloned and designated 2.5PCR (SEQ ID NO: 7) and 3.5PCR (SEQ ID NOS: 8, 14 and 15 for clone numbers 2, 10 and 19 respectively). On Northern blots 2.5PCR and 3.5PCR (clone 2. 3.5PCR-2) hybridised with mRNA of about 3.5 kb (FIGS. 9 e, f) in the same pattern (with respect to age of Haemonchus used to obtain the mRNA) as M1, B1A, M1AUS and AustB1.
Full sequencing of clones was carried out by ‘oligonucleotide walking’, As shown in FIG. 1, the sequence for the 2.5 kb product (SEQ ID NO: 7) is part of H11-2 (SEQ ID NO: 20) and the sequence for the 3.5 kb product (SEQ ID NO: 8) is the major part of H11-3 (SEQ ID NO: 21). The amino acid translations of both these sequences (shown in FIG. 6) contain the zinc binding motif His Glu Xaa Xaa His Xaa Trp (HEXXHXW) characteristic of microsomal aminopeptidases.

Sequencing of 5′ End PCR Clones

cDNA was synthesised using a primer matching a conserved sequence in cDNA clone AustB1, 2.5PCR and 3.5PCR (SEQ ID NOS: 6, 7 and 8) which hybridises with the mRNA for these sequences about 1.3 Kb from the 5′ end. The cDNAs were C-tailed at the 5′ end and then PCR reactions carried out with a universal Anchor (A) primer for the 5′ end and three primers specific for each of the sequences AUSTB1, 2.5PCR and 3.5PCR-Clone 2 (SEQ ID NOS: 6, 7, 8) for the 3′ end. The reactions each gave a product of the predicted size, just under 1.3 kb: 1301 bp (SEQ ID NO: 9), 1280 bp (SEQ ID NO: 10) and 1292 (SEQ ID NO: 11) respectively. All three sequences have an untranslated region at the 5′ end (FIG. 2). All begin with the same 22 bp sequence (5′ GGTTTAATTACCCAAGTTTGAG 3′) which is known as the Spliced Leader Sequence 1 (SL1) and is present in the untranslated 5′ region of a wide variety of nematodes Huang et al., 1990. In SEQ ID NOS: 9 and 10, the SL1 sequence is immediately before the initiating ATG. In SEQ ID NO: 11 there are 13 bp between the SL1 and the initiating ATG. All three sequences have full ORFs.

Sequencing of AustB1 3′ End PCR Clone

Using a specific primer matching positions 1414-1438 in Aust B1 (SEQ ID NO: 6), the PCR product gave a band as predicted of about 1.3 kb. Sequencing of the cloned band yielded the sequences SEQ ID NOS: 12 and 13. They gave an ORF from 1-615 bp and a substantial untranslated region.

Sequence Analysis of Cloned PCR Products

Composites of the sequences described above, designated H11-1, H11-2 and H11-3, are shown in FIG. 2. The amino acid sequences predicted from these are shown in FIG. 6 a. The validity of the predicted translations of the DNA sequences presented is substantially confirmed by the matches with amino acid sequences determined by Edman degradation from CNBr and proteolytic cleavage fragments (FIG. 7). Thus 27 residues of the 29 residue N-terminal sequence of H11S (SEQ ID NO: 16) match H11-2 from residues 61-90 (FIG. 7 b). The matches of valine (V) at position 78 and glycine (G) at position 90 are characteristic of H11-2 since H11-3 has asparagine (N) at position 90 and H11-1 has leucine (L) at position 86 (which corresponds to position 78 in H11-2). Two residues of the H11S N-terminus amino acid sequence (SEQ ID NO: 16) do not match any of the three H110D sequences presented here. To be particularly noted are the exact matches of the very similar, but distinctive sequences Pep A and B (previously described in WO90/11086) with H11-2 and H11-1 respectively in the region of residues 540-555. Similarly in the 450-470 residue region, Pep D is an exact match for H11-2, while the similar but distinct Pep E matches more closely H11-3.
By way of example the translated amino acid sequence of one of the full-length sequences (H11-3) is compared in FIG. 6 b with two sequences for mammalian microsomal aminopeptidases. The homology of the H110D translation with these aminopeptidases is shown by boxing identical amino acids. A characteristic motif of microsomal aminopeptidases is the amino acid sequence HEXXHXW, which functions as the zinc binding site (Jongeneel et al., 1989; Vallee et al., 1990); this is shown by asterisks in FIG. 6. This sequence, which is shown to be present in the translations of H11-1, H11-2 and H11-3, is conserved in all the microsomal aminopeptidases. Other features common to the mammalian and Haemonchus microsomal aminopeptidases are the presence of a comparatively short intracellular region, a single transmembrane sequence adjacent to the N-terminus and several potential glycosylation sites. The levels of homology (similarities of 52-55% and identities of 30-32%) of H11-1, -2 and -3 to mammalian microsomal aminopeptidases are shown in Table 2.

