PRODUCTION AND USE OF ANTHELMINTIC AGENTS AND PROTECTIVE ANTIGENS

Abstract: The protein doublet H110D, the individual components thereof, and the production and use thereof in a vaccine against a nematode infection. This protein doublet is a plasma membrane-associated protein material of the intestinal microvilli of Haemonchus contortus. H110D has a molecular weight of about 110 kd and reacts with antibodies raised in animals injected with a contortin-enriched fraction. Injection of preparations of the protein doublet H110D or of its components induces the production of specific protective antibodies.

Description: PRODUCTION AND USE OF ANTHELMINTIC AGENTS AND PROTECTIVE ANTIGENS. This invention relates to production of materials for study as potential anthelmintic agents and as protective antigens. Nematodes are parasites which are found in farm animals and in man and are the cause of disease therein. Haemonchosis is a disease of ruminants due to the presence, in the gastro-intestinal tract, of the blood-feeding nematode Haemonchus. World-wide it is of very considerable economic importance having effects which range from reduction in weight gain, loss of production and agalactia through to death of domesticated animals. Ostertagiasis is caused by a related nematode Ostertaqia and has similar effects. Both diseases are characterised by wasting due 'in part to anorexia associated with severe infections. • In sheep and cattle, Ostertaqia is the most important in winter rainfall areas, while Haemonchus is more important in summer rainfall zones. In Australia, for example, it is estimated that approximately one third of the 300 million sheep in the country are likely to be infected with Haemonchus. The adult female of the nematode Haemonchus is up to 3 cm long and about 1 mm wide and has a diagonal red and white striped appearance. For this reason it is sometimes known as the barber's pole worm. The red colour is due to the presence of the host's blood in the intestine of the parasite. The parasitic stage of the worm lives attached to the wall of the stomach of the host animal, e.g. sheep, goat or cow, which it damages causing loss of blood in addition to that which it ingests. The animal becomes infected by ingesting infective larvae as it grazes on infested pastures. In the rumen the larva moults and passes into the abomasum where it develops. attaches itself to the stomach wall and commences ingesting blood. Over the following 7 days or so it continues to grow and then moults to give the young adult stage. About 18 to 21 days after infection it has become sexually mature and mates. The eggs pass out with the host faeces and hatch in due course into free-living larvae which develop into infective larvae. Ostertagia differs from Haemonchus in that it feeds on the host animal's plasma, not on its blood. It has a smaller intestine than Haemonchus. Although control of nematodes, such as Haemonchus and Ostertagia, can be achieved in sheep and cattle by administration of antheliriintic drugs, this approach is far from satisfactory as repeated dosing is necessary in order to achieve any degree of control over an infestation in a flock of sheep or herd of cattle. Numerous attempts have been made to develop vaccines to Haemonchus and to Ostertagia including use of attenuated live worms and extracts of the worms. However, so far a commercially successful vaccine has not yet been produced. Although injection of extracts of various kinds produced from Haemonchus or Ostertaqia has in some cases led to generation of antibodies directed against the parasite, such antibodies appear to be similar to those formed by natural infection and do not seem to enhance natural resistance to the parasite. I have previously reported in "Tissue & Cell", 1977, 9_ (D at pages 23 to 34, the existence of a helical, polymeric extracellular protein which is associated with the luminal surface of Haemonchus contortus intestinal cells. In the fourth stage larvae and adult nematodes (the parasitic stages) the miorovilli on the intestinal cells of Haemonchus contortus have associated therewith filaments in the form of helices about 400A in diameter and of variable pitch. These filaments proved to be p rimarily protein which I designated as contortin. Further investigation showed that the helical filament of contortin appears to be formed by lateral polymerization of a monomeric form which has a Y-shaped structure with arms about 45A long and 25A wide seen in negatively stained preparations. A further description of contortin appeared in "Proceedings of the Sixth European Congress on Electron Microscopy", Vol II, pages 515 and 516. Ostertaqia circumcincta also has contortin-like material associated with the microvilli in its intestine. A brief report that antibodies against contortin have been raised in rabbits with a view to testing whether contortin is antigenic and would act as a vaccine in