methods for evaluating and maximising the immune response are surveyed. ..... Trichostrongylus colubriformis in guinea pigs (52) and for Schistosoma in mice (55) ... homogenise the whole parasites and then to generate fractions for testing.
Rev. sci. tech. Off. int. Epiz., 1990,
9 (2), 577-595
Strategies for vaccination against gastro-intestinal nematodes T.S. SMITH and E.A. MUNN * Summary: A great diversity of gastro-intestinal nematodes parasitise man and his animals. Each nematode is antigenically highly complex with distinct developmental stages. The parasites occupy a range of niches in the host gut and have a variety offeeding habits. Hosts have evolved only partially successful local immune responses. The host and parasite factors modulating these responses are described. Strategies for vaccination are surveyed. It is argued that the best route to successful vaccines will be to seek parasite immunogens which are not normally seen by the host's immune system in the course of infection. Routes to be followed in identifying and characterising such immunogens are proposed and methods for evaluating and maximising the immune response are surveyed. The success of this strategy is illustrated by reference to an experimental molecular vaccine against Haemonchus contortus. Strategies for the use of vaccines and methods for their production in vitro are reviewed. KEYWORDS: Gastro-intestinal nematodes - Immune responses - Molecular vaccines - Parasites - Vaccination - Vaccines.
I N T R O D U C T I O N
Of the fifty or so genera of nematodes which are parasitic in the gastro-intestinal (GI) tracts of vertebrates (9, 16), vaccination has been envisaged primarily for those which infest mammals and birds of importance to man for reasons of economy or as domestic pets. Gastro-intestinal nematode infections of farm animals in particular are a source of great economic loss (19, 50). At present the only means for their control depends on pasture management and treatment with anthelmintic chemicals. There are a number of factors that combine to make vaccination an attractive alternative to the use of these agents. The foremost of these are: a) the appearance in many parts of the world of GI nematodes resistant to anthelmintics (75, 76), this resistance persisting even in the absence of continuous selection pressure (6) b) the need for frequent administration c) increasing pressure for a reduction in toxicity and persistence of chemical residues in the environment. * Institute of Animal Physiology and Genetics Research, Cambridge Research Station, Babraham Hall, Cambridge CB2 4AT, United Kingdom.
578 The ultimate aim must be a multivalent vaccine effective against a range of GI nematodes but initial efforts will be directed to individual genera. The strategies required for each genus will depend on the consideration of a number of factors involving the hosts, the parasites and their interactions.
HOST FACTORS The ability of the host to respond to assault by the parasites will depend on its genotype, age, previous exposure to the nematode, its plane of nutrition and hormonal status. The immune response of the host will further depend on the immunogenicity of the parasites and on their ability to suppress or modulate the activity of the host's immune system. Immunological background The rejection of GI nematodes is brought about through the combined action of specific antibodies, B and T lymphocytes and non-specific effector cells. Antigendependent immune responses occur principally in the peripheral lymphoid system. This consists of the peripheral lymph nodes and spleen which are involved in systemic immune responses, and the gut-associated lymphoid tissue (GALT) and other mucosal immune cell systems which are involved in local or mucosal immunity. A protein antigen arriving in the peripheral lymph node is taken up and processed by cells (particularly macrophages) which present fragments of it at the cell surface as complexes with proteins specified by immune response, class II Major Histocompatibility Complex (MHC) genes. T-lymphocytes (T-helper cells), activated when their surface membrane receptors interact with the appropriate epitopes (39) on the processed antigen-MHC protein complex, proliferate and interact with the B-lymphocytes expressing membrane-bound immunoglobulin specific for other presented epitopes of the antigen. These interactions lead to proliferation and differentiation of the B-lymphocyte population producing memory cells and plasma cells which secrete specific antibodies. The antibodies are released into the lymph and thence the blood. T-lymphocytes and B-memory lymphocytes pass into the blood vascular system and recirculate between it, the spleen and the peripheral lymph nodes. The immune response is regulated by the activities of suppressor T cells. The mucosal immune system samples antigens at the gut mucosal surface by way of M-cells. These transport the antigens to underlying lymphoid tissue which contains antigen-presenting cells, B and T lymphocytes. Antigen stimulates proliferation and movement of the lymphocytes to the mesenteric lymph nodes and thence to the blood vascular system. These lymphocytes home back to the GALT. The first antibodies secreted by B-cells in response to a primary antigenic stimulation belong to the immunoglobulin class IgM. They are followed by IgG, the most common immunoglobulin in serum. The subclass IgGl, which is the major immunoglobulin in ruminant colostrum, has been implicated in immune responses to several GI nematodes. IgA antibodies occur in serum at low levels but are especially important, in a secretory form (slgA), in mucosal immunity. These antibodies act by combining with antigens. This can lead to inactivation of enzymes and blocking of binding sites or can trigger the binding of complement which may itself lead to
