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Vaccination against gastrointestinal nematode parasites of ruminants using gut-expressed antigens
Veterinary Parasitology 100 (2001) 21–32
Vaccination against gastrointestinal nematode parasites of ruminants using gut-expressed antigens D.P. Knox∗ , W.D. Smith Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, Scotland EH26 0PZ, UK
Abstract To date, proteins isolated from the surface of the gut of gastrointestinal nematodes, particularly Haemonchus contortus, have generally proved to be useful protective antigens and several are being progressed towards recombinant protein-based vaccines. This paper describes the properties of some of the most promising antigens and summarises their performance in laboratory and field based trials. The antigens described include contortin, H11, H-gal-GP, GPI and cysteine proteinases. In addition, the discussion addresses the utility of selected antigens to protect against co-infecting nematode species such as Teladorsagia circumcincta and against related nematode infections such as Ostertagia ostertagi in cattle. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Vaccination; Gastrointestinal nematodes; Ruminants; Gut antigens
1. Introduction Anthelmintic resistance in nematode populations and concerns about the effects of drug residues on consumer health and the environment have focussed attention on the prospect of developing effective anti-nematode vaccines. These studies focus on characterising the host anti-parasite immune response, the nature of the protective components of the response, the parasite life-cycle stage targeted and, ultimately, with the aim of vaccine development, identifying the antigens responsible for protection. Vaccine development based on antigens that stimulate host immune responses during the course of infection is not straightforward. Partly so because no single component of this multifaceted response has been identified which mediates protective immunity and partly because, even if such a component were identified, the ability to induce it in the appropriate amount and location (i.e. the gastrointestinal mucosa) is currently lacking. An alternative approach is to ignore the natural host ∗ Corresponding author. Fax.: +44-131-4456111. E-mail address: [email protected] (D.P. Knox).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 4 8 0 - 0
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immune response and instead direct a response against parasite proteins which are essential to parasite survival and which are accessible to antibody such as excretory/secretory, cuticular or, in the case of blood-feeding species, gut-expressed proteins. This paper outlines the progress on vaccination against nematode parasites based on proteins isolated from the microvillar surface of the parasite enterocyte.
2. The nematode gut The nematode gut comprises, from anterior to posterior, a buccal capsule, a muscular pharynx, a fairly straight intestine and, finally, a rectum or cloaca. The pharynx and cloaca are, in part, lined with cuticle. The intestine is a syncytial or oligocytous structure with two cells in any circumference containing a lumen lined with a brush-border of millions of microvilli (Bird, 1971; Munn and Greenwood, 1984). In trichostrongylid nematodes, there is a fibrous sub-microvillar layer, known as the endotube, to which the filamentous cores of the microvilli attach (Munn, 1977) and this layer, together with the microvilli, can be dissected from the remainder of the intestine in most species (Munn and Greenwood, 1984).
3. Brush-border associated proteins from the intestinal cells of Haemonchus contortus Several proteins have now been characterised from the microvillar surface of Haemonchus. Since this parasite is a voracious blood feeder, these proteins are exposed to host immunoglobulin in the blood meal. Most, but not all, of these proteins have been found to be protective when used to immunise sheep against challenge infection with the parasite. What follows is an outline of the salient characteristics of these proteins together with a description of their protective antigen capability. 3.1. Contortin The microvillar surface is coated with a layer of electron dense amorphous material (glycocalyx). In Haemonchus contortus there are helical filaments, composed of contortin associated with this layer, which fill the spaces between the microvilli. Contortin is not attached to the plasma membrane and some lies free in the lumen of the intestine and is also present in large amounts in the pharynx although this may not be the case in vivo (Munn, 1977). Contortin, which can be purified from phosphate-buffered saline extracts of adult parasites by ultra-centrifugation, is composed of subunits with an apparent molecular weight of 60 kDa as judged by SDS-PAGE. Lambs vaccinated with a contortin-enriched preparation and subsequently challenged with Haemonchus infective larvae showed a mean reduction in worm burden of 78% (Munn et al., 1987). This result was particularly significant because it was the first to show that proteins expressed on the surface of the gut of a blood-feeding nematode, could induce high levels of protective immunity when used as an immunogen. Intestinal brush-border proteins are not normally accessible to the host immune system, even following repeated infection (Smith,
