Nematode 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 Stroncyloides stercoralis, Trichinella spiralis (infection in man and pigs), and the filarial worm Wuchereria bancrofti. Other important roundworm parasites include Ancylostoma caninum (infections of man), Stroncylus 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 that 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 may 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 nematode 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 antigen 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 antigen 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 is 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 10-60kD; 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 antigen 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 commuter.
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 antigen, antigen 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), Marcotl 52: Montanide 888 (Marcol is a Trademark of Esso. Montanide is a Trademark of SEPPIC, Paris), squalane or squalene, Adjuvant 65 (containing peanut oil, mannide 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 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, antigen 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, antigen, 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 Live 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 FaecesAnimal Day 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.g 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% (w/v) 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 6 MB (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 SE-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 of 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 of 88Adjuvant ControlsWorm Counts of 1096 2117 533sheep at slaughter% Protection of 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 7.5-9.3 356 .+-. 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 of 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 glycoproteins. The apparent molecular weight of these compounds compared with BRL high molecular weight protein standards were 100-12 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 alicuots 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 <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 mH 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 antigen may 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/lmM 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 chase 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 chase 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 ul/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.
The N-terminal amino acid sequence of the fractions was determined by gas phase sequencing on an Applied Biosystems model 470A amino acid sequencer. The following sequence was obtained (SEQ ID NO:1):
A F X P G S N N G M (T) D E I R Q I F (V) (D) (K) (Y)(H) (D)(N) (S) (G) (G) (P) (P) (P) (D)