Southern Blot Analyses

The results of H. contortus genomic DNA Southern blots probed with various H110D cDNA clones and PCR products are shown in FIG. 10. All probes show multiple bands of hybridisation; this is typical of a multigene family. As expected, B1A and AustB1 showed similar hybridisation patterns to each other, as did M1AUS and 3.5 kbPCR. However, these patterns were noticeably different from each other and from that seen with the 2.5 KbPCR probe, even under conditions of moderate stringency (FIG. 10A), reflecting the differing levels of homology between these three cDNAs.

Demonstration of Aminopeptidase Activities Associated With H110D

Microsomal aminopeptidase activity was found to associate with those fractions containing H110D, that is the supernatants from ultracentrifugation of Thesit extracts, ConA binding fraction (ConA H110D) and the fractions containing H110D obtained by ion exchange chromatography on a MonoQ column (Table 3). The specific activities with all substrates tested increased as the purity of the H110D increased.

TABLE 3

ENZYME ACTIVITIES OF FRACTIONS FROM
A TYPICAL H110D PREPARATION

	SPECIFIC ENZYME ACTIVITIES
	(O.D./minute/mg protein)

	Phenyl-			α-
	alanine-	Leucine-	Lysine-	Glutamic	Methionine-
FRACTION	pNA	pNA	pNA	acid-pNA	pNA

Phosphate	0.20	0.08	0.12	0.01	0.16
buffered
saline (PBS)
1% Tween	0.15	0.15	0.09	0.01	0.09
20/PBS
1% Thesit/PBS	1.94	1.25	0.57	0.54	1.91
ConA H110D	4.08	3.01	1.43	2.09	3.84
CamQ H110D	6.55	5.01	3.01	3.90	6.41

Effects of Inhibitors of Mammalian Aminopeptidases on H110D Aminopeptidase Activities

Addition of the inhibitor bestatin (which inhibits mammalian microsomal aminopeptidase) to ConA H110D at the concentration recommended by the supplier (Boehringer Mannheim) reduced the activity against leucine-p-nitroanilide by approximately 70%. A series of experiments were performed to test inhibition of activity by a range of protease inhibitors. Those inhibitors which were not specific for metalloproteases or aminopeptidases had no inhibitory effects on the reaction rate. Inhibitors that are known to affect metalloproteases or aminopeptidases did have an effect on reaction rates, as shown in Table 4. The most effective inhibitor was 5 mM phenanthroline.

TABLE 4

Inhibition of H110D aminopeptidase activities
using various protease inhibitors

		PERCENTAGE	PERCENTAGE
		INHIBITION	INHIBITION
		α-glutamic acid-	Leucine-p-
	CONCEN-	p-nitroanilide	nitroanilide
INHIBITOR	TRATION	substrate¹	substrate²

Amastatin	10	μM	0	61
	50	μM	0	66
Bestatin	50	μM	23	63
	100	μM	35	68
EDTA	1	mM	17	8
	10	mM	21	16
1,10 phenanthroline	1	mM	78	78
	10	mM	85	87
Phosphoramidon	10	μM	1	0.8
	50	μM	1	7

¹A mammalian aminopeptidase-A substrate.
²A mammalian aminopeptidase-M substrate.

