Main > IMMUNOLOGY > Vaccines > Parasite > Nematode Infection. Vaccine

Product Australia. BC

PATENT ASSIGNEE'S COUNTRY Australia
UPDATE 09.99
PATENT NUMBER This data is not available for free
PATENT GRANT DATE 07.09.99
PATENT TITLE Polynucleotides encoding excretory/secretory proteins of parasitic nematodes, host cells transformed therewith

PATENT ABSTRACT The invention provides excretory/secretory antigens derived from parasitic nematode species which are capable of inducing protective immunity against infection by parasitic nematode species, and related antigenic molecules. The invention also provides nucleotide sequences encoding the antigens and related molecules of the invention, recombinant DNA molecules comprising the nucleotide sequences, and transformed hosts carrying the recombinant DNA molecules. The invention further provides antibodies against the antigens and related molecules, and antibody compositions comprising the antibodies, vaccines comprising the antigens and/or related molecules and methods of treating or preventing nematode infections using the antigens and related molecules, vaccines, antibodies and/or antibody compositions of the invention.

PATENT INVENTORS This data is not available for free
PATENT ASSIGNEE This data is not available for free
PATENT FILE DATE 06.06.95
PATENT FOREIGN APPLICATION PRIORITY DATA This data is not available for free
PATENT REFERENCES CITED Chemical Abstracts, 81(13) (1974) Abstract No. 76156y.
Chemical Abstracts, 99(17) (1983) Abstract No. 137907q.
Biological Abstracts, 80 Abstract No. 61135.
Chemical Abstracts, 107(17) (1985) Abstract No. 149890a.
The J. of Immun., 132:898-904, 1984.
Journal of Parasitology, 67:592-593 (1981).
Molecul. & Biochem. Parasitology, 31:35-46 (1988).
Immunology, 58:515-522 (1986).
The Jour. of Parasitology, 48:562-571 (1962).
Reviews in Rural Science, 6:67-74 (1984).
"The Role of Immunologically Specific and Non-Specific Components of Resistance in Cross-Protection . . . ", Int. J. for Parasitol., 7: 211-215 (1977).
"Failure to Vaccinate Lambs Against Haemonchus Contortus With Functional Metabolic Antigens . . . ", Int. J. for Parasitol., 5: 427-430 (1975).
"Immunoregulation of Parasites in Natural Host-Parasite Systems . . . ", Biology and Control of Endoparasites pp. 297-323 (1982).
"Vaccination Against the Nematode Trichostrongylus Colubriformis 1. Vaccinations of Guinea-pigs With Worm Homogenates & Soluble Products . . . ", Int. J. Parasitol., 4: 293-299 (1974).
"Vaccination Against the Nematode Trichostrongylus Colubriformis III. Some Observations on Factors Influencing Immunity to Infection in Vaccinated Guinea-pigs", Int. J. Parasitol., 8: 33-37 (1978).
"Immune Expulsion of Parasitic Nematodes from the Alimentary Tract", Int. J. Parasitol., 19(2): 139-168 (1989).
"Attempts to Probe the Antigens and Protective Immunogens of Trichostrongylus Colubriformis In Immunoblots With Sera From Infected And Hyperimmune Sheep . . . ", Int. J. Parasitol., 15(2): 129-136 (1985).
"Partial Protection of Lambs Against Haemonchus contortus by Vaccination With A Fractionated Preparation of the Parasite", Vet. Parasitol., 23: 211-221 (1987).
"Oesophagostomum Radiatum: Successful Vaccination of Calves with High Molecular Weight Antigens", Int. J. Parasitol., 19(3): 271-274 (1989).
"Vaccination of Young Lambs by Means of a Protein Fraction Extracted from Adult Haemonchus contortus", Parasitology, 94: 385-397 (1987).
"Comparision of the Kinetics of Expulsion of Trichostrongylus colubriformis from Previously Uninfected, Reinfected, and Vaccinated Guinea Pigs", J. Parasitol., 63(4) 761-762 (1977).
"Immunity in the Guinea-pig to Trichostrongylus colubriformis-an Experimental System for the Study of Immunity", Parasitology, 55: 10P, 1965.
"Isolation, cDNA Cloning and Characterization of a 30KD Host-protective Antigen Secreted By The Sheep Parasite Trichostrongylus Colubriformis", Vaccines '88, G66 (1988).
Localization and Species Distribution of a 31 Kilodalton Glycoprotein Antigen (GP31) Of Ostertagia Circumcincta, Australian Society for Parasitol., Sydney Sep. 27-30 Program, 45, 1988.
"Isolation and Characterization of a Proteolytic Enzyme from the Adult Hookworm Ancylostoma Caninum", J. Biol. Chem., 260(12): 7343-7348 (1985).
A .lambda.gt11 cDNA Recombinant that Encodes Dirofilaria Immitis Paramyosin, Mol. Biochem. Parasitol., 35: 31-42 (1989).
"Molecular Cloning of an Immunodominant Antigen of Onchocerca Volvulus", J. Exp. Med., 168: 1199-1204 (1988).
"Construction of Onchocerca Volvulus cDNA Libraries and Partial Characterization of the cDNA for a Major Antigen", Mol. and Biochem. Parasitol., 34: 241-250 (1988).
"Cloning and Characterization of a Potentially Protective Antigen in Lymphatic Filariasis", Proc. Natl. Acad. Sci. USA, 85: 3604-3607 (1988).
"Attempts to Probe the Antigens and Protective Immunogens of Trichostongylus Colubriformis In Immunoblots With Sera . . . ", Int. J. Parasitol., 15(2) 129-136 (1985).
"Cloning in Single-stranded Bacteriophage as an Aid to Rapid DNA Sequencing", J. Mol. Biol., 143: 161-178 (1980).
"Versatile Cassettes Designed for the Copper Inducible Expression of Proteins in Yeast", Plasmid, 21: 147-150 (1989).
"Screening .lambda.gt Recombinant Clones by Hybridization to Single Plaques in situ", Science, 196: 180-182 (1977).
"Directed Deletion of a Yeast Transfer RNA Intervening Sequence", Science, 209: 1396-1400 (1980).
"Nucleotide Sequence Analysis of the Complement Resistance Gene from Plasmid R100", J. Bacteriology, 151(2): 819-827 (1982).
"Plasmid vectors for high-efficiency expression controlled by the pl promoter of coligphage lambda", Gene, 15: 81-93 (1981).
"The Murine Plasma Cell Antigen PC-1: Purification and Partial Amino Acid Sequence", J. Immunology, 134(1): 443-448 (1985).
"Effects on Trichinella spiralis of Host Responses to Purified Antigens", Science, 227: 948-950 (1985).
"A Search for a Simple Keratin-Fractionation and Peptide Mapping of Proteins from Feather Keratins", Aust. J. Biol. Sci., 26: 401-413 (1973).
"Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4", Nature, 227: 680-685 (1970).
"Constructing and Screening cDNA Libraries in .lambda.gt10 and .lambda.gt11", DNA Cloning, 1(2): 49-78 (1985).
"Basis for the Development of Vaccines for Control of Diseases Produced by Metazoan Parasites", Reviews in Rural Science, 6: 67-74 (1984).
"A Preliminary Evaluation of Factors Affecting An Experimental System for Vaccination-And-Challenge With Haemonchus Contortus In Sheep", Int. J. Parasitol., 19(2): 169-175 (1989).
"Shared Carbohydrate Epitopes on Distinct Surface and Secreted Antigens of the Parasitic Nematode Toxocara canis", J. Immunology, 139(1): 207-214 (1987).
"Mice Vaccinated against Nematospiroides dubius with Antigens Isolated by Affinity Chromatography From Adult Worms", Immunol. Cell. Biol., 65(3): 223-230 (1987).
"Glycoconjugate Antigens from Parasitic Nematodes", Molecular Paradigms for Eradicating Helminthic Parasites. UCLA Symposia vol. 60 (McInnes AJ ed) A.R. Liss Publication, New York 1987, pp. 267-279.
"Nematode Antigens", Current Topics in Microbiol. and Immonol., 120: 173-203 (1985).
Int. J. for Parasitology, 15:129-136 (1985).
Bas et al. (1986) JBC vol. 261, No. 2, 817-827.
O'Donnell et al. (1989) Int. J. Parasitol. vol. 19(3), 327-335.
Burgess et al. (1990) J. Cell. Biol. vol. 111, 2129-2138.
Lazar et al. (1988) Mol. Cell. Biol., 1247-1252.