579 lysis, or can stimulate binding of macrophages. Antibodies belonging to the immunoglobulin class IgE have been implicated in expulsion of some GI nematodes. IgE is a minor component of serum but is bound to the surface of many cells. It is cytophilic for mast cells and basophils and triggers antigen-related release of vasoactive substances, thus facilitating local infiltration of other immunoglobulins. Inflammatory responses in the gut wall lead to increased transport of sIgA and leakage of other classes of antibody (41, 42). This enteric response is not parasite-specific and can be triggered by the presence of another unrelated nematode. These effector mechanisms may reduce GI nematode burdens by expulsion of the parasites. This is never a complete process, but the surviving parasites may show reduced growth or fecundity. Age The most important age-related host factor is the non-responsiveness of the immune system of young animals to exposure to infective larvae of GI tract nematodes. Adult sheep repeatedly infected with Haemonchus for example, or dosed with infective larvae attenuated by irradiation, can develop good, local, protective immunity to larval challenge. Lambs, however, do not develop immunological reactivity to irradiated infective larvae of Haemonchus or their soluble extracts until between 7 and 24 months of age (35, 38, 69, 73, 74, 79). Similarly, irradiated larval Ostertagia vaccines will not work in 3- to 4-month-old calves, whereas animals a year old will develop strong resistance. The problem of non-responsiveness in young lambs seems to be due in part to the ability of the worms to suppress the immune response. One theory put forward to explain this phenomenon for Haemonchus involves induction of neonatal T-suppressor cells by exposure to larval antigens via the colostrum or by ingestion (72). Other mechanisms may operate. It has been suggested (42) that the inability of rats less than six weeks old to expel primary infections of Nippostrongylus brasiliensis, for example, is linked to the poor expression of MHC class II proteins (Ia antigens) on enterocytes (40). The only commercially available vaccine against a parasitic nematode, the attenuated larval vaccine against Dictyocaulus viviparus, the lungworm of cattle (54), is effective in calves only two months old. It is noteworthy, however, that this is attributable to the generation of a systemic response rather than a local or mucosal one. Similarly, the effective, but commercially unsuccessful attenuated larval vaccine against Ancylostoma caninum depended on a systemic response against migratory larvae. This problem too is age-related, although this case applies to older animals, which, by exposure to natural infections, have already acquired immunity. Thus, vaccination of dogs in endemic areas with irradiated larvae oí Ancylostoma caninum brought about hypersensitivity reactions which were damaging to their lungs (43). Genotype Whatever the mechanism, the lack of immune reponse to infective larvae of GI nematodes in young animals is genetically determined. Thus, the ability of merino lambs segregated into those most able (responders) and those least able (nonresponders) to mount an immune response to irradiated T. colubriformis larvae was shown to be heritable (13, 80). Similarly, the progeny of ewes which were 'responders' for H. contortus were less susceptible to infection with Haemonchus than those of 'non-reponders' (19, 64). In a wider context, breed of sheep can also have a significant
580 effect on the natural immune response even in adult animals. The indigenous breed of sheep (Red Masai) in Kenya was found to be more resistant to infection with the local strain of Haemonchus than imported breeds (in order of decreasing resistance: Merino, Dorper, Corriedale, and Hampshire Down) (56, 57). In the context of vaccination certain breeds may thus mount a more effective immune response than others. For some diseases and parasites this has been shown to be related to MHC gene restriction. Parturition and lactation Parturition and lactation can depress the level of acquired immunity to GI nematodes. This will lead to resumption of development of arrested fourth stage larvae, to increased fecundity of the adult worms already present and increased vulnerability of the host to new infections (10, 66). An explanation for the decrease in immunity to GI nematodes during this period is offered by experiments in laboratory animal models; in lactating rats, for example, failure to reject N. brasiliensis was shown to be due to failure in the response of antigen-stimulated lymphoid cells (12) attributed to increased levels of prolactin (31).
PARASITE FACTORS Pathogenicity The pathogenicity of GI nematodes depends on the site of infection, their mode of feeding and the total numbers present, including co-infection by other nematodes and other pathogens. For those nematodes with a direct life cycle the GI tract stage will be the most important; for those nematodes with migratory larvae it is this migratory stage which may be the more critical in terms of pathology. The main sites at which GI nematodes occur in mammals and birds are the lower gut (9,16) with only very few (e.g. Capillaria putorii, Haemonchus) living in the stomach and (e.g. Amidostomum, Capillaria contorta) in the oesophagus. The position in the gut will contribute to the pathophysiological effects. Thus, infections of the proximal gut may particularly affect digestion and absorption of nutrients whilst infections at distal sites will reduce the opportunity for resorption of leaked proteins (71). Irrespective of where the nematodes dwell in the GI tract they may be classified on the basis of their feeding habit. Parasites may ingest blood (e.g. Ancylostorna, Haemonchus), feed on the mucosa (e.g. Oesophagostomum) or dwell and feed within the lumen (e.g. Ascaris, Toxocara). Associated with a particular feeding habit may be both physical damage to the host and interference with physiological function. Immunogenicity and immunosuppression Many parasite antigens are recognised by the host during infection. In the case of Nippostrongylus infections of the rat, a popular laboratory model, the antigens generate a significant, rapid protective reponse in the host. In farm animals, however, many of the resulting immune responses to parasite infections seem to have little functional significance in terms of resistance to either the primary infection or subsequent re infection. Moreover, any immunity which does develop is slow to appear.
