Haemonchus contortus vaccine

TECHNICAL FIELD
The invention relates to antigens which confer protective immunity against infection by parasitic nematodes.
The invention also relates to vaccines conferring protective immunity against infection by parasitic nematodes, and to antibodies conferring passive immunity to infection by parasitic nematodes.
BACKGROUND ART
Nematodes (nema - thread; oides - resembling), which are unsegmented roundworms with elongated, fusiform, or saclike bodies covered with cuticle, are virtually ubiquitous in nature, inhabiting soil, water and plants, and are importantly involved in a wide range of animal and plant parasitic diseases.
The roundworm parasites of mammals belong to the phylum Nemathelminthes. The roundworms include the hookworm (e.g. Necator americanus and Ancylostoma duodenale), roundworm (e.g. the common roundworm Ascaris lumbricoides), whipworm (e.g. Trichuris trichiura), and the pinworm or threadworm (e.g. Enterobius vermicularus), as well as Strongyloides stercoralis, Trichinella spiralis (infection in man and pigs), and the filarial worm Wuchereria bancrofti. Other important roundworm parasites include Ancylostoma caninum (infections of man), Strongylus vulgaris (infections of horses), Trichostrongylus colubriformis (infections of sheep), Haemonchus contortus (infections of sheep and goats), Ostertagia ostertagi (infections of cattle), Ascaris suum (infections in pigs), Toxascaris leonia or Uncinaria stenocephala (infections of dogs), Toxocara spp (circulatory infections of man) and Dirofilaria immitis (circulatory infections of cats and dogs).
Even when symptom-free, parasitic worm infections are harmful to the host animal for a number of reasons; e.g. they deprive the host of food, injure organs or obstruct ducts, may elaborate substances toxic to the host, and provide a port of entry for other organisms. In other cases, the host may be a species raised for food and the parasite may be transmitted upon eating to infect the ingesting animal. It is highly desirable to eliminate such parasites as soon as they have been discovered.
More commonly, such infections are not symptom-free. Helminth infections of mammals, particularly by parasitic nematodes, are a source of great economic loss, especially of livestock and pets, e.g. sheep, cattle, horses, pigs, goats, dogs, cats and birds, especially poultry. These animals must be regularly treated with anthelminthic chemicals in order to keep such infections under control, or else the disease may result in anaemia, diarrohea, dehydration, loss of appetite, and even death.
The only currently available means for controlling helminth infections is with the use of anthelminthic chemicals, but these are only effective against resident worms present at the time of treatment. Therefore, treatment must be continuous since the animals are constantly exposed to infection; e.g. anthelminthic treatment with diethylcarbamazine is required every day or every other day most of the year to control Dirofilaria immitis or the dog heartworm. This is an expensive and labor intensive procedure. Due to the widespread use of anthelminthic chemicals, the worms may develop resistance and so new and more potent classes of chemicals must be developed. An alternative approach is clearly desirable.
The development of a vaccine against parasitic nematodes would overcome many of the drawbacks inherent in chemical treatment for the prevention and curing of helminthic infections. The protection would certainly last longer, only the vaccinated animal would be affected, and the problems of toxicity and persistence of residues would be minimized or avoided. Accordingly, there have been attempts, reported in the prior art, to develop such vaccines using parasitic nematodes; unfortunately, they have met with limited success and factors such as material availability and vaccine stability have precluded their large scale use.
These previous attempts are discussed in International Patent Application No. PCT/AU88/00239 (WO 89/00163) and PCT/AU89/00416 (WO 90/03433).
Recent advances in biotechnology and in particular recombinant DNA technology, realistically offer the opportunity to produce commercially-viable vaccines against a range of economically-important parasites of man and domestic animals. This approach would overcome many of the problems proposed to account for the lack of efficacy of killed vaccines using crude parasite preparations. For example, the vaccines produced by recombinant DNA techniques would not contain immunosuppressants or immunomodulators which may be found in crude extracts of parasitic nematode species. But it is necessary to first identify the antigens. Once identified and characterized, recombinant DNA technology could be used to construct microorganisms which synthesize those proteins or portions of the proteins containing protective epitopes and use the products synthesized by the recombinant organism in vaccines to protect animals from infection with the parasites.
In PCT/AU88/00239 it has been demonstrated that a recombinant DNA derived antigen shown to be nematode tropomyosin, gave 50% protection in sheep against Haemonchus contortus challenge. In PCT/AU89/00416 excretory/secretory antigens from adult Trichostrongylus colubriformis have been shown to give protection to vaccinated guinea pigs. For reasons which will become clear later in the specification, these antigens are different from the antigen identified in the current specification.
DESCRIPTION OF THE INVENTION
Definitions
The term "adjuvant" as used throughout the specification refers to an agent used to enhance the immune response of the immunised host to the immunising composition.
The term "parenteral" as used herein includes subcutaneous injections, intraperitoneal or intramuscular injection, or infusion techniques.
The term "homologue" refers to proteins or to DNA sequences coding for those proteins which are related in structure to a first protein or DNA sequence to such an extent that it is clear that the proteins are related in function. In the context of this invention, it is demonstrated that the DNA from H. contortus which codes for the antigen of the invention can be used in DNA hybridisation experiments to identify specific DNA sequences in other species of parasitic nematodes. The conditions under which the hybridisation experiments were carried out indicate that the related DNA sequences are at least 50% homologous in nucleotide sequence over 60 base pairs to that isolated from H. contortus. These related DNA segments code for antigens in those other species of parasitic nematodes which are also related in amino acid sequence to the protective antigen isolated from H contortus. It is contended that the related proteins will act as effective immunogens to protect animals from parasitism by the other species of parasites. These related DNA sequences are referred to as homologous genes and the related proteins are referred to as homologous antigens. Also, in the context of this invention, it has been demonstrated tht the protective antigen is a member of a gene family wherein the encoding polynucleotide and the gene product share an homology of the order of 50% over 60 nucleotides or 70% over 20 amino acids respectively with the encoding gene and protective antigen of this invention. These related genes and gene products are also homologues of this invention.
Homologues of the invention my also be generated in vitro as hereinafter described.
The term "derived" in the context of the antigens of the invention as used herein is intended to encompass antigen obtained by isolation from a parasitic nemanode life stage expressing the antigen, as well as antigen obtained by manipulation of and expression from nucleotide sequences prepared from a parasitic nematode life stage, including genomic DNA, mRNA, cDNA synthesized from mRNA and synthetic nucleotides prepared to have sequences corresponding to the antiget encoding sequences.
It is also intended to encompass synthetic peptide antigens prepared on the basis of the known amino acid sequences of the antigen as expressed by nematodes or cell lines expressing recombinant forms of the antigen.
Disclosure
It is preferable, if possible, to identify "novel" or "concealed" antigens i.e. components of the parasite which can act as effective and protective immunogens but which are not involved in naturally acquired immunity. To identify those components it is necessary to vaccinate the host with extracts from the parasite, identify fractions which give some protection, subfractionate the protective components using protein chemistry techniques, vaccinate animals with those subfractions and identify those subfractions which protect and continue with that process until a pure parasite component is used to vaccinate and protect the animal.
As much as possible, the natural host should be used in such experiments. As laboratory model animals are not the natural host for the parasite they are likely to be able to reject the parasite by mechanisms of which the natural host is incapable. Thus it is possible that antigens which protect laboratory animals against particular parasites may not be effective in the natural host of the parasite. This will be more likely to be the case in situations where the natural host develops immunity to the parasite very slowly.
The antigen characterised in the current work is derived from Haemonchus contortus but it is recognised that similar or related antigens, "homologues", could be identified from other species of parasitic nematode known to infect man or domestic animals and that these related antigens, would provide effective vaccines against parasitism by species of nematode. Species of parasites and hosts they may infect include for example: Trichinella spiralis or Ancylostoma caninum infections of man, Strongylus vulgaris infections of horses, Trichostrongylus colubriformis infections of sheep, Haemonchus contortus infections of goats, Ostertagia ostertagi infections of cattle, Ascaris suum or Trichinella spiralis infections of pigs, Toxascaris leonina or Uncinaria stenocephala infections of cats and Ancylostoma caninum or Trichuris vulpis infections of dogs as well as infections of the circulatory system of man by larvae of Toxocara spp and of the circulatory system of dogs by Dirofilaria immitis as well as infections of the circulatory system, urogenital system, respiratory system, skin and subcutaneous tissues of these and other species of animal. It should be noted that this list is by no means complete.
A preparation is described which gives protection against challenge infection by Haemonchus contortus. The protective components in the fraction are identified as being soluble in buffers containing low levels of zwitterionic detergents and as not being retained following chromatography on wheat-germ lectin sepharose columns. The protective antigens thus prepared are capable of being further purified by, for example, fractionation on lentil lectin (LL) chromatography or Helix pomatia lectin (HpL) chromatography, reverse phase HPLC, size exclusion chromatography or other purification methods known in the art in the presence of solubilizing detergents.
According to a first embodiment of the present invention there is provided a substantially purified antigen derived from a first species of parasitic nematode, which antigen is capable of providing protection to a host from parasitism by a second nematode species, which may be the same as or different from the first nematode species, following vaccination of the host with the antigen, characterized in that the antigen is proteinaceous, has a pI in the range of 3.8-4.4, binds to lentil lectin and Helix pomatia lectin and has a molecular weight of approximately 45 kD as determined by reducing SDS-PAGE. Typically the antigen is at least 90% pure. This level of purity is demonstrated with respect to the cleanliness of the preparations used in amino acid sequencing.
The invention encompasses the antigen in both glycosylated and non-glycosylated form.
Typically, the first parasitic nematode species is selected from species of the genera Trichinella, Ancylostoma, Strongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator, Enterobius, Strongyloides and Wuchereria. Examples of such species include Trichinella spiralis, Ancylostoma caninum, Strongylus vulgaris, Trichostrongylus colubriformis, Haemonchus contortus, Ostertagia ostertagi, Ascaris suum, Toxascaris leonina, Uncinaria stenocephala, Trichuris vulpis, Dirofilaria immitis, Toxocara spp, Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercoralis and Wuchereria bancrofti.
Typically the protection conferred on the host is protection against a parasitic nematode species selected from species of the genera Trichinella, Ancylostoma, Strongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator, Enterobius, Strongyloides and Wuchereria. Examples of such species include Trichinella spiralis, Ancylostoma caninum, Strongylus vulgaris, Trichostrongylus colubriformis, Haemonchus contortus, Ostertagia ostertagi, Ascaris suum, Toxascaris leonina, Uncinaria stenocephala, Trichuris vulpis, Dirofilaria immitis, Toxocara spp, Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides Stercoralis and Wuchereria bancrofti.
Preferably the first and second nematode species are selected from the genera Haemonchus, Trichostrongylus and Ostertagia.
More preferably the first and second nematode species are from the genus Haemonchus.
Preferred first and second nematode species are Haemonchus contortus, Trichostrongylus colubriformus and Ostertagia circumcincta.
More preferably the first and second nematode species are Haemonchus contortus.
The present inventors have determined that the antigen of the first embodiment is most likely a proteolytic cleavage product of a higher molecular weight nematode glycoprotein.
The nematode glycoprotein could be prepared from native sources by antibody affinity chromatography using antibodies raised to the expression product of the cloned gene.
The higher molecular weight glycoprotein in glycosylated and unglycosylated form is also encompassed by the present invention and is termed hereinafter the "antigen precursor".
The antigen precursor in substantially purified form is also part of the first embodiment of the present invention.
Typically the antigen precursor comprises the amino acid sequence illustrated in FIG. 8 and in SEQ ID No: 12.
According to a second embodiment of the present invention there is provided a homologue of the antigen or antigen precursor of the first embodiment.
Typically the homologue is at least 70% homologous over 20 amino acids to the amino acid sequence illustrated in FIG. 8 and in SEQ ID No:12.
According to a third embodiment of the present invention there is provided a polynucleotide molecule, excluding polynucleotide molecules as they exist in nature which encodes an antiget or an antigen precursor of the first embodiment, or a homologue of the second embodiment.
Typically the polynucleotide molecule is a DNA molecule.