PATENT PARENT CASE TEXT This data is not available for free
PATENT CLAIMS We claim:

1. An isolated polynucleotide segment comprising a nucleotide sequence encoding an excretory/secretory protein, obtainable from a parasitic stage of a parasitic nematode,

wherein said excretory/secretory protein has a molecular weight selected from the group consisting of about 11 kD, about 17 kD, about 30 kD, about 37 kD and about 81 kD, as estimated by SDS-PAGE,

wherein said protein confers protective immunity on a host against infection by a parasitic nematode,

wherein said 11 kD protein comprises SEQ ID NO: 20,

wherein said 17 kD protein comprises SEQ ID NO: 15,

wherein said 30 kD protein comprises SEQ ID NO: 31,

wherein said 37 kD protein comprises SEQ ID NO: 7,

wherein said 81 kD protein comprises SEQ ID NO: 26.

2. A polynucleotide according to claim 1, wherein said protein is a 17 kD protein that comprises SEQ ID NO: 19.

3. A polynucleotide according to claim 2, wherein said 17 kD protein comprises SEQ ID NO: 4.

4. A polynucleotide according to claim 2, wherein said polynucleotide comprises SEQ ID NO: 18.

5. A polynucleotide according to claim 4, wherein said polynucleotide comprises SEQ ID NO: 3.

6. A polynucleotide according to claim 1, wherein said protein in an 11 kD protein that further comprises SEQ ID NO: 21.

7. A polynucleotide according to claim 6, wherein said 11 kD protein comprises SEQ ID NO: 25.

8. A polynucleotide according to claim 7, wherein said polynucleotide comprises SEQ ID NO: 24.

9. A polynucleotide according to claim 1, wherein said protein is a 30 kD protein that comprises SEQ ID NO: 31 and further comprises SEQ ID NOs. 1 and 2.