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1993), hence they are termed hidden or concealed antigens and the immunity conferred by them is described as artificial immunity. 3.2. H11 Contortin-enriched preparations contained a 110 kDa contaminant as judged by Western blotting despite only faint staining being evident in protein gels (Smith and Munn, 1990). This protein was purified using concanavalin A lectin-affinity chromatography combined with, on some occasions, Mono Q anion-exchange chromatography and it migrated as a doublet at 110 kDa as judged by SDS-PAGE, hence the short-hand designation, H11. It is the most effective immunogen isolated from a parasitic nematode to date, inducing high levels of protection (>90% reductions in worm burdens) which closely correlate with serum antibody titre (Smith et al., 1993). The immune response in sheep primed by immunisation with H11 can be boosted following challenge, presumably by H11 released from dead or dying parasites (Andrews et al., 1997), suggesting that this glycoprotein may not in fact be a completely hidden antigen. H11 is an integral membrane glycoprotein only expressed on the intestinal microvilli of the parasitic stages and homologues have been identified in Teladorsagia circumcincta and Ostertagia ostertagi (McMichael-Philips et al., 1995; Smith et al., 2000a, 2001). It shows microsomal aminopeptidase A and M activities and is encoded by at least three genes (Graham et al., 1993; McMichael-Philips et al., 1995; Smith et al., 1997). H11 has the predicted structure of a type II integral membrane protein with a short N-terminal cytoplasmic tail, a transmembrane region and an extracellular region organised into four domains (Smith et al., 1997; Newton and Munn, 1999). Enzyme activity is localised exclusively to the intestinal brush-border and is inhibited by H11 antisera in vitro (Smith et al., 1997), the level of inhibition observed being correlated to protection (Munn et al., 1997). 3.3. H-gal-GP Smith et al. (1994) used several different techniques, including lectin screening of worm sections and radiolabelling, to target integral membrane glycoprotein on the luminal surface of H. contortus intestinal cells. These proteins were then purified from detergent extracts of whole worms and evaluated in protection trials. One fraction, which selectively bound to lectins with specificity for N-acetylgalactosamine, reduced mean challenge worm burdens by up to 72% and mean faecal egg counts by up to 93%. This fraction was termed Haemonchus galactose-containing glycoprotein complex (H-gal-GP). The microvillar surface of the intestinal cells of worms retrieved from vaccinated lambs was coated with sheep immunoglobulin and protection was correlated with systemic antibody titre (Smith et al., 1999). H-gal-GP can be visualised as a single diffusely staining band after Blue Native PAGE with an estimated molecular weight of about 1000 kDa and resolves as four major protein zones, designated A, B, C and D, at ∼230, 170, 45 and <35 kDa, respectively, under non-reducing SDS-PAGE (Smith et al., 1999). Each zone resolved as more than one band after reduction. Biochemical analyses indicated that H-gal-GP exhibited aspartyl-,
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metallo- and, on occasion, cysteine protease activities. Initial N-terminal sequence (Smith et al., 1999) and cDNA analysis showed that the complex contained an aspartyl protease with homology to pepsin (Longbottom et al., 1997) and a metalloprotease (MEP, Redmond et al., 1997). Further molecular analysis has now shown that H-gal-GP contains at least 4 distinct MEPs with 38–54% similarity at the amino acid level to each other, these proteases being associated with the prominent 230 (MEP 1, 2 & 4) and 170 kDa (MEP 3) protein zones (Smith et al., 1999). It also contains a thrombospondin homologue, which migrates between the 170 and 230 kDa zones, this protein being related to a variety of “adhesin-type” proteins (Skuce et al., unpublished). The pepsin is the major component of the 45 kDa zone, a galectin is associated with the 35 kDa zone (Newlands et al., 2001) and a low molecular weight (∼13 kDa) inhibitor of Haemonchus cysteine proteases, cystatin has also been identified (Newlands et al., in press). H-gal-GP has proved resistant to fractionation using a variety of chromatographic techniques under native conditions. However, a closely related, somewhat simpler complex has been isolated using jacalin lectin. Unlike peanut lectin, jacalin can bind glycoproteins with sialylated forms of  1–3 N-acetylgalactosamine; a feature that resulted in this newer complex being designated Haemonchus sialylated galactose-containing glycoprotein complex (H-sialgal-GP). H-sialgal-GP is just as protective against Haemonchus as H-gal-GP (Smith et al., 2000a). The complexes appear identical except that the 230 kDa zone is missing in H-sialgal-GP, suggesting that MEPs 1, 2 and 4 are not essential for protection (Smith et al., 2000a). The precise function of these complexes remains undefined, but strong haemoglobinase activity associated with the aspartyl protease suggests that they play a role in digestion of the blood meal.