Preferably the polynucleotide molecule is a cDNA molecule.
A preferred polynucleotide molecule of the invention is a cDNA molecule having substantially the sequence illustrated in FIG. 7 (SEQ ID No: 9) or 8 (SEQ ID No: 11).
The invention includes within its scope DNA molecules having at least 50% homology over 60 nucleotides with the sequence illustrated in FIG. 8 (SEQ ID No: 11), and encoding a protective molecule capable of conferrring immunity against parasitic nematode infection.
According to a fourth embodiment of the present invention there is provided a recombinant DNA molecule comprising a DNA molecule of the third embodiment and vector DNA.
Typically the vector DNA comprises plasmid, phage or viral DNA.
Preferred vectors include lambda gt11, pUR290, pUR291, pUR282, pUK270, pUC8, pUC9, pZipNeo, an SV40 based vector, lambda gt10, an EMBL vector, pBR327, pBR329, or pBR329 containing a par locus, baculovirus or vaccinia virus.
According to a fifth embodiment of the invention there is provided a transformed host, carrying at least one recombinant DNA molecule according to the fourth embodiment.
Typically the host is selected from bacteria, yeasts, other fungi, insect, plant and mammalian cell lines. Preferred host cells are E. coli K12 derivatives.
According to a sixth embodiment of the present invention there is provided an expression product of a transformed host of the fifth embodiment comprising an antigen or antigen precursor of the first embodiment or a homologue of the second embodiment.
The expression product may be a fused expression product.
According to a seventh embodiment of the present invention there is provided a synthetic polypeptide corresponding to all or part of an antigen, precursor, homologue or expression product of the invention which synthetic polypeptide when administered to a host animal is capable of inducing protective immunity against infestation of the host animal by a parasitic nematode.
According to an eighth embodiment of this invention, there is provided a vaccine comprising an effective amount of at least one antigen and/or antigen precursor of the first embodiment and/or a homologue of the second embodiment and/or expression product of the sixth embodiment and/or synthetic polypeptide of the seventh embodiment together with a pharmaceutically and/or veterinarally acceptable carrier, diluent, excipient and/or adjuvant. The vaccines of the invention could alternatively comprise at least one anti-idiotypic antibody capable of protecting a host from infection by a parasitic nematode by mimicking the antigen, antigen precursor, homologue, expression product and/or synthetic polypeptide. A pharmaceutically and/or veterinarally acceptable carrier, diluent, excipient and/or adjuvant may be added to the active component.
As a further alternative, the vaccine may be a whole cell vaccine comprising a transformed host of the fifth embodiment together with a pharmaceutically and/or veterinarally acceptable carrier, diluent, excipient and/or adjuvant. The cells may be live or killed.
The transformed cells include those capable of expressing the expression product for mucosal presentation to a host to be vaccinated, such as, as a cell surface fusion product.
According to a ninth embodiment of this invention there is provided a process for the preparation of an antigen of the first embodiment, which process comprises:
a) homogenizing young adults of a parasitic nematode species to produce an homogenate;
b) obtaining membranous material from the homogenate;
c) extracting the membranous material with a buffer containing low levels of a zwitterionic detergent to obtain a detergent extract;
d) chromatographing the detergent extract on a wheat-germ lectin sepharose column; and
e) collecting flow-through from the column.
Preferably, the process also comprises:
f) fractionation by preparative iso-electricfocussing and collection of fractions having a pI in the range 3.8-4.4, or more preferably 4.0-4.3;
g) fractionation by gel filtration chromatography to collect fractions with molecular weights in the range 10-60 kD; and
h) fractionation by lentil lectin and/or Helix pomatia lectin chromatography and collecting bound material.
According to a tenth embodiment of this invention, there is provided a process for the preparation of a vaccine of the eighth embodiment which process comprises:
admixing an effective amount of at least one antigen and/or antigen precursor of the first embodiment and/or homologue of the second embodiment and/or expression product of the sixth embodiment and/or synthetic polypeptide of the seventh embodiment and/or transformed host of the fifth embodiment and/or antiidiotype antibody with a pharmaceutically and/or veterinarally acceptable carrier, diluent, excipient and/or adjuvant.
According to an eleventh embodiment of this invention, there is provided a method of protecting a host against infection by at least one parasitic nematode species which method comprises administering an effective amount of an antigen and/or antigen precursor of the first embodiment and/or homologue of the second embodiment and/or expression product of the sixth embodiment and/or synthetic polypeptide of the seventh embodiment and/or a vaccine of the eighth embodiment to the host.
According to a twelfth embodiment of this invention, there is provided an antibody raised against an antigen and/or antigen precursor of the first embodiment and/or homologue of the second embodiment and/or expression product of the sixth embodiment and/or synthetic polypeptide of the seventh embodiment and/or a vaccine of the eighth embodiment. The antibody of the invention may be monoclonal or polyclonal. The invention also provides other compounds which behave in a similar manner to the antibodies of the twelfth embodiment, by binding to and altering the structure and/or function of an antigen or antigen precursor of the first embodiment, or homologue of the second embodiment, expression product of the sixth embodiment, or synthetic polypeptide of the seventh embodiment.
According to a thirteenth embodiment of this invention, there is provided an antibody composition comprising an antibody of the twelfth embodiment together with a pharmaceutically and/or veterinarally acceptable carrier, diluent and/or excipient.
According to a fourteenth embodiment of this invention, there is provided a process for the preparation of an antibody of the twelfth embodiment which process comprises vaccinating an immunoresponsive host with an antigen and/or antigen precursor of the first embodiment and/or homologue of the second embodiment and/or expression product of the sixth embodiment and/or synthetic polypeptide of the seventh embodiment and/or a vaccine of the eighth embodiment.
According to a fifteenth embodiment of this invention, there is provided a process for the preparation of an antibody composition of the thirteenth embodiment which process comprises admixing an effective amount of an antibody of the twelfth embodiment with a pharmaceutically and/or veterinarally acceptable carrier, diluent, and/or excipient.
According to a sixteenth embodiment of this invention, there is provided a method of passively vaccinating a host in need of such treatment against a parasitic nematode, which method comprises administering an effective amount of an antibody of the twelfth embodiment and/or an antibody composition of the thirteenth embodiment to the host.
According to a seventeenth embodiment of the present invention there is provided a process for the preparation of a recombinant DNA molecule of the fourth embodiment which process comprises inserting a DNA molecule of the third embodiment into vector DNA.
According to an eighteenth embodiment of the present invention there is provided a process for the preparation of a transformed host of the fifth embodiment which process comprises making a host competent for transformation to provide a competent host and transforming the competent host with a recombinant DNA molecule of the fourth embodiment.
According to a nineteenth embodiment of the present invention there is provided a diagnostic kit comprising a sample of an antigen, antigen precursor, homologue, expression product or synthetic polypeptide of the present invention and/or an antibody of the present invention.
According to a twentieth embodiment of the present invention, there is provided a process for the biosynthesis of an expression product of the sixth embodiment which process comprises providing a transformed host of the fifth embodiment, culturing the host under suitable conditions to obtain expression of the expression product and collecting the expression product from the transformed host.
According to a twenty first embodiment of this invention there is provided an antiidiotype antibody corresponding to a portion of an antiget of the invention and capable of protecting a host immunised with the antiidiotype antibody from infestation by a parasitic nematode species.
It is recognised that variation in amino acid and nucleotide sequences can occur between different allelic forms of a particular protein and the gene(s) encoding the protein. Further once the sequence of a particular gene or protein is known, a skilled addressee, using available techniques, would be able to manipulate those sequences in order to alter them from the specific sequences obtained to provide a gene or protein which still functions in the same way as the gene or protein to which it is related. These molecules are referred to herein as "homologues" and are intended also to be encompassed by the present invention.
In this regard, a "homologue" is a polypeptide that retains the basic functional attribute, namely, the protective activity of an antigen of the invention, and that is homologous to an antigen of the invention. For purposes of this description, "homology" between two sequences connotes a likeness short of identity indicative of a derivation of the first sequence from the second. In particular, a polypeptide is "homologous" to an antigen of the invention if a comparison of amino-acid sequences between the polypeptide and the antigen, reveals an identity of greater than about 70% over 20 amino acids. Such a sequence comparison can be performed via known algorithms, such as the one described by Lipman and Pearson, Science 227: 1435 (1985), which are readily implemented by computer.
Homologues can be produced, in accordance with the present invention, by conventional site-directed mutagenesis, which is one avenue for routinely identifying residues of the molecule that can be modified without rendering the resulting polypeptide biologically inactive. Oligonucleotide-directed mutagenesis, comprising [i] synthesis of an oligonucleotide with a sequence that contains the desired nucleotide substitution (mutation), [ii] hybridizing the oligonucleotide to a template comprising a structural sequence coding for an antigen of the invention and [iii] using T4 DNA polymerase to extend the oligonucleotide as a primer, is preferred because of its ready utility in determining the effects of particular changes to the antigen structural sequence. Its relative expense may militate in favour of an alternative, known direct-mutagenesis method.
Also exemplary of antigen homologues within the present invention are molecules that correspond to a portion of the antigen, or that comprise a portion of the antigen without being coincident with the natural molecule, and that display the protective activity of an antigen of the invention.
Other homologues of the present invention are fragments of the antigen that retain protective activity. Likewise within the present invention would be synthetic polypeptides that (i) correspond to a portion of the antigen amino-acid sequence and (ii) retain an activity characteristic of the antigen. Such synthetic polypeptides would preferably be between 6 and 30 amino residues in length.
Whether a synthetic polypeptide meeting criterion (i) also satisfies criterion (ii) can be routinely determined by assaying for protective activity, in an appropriate host.
The amount of antigen, antigen precursor, homologue, expression product and/or synthetic polypeptide to be combined with carrier, diluent, excipient and/or adjuvant to produce a single vaccine dosage form will vary depending upon the infection being vaccinated against, the host to be treated and the particular mode of administration.
It will be understood, also, that the specific dose level for any particular host will depend upon a variety of factors including the activity of the specific antiget, antiget precursor, homologue, expression product and/or synthetic polypeptide employed, the age, body, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the particular infection state being prevented.
The vaccines of the present invention may be administered parenterally or potentially via mucosal routes in dosage unit formulations containing conventional, non-toxic, pharmaceutically and/or veterinarally acceptable carriers, diluents, adjuvants and/or excipients as desired.
Injectable preparations, for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
At present alum is the only registered adjuvant for human use however, experimental work is being conducted on other adjuvants for human use and it is anticipated that these other adjuvants would be suitable for use in preparing compositions for human vaccination in accordance with this invention.
Suitable adjuvants for the vaccination of animals include but are not limited to oil emulsions such a Freund's complete or incomplete adjuvant (not suitable for livestock use), Marcol 52: Montanide 888 (Marcol is a Trademark of Esso. Montanide is a Trademark of SEPPIC, Paris), squalane or squalene, Adjuvant 65 (containing peanut oil, mantide monooleate and aluminium monostearate), mineral gels such as aluminium hydroxide, aluminium phosphate, calcium phosphate and alum, surfactants such a hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N', N'-bis(2-hydroxyethyl) propanediamine, methoxyhexadecylglycerol and pluronic polyols, polyanions such as pyran, dextran sulfate, polyacrylic acid and carbopol, peptides and amino acids such as muramyl dipeptide, dimethylglycine, tuftsin and trehalose dimycolate. The antigens, precursors, expression products and/or synthetic polypeptides of the present invention can also be administered following incorporation into liposomes or other micro-carriers, or after conjugation to polysaccharides, proteins or polymers or in combination with Quil-A to form "Iscoms" (Immunostimulating complexes). Other adjuvants suitable for use in the present invention include conjugates comprising the immunogen together with an integral membrane protein of prokaryotic origin, such as TraT. (See PCT/AU87/00107).
Routes of administration, dosages to be administered as well as frequency of injections are all factors which can be optimised using ordinary skill in the art. Typically, the initial vaccination is followed some weeks later by one or more "booster" vaccinations, the net effect of which is the production of vigorous immune response both cellular and humoral.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows gel filtration HPLC results for IEF 5 and 6 on SDS polyacrylamide gel stained with silver. Lanes 1-6 contain GF1-GF6; lane 7 contains Bio-Rad low and high molecular weight standards with sizes given in kilodaltons and lane 8 contains IEF 5 and 6 (pre GF HPLC).