10. A polynucleotide according to claim 9, wherein said 30 kD protein comprises SEQ ID NO: 12.

11. A polynucleotide according to claim 10, wherein said polynucleotide comprises SEQ ID NO: 11.

12. A polynucleotide according to claim 1, wherein said protein is a 37 kD protein comprising SEQ ID NO: 6.

13. A polynucleotide according to claim 12, wherein said polynucleotide comprises SEQ ID NO: 5.

14. A polynucleotide according to claim 1, wherein said polynucleotide is a DNA molecule.

15. A polynucleotide according to claim 1, wherein said polynucleotide is a recombinant DNA molecule further comprising an expression control sequence operatively linked to said nucleotide sequence.

16. A polynucleotide molecule according to claim 15, wherein said expression control sequence is selected from the group consisting of the tryptophan operon, the leftward promoter of bacteriophage lambda, the tac promoter, the Cup 1 promoter and the SV40 early promoter.

17. An isolated host, comprising a host cell transformed with a polynucleotide according to claim 1.

18. An isolated host according to claim 17, wherein the host is a bacterial cell, a yeast or other fungus, a vertebrate cell, an insect cell, or a plant cell.

19. An isolated host cell according to claim 18, wherein the host is a human cell.

20. An isolated host cell according to claim 18, wherein the host cell is transformed with a vaccinia virus or a baculovirus.

21. An isolated host cell according to claim 18, wherein the host is Saccharomyces cerevisiae.

22. An isolated host cell according to claim 18, wherein the host cell is selected from the group consisting of E. coli, an enteric organism other than E. coli, a Pseudomonas and a Bacillus species.

23. An isolated host cell according to claim 22, wherein the host is an E. coli K12 cell selected from the group consisting of JM109 and Y1090.

24. An isolated host cell according to claim 22, wherein the host is E. coli strain BTA 1689 (ATCC 68099).

25. An isolated host cell according to claim 22, wherein the host is E. coli strain BTA 1691 (ATCC 68098).

26. An isolated host cell according to claim 22, wherein the host is E. coli strain BTA 1690 (ATCC 68100).

27. An isolated polynucleotide segment that encodes a fusion protein wherein said fusion protein comprises a protein encoded by a polynucleotide according to claim 15.

28. A polynucleotide segment according to claim 27, wherein said fusion protein comprises the E. coli .beta.-galactosidase gene, the E. coli TraT gene, or the trp operon.

29. A method of preparing an expression product of a transformed host, comprising culturing a transformed host according to claim 17 and isolating an excretory/secretory protein of a parasitic stage of a nematode species,

wherein said protein induces protective immunity against nematode infestation when administered to a vertebrate host.
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PATENT DESCRIPTION TECHNICAL FIELD

The invention relates to the identification of antigens which induce protective immunity in a host against infection by parasitic nematode species, such as species of the genera Trichinella, Ancylostoma, Strongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator, Enterobius, Strongyloides and Wuchereria, especially the genera Trichostrongylus and Haemonchus. 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, the larvae of Toxocara spp., Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercoralis and Wuchereria bancrofti, particularly Trichostrongylus colubriformis and Haemonchus contortus.

The invention also relates to nucleotide sequences encoding these antigens, as well as to recombinant DNA molecules containing such nucleotide sequences and host cells expressing these nucleotide sequences.

The invention further relates to methods for the production of the antigens, nucleotide sequences, recombinant DNA molecules and hosts of the invention.

The invention relates to antibodies raised against the antigens of the invention and to compounds which act in a manner similar to those antibodies.

Additionally, the invention relates to vaccines which induce protective immunity against infection by parasitic nematodes such as species of the genera Trichinella, Ancylostoma, Strongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris, Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator, Enterobius, Strongyloides, and Wuchereria, especially the genera Trichostrongylus and Haemonchus. Examples of such species include Trichinella spiralis or Ancylostoma caninum in man, Strongylus vulgaris in horses, Trichostrongylus colubriformis in sheep and goats, Haemonchus contortus in sheep and goats, Ostertagia ostertagi in cattle, Ascaris suum or Trichinella spiralis in pigs, Toxascaris leonina or Uncinaria stenocephala in cats, Ancylostoma caninum or Trichuris vulpis in dogs, Dirofilaria immitis in dogs, or the larvae of Toxocara spp in man, or infection by Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercoralis or Wuchereria bancrofti, and particularly Trichostrongylus colubriformis or Haemonchus contortus.

BACKGROUND ART

Nematodes (nema--thread; oides--resembling), which are unsegmented roundworms with elongated, fusiform, or saclike bodies covered with cuticle, are virtually ubiquitous in nature, inhabiting soil, water and plants, and are importantly involved in a wide range of animal and plant parasitic diseases.