In addition, samples of the 45 kD antigen were purified by electrophoresis on SDS polyacrylamide gels, transferred to nitrocellulose, stained with Ponceau S, the areas of the nitrocellulose which contained the 45 kD antigen were cut out and incubated with Endoproteinase Lys-C. Peptides which resulted from the digestion were separated by reverse phase HPLC and sequenced as above. The following sequences were obtained:
(K) (SEQ ID NO:2) X X (P) (D) X E V E A N (T) A A Y A (N) E (E) (Y) (S) (K) (SEQ ID NO:3) D N E Y R S L I A X X (Q) (Q) (L) X (S) (E) (H) (G) (K) (SEQ ID NO:4) (L) (D) (G) F A P K X (D) (G) (D) (G) (A) (A) (K) (SEQ ID NO:5) (H) N E Y R (S) I (L) (A) (K) (P) X (L) (N) X (S) (I) (T) (G) (K) (SEQ ID NO:6) X (P) Y (D) X (D) V X A (D) X X X (T) (P) (P) X (G)(K)(P) (T) (T) (D) (E)
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.
A112/301 (SEQ ID NO:7)GCGAATTCCCGGG.GCA.TTT.CAT.CCG.GGG.AAC.AAC.AAC.GGG.ATG.ACG.GAC.G T C C A A T T T A A T C T T T T A112/302 (SEQ ID NO:8)GCGAATTCCCGGG.GCA.TTT.CAT.CCG.GGG.TCG.AAC.AAC.GGG.ATG.ACG.GAC.G T C C A A AGA T T A A T C T T T T T C C C C C
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 (A112/301), SEQ 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 prom 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 mm polyallomer tubes. The gradients were centrifuged at 31,000 rpm for 16 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 places using Luria top agar containing 10 mM MgSO.sub.4. A total of 6.times.10.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 Tag 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-oliao (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 mM DTT, 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 Tag 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 ID 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 poly (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. Of 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 0) 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 Endaproteinase 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 ID 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 to, 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 sheen 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., 1982) 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 T. 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 Jan. 29, 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 gastrointestinal 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 E F 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:#acids (A) LENGTH: 21 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (v) FRAGMENT TYPE: N-terminal- (vi) ORIGINAL SOURCE:#contortus(A) ORGANISM: Haemonchus- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/note= "May be Phe or Tyr"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 3#/note= "May be His"INFORMATION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 5#/note= "May be Gly, Asp or Pro"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 6#/note= "May be Ser or Asn"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 19#/note= "May be Val or