FIG. 2 shows lectin affinity chromatography results for IEF 5 and 6 on an SDS polyacrylamide gel stained with silver. Lane 1 contains molecular weight standards. Lanes 2 and 3 contain material bound and eluted from lentil lectin Sepharose (LLS); lane 4, material that was bound and eluted from Helix pomatia lectin Sepharose (HpLS) after initial binding and elution from LLS and lane 5 contains material that did not bind to HpLS after initial binding and elution from LLS.
FIG. 3 shows lectin affinity chromatography results for IEF 5 and 6 as a Western blot overlayed with Concanavalin A-HRP and reactive material visualized by enzymatic reaction. Lane 1 contains IEF 5 and 6; lane 2, material not binding to LLS; lane 3, material bound and eluted from LLS; lane 4, material bound and eluted from HpLS after initial binding and elution from LLS; lane 5, material not bound to HpLS after initial binding and elution from LLS; lane 6, material not bound to LLS or HpLS and lane 7, BRL high molecular weight standards.
FIG. 4 shows lectin affinity chromatography results for IEF 5 and 6 as a Western blot overlayed with Helix pomatia lectin - HRP and reactive material visualized by enzymatic reaction. Lane contents are as described for FIG. 3.
FIG. 5 shows reverse-phase HPLC results for IEF 5 and 6 on a PLRP-S column. The gradient used for elution consisted of 30% acetonitrile in water with 0.1% TFA to 60% acetonitrile in water with 0.1% TFA. The flow rate was 1 ml/min and the absorbance monitored at 220 nm. Peak fractions indicated by numbers were collected manually. FIG. 6(a) and FIG. 6(b) shows reverse-phase HPLC results for IEF 5 and 6, as an SDS polyacrylamide gel (9-22% gradient) stained with silver. Numbers refer to peak fractions shown in FIG. 5. Lane S, Bio-Rad low and high molecular weight standards in the sizes given in kilodaltons.
FIG. 7 shows the cloned DNA sequence (SEQ ID No: 9) of pBTA879 and the derived amino acid sequence (SEQ ID No: 10) of the cloned gene coding a homologue of the protective antigen.
FIG. 8 shows the cloned DNA sequence (SEQ ID No: 11) of pBTA963 and the derived amino acid sequence (SEQ ID No: 12) of the cloned gene coding for the protective antigen.
FIG. 9 shows a Southern blotting of DNA from H. contortus hybridised to the cDNA from pBTA879 and pBTA963 showing the presence of homologous genes within the parasite genome. Lane 1. Haemonchus contortus DNA, HinfI digest, probed with pBTA963. Lane 2. Haemonchus contortus DNA, HinfI digest, probed with pBTA879.
FIG. 10 shows Southern blotting of DNA from T. colubriformis, Ostertagia circumcincta and Ostertagia ostertagi with a 45 kD antigen probe demonstrating the presence of related genes in other species of nematode. Lane 1. Haemonchus contortus DNA, HinfI digest. Lane 2. Trichostrongylus colubriformis DNA, HinfI digest. Lane 3. Ostertagia ostertagi DNA, HinfI digest. Lane 4. Ostertagia circumcincta DNA, HinfI digest. Lane 5. Plasmid pBTA963, HinfI digest - positive control.
FIG. 11(a) and FIG. 11(b) shows SDS PAGE gels and Western blots of the recombinant 45 kD antigen expressed in E coli. FIG. 11(a) Expression of 45 kDa Antigen E. coli:Samples were electrophoresed on a 12.5% SDS polyacrylamide gel. The gel was then stained with Coomassie brilliant blue. Lane 1: uninduced control; Lane 2:1 hour post-induction; Lane 3:3 hours post-induction; Lane 4: Biorad prestained SDS PAGE standards. FIG. 11(b) Western Blot of Expressed 45 kDa Antigen: Samples were electrophoresed as in FIG. 11(a), then electrophoretically blotted onto a nitrocellulose filter. The filter was probed with rabbit serum raised against a peptide corresponding to the truncated N-terminus of the 45 kDa protein. The Promega Protoblot alkaline phosphatase system (product no. W3930) was used to develop colour.
FIG. 12 shows Western blots of extracts from H. contortus and the dog heart worm D. immitis showing that antigens immunologically related to that of the invention are expressed in other species of parasitic nematode. Lane 1, D immitis extract; Lane 2, H contortus extract; Lane 3, BRL high molecular weight standards.

BEST METHOD OF CARRYING OUT THE INVENTION
Young adults of Haemonchus contortus were homogenised in phosphate-buffered saline (PBS) and the homogenate was centrifuged in order to sediment the membranous material from the nematodes. This pellet was found to protect sheep from challenge infection following two vaccinations. The protective fraction was then extracted with a number of different detergents. Zwittergent 3-14 (Calbiochem) was found to be suitable although other detergents were also found to be effective. Efficiency of extraction was judged by the ability of the detergent used to solubilize protective antigens (as judged by vaccination/challenge experiments), with the highest specific activity (as estimated by the number of micrograms of solubilized material required to give protection) whilst leaving an insoluble residue which failed to give significant protection following the vaccination/challenge protocol employed (42%, see Table 3). It is acknowledged that the Zwittergent 3-14 extraction procedure may not have been completely efficient and some of the protective antigens may be found in the detergent insoluble fraction.
The recombinant DNA molecules and transformed host cells of the invention are prepared using standard techniques of molecular biology.
Expression products of the invention are obtained by culturing transformed host cells of the invention under standard conditions as appropriate to the particular host cell and separating the expression product from the culture by standard techniques. The expression product may be used in impure form or may be purified by standard techniques as appropriate to the expression product being produced.
Where appropriate, whole cells may be used in vaccines.
Synthetic polypeptides of the invention are prepared by standard techniques of peptide synthesis based on the known sequences of antigens, antigen precursors, homologues and expression products of the invention.
The homologues, expression products and synthetic polypeptides can be tested for protective activity as described in the following examples. Recombinant DNA technology can be used to provide a large amount of the protective antigen or homologues described herein. The DNA segment coding for the protective antigen or the precursor for the protective antigen or homologue can be inserted into any of a number of recombinant plasmid systems to enable the molecule to be synthesised in large amounts. The recombinant systems include E. coli, yeast, and baculovirus systems and viruses such as vaccinia. The recombinant organisms can be grown in large volumes in fermenters and the recombinant antigens purified by standard methods -
solubilisation in solutions containing urea and reducing agents such as DTT or mercaptoethanol, refolding in the presence of reagents such as reduced and oxidised glutathione, purification by ion exchange, filtration and/or gel permeation chromatography, terminally sterilised by filtration and adjuvanted in any of a number of adjuvants including oils.
The vaccine is prepared by mixing, preferably homogeneously mixing, an antigen, antigen precursor, homologue, expression product and/or synthetic polypeptide and/or transformed host and/or antiidiotype antibody of the invention with a pharmaceutically and/or veterinarally acceptable carrier, diluent, excipient and/or adjuvant using standard methods of pharmaceutical and/or veterinary preparation.
The amount of antigen, antigen precursor, homologue, expression product, synthetic polypeptide and/or transformed host and/or antiidiotype antibody required to produce a single dosage form will vary depending upon the infection being vaccinated against, host to be treated and the particular mode of administration. The specific dose level for any particular host will depend upon a variety of factors including the age, body weight, general health, sex and diet of the host, time of administration, route of administration, rate of excretion and drug combination.
The vaccine may be administered parenterally in unit dosage formulations containing conventional, non-toxic, pharmaceutically and/or veterinarally acceptable carriers, diluents, excipients and/or adjuvants as desired.
Antiidiotypes are raised by vaccinating a suitable host with an antigen, precursor, expression product, homologue and/or synthetic polypeptide of the invention and using the resulting antibodies to raise antibodies against the antigen binding region of the antibodies raised in the first vaccination.
Antibodies are raised using standard vaccination regimes in appropriate hosts. The host is vaccinated with an antigen, antiget precursor, homologue, expression product, synthetic polypeptide and/or vaccine of the invention. An immune response is generated as result of vaccination. The immune response may be monitored, for example, by measurement of the levels of antibodies produced.
The antibody composition is prepared by mixing, preferably homogeneously mixing, antibody with a pharmaceutically and/or veterinarally acceptable carrier, diluent, and/or excipient using standard methods of pharmaceutical and/or veterinary preparation.
The amount of antibody required to produce a single dosage form will vary depending upon the infection being vaccinated against, host to be treated and the particular mode of administration. The specific dose level for any particular host will depend upon a variety of factors including the age, body weight, general health, sex, and diet of the host, time of administration, route of administration, rate of excretion, drug combination and the severity of the infection undergoing treatment.
The antibody composition may be administered parenterally, in unit dosage formulations containing conventional, non-toxic, pharmaceutically and/or veterinarally acceptable carriers, diluents, and/or excipients as desired, to passively protect hosts against nematode infestation.
Diagnostic kits are prepared by formulating expression product, antibodies, antiget, antigen precursor, homologue or synthetic polypeptide at appropriate concentration to the substance(s) to be detected with a pharmaceutically and/or veterinarally acceptable carrier, diluent and/or excipient. A positive control standard of a known concentration of the substance to be detected is prepared similarly. The negative standard comprises carrier, diluent and/or excipient alone.
The invention is further described with reference to the following examples, which are not limiting on the scope of the present invention.
EXAMPLE 1
Young H. contortus nematodes were recovered from infected sheep. The nematodes were washed three times in PBS and homogenised in PBS. The homogenate was centrifuged at 500.times.g for 10 min to remove large and unbroken worm material. The supernatant was then centrifuged at 120,000.times.g av for 2 hours at 4.degree. C. The pelleted material was suspended in PBS and then used to vaccinate sheep subcutaneously in the absence of adjuvant on two occasions four weeks apart, each vaccination containing approximately 50 mg worm wet weight equivalent per kg body weight of the sheep. Three weeks after the second vaccination the sheep and five non-vaccinated infection controls were challenged with 10,000 infective larvae of Haemonchus contortus. On days 23, 27, 28, 33, 36 and 40 post infection, faecal egg counts were performed on all sheep. The results (eggs/g faeces) are presented in Table 1.
TABLE 1______________________________________Faecal Egg Counts Eggs/g Faeces DayAnimal 23 27 29 33 36 40______________________________________Controls1 1,433 4,300 2,800 6,467 4,267 5,9002 700 3,633 3,400 7,367 7,267 8,6003 600 4,467 4,767 4,033 4,367 7,8004 667 10,200 11,400 18,333 16,533 6,1005 3,200 5,333 5,133 7,367 4,433 6,700Vaccinates6 33 300 267 567 2,133 1,3507 6,167 14,434 8,000 22,434 11,133 16,5508 33 33 0 33 1,333 8509 0 0 67 67 767 1,35010 0 0 0 0 133 0______________________________________ It is clear that four of the five vaccinated animals were well protected from infection (p < 0.02 for the vaccinated group vs control group).
EXAMPLE 2
A series of experiments was conducted in which female guinea pigs were vaccinated with homogenates of adult H. contortus and with 120,000 .times.g pellets and supernatants derived from those homogenates (prepared essentially as described in Example 1). The guinea pigs received two vaccinations intraperitoneally in the absence of adjuvant three to four weeks apart and were challenged three to four weeks after the second vaccination with 1,000 infective larvae of H. contortus. Five to six days following infection, the animals were sacrificed and worm counts performed.
Table 2 summarises the results of these experiments showing the worms recovered from the vaccinates as a percentage of those recovered from control non-vaccinated animals receiving the same challenge infection.
TABLE 2______________________________________Fraction % Protection Experiment No.______________________________________Homogenate 61 163Homogenate (3 groups) 45, 55, 45 165120,000 .times. g pellet 59 196120,000 .times. g pellet 34* 171120,000 .times. g supernatant 29 196120,000 .times. g supernatant 15* 149______________________________________ *These animals only received one vaccination. It is clear from these results that fractions derived from homogenates of adult H. contortus are capable of giving significant protection to guinea pigs, particularly the particulate material found in the 120,000 .times. pellet.