The roundworm parasites of mammals belong to the phylum Nemathelminthes. The roundworms include the hookworm (e.g. Necator americanus and Ancylostoma duodenale), roundworm (e.g. the common roundworm Ascaris lumbricoides), whipworm (e.g. Trichuris trichiura), and the pinworm or threadworm (e.g. Enterobius vermicularus), as well as Strongyloides stercoralis, Trichinella spiralis and the filarial worm Wuchereria bancrofti. Other important roundworm parasites include Ancylostoma caninum (infections of man), Strongylus vulgaris (infections of horses), Trichostrongylus colubriformis, Ostertagia circumcincta (infections of sheep and goats), Haemonchus contortus (infections of sheep and goats), Ostertagia ostertagi, Haemonchus placei (infections of cattle), Ascaris suum (infections of pigs), Toxascaris leonina 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 (see CSIRO/BAE Report--"Socio-economic Developments and Trends in the Agricultural Sector: Implications for Future Research"). 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, diarrhoea, 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 labour 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 several reported attempts 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.

One such attempt described by J. K. Dineen, (1977) involves the use of irradiated larval vaccines. As with other such attempts, the utility of this method is restricted by the requirement to maintain viable nematodes for prolonged periods.

The failure of killed vaccine preparations to afford good anthelminthic protection has been thought to be due to a number of factors. For example, it has been considered by J. T. M. Neilson (1975) that parasitic nematodes may have evolved mechanisms by which they can secrete products which immunosuppress or immunomodulate the host's immune system, thereby both preventing the development of an effective immune response and rendering the host susceptible to other infections. It is believed by Dineen and Wagland (1982), that immunosuppressants or immunomodulators may be present in the crude preparations of parasitic nematodes which are used in the killed vaccines. A second problem suggested by this review article is that parasitic nematodes may have altered their antigen profile to one which resembles that of the host so that, in a natural infection, vigorous immunlogical reactions are not provoked by protective parasitic antigens. Such a phenomenon would also occur following vaccination with impure preparations of killed nematodes or extracts thereof.

Some workers have shown accelerated explusion of worms from host animals using whole homogenates of worms and impure subfractions see for example Rothwell and co-workers (1974, 1977, 1979), O'Donnell et at (1985), Neilson and Van de Walle (1987), Silverman: U.K. Patent 894603, Australian Patent 247 354, Adams (1989), East et al (1989), Munn and Greenwood (1987) (Australian Patent Application No. 77590/87), Connan (1965), Savin et al (1988) and McGillivery et al (1988).

In all of these studies, crude extracts of nematodes have been used to vaccinate animals, and no defined antigen or individual components of the extracts have been identified as being responsible for protection.

There have been some reports attempting to identify purified protective components, see for example Silberstein and Despommier (1985), Hotez et al (1985), Grandea et al (1989), Lucius et al (1988), Donelson et al (1988), Nilsen et al (1988). However, protection has either not been shown or not substantiated for the components described.

In only one natural host/parasitic nematode system has a purified cloned subunit been shown to be protective. In Australian Patent Application No. 19998/88, it was demonstrated that a recombinant DNA derived antigen shown to be nematode tropomyosin, gave 50% protection in sheep against Haemonchus contortus challenge. For reasons which will become clear later in this specification, this antigen is different to those identified in the current specification: the current antigens being found in the excretory/secretory fluids of nematodes following incubation in vitro.

The CSIRO/BAE working paper "Socio-economic Developments and Trends in the Agricultural Sector: Implications for Future Research" cited intestinal parasites as one of the three most urgent health problems in the Australian sheep industry and indicated that the development of vaccines holds great promise for better control of these infections.

It is well established that animals which are infected with parasitic nematodes develop an immunity which renders them less susceptible to subsequent infection (see Rothwell 1989 for review).

Although it has been demonstrated (e.g. O'Donnell et al 1985) that many parasite proteins are recognised by the immune system of infected host animals during parasitic infection, many of the immune responses will have no functional significance in terms of resistance to re-infection. The major step is to identify, from the many thousands of proteins present in the parasitic organism, the individual proteins which can induce immune responses in the host animal that protect it from re-infection.

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 characterised, 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.

The present inventors have studied in detail the excretory/secretory products from adult T. colubriformis and components from the mixture which are capable of giving protection following vaccination of target animals have been purified and characterised at the molecular level.

Definitions

The term "adjuvant" as used throughout the specification refers to an agent used in immunising compositions to enhance the immune response of an immunised host to the administered immunising composition.

The term "parenteral" as used herein includes subcutaneous injections, intraperitoneal or intramuscular injections, or infusion techniques.

The term "homologue" refers to proteinaceous molecules or to DNA sequences coding for those proteinaceous molecules which are related in structure to a first proteinaceous molecule or DNA sequence to such an extent that it is clear that the proteinaceous molecules themselves, or as encoded by the DNA, are related. Related DNA sequences are referred to as homologous genes and the related proteins are referred to as homologous antigens. The homology is expected to be at least 70% over 20 amino acids at the amino acid sequence level and at least 50% over 60 nucleotides at the DNA level.

It is recognised that the nematode population worldwide is genetically diverse as is the case for all organisms which reproduce sexually. Each individual of a population differs subtly from the others in the population and these differences are a consequence of differences in the sequence of the DNA which each individual inherits from its parents.