Ser"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 20#/note= "May be Asp, Gly or Pro"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 21#/note= "May be Lys, Gly, Pro or Asp"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- Ala Xaa Xaa Pro Xaa Xaa Asn Asn Gly Met Th - #r Asp Glu Ile Arg Gln# 15- Ile Phe Xaa Xaa Xaa 20- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 19 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (v) FRAGMENT TYPE: N-terminal- (vi) ORIGINAL SOURCE:#contortus(A) ORGANISM: Haemonchus- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2..3#/note= "Amino acids unknown"ON:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 4#/note= "May be Pro or Tyr"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 6#/note= "Amino acid unknown"ION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 17#/note= "May be Asn or Ser"TION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Lys Xaa Xaa Xaa Asp Xaa Glu Val Glu Ala As - #n Thr Ala Ala Tyr Ala# 15- Xaa Glu Glu- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 16 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (v) FRAGMENT TYPE: N-terminal- (vi) ORIGINAL SOURCE:#contortus(A) ORGANISM: Haemonchus- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/note= "May be Asp, Ser, His or Gly"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 11..12#/note= "Amino acids unknown"ON:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 16#/note= "Amino acid unknown"ION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- Lys Xaa Asn Glu Tyr Arg Ser Leu Ile Ala Xa - #a Xaa Glx Gln Leu Xaa# 15- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 9 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (v) FRAGMENT TYPE: N-terminal- (vi) ORIGINAL SOURCE:#contortus(A) ORGANISM: Haemonchus- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/note= "May be Leu, Asp, Gly or Ala"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 3#/label= RESIDUE-3R INFORMATION:#"AMINO ACID MAY BE ASP, GLY OR ALA"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 4#/label= RESIDUE-4R INFORMATION:#"AMINO ACID MAY BE GLY OR ASP"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 9#/note= "Amino acid unknown"ION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- Lys Xaa Xaa Xaa Phe Ala Pro Lys Xaa1 5- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 16 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (v) FRAGMENT TYPE: N-terminal- (vi) ORIGINAL SOURCE:#contortus(A) ORGANISM: Haemonchus- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/note= "May be His, Ser or Gly"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 9#/note= "May be Leu or Ile"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 10#/note= "May be Ala or Thr"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 13#/note= "Amino acid unknown"ION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 16#/note= "Amino acid unknown"ION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Lys Xaa Asn Glu Tyr Arg Ser Ile Xaa Xaa Ly - #s Pro Xaa Leu Asn Xaa# 15- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 18 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: peptide- (v) FRAGMENT TYPE: N-terminal- (vi) ORIGINAL SOURCE:#contortus(A) ORGANISM: Haemonchus- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 2#/note= "Amino acid unknown"ION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 3#/note= "May be Pro, Gly or Asp"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 4#/note= "May be Tyr or Lys"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 5#/note= "May be Asp or Pro"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 6#/note= "Amino acid unknown"ION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 7#/note= "May be Asp, Thr or Glu"- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 9#/note= "Amino acid unknown"ION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 11#/note= "May be Asp or Thr"TION:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 12..14#/note= "Amino acids unknown"ON:- (ix) FEATURE: (A) NAME/KEY: Modified-sit - #e (B) LOCATION: 18#/note= "Amino acid unknown"ION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- Lys Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Ala Xa - #a Xaa Xaa Xaa Thr Pro# 15- Pro Xaa- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 50 base (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:# 50CHTTYC AYCCNGGNAA YAAYAAYGGN ATGACNGAYG- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 50 base (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- (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: 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:# 50CHTTYC AYCCNGGNWS NAAYAAYGGV ATGACDGAYG- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1400 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: cDNA- (vi) ORIGINAL SOURCE:#contortus(A) ORGANISM: Haemonchus- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 17..1381- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:#CTG GCG CCA TTA 49 GCC TGC TTG GTT CTT#Thr Ile Ala Cys Leu Val Leu Leu Ala Pro L - #eu# 10- TGG GCG GCT GAT AAG TAT GTG ATT TGT CCT TC - #T GAC AAT GGC ATG ACA 97Trp Ala Ala Asp Lys Tyr Val Ile Cys Pro Se - #r Asp Asn Gly Met Thr# 25- AAT GAA GTT AGA AAT ATG TTC GTT GAT ACG CA - #C AAT AAA CTC CGA TCG 145Asn Glu Val Arg Asn Met Phe Val Asp Thr Hi - #s Asn Lys Leu Arg Ser# 40- CAG ACT GCT CAA GGA AAG GCT AAG AAC GCA TT - #C GGT GGA TTT GCT CCA 193Gln Thr Ala Gln Gly Lys Ala Lys Asn Ala Ph - #e Gly Gly Phe Ala Pro# 55- AAA GCA GCT CGA ATG TTA AAA GTG AGT TAT GA - #T TGC GAC ATG GAA GCT 241Lys Ala Ala Arg Met Leu Lys Val Ser Tyr As - #p Cys Asp Met Glu Ala# 75- AAC ATG ATG AAA TGG GCA AAG CAG TGT CAT TT - #C TAT CAC CCT CCA CCC 289Asn Met Met Lys Trp Ala Lys Gln Cys His Ph - #e Tyr His Pro Pro Pro# 90- GCA TAT AGG AAC TAC TGG GGA CAA AAT ATT TA - #T ATG GTG GGA GAT CGA 337Ala Tyr Arg Asn Tyr Trp Gly Gln Asn Ile Ty - #r Met Val Gly Asp Arg# 105- TAC TAC AAT TTC ACC TGG CCG TCA ATT GCA GA - #A ACG GCC GTC ATA TCA 385Tyr Tyr Asn Phe Thr Trp Pro Ser Ile Ala Gl - #u Thr Ala Val Ile Ser# 120- TGG TGG CAG GAG TTA CAG GTT TTT GGT