3.4. p46 and p52 Jacalin lectin, in combination with ion exchange and gel filtration chromatography, has also been used to isolate a pair of protective glycoproteins of 46 and 52 kDa from Haemonchus membranes. In combination these proteins reduced the egg output and worm count of immunised lambs by 78 and 33%, respectively (Smith et al., 2000b). The p46 molecule had been described earlier as a 45 kDa glycoprotein that protected guinea pigs against Haemonchus (Sharp et al., 1992). N-terminal sequence analysis of p46 revealed significant homology with that predicted from a cDNA fragment which encoded the 45 kDa protein of Sharp et al. (1992). Furthermore, the N-terminal data from p52 was almost identical to the predicted product of a second, closely related cDNA fragment (Sharp et al., 1992), now known to be part of a recently isolated full length gene, designated Hc40 (Rehman and Jasmer, 1998). Hc40 would be predicted to encode a protein of 51 kDa, in near agreement with the estimated mass of p52. The sequence of Hc40 suggests that the N and C terminal halves of p52 are closely related, with many motifs repeated in each half (Rehman and Jasmer, 1998). Intriguingly, many of these are also common to ES 24, a natural antigen discovered in Haemonchus excretory–secretory products which, in combination with a 15 kDa molecule, is also highly protective when used to vaccinate sheep (Schallig et al., 1997).
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3.5. Cysteine protease enriched extracts Extracts from adult H. contortus and excretory/secretory harvested by in vitro maintenance of adult parasites contain several strong cysteine protease activities with the capacity to degrade blood proteins such as haemoglobin and albumin, major constituents of the blood meal (Knox et al., 1993; Rhoads and Fetterer, 1995, 1997). Water-soluble extracts of adult H. contortus were shown to contain a 35 kDa cysteine protease which degraded fibrinogen and increased clotting time in sheep plasma in vitro (Cox et al., 1990a). Lambs immunised with extracts from the adult parasite enriched for this activity were claimed to be protected against challenge infection (Boisvenue et al., 1992) despite unconvincing raw data from two trials involving small numbers of sheep (Cox et al., 1990b). The cDNA (AC1) encoding this protease was isolated and showed 42% amino acid sequence identity to human cathepsin B. A cysteine protease enriched fraction (TSBP) was prepared from membrane extracts of adult H. contortus by passage over a Thiol-Sepharose affinity column and the proteins obtained were clearly localised to the microvillar surface of the intestinal cells (Knox et al., 1995, 1999). Lambs immunised with TSBP were substantially protected against a single challenge infection with H. contortus with reductions in daily faecal egg outputs of 77% and final worm burdens of 47% over three trials. Again, the microvillar surface of worms surviving in vaccinated lambs was coated with sheep immunoglobulin and recent experiments in our laboratory show that antibody harvested from vaccinated lambs functionally inhibits the cysteine protease components of TSBP (Knox, unpublished). Interestingly, TSBPs prepared in the same way from water-soluble parasite extracts were ineffective immunogens. TSBP comprised a prominent 60 kDa protein and several minor bands between 35 and 45 kDa and 97–120 kDa. Protease activity was mostly attributable to cysteine proteases although serine/metallo proteases were also identified (Knox et al., 1999). Lectinbinding studies showed that most of the TSBP proteins were glycosylated. Expression library immunoscreening revealed that the prominent 60 kDa component of TSBP is a glutamate dehydrogenase (GDH) homologue (Skuce et al., 1999a) and that the cysteine protease activity can be attributed to the protein products of three distinct genes, cDNAs derived from which have been designated hmcp1, 4 and 6 (Skuce et al., 1999b). Like AC1, these cDNAs showed closest homology to mammalian cathepsin B cysteine proteases. The expression of the GDH and cysteine protease encoding genes coincides with the onset of blood-feeding and immunolocalisation studies showed that the former was expressed in the cytoplasm of the intestinal cells (Skuce et al., 1999a) while the latter was expressed on the microvillar surface (Skuce et al., 1999b). This would argue against the GDH being the protective component of TSBP as it would be inaccessible to antibody and this, is indeed the case (Skuce et al., 1999a). TSBP can be further fractionated by anion exchange chromatography into a fraction which contains the bulk of the GDH protein and activity, fractions which contain the cysteine proteases and very little GDH and a protein fraction which does not bind to the column (Knox et al., 1995). Lambs immunised with the GDH fraction, or the unbound protein fraction, were completely unprotected against