581 The ability of GI nematodes (notably Haemonchus) (11, 53) to evade the host immune response is particularly evident in sheep where, because of the slow development of protective immunity, chronic debilitating illness often occurs. The fourth stage larvae of Haemonchus (and of other trichostrongylids) are able to persist in the mucosa in a metabolically dormant state but, in addition, the actively parasitic adult worms also persist (41). It has been proposed that both stages of the nematodes are able to secrete immunosuppressive or immunomodulatory factors which prevent development of an effective immune response; for example, by activation of host suppressor cells (1,2). Immunosuppressive factors also occur in homogenates and extracts of larvae and adults (3, 45) which probably accounts for the poor performance of impure preparations or extracts of GI nematodes as vaccines. The secretion of immunosuppressive factors which specifically block only effective responses is unlikely. Presumably these factors lead to a general suppression and, although specific antibodies are generated, protective levels of response are not achieved.
STRATEGIES Whilst it would clearly be highly beneficial to stimulate adequate mucosal immunity this is not possible with our present level of knowledge. There is plenty of evidence, however, that protective immunity against GI nematodes can be achieved by means of systemic immune responses and this therefore is the route to follow. It is necessary to stimulate in young animals the level of immune response which protects the adult; and in the adult to maintain that level even at times of pregnancy and lactation. There are three approaches. The simplest (but by no means easy) approach is to identify some component on the parasite which is accessible to the immune effector system. Antibody raised against that component, when bound, facilitates the activation of complement or cytotoxic cells. The generation of many antibodies during the course of natural and experimental infections without noticeable effect on the parasites suggests that this approach will be profitable only if the target immunogen is one not normally seen by the host in the course of an infection. The second approach is to induce antibodies to specific parasite molecules with the intention of interfering with the function of the target molecule. For this, the antibody will need to be directed at a very Umited number of epitopes, possibly only one. The target molecule for this kind of approach is likely to be an enzyme, or a transporter or carrier of some kind. That is, it will be a protein. It will probably have attached groups, particularly carbohydrate, not immediately involved in its functional role. The third approach, which may be a subset of the previous one, is to seek to overcome immune evasion mechanisms operated by the parasite so that the natural immunity of the host is fully operational. Whilst it might be ideal to induce both systemic and local immunity involving both antibody-dependent and cell-mediated protection, we propose that the first aim is to produce antibody-dependent protection by stimulating systemic responses. The presumption made here is that this aim can be achieved by use of a single, or at most very limited number, of immunogens. Out of the vast number of potential immunogens present in GI nematodes it is likely in any case that only one or two will generate a protective immune response. The crucial problem is to be able to identify these allimportant molecules in the first place. Subsequently, the need is to purify and characterise them, then produce them in sufficient quantity in vitro and finally present
582 them to the host's immune system in such a way as to induce a high, maintained immune effector response. Whichever route is pursued there are also technical and production factors to be taken into account, such as level of control required, storage and shelflife of the immunogen and mode of delivery to the host.
IDENTIFICATION OF PROTECTIVE ANTIGENS In the search for protective immunogens the two principle strategy decisions to be taken first are whether to try and identify immunogens involved in any natural immunity and seek to maximise the responses to these, or to try to identify some novel target for the immune effector system. Identification of immunogens involved in natural immunity depends principally on the presence in the circulation (or lymph) (68) of the appropriate antibodies which can then be used as screening reagents or in immunoprecipitations for antigens separated by fractionation of the parasites. Novel antigens may be identified in injected fractions by correlation of protection with presence of biochemically identified components as well as by serological criteria. Antigens identified by infection-induced antibodies To maximise the chance of finding such antibodies against larval antigens for experimental purposes, host animals may be made hyperimmune by repeated challenge with live infective larvae. Antibodies to the antigens of adult parasites are unlikely to provide selective reagents unless significant differences can be demonstrated in their specificities or isotypes between protected and non-protected hosts. It should be borne in mind that immuno-dominant antigens from larvae or adults are unlikely to generate antibodies which are either directly or indirectly protective. It would be best indeed to avoid such molecules. Sher (65) has called the latter approach 'Waksman's postulate' after the proposal of Dr B. Waksman that in the search for potential vaccine immunogens against the trematode Schistosoma, attention should be focused on those molecules against which little or no response is generated in the course of natural infection. His proposal was based on the suggestion that any antigen to which the host mounted a major immune response was unlikely to be important for the survival of the organism. A further logical development of this proposal is that the parasite antigen to be targeted should be accessible to the appropriate immune effector arm. This means in general that the antigen should be released from (the so-called ES, or excretory secretory antigens), or expressed at, an external surface of the parasite, i.e. either on the cuticle or its openings, or at the luminal surface of the gut. The two conditions, accessibility to the host's immune system but generating little or no response, seem to be irreconcilable. As will be described later, however, antigens may well be present at surface sites which normally do not produce a response by the host's immune system during the course of an infection. ES antigens include some surface molecules and molecules expressed by the parasite following invasion of the host. Molecules which have been identified and shown to be secreted at one time or another, by one or another GI nematode, include enzymes (53, 63), an anticoagulant (25), complement binding factors, eosinophil attractant (33) and immunosuppressants. Antibodies induced to these antigens may only act indirectly on the parasite (i.e. at some distance from the source of secretion) but if they impair function, for example by interfering with a protease activity and thus blocking larval