GTT CC - #A GAG AAC AAT ATC GTA 433Trp Trp Gln Glu Leu Gln Val Phe Gly Val Pr - #o Glu Asn Asn Ile Val# 135- GTC GCG CCA GAT GAA CAC AAA ACT GGT CAC TA - #C ATG CAG GTG GTC TGG 481Val Ala Pro Asp Glu His Lys Thr Gly His Ty - #r Met Gln Val Val Trp140 1 - #45 1 - #50 1 -#55- CAA TGG ACC TAC AAA ATT GGT TGC GCA ATT AA - #T TAT TGC ACA ATA AAC 529Gln Trp Thr Tyr Lys Ile Gly Cys Ala Ile As - #n Tyr Cys Thr Ile Asn# 170- AAG CCA TGG CCA TGG ACA ATC GCA GGA TGC AA - #C TAT AAC CCT GGT GGT 577Lys Pro Trp Pro Trp Thr Ile Ala Gly Cys As - #n Tyr Asn Pro Gly Gly# 185- GAT AAT GCT TAT TGG GTG GTC TAC GAG ATG GG - #A GAT CCA TGC ACA ACT 625Asp Asn Ala Tyr Trp Val Val Tyr Glu Met Gl - #y Asp Pro Cys Thr Thr# 200- GAC GCC GAC TGC AAA TGT GCT GGT TGC GTT TG - #C AGC CAA GAA GAG GCC 673Asp Ala Asp Cys Lys Cys Ala Gly Cys Val Cy - #s Ser Gln Glu Glu Ala# 215- CTT TGC ATT CCG CCA GAA TAC ACT CCC CTT CC - #A CCT ACT ACC ACT TCA 721Leu Cys Ile Pro Pro Glu Tyr Thr Pro Leu Pr - #o Pro Thr Thr Thr Ser220 2 - #25 2 - #30 2 -#35- ACC ACA ACA CCG AAG CCA ACT ACA ACA ACA AC - #C GTT GGG GTA CCT AAT 769Thr Thr Thr Pro Lys Pro Thr Thr Thr Thr Th - #r Val Gly Val Pro Asn# 250- GCT GGG TCG TGC CCT GAA CTT AAC AAT GGA AT - #G ACT GAC GAA GCT AGG 817Ala Gly Ser Cys Pro Glu Leu Asn Asn Gly Me - #t Thr Asp Glu Ala Arg# 265- AAG ATG TTT GTC GAC AAA CAT AAT GAA TAC CG - #A TCG CTC ATT GCT AAA 865Lys Met Phe Val Asp Lys His Asn Glu Tyr Ar - #g Ser Leu Ile Ala Lys# 280- GGG CAA GCC AAG GGT AAA CCT GGA CAA TTC GC - #C CCA AAG GCT GCC AGA 913Gly Gln Ala Lys Gly Lys Pro Gly Gln Phe Al - #a Pro Lys Ala Ala Arg# 295- ATG ATG AAA GTG AAC TAC GAT TGC GAT GTT GA - #A GCA AAT GCA ATG GAA 961Met Met Lys Val Asn Tyr Asp Cys Asp Val Gl - #u Ala Asn Ala Met Glu300 3 - #05 3 - #10 3 -#15- TGG TCC AAG ACT TGC ACA TTT GGA CTC AAC AC - #T GCT GCG ATG TTA AAG1009Trp Ser Lys Thr Cys Thr Phe Gly Leu Asn Th - #r Ala Ala Met Leu Lys# 330- CGA TGG GGG AAT AAC ATG CAC ATG ATG TCG TC - #C AAG GCT AAT AAC AAG1057Arg Trp Gly Asn Asn Met His Met Met Ser Se - #r Lys Ala Asn Asn Lys# 345- ACA GAG GCT GCA GCT GAG GCC GTC GCA GCC TG - #G TTC GGT GAT TTA CAA1105Thr Glu Ala Ala Ala Glu Ala Val Ala Ala Tr - #p Phe Gly Asp Leu Gln# 360- AAA TAT GGC GTA CCT GAG AAT AAC GTC TTC AC - #G ATG AAC GTT TAC ACG1153Lys Tyr Gly Val Pro Glu Asn Asn Val Phe Th - #r Met Asn Val Tyr Thr# 375- ACT TTA AGT AAA TAC AGT CAG TTA GCG TGG CA - #A TCG AGC GAC AGA ATT1201Thr Leu Ser Lys Tyr Ser Gln Leu Ala Trp Gl - #n Ser Ser Asp Arg Ile380 3 - #85 3 - #90 3 -#95- GGT TGT GTA GTT GTA CCT TGT TGG AGC TCA TG - #G ACG GTT GTG GTG TGT1249Gly Cys Val Val Val Pro Cys Trp Ser Ser Tr - #p Thr Val Val Val Cys# 410- GAA TAC AAT CCC GGA GGA GAC CTG CCT GGC GA - #G GCT ATC TAT GAC GTA1297Glu Tyr Asn Pro Gly Gly Asp Leu Pro Gly Gl - #u Ala Ile Tyr Asp Val# 425- GGA GAT CCC TGT ACG AAA GAC GCC GAC TGT CA - #G TGC CCC GGC TGC ACC1345Gly Asp Pro Cys Thr Lys Asp Ala Asp Cys Gl - #n Cys Pro Gly Cys Thr# 440- TGT AGC AGA GAT GAG GGC CTT TGC GTT GCT CC - #A TGAACACTGG CGGCCGCTTA1398Cys Ser Arg Asp Glu Gly Leu Cys Val Ala Pr - #o# 455# 