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challenge whilst lambs immunised with the pooled protease-containing fractions were protected against challenge to the same degree as positive controls which received unfractionated TSBP (Knox et al., 1995), supporting the view that protection is attributable to the cysteine proteases. 3.6. GA1 and P1 antigens Monoclonal antibodies were used to identify and purify a group of proteins, Mr 46, 52 and 100 kDa, which, collectively, induced reductions of 60 and 50% in worm and faecal egg outputs, respectively, in immunised goats (Jasmer et al., 1993). N-terminal protein sequence analyses of the three proteins, termed p46GA1 , p52GA1 and p100GA1 , and cDNA library immunoscreening showed that all three were encoded by the same GA1 gene and are initially expressed as a polyprotein (p100GA1 , Jasmer et al., 1996). Western blot analysis indicated that the GA1 proteins were expressed in adult worms but not in infective larvae, a result essentially confirmed by Northern blot analysis. The GA1 gene product showed closest homology with bacterial Tolb proteins that are associated with the bacterial membrane and periplasm and may be involved in transport. GA1 derived proteins were detected in ES products from adult worms and host abomasal mucus indicative of release from the microvillar surface. Following on from this, protective immunity stimulated by immunisation with these proteins may involve anamnestic and mucosal immune responses. This suggestion was supported in a later study (Karanu et al., 1997) which provided evidence for a contribution from CD4+ lymphocytes to gut antigen-induced immunity. Smith et al. (1993) identified a group of three peptides (P1) which were separated from H11 by ion-exchange chromatography. The constituent peptides (45, 49 and 53 kDa) showed some similarities to the GA1 proteins and induced mean reductions of 69% in faecal egg output and 38 and 20% reductions in female and male worm numbers, respectively (Smith et al., 1993). The proteins, designated p45, p49 and p53, form a complex (p150) which is a ubiquitous constituent of the microvillar membrane of the intestinal cells. p53 has a membrane anchor and associates non-covalently with a disulphide bridged dimer of p45 and p49. All three components share peptide epitopes (Rocha and Munn, 1997).
4. Protective antigen capacity of intestinal brush-border proteins for other helminth species 4.1. Cross-protective capacity of Haemonchus proteins Compared to Haemonchus, much less is known about the gut membrane proteins of the other important helminth parasites of livestock. Although, T. circumcincta possesses antigenically cross-reactive homologues of H11 and H-gal-GP (McMichael-Philips et al., 1995; Smith et al., 2001a), immunisation of sheep with a mixture of H11 and H-gal-GP did not provide any cross-protection (Smith et al., 2001a). Nor was any cross-protection recorded against Trichostrongylus axei or Cooperia oncophora (Smith et al., 2001a), endorsing
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earlier findings with Nematodirus battus, when detergent extracts of Haemonchus intestines were employed as antigen (Smith, 1993). 4.2. Protective capacity of homologous gut membrane proteins Such trials have provided mixed results. The most promising was from Fasciola hepatica, where 89% fewer flukes were recently reported from sheep immunised with aminopeptidases purified from the parasite (Piacenza et al., 1999), a finding which needs to be repeated and extended. An earlier report stated that vaccination with homologous aminopeptidases induced protection in pigs challenged with Ascaris suum (50% reduction in larval counts, Ferguson et al., 1969). Results from only two of the ruminant trichostrongyle species have been published, namely O. ostertagi and T. circumcincta. Calves immunised with a combination of H11 and H-gal-GP homologues from O. ostertagi showed 30–50% reduction in egg counts, but there was no effect on worm numbers and neither antigen protected when administered on its own (Smith et al., 2000a). With T. circumcincta, lambs vaccinated with homologous TSBP were highly protected (71 and 65% reduction in eggs and worms, respectively; Knox et al. (1995)), but this could not be reproduced in subsequent trials. Furthermore, lambs immunised with fractions of T. circumcincta containing a mixture of its H11 and H-gal-GP homologues were not protected at all (Smith et al., 2001a). Despite this, the same T. circumcincta and O. ostertagi preparations did cross-protect sheep against a Haemonchus challenge (Smith et al., 2000a, 2001), indicating that neither immunogen was defective, and suggesting that these species do not ingest sufficient antibody for the gut antigen approach to vaccination to be appropriate.