EXAMPLE 3
Young adult H. contortus were homogenised in Tris buffered saline and a 120,000.times. g pellet prepared as described in Example 2. The pellet was resuspended in Tris buffered saline (TBS) containing 1% (WV) Zwittergent SB-14 (Sigma) by sonication and shaking at 4.degree. C. for 1 hour. The extract was centrifuged at 50,000.times. gav for 30 minutes to pellet the detergent insoluble material. The supernatant fraction was passed over an affinity column of wheat germ lectin-sepharose 6MB (Sigma) on two occasions to separate glycoproteins containing terminal N-acetyl-glucosamine residues. The five fractions derived from this procedure were used to vaccinate guinea pigs as described in Example 2. The results (Table 3) clearly demonstrate that a significant portion of the protective material was solubilized in the detergent, but the majority of the protective antigen(s) failed to bind to wheat germ lectin.
TABLE 3______________________________________Fraction .mu.g/animal % Protection______________________________________SB-14 insoluble material 800 .mu.g 42%SB-14 soluble material 700 .mu.g 71%SB-14 soluble WGA.sup.+ 10 .mu.g 22%SB-14 soluble WGA.sup.- 700 .mu.g 77%______________________________________ WGA.sup.+ is wheat germ agglutinin binding material WGA.sup.- is wheat germ agglutinin nonbinding material SB14 is Zwittergent SB14 (Calbiochem).
Material contained in a particulate preparation derived from a homogenate of H. contortus, when used to vaccinate guinea pigs or sheep, is capable of promoting an immune response in the vaccinated animals which gives rise to reduced parasitism in those animals when they are infected with the parasite. The protective antigens are largely soluble in detergents such as Zwittergent SB-14 and do not bind to wheat germ agglutinin under the conditions employed in the above examples.
EXAMPLE 4
A Zwittergent SB-14 extract was prepared from 10-15 g wet weight of young adult H. contortus as described in Example 3, and used to vaccinate sheep in two experiments.
The animals received two vaccinations three weeks apart, the first in Freund's complete adjuvant and the second in Freund's incomplete adjuvant. Three weeks after the second vaccination, the sheep were challenged per os with 10,000 larvae of H. contortus. Faecal egg counts were performed twice per week starting 21 days post infection for the next 4 weeks at which time the animals were slaughtered for worm counts (Table 4a and 4b). It is clear that the Zwittergent SB-14 extract contained antigens which were capable of giving significant protection in sheep against infection as measured by either faecal egg counts or worm counts, in spite of the fact that the amount of protein used to vaccinate each animal in the experiment was very small (10.+-.2.5 mg) and the extract contained a large number of components as judged by SDS polyacrylamide gel electrophoresis.
TABLE 4a______________________________________ SB-14 SB-14 in Extract of Challenge Adjuvant H.c. L5 in Controls Controls Adjuvant______________________________________Group average faecal 21080 18623 7844egg counts% Protection cf 62.8adjuvant controlsWorm counts of sheet 3874 3774 1422at slaughter% Protection of 63.3adjuvant controls______________________________________
TABLE 4b______________________________________ SB-14 SB-14 in Extract of Challenge Adjuvant H.c. L5 in Controls Controls Adjuvant______________________________________Group average 36643 75600 9114faecal egg counts% Protection cf 88Adjuvant ControlsWorm Counts of 1096 2117 533sheep at slaughter% Protection cf 75adjuvant controls______________________________________
EXAMPLE 5
For the same experiment as in Example 3, five times more material was prepared than that used in that Example. The remaining Zwittergent SB-14 extract (approximately 63 mg of protein) was fractionated by preparative isoelectric focussing in a 4% Ultrodex resin (LKB) containing Pharmalyte 3-10 ampholines and 0.5% CHAPS detergent (Calbiochem). Following electrophoresis for 10000 Vh, the bed was scraped into 30 fractions and the material in each fraction was eluted in 0.1% CHAPS. The pH of each fraction was determined and each fraction was then concentrated to 1 ml on a YM10 membrane. An aliquot of each fraction was analysed by SDS polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Aliquots (approximately 1/8) of each fraction were pooled based on the components visualised on the SDS gel giving a total of 5 fractions which were used to vaccinate guinea pigs as described in Example 2. Worm counts (Table 5) show that the majority of the protective antigen(s) is contained in Fractions 1-10 of the IEF gel which covers the pI range of 3.3-4.6. Other fractions also contained material which resulted in a decrease in worm burdens and these are also of interest in this application.
TABLE 5A______________________________________Experiment #229Fraction pI Worm Counts % Protection______________________________________Controls 467 .+-. 190 --SB-14 Extract 226 .+-. 62 52%IEF 1-10 3.3-4.6 233 .+-. 155 50%IEF 11-14 4.7-5.3 341 .+-. 112 37%IEF 15-18 5.4-6.2 323 .+-. 107 31%IEF 19-22 6.6-7.5 528 .+-. 33 0IEF 23-30 7.7-9.3 347 .+-. 247 26%______________________________________
TABLE 5B______________________________________Experiment #250Fraction pI Worm Counts % Protection______________________________________Controls 664 .+-. 152 --SB-14 Extract 254 .+-. 111 62%IEF 1-10 3.3-4.6 238 .+-. 67 64%IEF 11-14 4.7-5.3 274 .+-. 287 59%IEF 15-18 5.4-6.2 455 .+-. 251 31%IEF 19-22 6.6-7.5 474 .+-. 427 28%IEF 23-30 7.7-9.3 356 .+-. 100 46%______________________________________
For the second experiment in Example 4, the results of which are presented in Table 4b, groups of sheep were vaccinated with similar broad range IEF fractions as in the above example. The results are presented in Table 5c.
TABLE 5c______________________________________ % Protection cf Group Average adjuvant Faecal controls egg Worm EggFraction counts counts Counts Worms______________________________________Adjuvant controls 75600 2117 -- --SB-14Extract 9114 533 88 75IEF 1-10 26314 1440 65 32IEF 11-14 27814 395 63 81IEF 15-18 24271 503 68 76IEF 19-22 16917 581 78 73IEF 23-30 27329 956 64 55______________________________________
This data corroborates that obtained in the guinea pig vaccination and challenge experiments in that it clearly demonstrates that there are antigens in the IEF fractions 1-10 which are capable of providing significant degrees of protection to sheep against H. contortus challenge infections. It is also clear from these data that there are additional protective components in the other IEF fractions examined in this experiment and these are of interest in this application as well as those contained in IEF fractions 1-10.
EXAMPLE 6
Half of the first 10 IEF fractions used in Example 5 were pooled in pairs and used to vaccinate guinea pigs. The worm counts (Table 6) show the majority of the protective material was contained in fractions 5 and 6 from the original IEF gel which contains material with a pI of 3.8-4.4.
TABLE 6______________________________________Fraction pI Worm Counts % Protection______________________________________Controls 589 .+-. 194 --IEF 1-10 3.3-4.6 183 .+-. 85 69%IEF 1 & 2 3.3-3.5 509 .+-. 122 13%IEF 3 & 4 3.7-3.8 373 .+-. 143 37%IEF 5 & 6 4.0-4.3 225 .+-. 103 62%IEF 7 & 8 4.4-4.5 321 .+-. 115 45%IEF 9 & 10 4.5-4.6 335 .+-. 223 43%______________________________________
When aliquots of the IEF fractions were electrophoresed on SDS polyacrylamide gels and stained with silver, a number of components could be visualized which seemed to be enriched in the pI 3.8-4.4 fractions. Some of these were not sharp bands and are presumably 15 glycoproteins. The apparent molecular weight of these compounds compared with BRL high molecular weight protein standards were 100-120 kD, 40-55 kD, a cluster of perhaps 5 components in the 14-20 kD range and material not resolved by the gel at molecular weight less than 15 kD. In addition there was a sharp doublet of proteins with apparent molecular weights of 32-36 kD which were more abundant however in neighbouring less protective fractions and are therefore considered unlikely to be the antigens responsible for the protective immune responses. The IEF fractions were also used in "Western blots" using serum from sheep vaccinated in Example 4 as indicator serum. All of the components in the pI 3.8-4.4 range which were observed in the silver stained gels reacted with the sheep serum and are therefore capable of eliciting an immune response in sheep under the vaccination regime carried out in that experiment. These results indicate that potentially protective antigens can be isolated from young adult H. contortus which are particulate, solubilized at least in part with 1% Zwittergent SB-14, have a pI in the range of 3.8-4.4 and may have apparent molecular weights of 100-120 kD, 40-55 kD, 14-20 kD or less than 15 kD as estimated by SDS gel electrophoresis and compared with BRL high molecular weight markers.
EXAMPLE 7
IEF fractions 5 and 6 were dialysed against 50 mM sodium phosphate buffer pH 6.6 containing 0.6% CHAPS and concentrated six-fold on a YM-10 membrane. 0.2 ml aliquots were fractionated by HPLC gel filtration using a Bio-Sil TSK-400 column (30.times.0.75 cm.) (Bio-Rad) in the same buffer at a flow rate of 0.2 ml/min and eluent absorbance monitored at 280 nm. Fractions were collected and pooled on the basis of eluent absorbance profile. The fractions present in the last eluting peak were pooled and re-chromatographed on a TSK-SW 3000 G gel filtration column (Toyo Soda) using the conditions described above. A total of six pools were used to vaccinate guinea pigs as described in Example 2; each pool contained the equivalent of 4 ml IEF 5 and 6. Worm counts (Table 7) show that the majority of the protective antigen(s) is contained in GF 5 and GF 6 which include antigens in an approximate molecular weight range from <10 kD to 60 kD.
TABLE 7______________________________________ M.W. RangeFraction (kD) Worm Counts % Protection______________________________________Controls -- 436 .+-. 123 --IEF 5 and 6 <10->250 224 .+-. 125 49GF 1 >250 369 .+-. 104 15GF 2 100->250 344 .+-. 158 21GF 3 55->250 341 .+-. 168 22GF 4 55 387 .+-. 105 11GF 5 <10-60 233 .+-. 141 47GF 6 <10-44 237 .+-. 170 46______________________________________
When aliquots of the gel filtration pools were electrophoresed on SDS polyacrylamide gels and stained with silver, a number of components could be visualized which seemed to be enriched in GF 5 and GF 6 (FIG. 1). The apparent molecular weight of these compared with Bio-Rad low molecular weight standards were 45 kD, perhaps four indistinct components in the 25-30 kD range and perhaps five proteins in the 14-20 kD range. There are also other components that are unresolved on this gel and co-migrate at the buffer front.
Components resident in GF 5 and GF 6 could be more highly resolved by several means. The methods described in Examples 8 and 9 are by way of illustration only.
EXAMPLE 8
6 ml IEF 5 and 6 was diluted with an equal volume of 100 mM Tris/250 mM sodium chloride pH 7 and stirred with 0.5 ml of lentil lectin-Sepharose 4B (Pharmacia) for 16 h at 4.degree. C. The Sepharose conjugate was removed by centrifugation for 1 min at 3,000.times.g, washed four times with 50 mM Tris-saline buffer pH 7 containing 0.1% Zwittergent SB-14 (TSZ buffer) then eluted with the same buffer to which had been added 0.3M methyl-.alpha.-D-mannopyranoside. Half of the lentil lectin bound fraction was stirred with 0.5 ml Helix pomatia lectin-Sepharose 6MB (Pharmacia) for 16 h at 4.degree. C., the conjugate washed four times in TSZ buffer and then eluted in TSZ buffer containing 0.2M N-acetyl-D-galactosamine. Aliquots of all fractions were run in triplicate on SDS polyacrylamide gels before 1) staining with silver (FIG. 2) or Western transfer and 2) staining with Concanavalin A horse radish peroxidase (HRP) conjugate (Sigma) (FIG. 3) or 3) staining with Helix pomatia lectin-HRP (Sigma) (FIG. 4). The silver stained gel shows a predominant protein of 45 kD which binds to both lentil and Helix pomatia lectins; the latter does not bind minor contaminant proteins. These observations are confirmed by the two lectin blots (Concanavalin A has the same sugar specificity as lentil lectin).