Further, random mutational events which can occur in either sexually or asexually reproducing organisms are a further source of genetic variation.

Thus, for each gene encoding a particular protein, there are likely to be differences in the sequence among the population of individuals.

Such related molecules are referred to herein as homologues.

Further homologous antigens may be defined as antigens related by evolution but not necessarily by function. Similar but not necessarily identical DNA or protein sequences may be provided. It should be noted however that function in this sense relates to the natural in vivo function of the protein.

Illustration of this point is provided by considering:

1. Tc Ad ESA 1-5 from Trichostrongylus colubriformis and other nematode species.

2. Tc Ad ESA 1-5 from variants or different individuals of the T. colubriformis population.

3. Tc Ad ESA 1-5 and related proteins from nematodes, which are homologues of Tc Ad ESA 1-5 as defined herein.

It is stressed that for the purposes of this invention, the homologues of antigens encompassed include only those molecules which share the immunological function of the antigens as defined herein.

Such homologous molecules may exist in the nematode population worldwide and will be capable, when incorporated into a vaccine either alone or in combination with other antigens, of eliciting in animals vaccinated with those molecules protective immune response.

In the context of this invention, the DNA from T. colubriformis which codes for an antigen of the invention can be used in DNA hybridisation experiments to identify specific DNA sequences in other species of parasitic nematodes. The conditions used for the hybridisation experiments will indicate the approximate % homology of the related DNA sequences to the DNA isolated from T. colubriformis. Typically, the conditions will be such that the related DNA sequences hybridising to the DNA isolated from T. colubriformis are at least 50% homologous in nucleotide sequence. 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 antigens isolated from T. colubriformis. It is contended that the related proteins will act as effective immunogens to protect animals from parasitism by the other species of parasitic nematodes with the possibility also of cross-species protection. These related DNA sequences are referred to as homologous genes and the related proteins are referred to as homologous antigens. Homologues of the invention may also be generated in vitro as herein described.

The term "derived" in the context of the antigens of the invention as used herein is intended to encompass antigens obtained by isolation from a nematode life stage expressing the antigen, as well as antigens obtained by manipulation of and expression from nucleotide sequences prepared from nematodes, 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 antigens as expressed by nematodes or cell lines expressing recombinant forms of the antigens.

Further, it should be recognised that it is possible to generate molecules which are not related to the Tc Ad ESA 1-5 antigens by evolution or necessarily by structure but which may serve as immunogens to generate an immune response against protective epitopes on the Tc Ad ESA 1-5 antigens and thereby act as effective vaccines. These molecules are referred to herein as "analogues" and, to the extent that they fulfil the functions of immunogens as defined herein, they are included within the scope of the invention. Such analogues include chemically synthesized oligopeptide molecules with sequences corresponding to portions of the amino acid backbone of the Tc Ad ESA 1-5 molecules, oligopeptides which when used as immunogens elicit an immune response which recognises native Tc Ad ESA 1-5 antigens in nematodes, and anti-idiotype antibodies raised against the variable region of antibodies which recognise the epitope(s) of the Tc Ad ESA 1-5 antigens.

Derivatives of antigens of the invention are molecules made from the antigens or molecules which are related to the antigens in a manner which suggests their preparation from the antigens.

DESCRIPTION OF INVENTION

The present inventors have found that protective immunity against infection by parasitic nematodes can be induced by immunization with excretory/secretory products of a parasitic nematode species. Five molecules termed Tc Ad ESA1 (SEQ ID NO: 12), Tc Ad ESA2 (SEQ ID NO: 6), Tc Ad ESA3 (SEQ ID NO: 4), Tc Ad ESA4 (SEQ ID NO: 25) and Tc Ad ESA5 are described which have been purified from the excretory-secretory fluids of mature adults of T. colubriformis and characterized. The present inventors have found that on vaccination, these proteins induce protective responses in guinea pigs against infection with T. colubriformis.

Adult worms were recovered from sheep 21 days after infection, washed and maintained in RPMI 1640 culture medium, containing antibiotics at 37.degree. C. for 16 hours. This culture medium which contains the excretory/secretory fluids from T. colubriformis, was concentrated over Diaflo membranes, and fractionated by adsorption to a lentil lectin-Sepharose 4B column.

The unbound fraction (LL.sup.-) and the bound fraction (LL.sup.+, eluted with methylmannoside) each contained only a few protein bands and were fractionated further by polyacrylamide gel electrophoresis and electroelution. Three proteins, designated Tc Ad ESA1 (SEQ ID NO: 12), Tc Ad ESA2 (SEQ ID NO: 6) and Tc Ad ESA5 have been isolated from the lentil lectin bound fraction and a further two proteins designated Tc Ad ESA3 (SEQ ID NO: 4) and Tc Ad ESA4 (SEQ ID NO: 25) were isolated from the unbound fraction. All five proteins confer immunity to T. colubriformis infection following intraperitoneal injection of guinea pigs, a laboratory model for sheep.