583 migration, or impairing nutritional processes of the parasite, they may well be good targets for direct acting antibodies. Several components of the outermost layer of the cuticle, the epicuticle, induce the formation of antibodies during infections and for N. brasiliensis and Nematospiroides, for example, there are stage-specific surface antigens (20, 37, 58, 59). The components of the basal layers may also be immunogenic and if they could be exposed to the host's immune system they might constitute a useful source of protective antigens (60). Many surface and ES antigens (for example, those secreted by Toxocara canis) (36) are glycoproteins and the carbohydrate moieties are often highly antigenic. As shown by binding of monoclonal antibodies, these compose epitopes which may be common to several proteins (23). It has been proposed that complex carbohydrates as components of glycoproteins (and glycolipids and proteoglycans) may be useful components of vaccines (24). Despite their ubiquity and immunogenicity, however, many surface glycoproteins do not generate natural protective immune responses. (Thus, the antigenic glycoprotein may be considered a microcosm of the antigenic nematode.) That the search for protective antigens should not be restricted to surface molecules is indicated by the observation that tropomyosin and paramyosin, which are usually considered to be intracellular proteins, are partially protective antigens for Trichostrongylus colubriformis in guinea pigs (52) and for Schistosoma in mice (55) respectively. Paramyosin epitopes are presented by living schistosomula in culture in a form which stimulates T cells, but as judged by binding of a fluorescently labelled monoclonal antibody, tropomyosin occurs only in the wall muscle of Trichostrongylus. A great many parasite molecules which are potential antigens will be expressed at sites, for example intracellularly, which remain both hidden from, and totally inaccessible to, the host's immune system throughout the life of the worm. These molecules become important only as components of homogenates and sub-cellular fractions in which their presence can mask antigens which may be significant in generating a protective immune response when injected. Antigens identified by experimentally induced antibodies The injection of killed worms, either intact or homogenised, has not given any significant protection and thus is unlikely to yield useful antibodies. This may be because any single antigen capable of inducing a protective immune response is not present in sufficient amount and because of the effects of the immuno-suppressive components which are probably present. On the other hand, injections of partially fractionated extracts of living or killed worms have in some cases proved very promising in inducing systemic immunity. These extracts provide the starting material for progressively more refined fractions which with repeated testing will provide more and more specific sera. Because most GI nematodes are relatively small it seems to be usual practice to homogenise the whole parasites and then to generate fractions for testing. However, the adults of most genera may be dissected to obtain individual organs or tissues on a scale sufficient to yield composition data and to generate antibodies in laboratory animals. This approach may even be used to look directly for the presence of protective antigens. Thus, it was shown that calves injected with homogenates of excretory glands from adult Oesophagostomum (1,350 were needed per calf) were protected 92% compared to controls (30).
584 ES antigens may be regarded as constituting a selected fraction since they are a defined subset of the total antigens present in the parasites. ES antigens released in vitro from cultured infective larvae are a classical source of material for attempting to induce protective antibodies. Once an antigen of interest has been located within a selected tissue and some characteristics determined (e.g. solubility, M on SDS-PAGE, or reaction with antiserum), then its purification from homogenates of the whole parasite can be undertaken on a larger scale. r
To illustrate the kinds of approach outlined above, examples are given here of work on protective antigens from three GI nematodes, Trichinella spiralis, Oesophagostomum radiatum and Haemonchus contortus. The particular significance here of the work on Trichinella is that it resulted in the first isolation of a protective antigen from a GI nematode purified to homogeneity (67). The migratory larvae of Trichinella induce significant levels of protective antibodies. Monoclonal antibodies derived from infected mice were selected by strength of binding to antigens soluble in 0.1% Triton X-100. The monoclonal antibodies were used to screen fractions of infective larvae (3 X 10 were needed) and then used for immuno-affinity chromatography. Three proteins were purified. As little as 0.1 ug of one of these (M 48 kd) injected into mice intraperitoneally in Freund's adjuvant substantially inhibited the establishment of muscle larvae after an oral challenge. This protein occurs in the ß-stichocytes, the lining of the gut and on the cuticle of infective larvae. 7
r