Sub-Fractionation of H110D

The distribution of activities associated with fractions from ion exchange chromatography of ConAH110D on MonoQ are shown in FIG. 13 a and SDS-PAGE of the fractions in FIG. 13 b.
Further, enzymatic activity was associated with sub-fractions of H110D separated by re-cycling free flow isoelectric focussing (FIGS. 14 and 15). At lower pI values (pH 4.5) these sub-fractions contain only the larger of the bands which make up the H110D doublet seen on SDS-PAGE and at higher values (pH 6.5) they contain only the smaller of bands which make up the H110D doublet. Intermediate fractions contain both these bands. The smaller band may also be obtained as a separate fraction by ion exchange chromatography on MonoQ-using the Pharmacia SMART® apparatus. All these sub-fractions bind sheep antibodies to H110D affinity purified on the protein expressed by the λgt11 clone M1 whereas antibodies eluted from λgt11 with no insert do not bind (FIG. 14 c). All the sub-fractions bind mouse monoclonal antibodies designated TS 3/19.7 (FIG. 14 c) which also bind to the recombinant protein expressed by clone M1. All the sub-fractions show microsomal aminopeptidase activity (FIG. 15 c) although this activity is comparatively low in the fractions obtained at the highest and lowest pIs. This lower activity may be attributed to lowered protein concentrations, effects of extremes of pH during sub-fractionation or a requirement for the presence of both larger and smaller bands for maximal activity.
Vaccination with Separated Components of H110D
The separated upper and lower bands obtained by free flow isoelectric focussing or by ion exchange chromatography induce the formation of protective antibodies when injected into sheep as exemplified in the following experiment. Thirty sheep approximately six months old were assigned to 5 groups of 6 so that each group was matched for range of weights of sheep. Each animal was injected with a total of 150 μg protein given in 3 equal doses as described in Munn et al. (1992) and Tavernor et al. (1992a, b) over a period of 54 days. The animals in group L were injected with the lower (smaller) band of the H110 Doublet, those in group U with the upper band, U+L with recombined upper and lower bands, D with the two (unseparated) bands obtained by free-flow isoelectric focussing at intermediate pH values and as a control (group C) horse spleen ferritin (an antigenic unrelated protein). The sheep were challenged with 10,000 infective larvae three weeks after the third injection and the experiment terminated 29-31 days post infection. The outcome of the experiment is summarised in FIG. 16. Injection of any of the sub-fractions reduced parasite egg output throughout the trial by some 90% and reduced total worm numbers by 63-84%, all showing a significant difference (p<0.05) to the controls using non-parametric statistical analyses. Reductions (70-88%) in the numbers of female worms were greater than the reductions in the numbers of male worms, and (except for the reduction in male worm numbers in the sheep injected with the recombined upper and lower bands where p<0.07) for both sexes the reductions were significant (p<0.05).
Vaccination with H11S and H11A
A truncated, water-soluble form of H110D (H11S; which retains its enzymic activity) may be obtained from the native molecule by treatment with elastase. This form was found to contain predominantly ApM-like enzyme activity and a Thesit extract of the elastase digested pellet (H11A) was enriched for ApA-like activity (see Table 5).
TABLE 5

Ratio

Aminopeptidase-M:Aminopeptidase-A

(leucine-pNA) (α-glutamic acid-pNA)

H110D 1.44:1

H11S 26.0:1

H11A 0.48:1

The following experiment shows that vaccination of sheep with either H11S or H11A gives protection against Haemonchus challenge or infection. Eighteen sheep approximately eight months old were assigned to 3 groups of 6 so that each group was matched for range of weights of sheep. Each animal was injected with a total of 100 μg protein given in 2 equal doses as described in Munn et al. (1992) and Tavernor et al. (1992a, b) over a period of 23 days. The animals in group A were injected with H11A, those in group S with H11S and those in group C with horse spleen ferritin (an antigenically unrelated protein) as a negative control. The sheep were then challenged with 10,000 infective larvae 25 days after the second injection and the experiment terminated at 34-36 days post infection. The outcome of the experiment is summarised in FIG. 17. Injection of H11S reduced parasite egg output throughout the trial by 89% and reduced total worm numbers by 76%. Injection of H11A reduced parasite egg output throughout the trial by 98% and reduced total worm numbers by 84%. These showed a significant difference (p<0.05) from the controls using non-parametric statistical analyses.