1400- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 454 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Thr Ile Ala Cys Leu Val Leu Leu Ala Pro Le - #u Trp Ala Ala Asp Lys# 15- Tyr Val Ile Cys Pro Ser Asp Asn Gly Met Th - #r Asn Glu Val Arg Asn# 30- Met Phe Val Asp Thr His Asn Lys Leu Arg Se - #r Gln Thr Ala Gln Gly# 45- Lys Ala Lys Asn Ala Phe Gly Gly Phe Ala Pr - #o Lys Ala Ala Arg Met# 60- Leu Lys Val Ser Tyr Asp Cys Asp Met Glu Al - #a Asn Met Met Lys Trp# 80- Ala Lys Gln Cys His Phe Tyr His Pro Pro Pr - #o Ala Tyr Arg Asn Tyr# 95- Trp Gly Gln Asn Ile Tyr Met Val Gly Asp Ar - #g Tyr Tyr Asn Phe Thr# 110- Trp Pro Ser Ile Ala Glu Thr Ala Val Ile Se - #r Trp Trp Gln Glu Leu# 125- Gln Val Phe Gly Val Pro Glu Asn Asn Ile Va - #l Val Ala Pro Asp Glu# 140- His Lys Thr Gly His Tyr Met Gln Val Val Tr - #p Gln Trp Thr Tyr Lys145 1 - #50 1 - #55 1 -#60- Ile Gly Cys Ala Ile Asn Tyr Cys Thr Ile As - #n Lys Pro Trp Pro Trp# 175- Thr Ile Ala Gly Cys Asn Tyr Asn Pro Gly Gl - #y Asp Asn Ala Tyr Trp# 190- Val Val Tyr Glu Met Gly Asp Pro Cys Thr Th - #r Asp Ala Asp Cys Lys# 205- Cys Ala Gly Cys Val Cys Ser Gln Glu Glu Al - #a Leu Cys Ile Pro Pro# 220- Glu Tyr Thr Pro Leu Pro Pro Thr Thr Thr Se - #r Thr Thr Thr Pro Lys225 2 - #30 2 - #35 2 -#40- Pro Thr Thr Thr Thr Thr Val Gly Val Pro As - #n Ala Gly Ser Cys Pro# 255- Glu Leu Asn Asn Gly Met Thr Asp Glu Ala Ar - #g Lys Met Phe Val Asp# 270- Lys His Asn Glu Tyr Arg Ser Leu Ile Ala Ly - #s Gly Gln Ala Lys Gly# 285- Lys Pro Gly Gln Phe Ala Pro Lys Ala Ala Ar - #g Met Met Lys Val Asn# 300- Tyr Asp Cys Asp Val Glu Ala Asn Ala Met Gl - #u Trp Ser Lys Thr Cys305 3 - #10 3 - #15 3 -#20- Thr Phe Gly Leu Asn Thr Ala Ala Met Leu Ly - #s Arg Trp Gly Asn Asn# 335- Met His Met Met Ser Ser Lys Ala Asn Asn Ly - #s Thr Glu Ala Ala Ala# 350- Glu Ala Val Ala Ala Trp Phe Gly Asp Leu Gl - #n Lys Tyr Gly Val Pro# 365- Glu Asn Asn Val Phe Thr Met Asn Val Tyr Th - #r Thr Leu Ser Lys Tyr# 380- Ser Gln Leu Ala Trp Gln Ser Ser Asp Arg Il - #e Gly Cys Val Val Val385 3 - #90 3 - #95 4 -#00- Pro Cys Trp Ser Ser Trp Thr Val Val Val Cy - #s Glu Tyr Asn Pro Gly# 415- Gly Asp Leu Pro Gly Glu Ala Ile Tyr Asp Va - #l Gly Asp Pro Cys Thr# 430- Lys Asp Ala Asp Cys Gln Cys Pro Gly Cys Th - #r Cys Ser Arg Asp Glu# 445- Gly Leu Cys Val Ala Pro 450- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 1386 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown- (ii) MOLECULE TYPE: cDNA- (vi) ORIGINAL SOURCE:#contortus(A) ORGANISM: Haemonchus- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 17..1339- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:#TGC CTG AAA GCC 49 CTT CTG ACG CCA TCA#Ser Val Leu Leu Thr Pro Ser Cys Leu Lys A - #la# 10- GCG TTT TGC CCC ACA TCG GAC AAT GGC ATG AC - #C GAT GAA ATT AGG CAG 97Ala Phe Cys Pro Thr Ser Asp Asn Gly Met Th - #r Asp Glu Ile Arg Gln# 25- ATT TTC GTT GAT AAG CAC AAT GAG TAT CGA TC - #T ATT ATT GCT AAA GGA 145Ile Phe Val Asp Lys His Asn Glu Tyr Arg Se - #r Ile Ile Ala Lys Gly# 40- CAG GCC AAG AAT AAA CTT GGA GGA TTC GCT CC - #G AAA GCA GCT CGA ATG 193Gln Ala Lys Asn Lys