Examples of the antigens of the invention are the purified proteins Tc Ad ESA1 (SEQ ID NO: 12), Tc Ad ESA2 (SEQ ID NO: 6), Tc Ad ESA3 (SEQ ID NO: 4), Tc Ad ESA4 (SEQ ID NO: 25) and Tc Ad ESAS having molecular weights of 30, 37, 17, 11 and 81 kD respectively as estimated by SDS-PAGE.

According to a first embodiment of this invention there is provided an antigen comprising:

an excretory/secretory protein derived from a first parasitic nematode species and capable of inducing protective immunity against infection of a host by a second parasitic nematode species, which may be the same as or different from the first nematode species; or a protein molecule comprising all, part, an analogue, homologue, derivative or combination thereof of the excretory/secretory protein, which protein molecule is capable of inducing protective immunity in a host against infection by a parasitic nematode.

Preferably, the excretory/secretory protein has an approximate molecular weight of 11, 17, 30, 37 or 81 kD as estimated by SDS-PAGE.

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 second 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 vulqaris, 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 parasitic nematode species is T. colubriformis.

Preferably, the second parasitic nematode species is T. colubriformis or H. contortus.

According to a second embodiment of this invention there is provided: a first nucleotide sequence encoding the amino acid sequence of an antigen of the first embodiment; a nucleotide sequence which hybridizes to the first nucleotide sequence; or a nucleotide related by mutation including single or multiple base substitutions, insertions or deletions to the first nucleotide sequence.

Preferred nucleotide sequences of the invention are those encoding the excretory/secretory proteins of the first embodiment having approximate molecular weights of 11, 17, 30, 37 and 81 kD as estimated by SDS-PAGE.

Preferably, the nucleotide sequence is a DNA sequence. The DNA sequences embraced by the present invention can be prepared, for example, from T. colubriformis cells by extracting total DNA therefrom and isolating the sequences by standard techniques. Alternatively, the DNA may be prepared in vitro, synthetically or biosynthetically, such as by the use of an mRNA template.

According to a third embodiment of this invention there is provided a process for selecting a DNA or RNA sequence coding for an antigen according to the first embodiment which process comprises providing one or more DNA or RNA sequences and determining which of the sequences hybridizes with a DNA or RNA sequence known to code for an antigen of the first embodiment or providing an antiserum to the antigen and identifying host-vector combinations that express the antigen.

The sequences may be from natural sources, may be RNA sequences, synthetic sequences, DNA sequences from recombinant DNA molecules or combinations of such sequences.

Preferably, the process used to identify and characterize DNA coding for the antigen involves the extraction of mRNA species from cells producing the antigen, their conversion to double stranded DNA (cDNA) and the insertion of these into an autonomously replicating factor, such as a plasmid or phage vector. This is followed by transformation of a host cell such as a bacterial strain with the factor and screening of the library produced with synthetic DNA probes which are complementary to the antigen encoding mRNA or DNA sequences in order to detect those clones which contain DNA coding for the antigen as opposed to any other cell proteinaceous components.

According to a fourth embodiment of this invention, there is provided a recombinant DNA molecule comprising a DNA sequence of the third embodiment and vector DNA.

The DNA sequence may be a natural, synthetic or biosynthetic DNA sequence.

Preferred recombinant DNA molecules of the invention include an expression control sequence operatively linked to the DNA sequence.

In one preferred form of the invention, the DNA sequence is operatively linked to the .beta.-galactosidase gene of E. coli. Other preferred control systems include those of the tryptophan (Trp) operon, the Tra-T gene of E. coli, the leftward promoter of bacteriophage lambda, the Cup 1 promoter and hybrid promoters such as tac or viral promoters such as the SV40 early promoter.

Preferably, the vector DNA is plasmid DNA. Suitable plasmid vectors include pUR290, pUC18, pYEUC114 and derivatives thereof.

Alternatively, the vector DNA may be bacteriophage DNA such as bacteriophage lambda and derivatives thereof, such as lambda gt11 and lambda gt10.

According to a fifth embodiment of this invention there is provided a fused gene comprising a promoter, a translation start signal and a DNA sequence of the third embodiment.

According to a sixth embodiment of this invention there is provided a process for the preparation of a recombinant DNA molecule of the fourth embodiment which process comprises providing a DNA insert comprising a DNA sequence of the third embodiment and introducing the DNA insert into a cloning vector.

Preferably, the DNA insert is introduced into the cloning vector in correct spacing and correct reading frame with respect to an expression control sequence.

According to a seventh embodiment of this invention there is provided a transformed host, transformed with at least one recombinant DNA molecule of the fourth embodiment.

Preferably, the transformed host is capable of expressing an antigen of the first embodiment.

Suitable hosts include bacterial cells, yeasts such as Saccharomyces cerevisiae strain CL13-ABSY86 , other fungi, vertebrate cells, insects cells, plant cells, human cells, human tissue cells, live viruses such as vaccinia and baculovirus, and whole eukaryotic organisms.

Suitable bacterial hosts include E. coli and other enteric organisms, Pseudomonas, and Bacillus species.

Preferred hosts are E. coli K12 derivatives; in particular JM109 and Y1090.

According to an eighth embodiment of this invention there is provided a process for transforming a host to provide a transformed host of the seventh embodiment which process comprises providing a host, making the host competent for transformation, and introducing into the host a recombinant DNA molecule of the fourth embodiment.