Inhibition of H110D Aminopeptidase Activities by Antibodies

Solutions containing H110D were incubated with sera from individual sheep injected with fractions containing H110D or from control sheep. The solutions were then assayed for aminopeptidase activities using phenylalanine and α-glutamic acid pNAs as substrates. The degree of inhibition of activity (maximally 80%) correlated with the level of protection shown by the individual sheep from which the sera were obtained (see FIG. 18).

Localisation of Enzyme Activity

Frozen sections of adult Haemonchus contortus were examined for aminopeptidase activity. As shown in FIG. 19, aminopeptidase enzyme activities are localised to the luminal surface of the intestine. H110D protein is also specifically found in this location.

Expression of H110D (3.5 PCR Clone 2) Using the Eukaryotic Baculovirus-Insect Cell System

Expression of Aminopeptidase Activity in Insect Cells

Infected cells were harvested and assayed for aminopeptidase activities using phe-, leu-, met- and lys-pNA as substrates. The assay was complicated by the observation that the insect cells possess an aminopeptidase activity with a marked preference for lys-linked amide bonds. However, cell extracts containing the expressed H110D additionally cleaved leu-, met- and phe-pNA in that order of preference.
Molecular Weight and Immunoreactivity of the Expressed H110D (3.5 PCR clone 2) Protein
Samples of infected or control cell extracts were electrophoresed on a 7.5% SDS-polyacrylamide gel, which was then stained with Coomassie Blue. The 3.5-2-P3A and 3.5-2-P4A infected cell lysates both had a band at the same size as H110D, 110 kd, which migrated directly beneath the co-expressed β-gal, which has a molecular weight of 120 kd. This 110 kd band was not present in any of the negative control lysates. (It was also absent from the P2A lysate, which did not express enzyme activity).
A duplicate gel was Western blotted and probed with anti-H110DN (FIG. 21). A very strong, specific positive immunoreaction was obtained to the 110 kd band expressed by 3.5-2-P3A and 3.5-2-P4A, and to the native H110D doublet in a control track containing ConA H110D, while no reaction was seen in any of the negative control tracks.

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Claims

1. A synthetic polypeptide comprising an amino acid sequence encoded by a nucleotide sequence which encodes a helminth aminopeptidase or an antigenic portion thereof, said nucleic acid selected from the group consisting of i) any sequence of SEQ ID NOs:1-15 or 19-21, ii) a sequence fully complementary to said sequence of (i), iii) a portion of said sequence of (i) wherein said portion of said sequence encodes an antigenic peptide of said helminth aminopeptidase, and (iv) a sequence which hybridizes with said sequence of (i) or (ii) under conditions of 2 times SSC, 65 C (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least 60% identity with said sequence of (i), other than a synthetic polypeptide corresponding to the protein doublet H110D or a synthetic polypeptide selected from:

(SEQ ID NO: 25) (a) Met Gly Tyr Pro Val Val Lys Val Glu Glu Phe, (SEQ ID NO: 26) (b) Met Gly Phe Pro Val Leu Thr Val Glu Ser, (SEQ ID NO: 27) (c) Met Gly/Phe Asn Phe Lys Ile Glu/Val Thr/Glu Ala Gly, (SEQ ID NO: 28) (d) Met Lys Pro/Glu Thr/Val Lys Asp/Ala Thr/Lys Leu - Ile Thr, (SEQ ID NO: 29) (e) Met Leu Ala Leu Asp Tyr His Ser - Phe Val, (SEQ ID NO: 30) (f) Met Leu Ala Glu/Tyr Asp Gln/Ala Glu Asp Val, (SEQ ID NO: 31) (g) Met Gly Phe Pro Leu Val Thr Val Glu Ala Phe Tyr, (SEQ ID NO: 32) (h) Met Lys Thr Pro Glu Phe Ala Val/Leu Gln Ala Phe/Thr Ala Thr Ser/Gly Phe Pro, (SEQ ID NO: 33) (i) Lys His/Tyr Asn/Val Ser Pro Ala Ala Glu Asn/ Leu Leu Asn/Gly, (SEQ ID NO: 34) (j) Lys - Thr Ser Val Ala Glu Ala Phe Asn, (SEQ ID NO: 35) (k) Lys Ala Ala Glu Val Ala Glu Ala Phe Asp - Ile - - - Lys Gly, (SEQ ID NO: 36) (l) Lys Ala Val Glu Val/Pro Ala Glu Ala Phe Asp Asp Ile Thr? Tyr - - Gly Pro Ser, (SEQ ID NO: 37) (m) Lys - Glu Glu Thr Glu Ile Phe Asn Met, (SEQ ID NO: 38) (n) Lys - - - Pro Phe Asn/Asp Ile Glu Ala Leu, (SEQ ID NO: 39) (o) Asp Gln Ala Phe Ser Thr Asp Ala Lys, (SEQ ID NO: 40) (p) Met Gly Tyr Pro Val Val Lys Val Glu Glu Phe - Ala Thr Ala Leu, (SEQ ID NO: 41) (q) Met Gly Phe Pro Val Leu Thr Val Glu Ser - Tyr? - Thr, (SEQ ID NO: 42) (r) Met Glu/Phe Asn Phe Leu Ile Glu/Val Thr/Glu Ala Gly - Ile Thr, (SEQ ID NO: 43) (s) Met Gly Phe Leu Val Thr Val Glu Ala Phe Tyr - Thr Ser, (SEQ ID NO: 44) (t) Met Lys Thr Pro Glu Phe Ala Val/Leu Gln Ala Phe/Thr Ala Thr Ser/Gly Phe Pro (SEQ ID NO: 45) (u) Met Lys Pro/Glu Thr/Val Leu Asp/Ala Thr/Lys Leu - Ile Thr - Gly, (SEQ ID NO: 46) (v) Met Leu Ala Leu Asp Tyr His Ser - Phe Val Gly?, (SEQ ID NO: 47) (w) Met Leu Ala Glu/Tyr Asp Gln/Ala Glu Asp Val, (SEQ ID NO: 48) (x) Lys His/Tyr Asn/Val Ser Pro Ala Ala Glu Asn/ Leu Leu Asn/Gly, (SEQ ID NO: 49) (y) Lys - Thr Ser Val Ala Glu Ala Phe Asn, (SEQ ID NO: 50) (z) Lys Ala Ala Glu Val Ala Glu Ala Phe Asp - lIe - - - Lys Gly, (SEQ ID NO: 51) (aa) Lys Ala Val Glu Val/Pro Ala Glu Ala Phe Asp Asp Ile Thr? Tyr - - Gly Pro Ser, (SEQ ID NO: 52) (bb) Lys - Glu Gln Thr Glu Ile Phe Asn Met, (SEQ ID NO: 53) (cc) Lys - - - Pro Phe Asn/Asp Ile Glu Ala Leu and (SEQ ID NO: 54) (dd) Asp Gln Ala Phe Ser Thr Asp Ala Lys.

2. A synthetic polypeptide comprising an amino acid sequence encoded by a nucleotide sequence which encodes a polypeptide which raises protective antibodies against helminth parasites, which nucleotide sequence incorporates at least one aminopeptidase-encoding sequence, selected from the group consisting of i) any sequence of SEQ ID NOS: 1-15 or 19-21, ii) a sequence fully complementary to said sequence of (i), iii) a portion of said sequence of (i), and iv) a sequence which hybridizes with said sequence of (i) or (ii) under conditions of 2 times SSC, 65 C (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least 60% identity with said sequence of (i), other than a synthetic polypeptide corresponding to the protein doublet H110D or a synthetic polypeptide as defined in (a) to (dd) in claim 1.