Leu Gly Gly Phe Ala Pr - #o Lys Ala Ala Arg Met# 55- TTG AAA GTG GGT TAC GAT TGC GAA GTT GAG GC - #A AAT ACG GCG GCA TAT 241Leu Lys Val Gly Tyr Asp Cys Glu Val Glu Al - #a Asn Thr Ala Ala Tyr# 75- GCA AAA GAG TGC AAG TTC GAA CAT GAT CCA CC - #C GAG CAA AGG AAT TAT 289Ala Lys Glu Cys Lys Phe Glu His Asp Pro Pr - #o Glu Gln Arg Asn Tyr# 90- TGG GGG CAG AAC CTG TGG ATG CTA GGA GGG AC - #T AAT TAC AGC AAG ACG 337Trp Gly Gln Asn Leu Trp Met Leu Gly Gly Th - #r Asn Tyr Ser Lys Thr# 105- GAA TCT GCA AAA TTA AGT GTC CAA GCT TGG TA - #C TGG GAA TTG AAG ATG 385Glu Ser Ala Lys Leu Ser Val Gln Ala Trp Ty - #r Trp Glu Leu Lys Met# 120- TTT GGA GTG CCC GAT GAA AAT ATC CTG ACA AT - #G GAA GTC TTC GAT CGG 433Phe Gly Val Pro Asp Glu Asn Ile Leu Thr Me - #t Glu Val Phe Asp Arg# 135- GGT GTT GGC CAC TAC ACA CAG GTA GCC TGG CA - #G TCT AGC GAC AAA ATC 481Gly Val Gly His Tyr Thr Gln Val Ala Trp Gl - #n Ser Ser Asp Lys Ile140 1 - #45 1 - #50 1 -#55- GGC TGC GCA GTT GAA TGG TGC CCA ACC ATG AC - #A CTT GTA GCA TGC GAG 529Gly Cys Ala Val Glu Trp Cys Pro Thr Met Th - #r Leu Val Ala Cys Glu# 170- TAC AAC CCT GCA GGA AAT AGG ATC AAT CAT TA - #T ATT TAC GAC ATC GGA 577Tyr Asn Pro Ala Gly Asn Arg Ile Asn His Ty - #r Ile Tyr Asp Ile Gly# 185- GAT CCA TGC ACA ACT GAT GAA GAC TGT CAA TG - #C ACT GGC TGC ACT TGT 625Asp Pro Cys Thr Thr Asp Glu Asp Cys Gln Cy - #s Thr Gly Cys Thr Cys# 200- AGT AAA GAT GAG GCC CTT TGT ATT CCT CCA GG - #A TAT ACT ACC GTC ATG 673Ser Lys Asp Glu Ala Leu Cys Ile Pro Pro Gl - #y Tyr Thr Thr Val Met# 215- CCA CCG ACT ACA GAG AAA CCT ACT ACA ACA CC - #T AAA ATA TAC CAT CCA 721Pro Pro Thr Thr Glu Lys Pro Thr Thr Thr Pr - #o Lys Ile Tyr His Pro220 2 - #25 2 - #30 2 -#35- GGT GGG ATG TGC CCT GAG AAT AAT AAC GGA AT - #G ACA GAT GAA GCT AGG 769Gly Gly Met Cys Pro Glu Asn Asn Asn Gly Me - #t Thr Asp Glu Ala Arg# 250- CAG ATG TTC GTC GAC AAA CAC AAT GAG TAT CG - #A TCC CTC ATA GCT AAA 817Gln Met Phe Val Asp Lys His Asn Glu Tyr Ar - #g Ser Leu Ile Ala Lys# 265- GGA CTA GCT CAT AAC AAT CTT GGA GGG TTT GC - #T CCA AAA GCG GCT AGA 865Gly Leu Ala His Asn Asn Leu Gly Gly Phe Al - #a Pro Lys Ala Ala Arg# 280- ATG ATG AAA GTG AGC TAC AAT TGC GAA ATC GA - #A GCG AAT CGA GTG GAG 913Met Met Lys Val Ser Tyr Asn Cys Glu Ile Gl - #u Ala Asn Arg Val Glu# 295- TGG GCG AAG GAT TGC ACG CTT GGG TAC AAC TC - #T GTT GCT CAA AAT AAC 961Trp Ala Lys Asp Cys Thr Leu Gly Tyr Asn Se - #r Val Ala Gln Asn Asn300 3 - #05 3 - #10 3 -#15- CAA TGG GGT TAT AAT GTA CAT TCA CTA CTG CC - #G CAT ATT AAC AAG ACG1009Gln Trp Gly Tyr Asn Val His Ser Leu Leu Pr - #o His Ile Asn Lys Thr# 330- GTA GCA GCA GCA GAG AGT GTC GAG GCC TGG TT - #C AAT GAA CTA CAG ACA1057Val Ala Ala Ala Glu Ser Val Glu Ala Trp Ph - #e Asn Glu Leu Gln Thr# 345- TAT GGT GCA CCT CAG GAT AAC GTT TTC AGT AT - #G GAG GTT TTC AAT CAA1105Tyr Gly Ala Pro Gln Asp Asn Val Phe Ser Me - #t Glu Val Phe Asn Gln# 360- AAC GTA ATA CAG GAA TAC GCT CAG TTG GCG TG - #G CAA TCG AGC AAC CAG1153Asn Val Ile Gln Glu Tyr Ala Gln Leu Ala Tr - #p Gln Ser Ser Asn Gln# 375- ATT GGT TGT GGA ATT TTT TCT TGC TGG GGT GG - #C GCC TCT ACA TTT