Guinea pigs were vaccinated as described in Example 2 with samples of the fractions of IEF 5 and 6 following fractionation by lectin affinity chromatography as outlined above. Worm counts at slaughter (Table 8) showed that protection was afforded by the lectin-bound fraction. Other fractions also afforded some protection in this experiment. This could be due to incomplete removal of the 45 kD component from the IEF fraction 5 & 6 by the lectins as the antigenmay exist in various forms with different degrees of glycosylation (although little material of this molecular weight can be seen on the SDS gel profiles of the Helix pomatia lectin unbound fraction). The protection obtained with the other fractions could also be due to the presence in those fractions of other protective antigens. These other antigens are of interest, here.
TABLE 8______________________________________Fraction Worm Counts % Protection______________________________________Controls 462 .+-. 320 0IEF 5 and 6 273 .+-. 238 41Lentil lectin bound (LL.sup.+) 315 .+-. 127 32Lentil lectin unbound (LL.sup.-) 207 .+-. 288 55LL.sup.+, Helix pomatia lectin 221 .+-. 150 52boundLL.sup.+, Helix pomatia lectin 218 .+-. 114 53unbound______________________________________
EXAMPLE 9
IEF 5 and 6 was dialysed against 20 mM Tris/1 mM EDTA pH 7.5 and then concentrated ten-fold using a YM-10 membrane. 0.3 ml aliquots were acidified by the addition of trifluoroacetic acid (TFA) to a concentration of 0.1% and then centrifuged to remove any precipitate. The supernatant was injected onto a PLRP-S reverse phase HPLC column (Polymer Laboratories) equilibrated in 30% acetonitrile/0.1% TFA and developed with a linear gradient over 30 min until 60% acetonitrile/0.1% TFA was achieved. Eluent absorbance was monitored at 220 nm (FIG. 5) and fractions collected across the gradient. Selected fractions were run on SDS polyacrylamide gels and stained with silver (FIG. 6) to show that most of the proteins present in the starting material were recovered and were highly resolved.
These data demonstrate that the antigens in the protective fraction could be well resolved by reverse phase HPLC but further studies demonstrated that the material was no longer protective to guinea pigs following vaccination. Presumably treatment of the antigen fraction with the solvents used on the reverse phase HPLC denatured the antigens so that they no longer resembled the structure of the native antigens.
EXAMPLE 10
In order to remove the detergent, concentrate the sample, remove salts and exchange the antigen into a buffer suitable for amino acid sequence analysis, the lentil lectin bound, Helix pomatia bound antigen of approximately 45 kD was fractionated by reverse phase HPLC on a Polypore phenyl RP (30.times.2.1 mm) column and resolved over 20 minutes with a gradient of from 0.1% TFA/10% acetonitrile to 0.07% TFA/70% acetonitrile (flow rate 200 u l/min). The 45 kD antigen was eluted toward the centre of the gradient as a major peak with a small shoulder. The two fractions were collected separately but appear to be composed of the same polypeptide based on the N-terminal amino acid sequence data obtained. ##STR1##
The N-terminal amino acid sequence of the fractions was determined by gas phase sequencing on an Applied Biosystems model 470 l A amino acid sequencer. The following sequence was obtained (Seq ID No:1) ##STR2##
X indicates that no amino acid could be ascribed to that particular position. Residues in brackets are ascribed with less confidence than the other residues. In some cases, it was not possible to differentiate between two or three residues in a particular cycle in which case, they are listed beneath one another. The first residue in each peptide is assumed to be a lysine (K) based on the specificity of Endoproteinase Lys-C.
These sequences are suitable for the design of oligonucleotide sequences which would be suitable to use as hybridisation probes to identify the gene coding for the antigen in gene libraries generated using H. contortus RNA (complementary DNA libraries) or DNA (genomic libraries) or as primers for the polymerase chain reaction (PCR).
EXAMPLE 11
Molecular Cloning of the 45 kD gene
(a) Oligonucleotide synthesis
From the amino acid sequence described in Example 10, oligonucleotides were prepared that could be used to screen cDNA and genomic libraries to identify the gene(s) encoding the 45 kD antigen. In addition, the oligonucleotide could be used in conjunction with oligo (dT) in PCR (Saiki et al., 1988) to amplify the DNA encoding the 45 kD protein.
The following multiply-degenerate primers were designed and synthesized on an Applied Biosystem Model 380A automated DNA synthesizer. Nucleotides additional to those necessary to encode the required amino acid sequence were included on the 5' ends of the oligonucleotides. These sequences encode sites for the restriction enzymes, Eco RI and Sma I in order to assist in the cloning of PCR amplified DNA into appropriate vectors. An oligo (dT) primer for use in PCR was also synthesized. The primer also contained Eco RI and Sma I restriction sites. ##STR3##
A112/302 (SEQ ID No: 8) is identical to A112/301 (SEQ ID No: 7) except for the sixth codon from the 5' end. This was altered from AAC/T to T/AC/GG/A/T/C to account for the mixed signal seen in the amino terminal sequence, viz. asparagine (A112/301SEQ ID No: 7) or serine (A112/302, SEQ ID No: 8).
(b) RNA isolation and cDNA library construction
Total RNA was isolated from 1 g (wet weight) of H. contortus using an RNA extraction kit purchased from Pharmacia (Cat #XY-016-00-01). Larvae were obtained from the abomasum of sheep 15 days after infestation with exsheathed L3 stage parasites and stored a -70.degree. C. after snap freezing in liquid nitrogen. In order to extract RNA, the frozen worms were pulverized under liquid nitrogen, added to 7 ml extraction solution (which is a buffered aqueous solution containing guanidine thiocyanate, N-lauryl sarcosine and EDTA; density at 25.degree. C. =1.15 g/ml) and then layered over 2.times.1.25 ml cushions of CsTFA (buffered aqueous solution containing CsTFA; density at 25.degree. C.=1.51 g/ml) in 13.times.51 mmpolyallomer tubes. The gradients were centrifuged at 31,000 rpm for 6 hours at 15.degree. C. using an SW 50.1 rotor in an L8-70 Beckman ultracentrifuge. After centrifugation, pellets of RNA were dissolved in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) and reprecipitated from ethanol at -20.degree. C. The sedimented RNA was then dissolved again in TE and further purified by centrifugation through an oligo (dT)-cellulose column (Pharmacia mRNA Purification Kit Cat, #XY-012-00-02) as described by the manufacturer.
The resulting purified poly(A).sup.+ RNA was used to construct cDNA libraries using a Pharmacia cDNA Synthesis Kit (Cat #XY-003-00-03). Briefly, polyadenylated RNA purified from 325 .mu.g total RNA was treated with the Moloney Murine Leukaemia Virus Reverse Transcriptase in the presence of oligo (dT).sub.12-18. Second strand synthesis was accomplished using RNase H and E. coli DNA polymerase I. The double stranded cDNA was treated with the Klenow fragment of DNA polymerase and ligated to Eco RI/Not I adaptors. The cDNA was then treated with T4 polynucleotide kinase to phosphorylate it and ligated into Eco RI digested, dephosphorylated lambda gt 10 arms and packaged in vitro into infectious bacteriophage particles (Protoclone lambda gt 10 System and Packagene System, Promega) as described by the supplier. The packaged cDNA was transfected into E. coli C600 Hfl and plated on Luria agar plates using Luria top agar containing 10 mM MgSO.sub.4. A total of 6.times.107.sup.7 p.f.u. were obtained of which 98 % were recombinants. The average insert size was 2.0 kbp.
(c) Preparation of DNA probes for screening recombinant libraries.
A 45 kD antigen-specific double stranded DNA probe was prepared using PCR. The procedure used was based on that described by Saiki et al. (1985) and used a cloned form of Taq polymerase (Perkin Elmer Cetus). 2 .mu.g total RNA was annealed to 100 ng oligo (dT) PCR primer in 6 .mu.l water by heating to 70.degree. C. for 5 minutes and then leaving to cool to room temperature. The annealed RNA-oligo (dT) was then incubated with 200 units reverse transcriptase (BRL) in the presence of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM MgCl.sub.2, 5 mM spermidine, 10 mMDTT, 1 unit RNasin for 1 hour at 37.degree. C. in a final volume of 25 .mu.l. A similar reaction from which reverse transcriptase was omitted served as a negative control for the PCR reaction.
PCR was performed on cDNA produced as described above. The reaction mixture contained first strand cDNA synthesized from 1 .mu.g total RNA, 1 .mu.M each of one of A112/301 (SEQ ID No. 7 or A112/302 (SEQ ID No: 8) and 1 .mu.M oligo (dT) PCR primer, 200 .mu.M of each dNTP, 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 2 mM MgCl.sub.2, 0.01% gelatin, 0.01% Triton X-100 and 2 units of Taq polymerase in a total volume of 100 .mu.l. Amplification was carried out over 25 cycles, each of which consisted of denaturation for 1 minute at 94.degree. C., annealing for 2 minutes at 40.degree. C. and extension for 3 minutes at 72.degree. C.
Samples of each PCR reaction were analyzed on a 0.8% agarose gel at the end of the reactions.
In the reaction containing primer A112/301 (SEQ ID No: 7) and oligo (dT), a unique band of approximately 650 bp was observed. Several other bands were present but these were also seen in the reaction in which oligo (dT) only was used. The approximately 650 bp band was not seen when primer A112/302 (SEQ No: 8) was used. No bands were seen in the negative control reaction from which reverse transcriptase was omitted.
The approximately 650 bp PCR product was digested with Eco RI, purified by electrophoresis on an agarose gel, ligated into pBluescript SK- (Stratagene) using conventional techniques (Maniatis et al., 1982) and sequenced using the dideoxy chain termination procedure (Amersham Microtitre Plate Sequencing Kit, Cat #RPN.1590). Sequence analysis of the ends of the clone confirmed that it contained the sequence of primer A112/301 (SEQ ID No: 7) at the 5' end and a poiy (A) stretch at the 3' end. Furthermore, 14 out of the 18 nucleotides immediately downstream from the 3' end of the region corresponding to the primer A112/301 (SEQ ID No: 7) corresponded to those predicted from the amino acid sequence of the purified protein. This is an homology of 86% at the amino acid level (18 amino acids out of the 21 returned from N terminal sequencing). The differences between the amino acid sequence obtained from the purified antigen and that predicted from the DNA sequence of the PCR clone could be accounted for by ambiguities in the amino terminal sequence analysis of the purified protein and/or PCR incorporation errors although this is an unlikely explanation given the large number of differences. The PCR clone was grown, DNA was isolated, digested with Eco RI and the insert purified for use as a hybridisation probe to screen the cDNA library described in (b) of this example.
Approximately 10.sup.5 p.f.u. were screened by hybridization of nitrocellulose filter replicas of the library at 55.degree. C. in a solution containing 2.times.10.sup.5 cpm/ml probe, 5.times.SSPE, 5.times.Denhardt's solution, 0.5% (w/v) SDS and 20 .mu.g/ml sheared, denatured salmon sperm DNA. After washing the filters at 60.degree. C. in 0.5.times.SSC, 0.1% SDS and autoradiography, 16 positive plaques were identified. 0f these, 8 were picked for subsequent purification and analysis. Eco RI inserts were isolated from the purified phage DNA and subcloned into pBluescript SK- for further analysis. The sequence of one of these clones, pBTA879, is shown in FIG. 7 and in SEQ ID No: 9.
There is a single long open reading frame of 1336 nucleotides followed by a translation stop codon, TGA. The open reading frame of this clone extends from the 5' end of the clone. There is no methionine initiation codon present in this region of the sequence so this clone probably does not represent the complete coding region.
Close examination of the amino acid sequence derived from the cDNA sequence (FIG. 7, SEQ ID Nos. 9 and 10) reveals a region of homology with the amino terminal sequence of the purified 45 kD protein commmencing at nucleotide 65 from the 5' end of the cDNA clone where 11 of the predicted amino acids occur over the following 16 residues. In addition, there is a second region with homology with the N-terminal sequence commencing at nucleotide 773 of the sequence (SEQ ID No: 9). Of 19 amino acids, 14 residues are identical with those determined from the protein sequence. The level of homology was 73.7% at the amino acid level. It appears that there may be repetitive domains within the molecule. Both of these regions of homology are indicated in FIG. 7 by double underlining.