According to a ninth embodiment of this invention there is provided an expression product of a transformed host of the seventh embodiment which product comprises an antigen of the first embodiment.

Preferably, the expression product is provided in substantially pure form.

Preferably, the expression product comprises a first polypeptide sequence homologous to the host and a second polypeptide sequence which is an amino acid sequence coding for an antigen of the first embodiment.

More preferably, the first amino acid sequence is part or all of .beta.-galactosidase or Tra-T and the host cell is E. coli.

According to a tenth embodiment of this invention there is provided a process for the biosynthesis of a proteinaceous product comprising an antigen of the first embodiment which process comprises:

transforming a host with a recombinant DNA molecule of the fourth embodiment so that the host is capable of expressing a proteinaceous product which includes an antigen of the first embodiment; culturing the host to obtain expression; and collecting the proteinaceous product.

According to an eleventh embodiment of this invention there is provided an epitope of an antigen of the first embodiment which is responsible for the protective immune response. The epitope may be created artificially by the synthetic production of oligopeptides which contain sequences of portions of the antigen which can be predicted from the results of immunochemical tests on fragments of the proteins produced in bacteria or generated as a result of chemical or enzymatic cleavage of the native or recombinant peptides.

According to a twelfth embodiment of this invention there is provided an antibody generated against an epitope of the eleventh embodiment. These antibodies or idiotypes can be used for passive protection of animals.

According to a thirteenth embodiment of this invention there is provided an antibody generated against the variable region of an antibody of the twelfth embodiment, a so called anti-idiotype antibody, which mimics a protective epitope of the antigen and may be used as an effective vaccine in active immunization of animals.

According to a fourteenth embodiment of this invention there is provided a vaccine comprising an effective amount of one or more antigens of the first embodiment, expression products of the ninth embodiment, epitopes of the eleventh embodiment and/or anti-idiotype antibodies of the thirteenth embodiment, together with a pharmaceutically acceptable excipient, carrier, adjuvant and/or diluent.

Preferred vaccines include those suitable for injectable or oral administration. Preferably, injectable vaccines include a pharmaceutically acceptable adjuvant.

According to a fifteenth embodiment of this invention there is provided an antibody prepared as a result of vaccination of a host by administration of one or more antigens, expression products, epitopes, anti-idiotype antibodies and/or vaccines of the present invention to the host. Such antibodies include polyclonal and monoclonal antibodies. It is recognised that there are compounds which act in a manner similar to the antibodies of the fifteenth embodiment. Although these compounds are not antibodies their presence in the host can produce a similar protective effect to the antibodies. Throughout the specification and claims, reference to antibodies of the fifteenth embodiment should be construed as extending to these compounds.

According to a sixteenth embodiment of this invention there is provided: an antibody composition comprising at least one antibody of the twelfth and/or fifteenth embodiment together with a pharmaceutically acceptable carrier, diluent and/or excipient.

According to a seventeeth embodiment of this invention, there is provided a process for the preparation of an antigen of the first embodiment which process comprises: collecting excretory-secretory fluids from a parasitic nematode species; fractionating the fluid by lentil lectin chromatography with methylmannoside as eluent; collecting the bound and unbound fractions; further fractionating by SDS-gel electrophoresis; and electroeluting the antigen.

According to an eighteenth embodiment of this invention there is provided a process for the preparation of a fused gene of the fifth embodiment which process comprises providing a promoter, a translation start signal and a DNA sequence of the third embodiment and operatively linking the promoter, translation start signal and DNA sequence.

According to a nineteenth embodiment of this invention there is provided a process for the preparation of a vaccine of the fourteenth embodiment which process comprises admixing an effective amount of at least one antigen of the first embodiment and/or expression product of the ninth embodiment and/or epitope of the eleventh embodiment and/or anti-idiotype antibody of the thirteenth embodiment with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

According to a twentieth embodiment of this invention there is provided a process for the preparation of an antibody of the fifteenth embodiment which process comprises immunizing an immunoresponsive host with an antigen of the first embodiment and/or expression product of the ninth embodiment and/or epitope of the eleventh embodiment and/or anti-idiotype antibody of the thirteenth embodiment and/or a vaccine of the fourteenth embodiment.

According to a twenty-first embodiment of this invention there is provided a process for the preparation of an anti-idiotype antibody of the thirteenth embodiment which process comprises immunizing an immunoresponsive host with an antibody of the twelfth embodiment.

According to a twenty-second embodiment of this invention there is provided a process for the preparation of an antibody composition of the sixteenth embodiment which process comprises: admixing an effective amount of at least one antibody of the twelfth and/or fifteenth embodiment with a pharmaceutically acceptable carrier, diluent and/or excipient.

According to a twenty-third embodiment of this invention there is provided a method of protecting a host in need of such treatment from infection by a parasitic nematode species which method comprises vaccinating the host with an antigen, expression product, vaccine, epitope and/or anti-idiotype antibody of the invention.