Although older animals develop resistance to Oesophagostomum, young calves are susceptible. Three-month-old calves injected with homogenates of L4 larvae were protected 85% against oral challenge (30). A saline-soluble fraction from larvae cultured to the L4 stage in vitro, injected sub-cutaneously in Freund's adjuvant induced circulating specific antibodies and gave a 99% reduction in adult worm burden when used at a dose of 2 mg total protein per calf and a reduction of 80% at 0.4 mg per calf (17). The fraction clearly contains a mixture of proteins and by comparison with the Trichinella studies it may be calculated that essentially total protection could be obtained with between 10 and 20 ug of a purified antigen. The luminal surface of the intestine of fourth stage larvae and adults of Haemonchus contortus is bathed in host blood. The blood of infected sheep contains antibodies to many Haemonchus antigens but none is specific for the parasite's intestine. Clearly, however, if antibodies were to be induced to antigens at the luminal surface they might well act to the detriment of the parasite. Electron microscopy revealed that the surface of the microvilli of blood-ingesting stages is coated with material with characteristic structure (subsequently shown to be a polymeric protein) which has been named contortin (46). By means of electron microscopy, using the rapid negative staining technique to track the protein, a simple differential centrifugation scheme was developed which yielded a fraction substantially enriched in contortin from homogenates of adult worms. Injection of the contortin-enriched preparation (CEP) leads to the formation of circulating precipitating antibodies to contortin (and to some other antigens of which the bulk were present in homogenates of the adult parasite's intestines). When challenged, the lambs with these antibodies were substantially protected against infection, their worm burdens being reduced on average about 70%. Further, many of the animals without antibodies died of haemonchosis (48). It is noteworthy that this protection was achieved even though the CEP contained some component that inhibited the antibody response to horse ferritin injected at the same time (45) and
585 may thus have brought about a general reduction of immune response. Since this approach seemed to work, other antigens were sought at the luminal surface of the intestine, particularly in the microvilli plasma membrane. This search was facilitated by the discovery of an entity, the endotube, which may be dissected from the intestine as a complex with the microvilli. All strongylid GI nematodes possess an endotube which may be dissected from the intestine (46). A major component of the microvillar membrane is an integral membrane glycoprotein (designated H11) with M of 110,000 (47). Antisera to CEP contained antibodies which bound to a component of CEP with this M even though on gels stained for protein, only traces of protein were detectable. Although, like contortin, antibodies to H11 were not generated during infections it seemed likely that this was a very strong antigen (particularly in view of the apparent presence of an immunoinhibitory component in the CEP). H11 is present in L4 and fifth stage Haemonchus. Solubilised in detergent after extraction of homogenates of adult Haemonchus with saline solutions, progressively purified preparations of H11 were obtained and tested for ability to induce protection against experimental haemonchosis in several breeds of sheep and in young lambs. The protective property co-purified with H11. For essentially pure H11 (the trace amounts of contaminating proteins present having been shown to have only modest protective ability), injected into sheep in microgram amounts, there were on average 93% and 86% reductions in female and male worm burdens respectively and a 94% reduction in faecal egg count over the patent period (49). Protection correlated with specific antibody titre. r
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P R O D U C T I O N
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P R O T E C T I V E
I M M U N O G E N S
For preliminary experimental purposes the larval and adult stages of the nematodes will be the obligatory sources of the immunogens. The larval, invasive stages of GI nematodes are usually fairly easy to obtain and thus, in theory, are a ready source of starting material for isolation of potential protective antigens. Because of the small size of the infective larvae, however, the isolation of workable amounts of antigens will require vast numbers with correspondingly large-scale facilities for their production. For ES antigens the appropriate culture conditions will be required. Usually the adults are very large relative to the invasive forms and therefore may represent a more convenient source of antigen than the larvae. Nonetheless, to obtain sufficient immunogen for analytical and trial purposes, the infection of often quite large numbers of host animals may well be required. Although the expression of antigens in adult nematodes is usually long-term rather than transient, the time of maximum rate of expression (also implying maximum level of mRNA, see below) for somatic antigens is most likely to be during development of the adult stage. Expression of antigens in the sexually mature adults is dominated by egg production and, to a lesser extent, by sperm production. On anything above the experimental trial scale a continuous, large, reproducible source of antigen will be needed and only material which can be produced in vitro is going to be of any practical use. Production of antigen in vitro will depend on whether it is protein or carbohydrate. If protein, the first choice will probably be to try to produce the whole molecule, or as much of it as possible, by recombinant DNA procedures
586 in Escherichia coli. A recombinant eukaryotic cell will be required if carbohydrate is essential. Alternatively, transgenic free-living nematodes which are easily grown in bulk in vitro, would be more likely to faithfully produce the structure of a glycoprotein from the parasitic nematode. Failing this it will be necessary to try anti-idiotype monoclonal antibodies, or synthesis of a shape that mimics that of the protective epitope (18). Recombinant DNA For in vitro production, recombinant DNA procedures will be the preferred method and the sooner this is started the better. cDNA libraries derived from mRNA and inserted into an expression vector in a prokaryote will be the method of choice and such libraries may be probed by means of antisera or monoclonal antibodies as well as DNA probes. For cDNA production, stage-specific expression, and hence the presence of the appropriate mRNA, may limit the usefulness of different stages. The metabolically inert infective larvae will have little