5. Glycosylation, conformation and protective efficacy of Haemonchus brush-border proteins With the possible exception of contortin, all of the proteins described above are known to be, or predicted from gene sequence data to be, glycosylated. Because several (H11, H-gal-GP, P1, p46/42 and GA1) can be separated from each other by lectin-affinity chromatography, their sugar residues must differ. The nature of the N-linked oligosaccharides of glycoproteins in detergent extracts of adult Haemonchus and of H11 specifically has been investigated in detail (Haslam et al., 1996). These experiments identified a core fucosylation of a type not previously observed in eukaryotic glycoproteins. The major N-linked glycans had up to three fucose residues attached to chitobiose cores. These were found at the 3- and/or 6-positions of the proximal N-acetylglucosamine (GlcNAc) and at the 3-position of the distal GlcNAc. The latter substitution was stated to be unique in N-glycans (Haslam et al., 1996). It was proposed that these multifucosylated core structures could be highly immunogenic. Protection is reduced when either H11 or H-gal-GP is progressively denatured by treatment with SDS alone or SDS+ dithiothreitol (Munn et al., 1997; Smith and Smith, 1996), suggesting that conformational epitopes are essential for the full expression of protective immunity.
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6. Expression of Haemonchus protective antigens by recombinant DNA technology The cDNAs encoding H11 as well as most of the components of H-gal-GP and TSBP have all been expressed in E. coli and, to date, none of these recombinant proteins have been reported to be protective. These findings tend to confirm the need for conformational epitopes suggested by the dissociation and reduction experiments described above. However, enzymatically active recombinant protein versions of H11 and a cysteine protease component of TSBP have been expressed in the baculovirus-Sf9 insect cell system (Smith et al., 1997) and the yeast Pichia pastoris, respectively (Skuce et al., unpublished). The fact that these proteins show enzymatic activity suggests that they are folded correctly and the outcome of protection trials is awaited. Moreover, recent work has demonstrated the feasibility of expressing heterologous nematode gut-expressed genes in the free-living nematode Caenorhabditis elegans in a tissue-specific manner, this work being conducted with the gene encoding the pepsin component of H-gal-GP (Redmond et al., in press). In theory, the post-translational processing in C. elegans should be very close to that found in parasitic nematodes. Given that C. elegans can be cultivated in large-scale cultures, this may provide the best method for the large-scale production of nematode glycoprotein antigens.
7. Practical considerations of vaccination against Haemonchus with gut membrane proteins 7.1. Laboratory trials Many trials using worm-free, housed sheep have shown that H11 and H-gal-GP are effective antigens, stimulating high levels of protection single a challenge infection of Haemonchus after administration of two or three injections with QuilA, a commercially acceptable adjuvant (Newton and Munn, 1999; Smith et al., 2000a,b). Both were also effective in young lambs (Tavernor et al., 1992; Smith et al., 1994), demonstrating that the hidden antigen approach to vaccination was not restricted by the age-related unresponsiveness phenomenon. Such unresponsiveness prevented the development of irradiated larval vaccines for Haemonchus (Urquhart et al., 1966; Smith and Angus, 1980) and could also prove to be problematic with the natural antigen approach to vaccination (Vervelde, 2000). Worms begin to be shed from immunised lambs between 7 and 14 days after challenge soon after the onset of blood-feeding (Smith and Smith, 1993). A consistent trend after vaccination with H11, H-gal-GP or TSBP is that the female worms are more susceptible than the males, possibly reflecting higher metabolic activity associated with egg production. Protection afforded by vaccination with H11 in Freunds adjuvant persisted for at least 23 weeks after vaccination (Andrews et al., 1997) and did not interfere with the development of acquired immunity (Smith and Smith, 1993). Immunised lambs given a trickle infection were largely protected against the anaemia normally associated with infection and grew as fast and as efficiently as uninfected lambs (Smith and Smith, 1993). Vaccination with H11 reduced worm egg output (98% reduction) from pregnant ewes challenged with 10,000 H. contortus L3 during the third trimester (Andrews et al., 1995), almost eliminating the increase in