Other regions within the predicted amino acid sequence which share homology with the Endoproteinase Lys-C peptide sequences derived from the purified protein are indicated in FIG. 7 (by single underlining). These regions all lie within the carboxy-terminal half of the molecule; all in the portion which is carboxy-terminal to amino acid 253 of SEQ ID No: 10.
In addition, there is a region immediately preceding the sequence starting at nucleotide 773 of SEQ ED No: 9 which is similar to peptide sequences which have been hypothesised as being highly susceptible to proteinase digestion.
The amino acid sequence N-terminal to that starting at nucleotide 65 of SEQ ID No: 9 is very hydrophobic and contains amino acids similar to those recognised by signal peptidases.
The most likely explanation of these analyses is that the cDNA clone which is described in FIG. 7 (SEQ ID No: 9) encodes a glycoprotein which is related to, but not identical the native glycoprotein isolated from H. contortus. The cDNA clone does not code for the full length protein; at least a complete signal peptide precedes that shown in FIG. 7 (SEQ ID No: 10) and it is possible that further amino acids may also be present in the full-length native molecule.
In order to isolate a cDNA clone coding for the full length native 45 kD antigen, cDNA libraries were screened with the fragment isolated from pBTA879. The cDNA library described in (b) of this example was again screened using the Eco RI band of pBTA 879 as hybridisation probe. Approximately 10.sup.5 p.f.u. were screened using the same conditions as outlined earlier. After washing the filters at 60.degree. C. in 0.5.times.SSC, 0.1% SDS and autoradiography, 15 positive plaques were identified. Of these 11 Eco RI inserts were purified and subcloned into pBluescript SK- for further analysis. The sequence of one of these clones, pBTA 963, is shown in FIG. 8 and in SEQ ID No: 11. This contains an open reading frame of 1320 nucleotides followed by a translation stop codon, TAA.
Once again, this clone does not contain an initiation methionine, however, 16 out of 18 amino acids coded for by the 5' sequence of this clone are identical to the original N-terminal amino acid sequence (SEQ ID No: 1). This region of homology starts at base 50 of SEQ ID No: 11 or amino acid 12 of SEQ ID No: 12 and a second repeat can be found starting at base 722 of SEQ ID No: 11. Both regions are shown with double underlining. The other peptide sequences (SEQ ID Nos. 2, 3, 4, 5, and 6) can also be located in the translated amino acid sequence from pBTA 963 (SEQ ID Nos. 11 and 12.)These have a much greater homology with the peptide sequences than is seen with pBTA 879 (SEQ ID Nos. 9 and 10). In most cases this homology is 100%.
The other characteristics of the two protein sequences are very similar. Both contain a hydrophobic leader sequence segment, both contain the peptidase sensitive region toward the middle of the molecules and both are of similar molecular weight and overall charge. However, the two share only approximately 54% homology in amino acid sequence overall although the homology is much higher in some places, particularly those from which the peptide sequences were derived.
To try to ensure that the cloned gene codes for the purified antigen, the Eco RI fragment was isolated from the original PCR clone and was inserted into an IPTG inducible bacterial expression vector. The gene product was isolated, purified and used to vaccinate sheep. Sera obtained from these sheep were used to probe Western blots of the purified 45 kD glycoprotein and homogenates of H. contortus. In both instances, an antigen with an apparent molecular weight of 45 kD in IEF fractions 1-10 was found to react specifically with the antisera. Antibodies generated against a 24 amino acid long peptide with a sequence predicted from the DNA sequence also reacted with the 45 kD antigen in IEF fractions 1-10.
Close inspection of the Western blots of whole parasite homogenates and each broad range IEF fraction resolved with serum from sheep vaccinated with the recombinant antigen reveals that there are in fact higher molecular weight parasite components which specifically react with the post vaccination serum. These higher molecular weight components are likely to be products of the same gene family to that of the isolated antigen. They are located in IEF fractions 2, 3 and 4 in particular and appear to smear across the isoelectric focussing gels. This phenomenon of not focussing as sharp band upon fractionation by IEF is a characteristic of glycoproteins; the variable carbohydrate residues introduce a heterogeneity of charge on the molecules and a heterogeneity in pI of the population of molecules.
It therefore appears that the native 45 kD antigen isolated from the parasite is a member of a class of proteins which share similar amino acid sequences over portions of the molecules. This is further supported by Southern blot analysis of H. contortus genomic DNA using both Eco RI inserts (FIG. 9). These show pBTA 963 hybridising to many bands with varying intensity. The bands at 1480, 890 and 740 base pairs are the most strongly hybridising. The same blot screened with pBTA 879 however lights up a band at 1350 base pairs with the 890 and 740 bands still binding weakly with this probe.
From the Western blots it appears that some members of the protein family may have a molecular weight in excess of 65 kD. It is also possible that the protection afforded to vaccinated sheep and guinea pigs by the IEF fraction 2, 3 and 4 in Examples 4 and 5 of this specification are due to the presence of the larger molecular weight forms of the protective antigen described herein.
No DNA or protein sequences with significant homology to that of this clone could be found after searching the Genbank, EMBL or PIR computer data bases.
EXAMPLE 12
Homologous genes related to that of the protective antigen are present in other species of parasitic nematode
DNA hybridisation (or Southern blot analysis) was carried out using standard techniques (Maniatis et al., 982) to determine whether other species of parasitic nematodes have genes which are "homologous" to those coding for the H. contortus protective antigen. The Eco RI insert of the cDNA clone pBTA963 described in Example 11 (FIG. 8, SEQ ID No: 11) was used as a hybridisation probe to DNA isolated from a number of other species of parasitic nematodes. As well as hybridizing strongly to several restriction fragments in the DNA isolated from the homologous species, i.e., H. contortus, as expected, the probe also hybridized to specific restriction fragments in DNA isolated from Ostertagia circumcincta, Ostertagia ostertagi and Trichostrongylus colubriformis. The blots were washed at a stringency that suggested that the level of homology was about 70%. (FIG. 10).
This clearly demonstrates that there are genes which are closely related to that coding for the protective antigen in these other species of parasitic nematodes and, by extension, in all species of parasitic nematodes. These genes could be isolated using standard molecular biological techniques, and recombinant organisms could be made which synthesise those related or homologous antigens from the other species of parasitic nematode. The present inventors consider that the related antigens will serve as effective immunogens to provide protection to vaccinated animals against infection by the other species of parasite. In addition, related antigens isolated from a broad range of parasitic nematodes could be isolated and provide effective protective immunogens to protect animals against infestation by an extensive range of such nematodes.
It has already been demonstrated that this approach can be successful (International application No. PCT/AU88/00239). That patent application describes how an antigen was purified from an homogenate of T. colubriformis based on the ability of that antigen to provide protection to guinea pigs against challenge infections by T. colubriformis. Amino acid sequence information was determined for this antigen which enabled the gene coding for the antigen to be isolated from recombinant DNA libraries. The DNA coding for the T. colubriformis gene was then used as a hybridisation probe to identify recombinant organisms coding for the "homologous " gene from H. contortus. Recombinant organisms were then constructed which synthesised the H. contortus antigen which was then used in vaccination and challenge trials in sheep and guinea pigs. The H. contortus recombinant antigen provided protection to vaccinated sheep against infestation by H. contortus and provided protection to guinea pigs against challenge infection by I. colubriformis.
This demonstrates that it is possible, given the DNA sequence homology demonstrated in the above hybridisation experiments, to use the cloned DNA sequence coding for protective antigen from one species of parasitic nematode to identify clones coding for the homologous gene products from other species of parasitic nematodes, engineer those recombinant organisms to express the homologous antigen and use this in a vaccine to provide protection against the other species of parasitic nematode. It is considered that a natural extension of the results presented here is to do so with the DNA sequences of the present invention.
It is to be understood that the nucleotide sequence of the homologous genes and the amino acid sequence of the homologous antigens may not be identical to those of the first target species but will be related by at least 50% over a stretch of at least 60 base pairs and preferably the relationship would be 70% or more over this same region with the homology at the amino acid level being at least 70% over 20 amino acids. In most cases, this degree of homology would be sufficient to enable an unambiguous identification of the relatedness of the two genes or proteins.
EXAMPLE 13
Expression of the 45 kD Antigen in E. coli
A number of systems could be used to express the recombinant 45 kD antigen, e.g. mammalian cells, virus-infected insect cells, yeasts, or bacteria. As an example, the gene was expressed in E. coli. The full cDNA fragment from pBTA 963 (SEQ ID No: 9) was isolated as a 1386 base pair Bam HI/Hind III fragment, and was subcloned into E. coli expression vector pBTA 954. pBTA 954 is a pUR-based vector containing the lac promoter, the initiating amino acids for the .beta.-galactosidase gene and multiple cloning sites. When expression was induced by addition of 1 mM IPTG, this yielded a fusion protein with 11 N-terminal amino acids encoded by the vector, fused to the 45 kD protein. The apparent molecular weight of the fusion protein was approximately 61 kD (FIG. 11a). The fusion protein was recognised by rabbit serum raised against a peptide corresponding to the truncated N-terminus of the 45 kD protein (FIG. 11b). This serum had previously been shown to recognise the dominant protein in the native extract, IEF fraction 1.
The fusion protein could be purified by standard techniques known in the art, formulated with a suitable adjuvant and used to vaccinate the host such as sheep and provide protection against parasite infection.
EXAMPLE 14
Immunological Cross-Reactivity between Antibodies to the 45 kD antigen and Dirofilaria immitis proteins
Rabbits were vaccinated with a synthetic peptide derived from the sequence of the N-terminal region of the 45 kD antigen. Antisera from these rabbits was used to probe a Western blot of extracts from both adult D. immitis and H. contortus. After development with a second antibody conjugated to alkaline phosphatase two antigens from each species were shown to be reactive. Components of 45 kD and 27 kD were detected in the H. contortus extract and 52 and 32 kD in the D. immitis extract (FIG. 12). This result supports other observations (Example 12) using DNA hybridisation techniques, that proteins related to the 45 kD antigen are present and are expressed in D. immitis.
EXAMPLE 15
Scale up of ManufactUring for Commercial Vaccines
The production and purification techniques so far described are carried out at laboratory scale. For commercial production of the antigens of the invention, large scale fermentation of transformed hosts is required.
The large scale fermentations are performed according to standard techniques, the particular techniques selected being appropriate to the transformed host used for production of the antigen.
DEPOSITION OF MICROORGANISMS
Strain BTA 2033, which is E. coli JM101 containing the plasmid pBTA 879, has been deposited with Australian Government Analytical Laboratories of 1 Suakin Street, Pymble 2073, New South Wales, Australia in accordance with the provisions of the Budapest Treaty on 29 Jan. 1992 under accession number N92/4387.
The genotype of E- coli JM101 is: .DELTA.(pro-lac). F' lacI.sup.q .DELTA.M15, traD1, .lambda..sup.-. pBTA 879 is pBLUESCRIPT-SK-minus (Stratagene, San Diego, Calif., USA) containing a 1400 base pair Eco RI insertion coding for a portion of an antigenic protein from the gasto-intestinal nematode,
Haemonchus contortus.
Strain BTA 2125, which is E. coli SURE strain containing the plasmid pBTA 963, has been deposited with Australian Government Analytical Laboratories of 1 Suakin Street, Pymble 2073, New South Wales, Australia in accordance with the provisions of the Budapest Treaty on 9 Jan. 1992 under accession number N92/4388.
The genotype of E. coli SURE is BTA2125 is: E. coli SURETM strain (Stratagene, San Diego, Calif., USA), genotype: mcrA, .DELTA.(mrr, hsd RMS mcrBC), endA1, supE44, thi-1, .lambda..sup.-, gyrA96, relA1, lac, recB, recJ, sbcC, umuC::Tnr(kan.sup.R), uvrC, {F' proAB, lacI.sup.q Z.DELTA.M15, Tn10(tet.sup.R)}.