mRNA but culture under conditions in which they develop to the next stage should generate the necessary messenger. By the same token, mRNA for ES antigens is likely to be present only under the appropriate conditions of culture. As noted above, for adults the time of maximum expression of somatic antigens and hence the relevant mRNA is likely to be during the growth stages. Cloning of antigens from only three GI nematodes has been described to date; one a protease from a hookworm and the others a muscle protein from two trichostrongylids. The hookworm work has been described in a brief report (26). A cDNA expression library was screened with an antiserum raised against a purified elastase (25) from adult Ancylostoma and a positive clone identified from which a fusion protein was produced and purified. Serum monospecific for the protein from an infected dog cross-reacted with the fusion protein. Its use in vaccine trials has not been described. The muscle protein, tropomyosin, was purified from sodium deoxycholate extracts of third stage larvae of Trichostrongylus colubriformis. It was identified on the basis of amino acid sequence homology (52). Messenger RNA from fourth stage larvae was used to produce a cDNA expression library. This was screened with oligonucleotide probes synthesised on the basis of partial amino acid sequence data. One of the positive clones thus identified was itself used to screen further libraries. A fusion protein with B-galactosidase was obtained and purified. This gave protection against the nematode in guinea pigs comparable to that obtained with the protein isolated from third stage larvae. This is thus the first description of protection against a GI nematode by a recombinant protein. The homologous recombinant protein obtained from a cDNA library from young adult Haemonchus contortus gave only marginal protection in sheep, but the prospects for success with this kind of approach must be high. This is shown by the following examples for vaccines against the tapeworm Taenia ovis and the tick Boophilus microplus. Hatched and activated oncospheres of T. ovis secrete potent host-protective antigens (unlike most GI nematode larvae). Sera from immune sheep showed strong antibody binding to oncosphere proteins with M by SDS-PAGE of 47-52 kd. These proteins, obtained by cutting out this region of the gel, protected lambs against challenge infection (this may or may not work with antigens from GI nematodes). Rabbit antibodies purified by binding to the 47-52 kd region were eluted and used to screen a cDNA expression library prepared from oncosphere mRNA. Positive clones were selected and antigen-ß-galactosidase fusion proteins were obtained. These were immunogenic in sheep, but failed to give protection. However, r
587 one of two fusion proteins prepared with the enzyme glutathione S-transferase (from S. japonicum) was strongly protective, especially when used in saponin as adjuvant when 50 ug gave 94% protection (27). The tick example is in many ways analogous to that of GI nematodes. Cattle acquire only a partial immunity based on an immediate hypersensitivity reaction to the ticks after extensive natural infection. This is insufficient to prevent serious losses to cattle production. Injection of partially fractionated extracts of adult ticks gave a degree of protection (28) which led to damage of the tick gut in a process involving complement (32). (This process is quite different to that involved in the limited natural protection.) Further fractionation and re-testing of the extracts led to identification of a membranebound glycoprotein with a M of 86 kd (77, 78). This was successfully produced from a cDNA expression library in E. coli and the recombinant product as a fusion protein with ß-galactosidase has yielded 77% reduction in tick egg output (61). r
Monoclonals and anti-idiotype antibodies Monoclonal antibodies will be of use to identify potential protective antigens, or the epitopes thereof, for purification on immuno-affinity columns, as probes for gene expression libraries and in the production of anti-idiotype antibodies. The antigen-binding site of a specific antibody molecule is complementary in its shape to the epitope to which it binds. This antigen-binding site can itself function as an antigen and is then known as an idiotype. The antigen-binding site of an anti idiotype antibody will mimic the shape of the original epitope. Given a supply of antibodies, in particular a monoclonal antibody, to an epitope of interest on a parasite antigen it is possible, therefore, to use these to generate further antibodies which mimic in their antigen-binding sites, the shape of the original parasite epitope. In principle these anti-idiotype antibodies may be used as surrogate antigens (15). The applicability of this approach has been demonstrated in experiments with Schistosoma mansoni in rats. A monoclonal antibody (IPLSm1) was raised against a schistosomulum membrane glycoprotein of M 38 kd. Rat anti-idiotype antibodies produced against IPLSml which inhibited the binding of IPLSml to the 38 kd target antigen were selected. When these anti-idiotype monoclonals were, in turn, injected into rats they induced the formation of specific anti-schistosome antibodies which bound to the 38 kd antigen. Animals with these antibodies were 50-76% protected against challenge infections (22). r
To minimise the production of undesirable antibodies to the anti-idiotype antibody they should be of the same species as the host animal to be vaccinated. Although there are no published data on their use for anti-idiotype work, bovine (4), ovine (21) and porcine (62) monoclonal antibodies have been produced from hetero-hybridomas made by fusing lymphocytes from immunised animals with murine cell lines. Ovine monoclonal antibodies have been obtained to antigens of two ovine GI nematodes. The first were from lymphocytes of sheep made hyperimmune by repeated infection with larval Ostertagia (5) and the second were from sheep injected with partially purified antigens from adult Haemonchus (14). Clearly the anti-idiotype approach is very laborious and technologically very demanding. Probably it would only be undertaken where the original epitope is conformational and cannot be obtained by recombinant DNA procedures, or where the antigen is unknown but reacts with a monoclonal antibody which possesses neutralising activity against the parasite (50).