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egg output normally associated with the peri-parturient relaxation of immunity. In addition, lambs born and reared on vaccinated ewes had high antibody titres to H11 as a result of colostral transfer and this antibody conferred moderate protection against a single challenge infection of 3000 infective larvae (Andrews et al., 1995). This latter result indicated that protection was antibody mediated, a conclusion supported by serum transfer experiments (Smith, 1993) and observations that antisera from vaccinated lambs inhibited the microsomal aminopeptidase activity and that the degree of inhibition was highly correlated to the level of protection obtained (Munn et al., 1997). 7.2. Field trials Sterner, more realistic tests of potential vaccine efficacy can only be obtained from field trials where sheep grazing Haemonchus contaminated pasture may be continuously or sporadically exposed to variable dose rates of infective larvae depending on the prevailing climate. Several trials have recently been completed using a combination of native H11 and H-gal-GP as antigen formulated with QuilA. Most of these experiments were conducted in Louisiana, USA in the summer, a fairly extreme test because the warm humid conditions were highly favourable to the development of Haemonchus (Bahirathan et al., 1996), providing a heavy and continuous larval exposure. One trial, was complicated by the fact a proportion of the ewes had already acquired natural immunity, nevertheless, immunisation did reduce the egg counts of those deemed to be susceptible by 65% (Kabagambe et al., 2000). However, further trials with lambs indicated that these antigens were unable to control the infection before the animals succumbed to potentially fatal anaemia (Miller and Smith, unpublished data). Another 11-month long experiment was conducted in yearling sheep, near Pretoria, South Africa where the relatively dry climate with sporadic, seasonal rain ensured a discontinuous challenge of parasites more normal to conditions encountered by commercial sheep farmers in the tropics and sub-tropics. Here, vaccination reduced egg output by more than 82% on average during one 4-month period of the trial and simultaneously reduced the degree of anaemia and deaths due to haemonchosis (Smith et al., 2001b). Although vaccine immunity was not sufficiently long lasting to prevent a surge in egg output which occurred after the onset of a period of irrigation, re-vaccinating the sheep at this point cleared their newly acquired infection and rapidly restored protection to approximately the level observed beforehand. It was clear that in this situation a vaccine based on parasite gut membrane proteins could offer substantial benefits in the control of natural haemonchosis.
8. Concluding remarks Obviously the biggest single barrier to the commercialisation of a vaccine against haemonchosis is the production of recombinant proteins which approach the efficacy of the best native antigens. To date, recombinant proteins expressed in E. coli have not induced significant protective immunity but work is progressing in a variety of expression systems to overcome this problem. It will be disappointing if these difficulties cannot be overcome in the short-term.
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In addition, further work is required to determine whether other adjuvant formulations can give longer lasting responses than QuilA. More field trials to optimise when and how often such a vaccine would be administered are needed, bearing in mind the age and reproductive status of the sheep as well as the likely level of parasite exposure, before the potential benefits and limitations of a vaccine can be defined. Gut antigen-based vaccines (based on the antigens described above, at least) and mediated by high systemic antibody responses, are unlikely to be useful against non-blood-feeding nematodes. Different antigens may be required and may need to be delivered in such a way as to stimulate specific local