GTG1201Ile Gly Cys Gly Ile Phe Ser Cys Trp Gly Gl - #y Ala Ser Thr Phe Val380 3 - #85 3 - #90 3 -#95- GCT TGC GAA TAC AAT CCT GGA GGA AAC TTC AT - #C GGC GAA TTG ATT TAT1249Ala Cys Glu Tyr Asn Pro Gly Gly Asn Phe Il - #e Gly Glu Leu Ile Tyr# 410- ACG ATG GGA GAT CCG TGC TCA ACT GAC GAA GA - #C TGT CAG TGC GCT GGT1297Thr Met Gly Asp Pro Cys Ser Thr Asp Glu As - #p Cys Gln Cys Ala Gly# 425- TGC GTC TGT AGC AAA GAT GAA GCA CTC TGT AT - #T GCT CCT TAAATGCTTG1346Cys Val Cys Ser Lys Asp Glu Ala Leu Cys Il - #e Ala Pro# 440# 1386 TGAA AGAAAAGCGG CCGCGAATTC- (2) INFORMATION FOR SEQ ID NO:12:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 440 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:- Ser Val Leu Leu Thr Pro Ser Cys Leu Lys Al - #a Ala Phe Cys Pro Thr# 15- Ser Asp Asn Gly Met Thr Asp Glu Ile Arg Gl - #n Ile Phe Val Asp Lys# 30- His Asn Glu Tyr Arg Ser Ile Ile Ala Lys Gl - #y Gln Ala Lys Asn Lys# 45- Leu Gly Gly Phe Ala Pro Lys Ala Ala Arg Me - #t Leu Lys Val Gly Tyr# 60- Asp Cys Glu Val Glu Ala Asn Thr Ala Ala Ty - #r Ala Lys Glu Cys Lys# 80- Phe Glu His Asp Pro Pro Glu Gln Arg Asn Ty - #r Trp Gly Gln Asn Leu# 95- Trp Met Leu Gly Gly Thr Asn Tyr Ser Lys Th - #r Glu Ser Ala Lys Leu# 110- Ser Val Gln Ala Trp Tyr Trp Glu Leu Lys Me - #t Phe Gly Val Pro Asp# 125- Glu Asn Ile Leu Thr Met Glu Val Phe Asp Ar - #g Gly Val Gly His Tyr# 140- Thr Gln Val Ala Trp Gln Ser Ser Asp Lys Il - #e Gly Cys Ala Val Glu145 1 - #50 1 - #55 1 -#60- Trp Cys Pro Thr Met Thr Leu Val Ala Cys Gl - #u Tyr Asn Pro Ala Gly# 175- Asn Arg Ile Asn His Tyr Ile Tyr Asp Ile Gl - #y Asp Pro Cys Thr Thr# 190- Asp Glu Asp Cys Gln Cys Thr Gly Cys Thr Cy - #s Ser Lys Asp Glu Ala# 205- Leu Cys Ile Pro Pro Gly Tyr Thr Thr Val Me - #t Pro Pro Thr Thr Glu# 220- Lys Pro Thr Thr Thr Pro Lys Ile Tyr His Pr - #o Gly Gly Met Cys Pro225 2 - #30 2 - #35 2 -#40- Glu Asn Asn Asn Gly Met Thr Asp Glu Ala Ar - #g Gln Met Phe Val Asp# 255- Lys His Asn Glu Tyr Arg Ser Leu Ile Ala Ly - #s Gly Leu Ala His Asn# 270- Asn Leu Gly Gly Phe Ala Pro Lys Ala Ala Ar - #g Met Met Lys Val Ser# 285- Tyr Asn Cys Glu Ile Glu Ala Asn Arg Val Gl - #u Trp Ala Lys Asp Cys# 300- Thr Leu Gly Tyr Asn Ser Val Ala Gln Asn As - #n Gln Trp Gly Tyr Asn305 3 - #10 3 - #15 3 -#20- Val His Ser Leu Leu Pro His Ile Asn Lys Th - #r Val Ala Ala Ala Glu# 335- Ser Val Glu Ala Trp Phe Asn Glu Leu Gln Th - #r Tyr Gly Ala Pro Gln# 350- Asp Asn Val Phe Ser Met Glu Val Phe Asn Gl - #n Asn Val Ile Gln Glu# 365- Tyr Ala Gln Leu Ala Trp Gln Ser Ser Asn Gl - #n Ile Gly Cys Gly Ile# 380- Phe Ser Cys Trp Gly Gly Ala Ser Thr Phe Va - #l Ala Cys Glu Tyr Asn385 3 - #90 3 - #95 4 -#00- Pro Gly Gly Asn Phe Ile Gly Glu Leu Ile Ty - #r Thr Met Gly Asp Pro# 415- Cys Ser Thr Asp Glu Asp Cys Gln Cys Ala Gl - #y Cys Val Cys Ser Lys# 430- Asp Glu Ala Leu Cys Ile Ala Pro# 440__________________________________________________________________________

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

Nematode vaccine

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Entry
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