588 It is noteworthy in the context of neonatal tolerance to GI nematodes that neonatal mice which are normally unresponsive to E. coli Kl 3 polysaccharide may be vaccinated against it with an anti-idiotypic antibody (70). The usefulness of monoclonal antibodies as probes of gene expression libraries is illustrated by reference to a monoclonal antibody which protects mice against Plasmodium chabaudi infection. The antibody has been used to select a cDNA clone which showed sequence homology with other species of Plasmodium. The epitope of the monoclonal antibody was then mapped to a linear sequence of five amino acids (34). Synthetic peptides The minimal requirement for induction of a protective immune response is a sufficiently large piece of the protective antigen to give the shapes of the critical epitopes suitably presented to the host's immune system. Bearing in mind, as noted earlier, that B and specific T helper cells recognise different epitopes (conformational and linear respectively, occupying non-overlapping areas) on the antigenic molecule, it follows that for a synthetic peptide antigen the sequence of the protein will need to be known and the epitopes characterised. The development of peptide vaccines is still at a very early stage and the prospect of producing synthetic peptides for use in vaccines against GI nematodes is remote. It is likely also to be a very expensive exercise, but the possibility of generating polyvalent vaccines (29) will undoubtedly encourage effort in this area. In following this route the experience gained in the development of a peptide vaccine for foot and mouth disease virus (7) will be invaluable. The lessons learned from the virus work are in many ways applicable in a more general sense to attempts to develop vaccines against GI nematodes. They are: 1) the biology of the pathogen should be well known 2) the availability of laboratory models is very helpful but it may not be possible to extrapolate from them to the true host 3) a simple in vitro test for antibody activity would be extremely valuable (what are needed for GI nematodes are reliable in vitro culture systems for the parasitic stages) 4) a limited portion of a protein fragment will generate a protective response, but antibodies to adjacent portions may be without effect 5) configuration of the epitope is critical.
PRESENTATION OF IMMUNOGEN TO IMMUNE SYSTEM Whichever type or combination of types of vaccine is chosen, the intention will be to establish protection as early as possible in the young animal and then either to maintain that level of protection or to have induced immunological memory such that infections over ensuing months will stimulate the level of immune response once again to protective levels.
589 Simulation of an effective, natural immune response would equip the animal to re-prime its defences on exposure to subsequent infections. Depending on the duration of the effective level of response, the use of 'novel' antigens (i.e. those not normally associated with the natural immune response) may, however, require that the animal has to be re-boosted at regular intervals although a reduction in the severity of infection may enable an effective natural response to develop at the same time. As noted above, some antigens when injected induce protective responses, even though they do not induce an immune response during infection. The site of injection and nature and composition of the adjuvant used will modulate the response and it may be that the route by which these molecules are presented to the host's immune system is of critical importance. Two recent developments - ISCOM's (immune stimulating complexes) and SAF-1 (Syntex adjuvant formulation), the former of which is already available as an equine influenza vaccine in Denmark - offer great promise as aids in vaccination. ISCOM's (44) are complexes of the adjuvant Quil-A and the antigen and can include membrane proteins which are presented on their surface rather as they would be in the native membrane. They are good stimulators of antibody and T-cell mediated responses and are self adjuvanting. SAF-1 includes a surfactant polymer composed of alternating hydrophilic and hydrophobic blocks which bind proteins to the surface of biodegradable oil drops (squalane). The formulation also includes MDP (muramyl di-peptide) which is an excellent stimulator of T-cell response (8). Finally, there is the possibility of producing recombinant viruses or bacteria that can be given directly to the host to form the protective antigen in situ (51). Each of these strategies will undoubtedly prove to have disadvantages as well as advantages in practice and the exciting potential for any of these approaches for use against GI nematodes obviously has yet to be fully explored. Stage of parasite to attack, and level of control In looking for protective antigens each nematode will have been considered in the context of factors such as feeding habits and whether one stage is more directly accessible to the host's immune system than another and if so, which site or tissue of the parasite is most open to attack. Successful immunisation against invasive stages immediately they arrive at or penetrate the mucosal surface is of particular appeal because if achieved it would prevent subsequent development of more severely pathogenic forms. Failing this, the early developmental stage of the nematode should be targetted. In practice, the stage immunised against may be determined by the stage-specificity of the protective antigen obtained. Whilst 100% protection might be considered ideal, a lower level of infection (if the pathological effects are minimal), may be acceptable. With due regard, one hopes, for animal welfare, the aim of the animal producer is to raise stock to produce a marketable product in the most cost-effective manner. The health of the animal may be considered partially as a matter of welfare, but primarily will be considered in terms of cost-benefit and this will largely dictate the level of control sought. A residual sub clinical infection might actually be beneficial if it was sufficient to stimulate the animal's natural immune response. Monovalent and polyvalent vaccines Since anthelmintics have a broad spectrum of activity against GI nematodes there has been little incentive to develop a monovalent vaccine. With the advent of resistance
590 to even the most recently developed anthelmintics by nematodes like Haemonchus (75), which are significant pathogens, this view must now be re-assessed. This may mean having to use genus-specific protective antigens, although for those vaccines which depend on an antibody directed to a functional epitope there is a good possibility of cross-genus protection. Even if there is no cross protection, antigens suitable to protect each parasite could be sought and combined to make a multivalent recombinant vaccine. In some cases, genetic restriction to one species of GI nematode by the host may force the production of a vaccine including a number of recombinant proteins to a single parasite. This solution could also be adopted if resistance to immune effector mechanisms developed or seemed likely to develop.