sheep appeared in the "Report for 1974-75" of the Agricultural Research Council Institute of Animal Physiology, Babraham, in June 1976. In the corresponding "Report for 1976-77", published in 1978, there were summarised on page 94 the results of further work by me in which I showed that intramuscular injection of contortin-enriched fractions into young lambs gave a substantial measure of protection against haemonchosis when they were subsequently challenged with larvae of Haemonchus contortus. This work on the use of a contortin-enriched preparation for protection against haemonchosis has been published in "Parasitology", 9_4, at pages 385 to 397. Although a contortin-enriched fraction can be used to protect lambs against infection by Haemonchus contortus, such a fraction is not commercially useful as it has generally required injection of a large quantity of such a fraction in order to produce in the animal a suitable immune response. Moreover it is difficult to obtain either the polymeric or monomeric form of contortin in purer form and in sufficient quantity to facilitate further study of their antigenic characteristics. There is also present in the supernatant liquor from- which contortin is obtained by the procedure described in the afore-mentioned article in "Tissue & Cell-" 1 and "Parasitology", a further material which appears to be primarily protein, which has a molecular " weight of approximately 61,000, and which reacts with antibodies to contortin. This protein I have named pre-contortin. There is a need to provide proteins which can be used as protective antigens to form the basis of vaccines against nematode infections in sheep, cattle and man. = The present invention accordingly seeks to provide novel protein extracts from nematodes which have potential as protective antigens against such nematode infections. The present invention is based upon the surprising discovery that a membrane associated protein material present in the icrovilli of Haemonchus contortus exhibits ^ a strong antibody reaction with antibodies raised in sheep using a contortin-enriched fraction. This material appears as a double band upon SDS (so ' dium dodecyl sulphate)-polyacrylamide gel electrophoresis of a protein fraction obtained by extraction of the microvilli of Haemonchus contortus with a solution of a non-ionic detergent. By comparison with the mobility of proteins of known molecular weight the apparent molecular weight of " this double band is approximately 110 Kilodaltons. I have given this protein doublet the name H110D. According to one aspect of the present invention I provide the protein doublet H110D. I also provide the individual components thereof. Such components include both the protein present in the protein doublet that has the higher molecular weight of the two bands apparent upon subjection to SDS-polyacrylamide gel electrophoresis and also the protein of the other band with the lower apparent molecular weight. The invention further provides a method of preparing a membrane associated protein comprising homogenizing nematodes in a liquid carrier, centrifuging resulting homogenate to separate suspended cell debris, extracting the separated cell debris with a solution of a non-ionic detergent to solubilise membrane protein material, separating the extracted cell debris from a first supernatant liquor, subjecting resulting first supernatant liquor to ultracentrifugation, separating sedimented material, recovering a second supernatant liquor containing membrane associated proteins, and separating from the second supernatant liquor, a protein or proteins having an apparent molecular weight, upon subjection to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, of approximately 110 kd. The protein or proteins prepared in this way exhibit a strong positive reaction _n vitro with antibodies raised in sheep by injection of a contortin-enriched fraction. When the nematodes used are Haemonchus contortus, the protein doublet HllOD can be isolated from the supernatant liquor from the ultracentrifugation step by conventional methods such as gel electrophoresis, iso-electric focussing, gel exclusion chromatography, ion exchange chromatography, and methods based on hydrophobicity. It can be identified as a double band which corresponds to a molecular weight of approximately 110 kd by SDS- polyacrylamide gel electrophoresis. The protein doublet HllOD is further characterised by the existence of a strong positive reaction jj vitro with antibodies raised in sheep by injection of a contortin-enriched fraction obtained from a homogenate of Haemonchus contortus. The strength of the antibody response indicates that the protein doublet HllOD, or one of its components, may be a beneficial protective antigen which could form a basis for a vaccine against Haemonchus and possibly also against other nematodes such as Ostertagia. I have demonstrated the value of preparations