Plasmid pBTA 963 is pBLUESCRIPT-SK-minus (Stratagene, San Diego, Calif., USA), containing a 1386 base pair Eco RI insertion coding for a portion of an antigenic protein from the gastro-intestinal nematode Haemonchus contortus.
INDUSTRIAL APPLICATIONS
The present invention is of use in providing antigens, vaccines, and antibodies suitable for protecting animals against infection by parasitic nematodes.
REFERENCES
Maniatis T, Fritsch EF and Sambrook J (eds) (1982) Molecular cloning: A laboratory manual CSH Laboratory, Cold Spring Harbor.
Saiki R. K., Gelfand D. H., Stoffels, Scharf S. J. Higuchi R., Horn G. T., Mullis K. B. and Erlich H. A. (1988) Primer directed amplification of DNA with a thermostable DNA polymerase Science 239: 487-491.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 12(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 21 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(v) FRAGMENT TYPE: N-terminal(vi) ORIGINAL SOURCE: (A) ORGANISM: Haemonchus contortus(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 2(D) OTHER INFORMATION: /note="May be Phe or Tyr"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 3(D) OTHER INFORMATION: /note="May be His"(ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 5(D) OTHER INFORMATION: /note="May be Gly, Asp or Pro"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 6(D) OTHER INFORMATION: /note="May be Ser or Asn"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 19(D) OTHER INFORMATION: /note="May be Val or Ser"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 20(D) OTHER INFORMATION: /note="May be Asp, Gly or Pro"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 21(D) OTHER INFORMATION: /note="May be Lys, Gly, Pro orAsp"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:AlaXaaXaaProX aaXaaAsnAsnGlyMetThrAspGluIleArgGln151015IlePheXaaXaaXaa20(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: N-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Haemonchus contortus(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 2..3(D) OTHER INFORMATION: /note="Amino acids unknown"(ix) FEATURE: (A) NAME/KEY: Modified-site(B) LOCATION: 4(D) OTHER INFORMATION: /note="May be Pro or Tyr"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 6(D) OTHER INFORMATION: /note="Amino acid unknown"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 17(D) OTHER INFORMATION: /note="May be Asn or Ser"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:LysXaaXaaXaaAspXaaGluValGluAlaAsnThrAlaAlaTyrAla151015XaaGluGlu(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: N-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Haemonchus contortus(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 2(D) OTHER INFORMATION: /note="May be Asp, Ser, His orGly"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 11..12(D) OTHER INFORMATION: /note="Amino acids unknown"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 16(D) OTHER INFORMATION: /note="Amino acid unknown"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:LysXa aAsnGluTyrArgSerLeuIleAlaXaaXaaGlxGlnLeuXaa151015(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 9 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: N-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Haemonchus contortus(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 2(D) OTHER INFORMATION: /note="May be Leu, Asp, Gly orAla"(ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 3(D) OTHER INFORMATION: /label=RESIDUE-3/note="AMINO ACID MAY BE ASP, GLY OR ALA"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 4(D) OTHER INFORMATION: /label=RESIDUE-4/note="AMINO ACID MAY BE GLY OR ASP"(ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 9(D) OTHER INFORMATION: /note="Amino acid unknown"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:LysXaaXaaXaaPheAlaProLysXaa15(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 16 amino acids(B) TYPE: amino acid (D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: N-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Haemonchus contortus(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 2(D) OTHER INFORMATION: /note="May be His, Ser or Gly"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 9 (D) OTHER INFORMATION: /note="May be Leu or Ile"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 10(D) OTHER INFORMATION: /note="May be Ala or Thr"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 13(D) OTHER INFORMATION: /note="Amino acid unknown"(ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 16(D) OTHER INFORMATION: /note="Amino acid unknown"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:LysXaaAsnGluTyrArgSerIleXaaXaaLysProXaaLeuAsnXaa151015 (2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: peptide(v) FRAGMENT TYPE: N-terminal(vi) ORIGINAL SOURCE:(A) ORGANISM: Haemonchus contortus(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 2 (D) OTHER INFORMATION: /note="Amino acid unknown"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 3(D) OTHER INFORMATION: /note="May be Pro, Gly or Asp"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 4(D) OTHER INFORMATION: /note="May be Tyr or Lys"(ix) FEATURE:(A) NAME/KEY: Modified-site (B) LOCATION: 5(D) OTHER INFORMATION: /note="May be Asp or Pro"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 6(D) OTHER INFORMATION: /note="Amino acid unknown"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 7(D) OTHER INFORMATION: /note="May be Asp, Thr or Glu"(i x) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 9(D) OTHER INFORMATION: /note="Amino acid unknown"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 11(D) OTHER INFORMATION: /note="May be Asp or Thr"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 12..14 (D) OTHER INFORMATION: /note="Amino acids unknown"(ix) FEATURE:(A) NAME/KEY: Modified-site(B) LOCATION: 18(D) OTHER INFORMATION: /note="Amino acid unknown"(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:LysXaaXaaXaaXaaXaaXaaValXaaAlaXaaXaaXaaXaaThrPro15 1015ProXaa(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base (B) LOCATION: 16(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 19(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 22(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 25(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 28(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 31(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 34(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 37(ix) FEATURE:( A) NAME/KEY: modified.sub.-- base(B) LOCATION: 40(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 46(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 49(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:GCGAATTCCCGGGGCHTTYCAYCCNGGNAAYAAYAAYGGNATGACN GAYG50(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 50 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(iv) ANTI-SENSE: YES(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 16(i x) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 19(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 22(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 25(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 28(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 29(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 30(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 31(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base (B) LOCATION: 34(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 37(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 40(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base(B) LOCATION: 46(ix) FEATURE:(A) NAME/KEY: modified.sub.-- base (B) LOCATION: 49(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:GCGAATTCCCGGGGCHTTYCAYCCNGGNWSNAAYAAYGGVATGACDGAYG50(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1400 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double (D) TOPOLOGY: unknown(ii) MOLECULE TYPE: cDNA(vi) ORIGINAL SOURCE:(A) ORGANISM: Haemonchus contortus(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 17..1381(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:GAATTCGCGGCCGCTTACGATTGCCTGCTTGGTTCTTCTGGCGCCATTA49ThrIleAlaCysLeuValLeuLeuAlaProLeu1510TGGGCGGCTGATAAGTATGTGATTTGTCCTTCTGACAATGGCATGACA97TrpAlaAlaAspLysTyrValIleCysProSerAspAsnGlyMetThr152025AATGAAGTTAGAAATATGTTCGTTGATACGCACAATAAACTCCGATCG145AsnGluValArgAsnMetPheValAspThrHisAsnLysLeuArgSer303540CAGACTGCTCAAGGAAAGGCTAAGAACGCATTCGGTGGATTTGCTCCA193Gl nThrAlaGlnGlyLysAlaLysAsnAlaPheGlyGlyPheAlaPro455055AAAGCAGCTCGAATGTTAAAAGTGAGTTATGATTGCGACATGGAAGCT241LysAlaAl aArgMetLeuLysValSerTyrAspCysAspMetGluAla60657075AACATGATGAAATGGGCAAAGCAGTGTCATTTCTATCACCCTCCACCC289As nMetMetLysTrpAlaLysGlnCysHisPheTyrHisProProPro808590GCATATAGGAACTACTGGGGACAAAATATTTATATGGTGGGAGATCGA337AlaTyrArgAsnTyrTrpGlyGlnAsnIleTyrMetValGlyAspArg95100105TACTACAATTTCACCTGGCCGTCAATTGCAGAAACGGCCGTCATATCA385TyrTyrAsnPheThrTrpProSerIleAlaGluThrAlaValIleSer110115120TGGTGGCAGGAGTTACAGGTTTTTGGTGTTCCAGAGAACAATATCGTA433Tr pTrpGlnGluLeuGlnValPheGlyValProGluAsnAsnIleVal125130135GTCGCGCCAGATGAACACAAAACTGGTCACTACATGCAGGTGGTCTGG481ValAlaPr oAspGluHisLysThrGlyHisTyrMetGlnValValTrp140145150155CAATGGACCTACAAAATTGGTTGCGCAATTAATTATTGCACAATAAAC529Gl nTrpThrTyrLysIleGlyCysAlaIleAsnTyrCysThrIleAsn160165170AAGCCATGGCCATGGACAATCGCAGGATGCAACTATAACCCTGGTGGT577LysProTrpProTrpThrIleAlaGlyCysAsnTyrAsnProGlyGly175180185GATAATGCTTATTGGGTGGTCTACGAGATGGGAGATCCATGCACAACT625AspAsnAlaTyrTrpValValTyrGluMetGlyAspProCysThrThr190195200GACGCCGACTGCAAATGTGCTGGTTGCGTTTGCAGCCAAGAAGAGGCC673As