According to a twenty-fourth embodiment of this invention there is provided a method of passively protecting a host in need of such treatment against infection by a parasitic nematode species which method comprises passively vaccinating the host with at least one antibody of the twelfth and/or fifteenth embodiment and/or antibody composition of the sixteenth embodiment.

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 (1985), which are readily implemented by computer.

Homologues can be produced in accordance with the present invention, by conventional site-directed mutagenesis, which is one avenue for routinely identifying residues of the molecule that can be modified without rendering the resulting polypeptide biologically inactive. oligonucleotide-directed mutagenesis, comprising ›i! synthesis of an oligonucleotide with a sequence that contains the desired nucleotide substitution (mutation), ›ii! hybridizing the oligonucleotide to a template comprising a structural sequence coding for an antigen of the invention and ›iii! using T4 DNA polymerase to extend the oligonucleotide as a primer, is preferred because of its ready utility in determining the effects of particular changes to the antigen sequence.

Also exemplary of antigen homologues within the present invention are molecules 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.

Also, encompassed by the present invention are synthetic polypeptides that (i) correspond to a portion of the antigen amino-acid sequence and (ii) retain protective 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, expression product, epitope and/or anti-idiotype antibody that may be combined with carrier, excipient, diluent and/or adjuvant to produce a single vaccine dosage form will vary depending upon the infection being treated or prevented, 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, expression product, epitope, anti-idiotype antibody and/or vaccine employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, the particular infection to be treated or prevented and the severity of the particular infection undergoing treatment or prevention.

The vaccine of the present invention may be administered orally or parenterally, in unit dosage formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents, adjuvants and/or excipients as desired.

Injectable preparations, for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to known arts 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, as 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.

The term "pharmaceutically acceptable adjuvant" can mean either the standard compositions which are suitable for human administration or the typical adjuvants employed in animal vaccinations. An appropriate adjuvant can be selected using ordinary skill in the art.

Suitable adjuvants for the vaccination of animals and humans include but are not limited to aluminium hydroxide and oil emulsions such as Marcol 52: Montanide 888 (Marcol is a Trademark of Esso. Montanide is a Trademark of SEPPIC, Paris.). Other adjuvants suitable for use in the present invention include conjugates comprising the expression product together with an integral membrane protein of prokaryotic or eukaryotic origin, such as TraT.

Routes of administration, dosages to be administered as well as frequency of injections are all factors which can be optimized 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 immunological responses such as high titres of antibodies against the antigen epitope, anti-idiotype antibody or expression product.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, antigens, epitopes, anti-idiotype antibodies and/or expression products may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include nanoparticles, microcapsules, LTB conjugates, cholera or its B subunit as a conjugate, in pharmaceutically acceptable emulsions, syrups, solutions, suspensions, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents or TraT as a conjugate, and sweetening, flavouring, and perfuming agents including sugars such as sucrose, sorbitol, fructose, etc., glycols such as polyethylene glycol, propylene glycol etc, oils such as sesame oil, olive oil, soybean oil etc., antiseptics such as alkylparahydroxybenzoate etc, and flavours such as strawberry flavour, peppermint etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of a Tra T--Tc Ad ESA 1 fusion (SEQ ID NOS. 28 and 29).

BEST MODE AND OTHER MODES OF CARRYING OUT THE INVENTION

The nucleotide sequences, fused genes, recombinant DNA molecules and transformed hosts of the invention are prepared using standard techniques of molecular biology such as those described in Maniatis et al (1982).

In preparing the nucleotide sequences of the invention, it is recognised that the genes of interest, and also cDNA copies made from the genes may be provided in low yield. PCR (polymerase chain reaction) techniques can be used to amplify the relevant DNA to facilitate detection and cloning.

Expression products of the invention are obtained by culturing the transformed hosts of the invention under standard conditions as appropriate to the particular host 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 and the particular host.

The vaccines of the invention are prepared by mixing, preferably homogeneously mixing, antigen, expression product, anti-idiotype antibody and/or epitope with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant using standard methods of pharmaceutical preparation.

The amount of antigen, expression product, anti-idiotype antibody and/or epitope required to produce a single dosage form will vary depending upon the infection to be treated or prevented, the 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 activity of the antigen, expression product, anti-idiotype antibody and/or epitope employed, the age, body weight, general health, sex, and diet of the host, time of administration, route of adminstration, rate of excretion, drug combination and the severity of the infection undergoing treatment.

The vaccine may be administered orally or parenterally in unit dosage formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants as desired.

Antibodies are raised using standard vaccination regimes in appropriate hosts. The host is vaccinated with an antigen, expression product, epitope, anti-idiotype antibody and/or vaccine of the invention.

The compounds acting in a similar manner to the antibodies of the invention may be purified naturally occuring compounds or synthetically prepared using standard techniques including standard chemical or biosynthetic techniques.

The antibody composition is prepared by mixing, preferably homogeneously mixing, antibody with a pharmaceutically acceptable carrier, diluent and/or excipient using standard methods of pharmaceutical preparation.

The amount of antibody required to produce a single dosage form will vary depending upon the infection to be treated or prevented, 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 activity of the antibody employed, 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 orally or parenterally in unit dosage formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents and/or excipients as desired.

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