mucosal immune responses. This will require continued efforts to define the key protective immune responses, to define the parasite antigens to which these responses are directed and to develop the means to deliver antigen to the host immune system in a manner to stimulate the correct response. New technologies will play a part. Already, the possibility of DNA vaccination is being explored, a topic which has recently been the subject of a comprehensive review (Alarcon et al., 1999). However, such an approach is quite likely to be met with consumer hostility towards the consumption of meat products contaminated with foreign DNA. We will be in a position to exploit genome-sequencing initiatives that should enable micro-array analysis of gene expression in parasite and host. These analyses should permit a very detailed analysis of the host–parasite interaction at the molecular level and allow us to target parasite gene-products associated with key events in the life-cycle such as the switch to the next developmental stage and moulting. In the future, it may be possible to generate a virulent mutant worms by targeted gene knock-out. The latter is already being applied extensively in C. elegans and veterinary parasitologists have much to learn from these experiments. References Alarcon, J.B., Waine, G.W., McManus, D.P., 1999. DNA vaccines: technology and application as anti-parasite and anti-microbial agents. Adv. Parasitol. 42, 344–410. Andrews, S.J., Hole, N.J.K., Munn, E.A., Rolph, T.P., 1995. Vaccination of sheep against haemonchosis with H11-prevention of the periparturient rise and colostral transfer of protective immunity. Int. J. Parasitol. 25, 839–846. Andrews, S.J., Rolph, T.P., Munn, E.A., 1997. Duration of protective immunity against ovine haemonchosis following vaccination with the nematode gut membrane antigen H11. Res. Vet. Sci. 62, 223–227. Bahirathan, M., Miller, J.E., Barras, S.R., Kearney, M.T., 1996. Susceptibility of Suffolk and Gulf Coast Native suckling lambs to naturally acquired strongylate nematode infection. Vet. Parasitol. 65, 259–268. Bird, A.F., 1971. The Structure of Nematodes. Academic Press, London. Boisvenue, R.J., Stiff, M.I., Tonkinson, L.V., Cox, G.N., Hageman, R., 1992. Fibrinogen-degrading proteins from Haemonchus contortus used to vaccinate sheep. Am. J. Vet. Res. 53, 1263–1265. Cox, G.N., Pratt, D., Hageman, R., Boisvenue, R.J., 1990a. Molecular cloning and sequencing of a cysteine protease expressed by Haemonchus contortus adult worms. Mol. Biochem. Parasitol. 41, 25–34. Cox, G.N., Pratt, D., Hageman, R., 1990b. Anticoagulant and anthelmintic proteins and methods for the production and use of the same. European Patent Application no. 90117879.8 Ferguson, D.L., Rhodes, M.B., Marsh, C.L., Payne, L.C., 1969. Resistance of immunised animals to infection by the larvae of the large roundworm of swine (Ascaris suum). Fed. Proc. 28, 497. Graham, M., Smith, T.S., Munn, E.A., Newton, S.E., 1993. Recombinant DNA molecules encoding aminopeptidase enzymes and their use in the preparation of vaccines against helminth infections. Patent no. WO 93/23542. Haslam, S.M., Coles, G.C., Munn, E.A., Smith, T.S., Smith, H.F., Morris, H.R., Dell, A., 1996. Haemonchus contortus glycoproteins contain oligosaccharides with novel highly fucosylated core structures. J. Biol. Chem. 271, 30561–30570.
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Jasmer, D.P., Perryman, L.P., Conder, G.A., Crow, S., McGuire, T.C., 1993. Protective immunity to Haemonchus contortus induced by immunoaffinity isolated antigens that share a phylogenetically conserved carbohydrate gut surface epitope. J. Immunol. 151, 5450–5460. Jasmer, D.P., Perryman, L.P., McGuire, T.C., 1996. Haemonchus contortus GA1 antigens: related phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection. Proc. Natl. Acad. Sci. U.S.A. 93, 8642–8647. Kabagambe, E.K., Barras, S.R., Li, Y., Pena, M.T., Smith, W.D., Miller, J.E., 2000. Attempts to control haemonchosis in grazing ewes by vaccination with gut membrane proteins of the parasite. Vet. Parasitol. 92, 15–23. Karanu, F.N., McGuire, T.C., Davis, W.C., Besser, T.E., Jasmer, D.P., 1997. CD4+ T lymphocytes contribute to protective immunity induced in sheep and goats by Haemonchus contortus gut antigens. Parasitol. Immunol. 