pAlaAspCysLysCysAlaGlyCysValCysSerGlnGluGluAla205210215CTTTGCATTCCGCCAGAATACACTCCCCTTCCACCTACTACCACTTCA721LeuCysIl eProProGluTyrThrProLeuProProThrThrThrSer220225230235ACCACAACACCGAAGCCAACTACAACAACAACCGTTGGGGTACCTAAT769Th rThrThrProLysProThrThrThrThrThrValGlyValProAsn240245250GCTGGGTCGTGCCCTGAACTTAACAATGGAATGACTGACGAAGCTAGG817AlaGlySerCysProGluLeuAsnAsnGlyMetThrAspGluAlaArg255260265AAGATGTTTGTCGACAAACATAATGAATACCGATCGCTCATTGCTAAA865LysMetPheValAspLysHisAsnGluTyrArgSerLeuIleAlaLys270275280GGGCAAGCCAAGGGTAAACCTGGACAATTCGCCCCAAAGGCTGCCAGA913Gl yGlnAlaLysGlyLysProGlyGlnPheAlaProLysAlaAlaArg285290295ATGATGAAAGTGAACTACGATTGCGATGTTGAAGCAAATGCAATGGAA961MetMetLy sValAsnTyrAspCysAspValGluAlaAsnAlaMetGlu300305310315TGGTCCAAGACTTGCACATTTGGACTCAACACTGCTGCGATGTTAAAG1009Tr pSerLysThrCysThrPheGlyLeuAsnThrAlaAlaMetLeuLys320325330CGATGGGGGAATAACATGCACATGATGTCGTCCAAGGCTAATAACAAG1057ArgTrpGlyAsnAsnMetHisMetMetSerSerLysAlaAsnAsnLys335340345ACAGAGGCTGCAGCTGAGGCCGTCGCAGCCTGGTTCGGTGATTTACAA1105ThrGluAlaAlaAlaGluAlaValAlaAlaTrpPheGlyAspLeuGln350355360AAATATGGCGTACCTGAGAATAACGTCTTCACGATGAACGTTTACACG1153Ly sTyrGlyValProGluAsnAsnValPheThrMetAsnValTyrThr365370375ACTTTAAGTAAATACAGTCAGTTAGCGTGGCAATCGAGCGACAGAATT1201ThrLeuSe rLysTyrSerGlnLeuAlaTrpGlnSerSerAspArgIle380385390395GGTTGTGTAGTTGTACCTTGTTGGAGCTCATGGACGGTTGTGGTGTGT1249Gl yCysValValValProCysTrpSerSerTrpThrValValValCys400405410GAATACAATCCCGGAGGAGACCTGCCTGGCGAGGCTATCTATGACGTA1297GluTyrAsnProGlyGlyAspLeuProGlyGluAlaIleTyrAspVal415420425GGAGATCCCTGTACGAAAGACGCCGACTGTCAGTGCCCCGGCTGCACC1345GlyAspProCysThrLysAspAlaAspCysGlnCysProGlyCysThr430435440TGTAGCAGAGATGAGGGCCTTTGCGTTGCTCCATGAACACTGGCGGCCGCTTA1398Cy sSerArgAspGluGlyLeuCysValAlaPro445450455AG1400(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 454 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:ThrIleAlaCysLeuValLeuLeuAlaProLeuTrpAlaAlaAspLys1510 15TyrValIleCysProSerAspAsnGlyMetThrAsnGluValArgAsn202530MetPheValAspThrHisAsnLysLeuArgSerGlnThrAlaGln Gly354045LysAlaLysAsnAlaPheGlyGlyPheAlaProLysAlaAlaArgMet505560LeuLysValSerTyrA spCysAspMetGluAlaAsnMetMetLysTrp65707580AlaLysGlnCysHisPheTyrHisProProProAlaTyrArgAsnTyr85 9095TrpGlyGlnAsnIleTyrMetValGlyAspArgTyrTyrAsnPheThr100105110TrpProSerIleAlaGluThrAlaVa lIleSerTrpTrpGlnGluLeu115120125GlnValPheGlyValProGluAsnAsnIleValValAlaProAspGlu130135140HisLysThrGlyHisTyrMetGlnValValTrpGlnTrpThrTyrLys145150155160IleGlyCysAlaIleAsnTyrCysThrIleAsnLysProTrpProTrp165170175ThrIleAlaGlyCysAsnTyrAsnProGlyGlyAspAsnAlaTyrTrp180185190ValValT yrGluMetGlyAspProCysThrThrAspAlaAspCysLys195200205CysAlaGlyCysValCysSerGlnGluGluAlaLeuCysIleProPro210 215220GluTyrThrProLeuProProThrThrThrSerThrThrThrProLys225230235240ProThrThrThrThrThrValGlyValPr oAsnAlaGlySerCysPro245250255GluLeuAsnAsnGlyMetThrAspGluAlaArgLysMetPheValAsp260265 270LysHisAsnGluTyrArgSerLeuIleAlaLysGlyGlnAlaLysGly275280285LysProGlyGlnPheAlaProLysAlaAlaArgMetMetLysValAsn290295300TyrAspCysAspValGluAlaAsnAlaMetGluTrpSerLysThrCys305310315320ThrPheGlyL euAsnThrAlaAlaMetLeuLysArgTrpGlyAsnAsn325330335MetHisMetMetSerSerLysAlaAsnAsnLysThrGluAlaAlaAla340 345350GluAlaValAlaAlaTrpPheGlyAspLeuGlnLysTyrGlyValPro355360365GluAsnAsnValPheThrMetAsnValTy rThrThrLeuSerLysTyr370375380SerGlnLeuAlaTrpGlnSerSerAspArgIleGlyCysValValVal385390395 400ProCysTrpSerSerTrpThrValValValCysGluTyrAsnProGly405410415GlyAspLeuProGlyGluAlaIleTyrAspValGlyAspProCys Thr420425430LysAspAlaAspCysGlnCysProGlyCysThrCysSerArgAspGlu435440445GlyLeuCysV alAlaPro450(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1386 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: unknown(ii) MOLECULE TYPE: cDNA(vi) ORIGINAL SOURCE:(A) ORGANISM: Haemonchus contortus(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 17..1339(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:GAATTCGCGGCCGCTTTCGGTGCTTCTGACGCCATCATGCCTGAAAGCC49SerValLeuLeuThrProSerCysLeuLysAla1 510GCGTTTTGCCCCACATCGGACAATGGCATGACCGATGAAATTAGGCAG97AlaPheCysProThrSerAspAsnGlyMetThrAspGluIleArgGln15 2025ATTTTCGTTGATAAGCACAATGAGTATCGATCTATTATTGCTAAAGGA145IlePheValAspLysHisAsnGluTyrArgSerIleIleAlaLysGly30 3540CAGGCCAAGAATAAACTTGGAGGATTCGCTCCGAAAGCAGCTCGAATG193GlnAlaLysAsnLysLeuGlyGlyPheAlaProLysAlaAlaArgMet45 5055TTGAAAGTGGGTTACGATTGCGAAGTTGAGGCAAATACGGCGGCATAT241LeuLysValGlyTyrAspCysGluValGluAlaAsnThrAlaAlaTyr6065 7075GCAAAAGAGTGCAAGTTCGAACATGATCCACCCGAGCAAAGGAATTAT289AlaLysGluCysLysPheGluHisAspProProGluGlnArgAsnTyr80 8590TGGGGGCAGAACCTGTGGATGCTAGGAGGGACTAATTACAGCAAGACG337TrpGlyGlnAsnLeuTrpMetLeuGlyGlyThrAsnTyrSerLysThr95 100105GAATCTGCAAAATTAAGTGTCCAAGCTTGGTACTGGGAATTGAAGATG385GluSerAlaLysLeuSerValGlnAlaTrpTyrTrpGluLeuLysMet110 115120TTTGGAGTGCCCGATGAAAATATCCTGACAATGGAAGTCTTCGATCGG433PheGlyValProAspGluAsnIleLeuThrMetGluValPheAspArg125 130135GGTGTTGGCCACTACACACAGGTAGCCTGGCAGTCTAGCGACAAAATC481GlyValGlyHisTyrThrGlnValAlaTrpGlnSerSerAspLysIle140145 150155GGCTGCGCAGTTGAATGGTGCCCAACCATGACACTTGTAGCATGCGAG529GlyCysAlaValGluTrpCysProThrMetThrLeuValAlaCysGlu160 165170TACAACCCTGCAGGAAATAGGATCAATCATTATATTTACGACATCGGA577TyrAsnProAlaGlyAsnArgIleAsnHisTyrIleTyrAspIleGly175 180185GATCCATGCACAACTGATGAAGACTGTCAATGCACTGGCTGCACTTGT625AspProCysThrThrAspGluAspCysGlnCysThrGlyCysThrCys190 195200AGTAAAGATGAGGCCCTTTGTATTCCTCCAGGATATACTACCGTCATG673SerLysAspGluAlaLeuCysIleProProGlyTyrThrThrValMet205 210215CCACCGACTACAGAGAAACCTACTACAACACCTAAAATATACCATCCA721ProProThrThrGluLysProThrThrThrProLysIleTyrHisPro220225 230235GGTGGGATGTGCCCTGAGAATAATAACGGAATGACAGATGAAGCTAGG769GlyGlyMetCysProGluAsnAsnAsnGlyMetThrAspGluAlaArg240 245250CAGATGTTCGTCGACAAACACAATGAGTATCGATCCCTCATAGCTAAA817GlnMetPheValAspLysHisAsnGluTyrArgSerLeuIleAlaLys255 260265GGACTAGCTCATAACAATCTTGGAGGGTTTGCTCCAAAAGCGGCTAGA865GlyLeuAlaHisAsnAsnLeuGlyGlyPheAlaProLysAlaAlaArg270 275280ATGATGAAAGTGAGCTACAATTGCGAAATCGAAGCGAATCGAGTGGAG913MetMetLysValSerTyrAsnCysGluIleGluAlaAsnArgValGlu285 290295TGGGCGAAGGATTGCACGCTTGGGTACAACTCTGTTGCTCAAAATAAC961TrpAlaLysAspCysThrLeuGlyTyrAsnSerValAlaGlnAsnAsn300305 310315CAATGGGGTTATAATGTACATTCACTACTGCCGCATATTAACAAGACG1009GlnTrpGlyTyrAsnValHisSerLeuLeuProHisIleAsnLysThr320 325330GTAGCAGCAGCAGAGAGTGTCGAGGCCTGGTTCAATGAACTACAGACA1057ValAlaAlaAlaGluSerValGluAlaTrpPheAsnGluLeuGlnThr335 340345TATGGTGCACCTCAGGATAACGTTTTCAGTATGGAGGTTTTCAATCAA1105TyrGlyAlaProGlnAspAsnValPheSerMetGluValPheAsnGln350 355360AACGTAATACAGGAATACGCTCAGTTGGCGTGGCAATCGAGCAACCAG1153AsnValIleGlnGluTyrAlaGlnLeuAlaTrpGlnSerSerAsnGln365 370375ATTGGTTGTGGAATTTTTTCTTGCTGGGGTGGCGCCTCTACATTTGTG1201IleGlyCysGlyIlePheSerCysTrpGlyGlyAlaSerThrPheVal380385 390395GCTTGCGAATACAATCCTGGAGGAAACTTCATCGGCGAATTGATTTAT1249AlaCysGluTyrAsnProGlyGlyAsnPheIleGlyGluLeuIleTyr400 405410ACGATGGGAGATCCGTGCTCAACTGACGAAGACTGTCAGTGCGCTGGT1297ThrMetGlyAspProCysSerThrAspGluAspCysGlnCysAlaGly415 420425TGCGTCTGTAGCAAAGATGAAGCACTCTGTATTGCTCCTTAAATGCTTG1346CysValCysSerLysAspGluAlaLeuCysIleAlaPro430435 440TGCAATAAATCTTCAGTGAAAGAAAAGCGGCCGCGAATTC1386(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 440 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:SerValLeuLeuThrProSerCysLeuLysAlaAlaPheCysProThr151015SerAspAsnGlyMetThrAspGluIleArgGlnIlePheVal AspLys202530HisAsnGluTyrArgSerIleIleAlaLysGlyGlnAlaLysAsnLys354045LeuGlyG lyPheAlaProLysAlaAlaArgMetLeuLysValGlyTyr505560AspCysGluValGluAlaAsnThrAlaAlaTyrAlaLysGluCysLys6570 7580PheGluHisAspProProGluGlnArgAsnTyrTrpGlyGlnAsnLeu859095TrpMetLeuGlyGlyThrAsnTy rSerLysThrGluSerAlaLysLeu100105110SerValGlnAlaTrpTyrTrpGluLeuLysMetPheGlyValProAsp115120 125GluAsnIleLeuThrMetGluValPheAspArgGlyValGlyHisTyr130135140ThrGlnValAlaTrpGlnSerSerAspLysIleGlyCysAlaValGlu1 45150155160TrpCysProThrMetThrLeuValAlaCysGluTyrAsnProAlaGly165170175AsnA rgIleAsnHisTyrIleTyrAspIleGlyAspProCysThrThr180185190AspGluAspCysGlnCysThrGlyCysThrCysSerLysAspGluAla195 200205LeuCysIleProProGlyTyrThrThrValMetProProThrThrGlu210215220LysProThrThrThrProLysIleTyrHisPr oGlyGlyMetCysPro225230235240GluAsnAsnAsnGlyMetThrAspGluAlaArgGlnMetPheValAsp245250 255LysHisAsnGluTyrArgSerLeuIleAlaLysGlyLeuAlaHisAsn260265270AsnLeuGlyGlyPheAlaProLysAlaAlaArgMetMetLys ValSer275280285TyrAsnCysGluIleGluAlaAsnArgValGluTrpAlaLysAspCys290295300ThrLeuGlyTyrA snSerValAlaGlnAsnAsnGlnTrpGlyTyrAsn305310315320ValHisSerLeuLeuProHisIleAsnLysThrValAlaAlaAlaGlu32 5330335SerValGluAlaTrpPheAsnGluLeuGlnThrTyrGlyAlaProGln340345350AspAsnValPheSerMetGluVa lPheAsnGlnAsnValIleGlnGlu355360365TyrAlaGlnLeuAlaTrpGlnSerSerAsnGlnIleGlyCysGlyIle370375 380PheSerCysTrpGlyGlyAlaSerThrPheValAlaCysGluTyrAsn385390395400ProGlyGlyAsnPheIleGlyGluLeuIleTyrThrMetGlyAsp Pro405410415CysSerThrAspGluAspCysGlnCysAlaGlyCysValCysSerLys420425430AspG luAlaLeuCysIleAlaPro435440__________________________________________________________________________

Number	Date	Country
4903590	Nov 1990	AUX
8900163	Jan 1989	WOX
9003433	Apr 1990	WOX
9011086	Oct 1990	WOX

Haemonchus contortus vaccine

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Non-Patent Literature Citations (11)

Entry
Bowie, A. V. et al. Science 247: 1306-1310 (1990).
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Scopes, R. K. Protein Purfication: Principles and practice Springer-Velag (1987) pp. 221-235.
Gerard, C. et al. "Purification of Glycoproteins" Methods of Enzymology 182:529-534 (1990).
Kennedy, M. W. and Queshi F., "Stage-specific secreted antigens of the parasitic larval stages of the nematode Ascaris", Immunology, vol. 58, 1986, pp. 516-522.
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