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. PA0 2. Tc Ad ESA 1-5 from variants or different individuals of the T. colubriformis population. PA0 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.