of HllOD as protective antigens by injection of both partially purified and highly purified preparations into lambs which responded by production of specific antibodies and were then very substantially protected against haemonchosis when they were subsequently challenged with larvae of Haemonchus contortus. The microvilli on the intestinal wall of Haemαnchus contortus are relatively long. Electron microscopy thereof reveals that each icrovillus has an axial core (which extends below the base of the microvillus to join a fibrous layer called the endotube), a plasma membrane, cross linker proteins which join the axial core to the membrane, and helically coiled contortin molecules adjacent to the external surface of the membrane. The endotube with the attached microvilli can be separated from the remaining, basal, cytoplasm of the intestine. The microvilli can be separated from the endotube of Haemonchus contortus by appropriate manipulative techniques. One method involves contacting the endotube and its attached microvilli with a solution of dithiothreitol and carefully scraping the microvilli off the endotube with a probe whilst observing the endotube surface under a microscope. As will be apparent to the skilled reader this technique is tedious and it is somewhat time consuming to obtain more than a small amount of material in this way. Analysis of a preparation of such microvilli indicated the presence of three major proteins, namely contortin, actin and a previously unidentified protein material which shows up as a doublet band with an apparent molecular weight of approximately 110 kd upon SDS-polyacrylamide gel electrophoresis. By analogy with microvilli from mammalian and avian species this unidentified protein material would be expected to correspond to the cross linker protein or proteins. However, further investigation shows surprisingly that this is not the case. Instead the 110 kd doublet band, which I have designated HllOD, proves to be a membrane associated protein material. This can be demonstrated by extraction of a sample with a non-ionic detergent solution, specifically either a 1% w/v Triton X-100, Thesit, CHAPS (i.e. 3-[(3-cholamidopropyDdimethyl- ammoniolpropanesulphonate), or n-octyl glucoside solution containing protease inhibitors (the words "Triton" and "Thesit" are trade marks). This extraction step can be carried out with or without pre-extraction with high salt and Tween 20 solutions (the word "Tween" is a trade mark). It dissolves the membrane protein material but, as can be seen by electron microscopy, leaves the cross linker proteins and axial core (presumably actin) intact. Close examination of microvilli of Haemonchus contortus by electron microscopy using a negative staining, technique reveals particles ' on the membrane surface which are immediately adjacent to the helically coiled contortin molecules. It seems likely that these particles correspond to the protein material of the doublet HllOD. It is further contemplated that, in an alternative method of preparing the protein doublet HllOD, Haemonchus contortus is replaced by another nematode, for example by another species of Haemonchus or by one or more species of Ostertaqia or some other nematode, which is then subjected to homogenization, extraction, centrifugation and the other steps outlined above for preparing the protein doublet HllOD from Haemonchus contortus. Also, the detergent .may be replaced by another detergent, preferably another non-ionic detergent. . The invention also provides a vaccine comprising the protein doublet HllOD, either alone or in conjunction with contortin. It also provides vaccines comprising the individual protein components of the protein doublet HllOD, either by themselves or in conjunction with contortin. Hence there is provided a vaccine based on the protein of the doublet HllOD that has the higher molecular weight of the two bands apparent upon subjection to polyacrylamide gel electrophoresis, either alone or in conjunction with contortin. Similarly there is provided a vaccine based upon the protein of the doublet HllOD that has the lower molecular weight of the two bands apparent upon subjection to polyacrylamide gel electrophoresis, either alone or in conjunction with contortin. It also includes vaccines based upon mixtures of these two proteins in any proportion, either alone or in conjunction with contortin. Such vaccines are useful in the treatment of haemonchosis, ostertagiasis and other diseases caused by nematodes in farm animals and in man. The invention is further illustrated in the following Example. •• ' - ' - Example Approximately 2g live weight adult Haemonchus contortus were homogenized in 60 ml lOmM phosphate buffered saline (pH 7.3). The resulting homogenate was centrifuged for 10 minutes at 10000 rp at 4°C. The supernatant liquid was decanted and' the pellets homogenized again and the homogenate re-centrifuged. The