3. A synthetic polypeptide comprising an amino acid sequence encoded by a nucleotide sequence which corresponds to or which is complementary to one or more sequences selected from the group of sequences consisting of i) M1(SEQ ID NO:1), ii) B1A (SEQ ID NO:2), iii) B1A-3′ (SEQ ID NO:3), iv) B2 (SEQ ID NO:4), v) M1AUS (SEQ ID NO:5), vi) AusB1 (SEQ ID NO:6), vii) 014-015 (2.5 PCR) (SEQ ID NO:7), viii) 014-872 (3.5 PCR clone 2) (SEQ ID NO:8), ix) A-648 (5′ END of B1) (SEQ ID NO:9), x) A-650 (5′-end of 2.5 PCR) (SEQ ID NO:10), xi) α-649 (5′ end of 3.5 PCR) (SEQ ID NO:11), xii) 014-178 (3′ end of AustB1 clone 2) (SEQ ID NO:12), xiii) 014-178 (3′ end of AustB1 clones 3 and 6) (SEQ ID NO:13) xiv) 014-872 (3.5 PCR clone 10) (SEQ ID NO:14), xv) 014-872 (3.5 PCR clone 19) (SEQ ID NO:15), xvi) H11-1 (SEQ ID NO:19), xvii) H11-2 (SEQ ID NO:20), xviii) H11-3 (SEQ ID NO:21), and xix) a sequence which hybridizes with any of said sequences of (i)-(xviii) under conditions of 2 times SSC, 65 C (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least 60% identity with said sequence of (i)-(xviii), other than a synthetic polypeptide corresponding to the protein doublet H110D or a synthetic polypeptide as defined in (a) to (dd) in claim 1.

4. A synthetic polypeptide as claimed in claim 1, 2 or 3 substantially free from other Haemonchus contortus components.

5. A synthetic polypeptide as claimed in claim 1, 2 or 3 in the form of a fusion polypeptide, comprising an additional polypeptide fused to said amino acid sequence as defined in any one of claims 1 to 3.

6. A synthetic polypeptide as claimed in claim 1, 2 or 3 linked to an oligosaccharide having the structure:

7. A synthetic polypeptide as claimed in claim 6 wherein said oligosaccharide has the structure:

8. A synthetic polypeptide as claimed in claim 1, 2 or 3 wherein said synthetic polypeptide is a recombinant polypeptide.

9. A synthetic polypeptide as claimed in claim 1, 2 or 3 prepared by a method comprising the steps:

a) culturing a prokaryotic or eukaryotic cell containing a nucleic acid molecule comprising at least one nucleotide sequence which encodes a helminth aminopeptidase or an antigenic portion thereof, said nucleic acid selected from the group consisting of i) any sequence of SEQ ID NOs:1-15 or 19-21, ii) a sequence fully complementary to said sequence of (i), iii) a portion of said sequence of (i) wherein said portion of said sequence encodes an antigenic peptide of said helminth aminopeptidase, and (iv) a sequence which hybridizes with said sequence of (i) or (ii) under conditions of 2 times SSC, 65 C (where SSC=0.15 M NaCl, 0.015 M sodium citrate, pH 7.2) and which has at least 60% identity with said sequence of (i), under conditions whereby said synthetic polypeptide is expressed, and

b) recovering said synthetic polypeptide thus produced.

10. A vaccine composition for stimulating immune responses against helminth parasites in a human or non-human animal, comprising at least one synthetic polypeptide as defined in claim 1, 2 or 3, or a virus or host cell having inserted therein a nucleic acid molecule encoding said polypeptide, for stimulation of an immune response to polypeptides encoded by the inserted nucleic acid molecule, together with a pharmaceutically acceptable carrier.

11. 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 in claim 10.