CONCLUSION The inability, so far, to produce a successful vaccine against GI nematodes is attributable in the first place to their complexity and their extensive interaction with, and adaptation to, their hosts. Thus, despite the ability of the hosts to mount potentially protective immune responses, the parasites in very many cases are able to evade these. As a result, stimulation of immune responses which are not part of the natural immune response appears to be the way forward. This means presentation of antigens in ways not normally encountered by the immune system of the host or, as suggested here, the identification and presentation of antigens to which the immune system does not normally respond. As illustrated, largely by reference to other groups of pathogens, realistic methods for the production and presentation of antigens are now available. The increasingly widespread occurrence of GI nematodes resistant to anthehnintics has provided the stimulus for tackling the major problem of identifying the critically important antigens. * * STRATÉGIES DE VACCINATION CONTRE LES NÉMATODES GASTRO-INTESTINAUX. - T.S. Smith et E.A. Munn. Résumé : Les nématodesgastro-intestinaux parasites de l'homme et des animaux domestiques sont extrêmement variés. Chaque nématode est d'une grande complexité antigénique et comporte différents stades de développement. Les parasites se logent dans toute une série de niches du tractus digestif et se nourrissent de diverses façons. Les réponses immunitaires locales élaborées par les hôtes ne sont qu'en partie efficaces. Les facteurs spécifiques de l'hôte et du parasite à l'origine de ces réponses sont décrits ainsi que les stratégies de vaccination. D'après les auteurs, la meilleure façon d'aboutir à des vaccins efficaces est de rechercher les éléments immunogènes des parasites qui ne sont pas normalement décelés par le système immunitaire de l'hôte lors de l'infestation. A cet égard, les auteurs proposent des méthodes pour identifier et caractériser ces éléments et examinent celles destinées à évaluer et à maximiser la réponse immunitaire. Le vaccin moléculaire contre Haemonchus contortus est cité à titre d'exemple de l'efficacité de cette approche. Les stratégies pour l'emploi de ces vaccins et les méthodes de production in vitro sont également passées en revue.
591 MOTS-CLÉS : Nématodes gastro-intestinaux - Parasites - Réponse immunitaire - Vaccination - Vaccins - Vaccins moléculaires. * * *
ESTRATEGIAS DE VACUNACIÓN CONTRA GASTROINTESTINALES. - T.S. Smith y E.A. Munn.
LAS
NEMATODOS
Resumen: Los nematodos gastrointestinales que son parásitos del hombre y de los animales son sumamente variados. La complejidad antigénica de cada tipo de nematodo es muy amplia y las fases de desarrollo muy distintas. Los parásitos se establecen en una serie de nichos del tracto digestivo y se alimentan de maneras diversas. Las respuestas inmunitarias locales de los huéspedes son sólo parcialmente eficaces. Los autores describen los factores específicos del huésped y del parásito que originan estas respuestas, así como las estrategias de vacunación. Consideran que la mejor manera de lograr vacunas eficaces consiste en buscar los elementos inmunógenos de los parásitos que no detecta el sistema inmunitario del huésped durante la infestación. Proponen métodos para identificar y caracterizar estos elementos y examinan los métodos de evaluación y optimación de la respuesta inmunitaria. Como ejemplo de la eficacia del enfoque citan la vacuna molecular contra Haemonchus contortus y, por último, pasan revista a las estrategias de utilización de las vacunas y las técnicas de su producción in vitro. PALABRAS CLAVE: Nematodos gastrointestinales - Parásitos - Respuesta inmunitaria - Vacunación - Vacunas - Vacunas moleculares. * * * REFERENCES - Time of onset and target of immune reactions in sheep with acquired immunity against Haemonchus contortus. Int. J. Parasit., 12, 439-443. 2. ADAMS D . (1983). - Investigation with dexamethasone on the process which moderates immunity against the nematode Haemonchus contortus in sheep. Aust. J. exp. Biol. med. 1. ADAMS D . (1982).
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