pellets were then extracted with 20 ml of a 1% Tween 20 solution and re-centrifuged. The supernatant liquor was discarded. The pellets were then extracted with 20ml of a 1% w/v Thesit solution. (The word "Thesit" is a trade mark: Thesit is a non-ionic detergent). The resultant extract was centrifuged at 10,000 g for 10 minutes in a refrigerated centrifuge. The supernatant liquor " was poured off the resulting sediment and then centrifuged for approximately 11 x 10 6 g.min. The sedimented material was discarded. Part of the supernatant liquor was subjected to electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate. The protein doublet, HllOD, was identified as a double band corresponding to a molecular weight of about 110 kd by a conventional staining technique and was eluted. The other part of the supernatant liquor was fractionated by gel exclusion and ion exchange chromatography and the fractions containing HllOD were pooled and concentrated. Small amounts of these materials injected into lambs induced the formation of specific antibodies and the subsequent protection of the animals against haemonchosis. This result demonstrates that the protein doublet HllOD is a protective antigen. The accompanying Figure is a photograph of the electrophoretogram revealed by staining for protein following electrophoresis of various extracts on polyacrylamide gel. The unlabelled pattern at the left of the ' Figure is given by a mixture of standard proteins of known molecular weight. The other designations are as follows: MV = microvilli SE = scraped endotube E. = endotube (with attached microvilli) Cyt = basal cytoplasm The HllOD doublet is indicated by the arrows.

Claims: CLAIMS : - 1. The protein doublet HllOD. 2. The protein present in the protein doublet HllOD that has the higher molecular weight of the two bands apparent upon subjection to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 3. The protein present in the protein doublet HllOD that has the lower molecular weight of the two bands apparent upon subjection to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. 4. A method of preparing a membrane associated protein comprising homogenizing nematodes in a liquid carrier centrifuging resulting homogenate to separate suspended cell debris, extracting the separated cell debris with a solution of a non-ionic detergent to solubilise membrane protein material, separating the extracted cell debris from a first supernatant liquor, subjecting resulting first supernatant liquor to ultracentrifugation, separating sedimented material, recovering a second supernatant liquor containing membrane associated proteins, and separating from the second supernatant liquor a protein or proteins having an apparent molecular weight, upon subjection to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, of approximately 110 kd. 5. A method according to claim 4, in which the nematodes are Haemonchus contortus. 6. A method according to claim 4 or claim 5, in which the step of separating the said protein or proteins from the second supernatant liquor includes use of gel electrophoresis, gel chromatography, ion exchange chromatography, iso-electric focussing, a method based on hydrophobicity or a combination of two or more of these techniques. 7. Proteins produced by a method according to any one of claims 4 to 6. 8. The protein doublet HllOD whenever prepared by a method according to any one of claims 4 t,o 6.ι 9. The use as protective antigens against nematodes of a protein or proteins according to any one of claims 1 to 3, 7' or 8. 10. A vaccine comprising the protein doublet HllOD. 11. A vaccine comprising the protein doublet HllOD and contortin. 12. A vaccine comprising the protein of claim 2. 13. A vaccine comprising the protein of claim 2 and contortin. 14. A vaccine comprising the protein of claim 3. 15. A vaccine comprising the protein of claim 3 and contortin. 16. A vaccine comprising a mixture of the proteins of claims 2 and 3. 17. A vaccine comprising a mixture of the proteins of claims 2 and 3 and contortin. 18. The use of the protein doublet HllOD in the production of a vaccine against a nematode infection. 19. The use of the protein of claim 2 in the production of a vaccine against a nematode infection. 20 " . The use of the protein of claim 3 in the production of a vaccine against a nematode infection. 21. The use of a mixture of the proteins of claims 2 and 3 in the production of a vaccine against a nematode infection. 22. " The use * of a protein according to claim 7 in the " production of a vaccine against a nematode inflection. 23. The use of the protein doublet of claim 8 in the production of a vaccine against a nematode infection. -

Inventors: MUNN EDWARD ALBERT, MUNN, EDWARD, ALBERT
Applicants: MUNN EDWARD ALBERT, MUNN, EDWARD, ALBERT
Application year: 1987
Main classification: A61K39
IPC codes: A61K39, A61P31, A61P33, C07K1, C07K4, C07K14