19, 435–445. Knox, D.P., Redmond, D.L., Jones, D.G., 1993. Characterisation of proteases in extracts of adult Haemonchus contortus, the ovine abomasal nematode. Parasitology 106, 395–404. Knox, D.P., Smith, S.K., Smith, W.D., Redmond, D.L., Murray, J.M., 1995. Thiol Binding Proteins. Patent Application no. PCT/GB95/00665. Knox, D.P., Smith, S.K., Smith, W.D., 1999. Immunization with an affinity purified protein extract from the adult parasite protects lambs against Haemonchus contortus. Parasitol. Immunol. 21, 201–210. Longbottom, D., Redmond, D.L., Russell, M., Liddell, S., Smith, W.D., Knox, D.P., 1997. Molecular cloning and characterisation of an aspartate protease associated with a highly protective gut membrane protein complex from adult Haemonchus contortus. Mol. Biochem. Parasitol. 88, 63–72. McMichael-Philips, D., Munn, E.A., Graham, M., 1995. Helminth parasite antigen with aminopeptidase-like activity. International patent application. WO 95/12671. Munn, E.A., 1977. A helical, polymeric extracellular protein associated with the luminal surface of Haemonchus contortus intestinal cells. Tissue Cell 9, 23–34. Munn, E.A., Greenwood, C.A., 1984. The occurrence of submicrovillar endotube (modified terminal web) and associated cytoskeletal structures in the inestinal epithelia of nematodes. Phil. Trans. R. Soc. London B 306, 1–18. Munn, E.A., Graham, M., Coadwell, W.J., 1987. Vaccination of young lambs by means of a protein fraction extracted from adult Haemonchus contortus. Parasitology 94, 385–397. Munn, E.A., Smith, T.S., Smith, H., Smith, F., Andrews, S.J., 1997. Vaccination against Haemonchus contortus with denatured forms of the protective antigen H11. Parasitol. Immunol. 19, 243–248. Newlands, G.F.J., Skuce, P.J., Knox, D.P., Smith, S.K., Smith, W.D., 1999. Cloning and characterisation of a -galactoside-binding protein (galectin) from the gastrointestinal nematode Haemonchus contortus. Parasitology 119, 483–490. Newlands, G.F.J., Skuce, P., Knox, D.P., Smith, W.D., 2001. Cloning and expression of cystatin, a potent cysteine protease inhibitor from the gut of Haemonchus contortus. Parasitology 122, 371–378. Newton, S.E., Munn, E.A., 1999. The development of vaccines against gastrointestinal nematodes, particularly Haemonchus contortus. Parasitol. Today 15, 116–122. Piacenza, L., Acosta, D., Basmadjan, I., Dalton, J.P., Carmona, C., 1999. Vaccination with cathepsin L proteases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep. Infect. Immunnol. 67, 1954–1961. Redmond, D.L., Knox, D.P., Newlands, G.F.J., Smith, W.D., 1997. Molecular cloning of a developmentally regulated putative metallo-peptidase present in a host protective extract of Haemonchus contortus. Mol. Biochem. Parasitol. 85, 77–87. Redmond, D.L., Clucas, C., Johnstone, I., Knox, D.P., 2001. Expression of Haemonchus contortus pepsinogen in Caenorhabditis elegans. Mol. Biochem. Parasitol. 112, 125–131. Rehman, A., Jasmer, D.P., 1998. A tissue specific approach for analysis of membrane and secreted protein antigens from Haemonchus contortus gut and its application to diverse nematode species. Mol. Biochem. Parasitol. 97, 55–68. Rhoads, M.L., Fetterer, R.H., 1995. Developmentally regulated secretion of cathepsin L-like proteases by Haemonchus contortus. J. Parasitol. 81, 505–512. Rhoads, M.L., Fetterer, R.H., 1997. Extracellular matrix: a toll for defining extracorporeal digestion of parasite proteases. Parasitol. Today 13, 119–122.
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Rocha, J., Munn, E.A., 1997. p150, a protective glycoprotein complex of the microvillar membrane of Haemonchus contortus. Conference Abstract from Parasitic Helminths — from Genomes to Vaccines, Edinburgh, UK, 6–9 September 1997. Schallig, H.D.F., Van Leeuwen, M.A., Verstrepen, B.E., Cornelissen, A.W., 1997. Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus. Mol. Biochem. Parasitol. 88, 202–213. Sharp, P.J., Wagland, B.M., Cobon, G.S., 1992. Nematode vaccine. International Patent WO 92/13889. Skuce, P.J., Stewart, E.M., Smith, W.D., Knox, D.P., 1999a. Cloning and characterisation of glutamate dehydrogenase (GDH) from the gut of Haemonchus contortus. Parasitology 118, 297–304. Skuce, P.J., Redmond, D.L., Liddell, S., Stewart, E.M., Newlands, G.F.J., Smith, W.D., Knox, D.P., 1999b. Molecular cloning and characterisation of gut-derived cysteine proteases associated with a host-protective extract from Haemonchus contortus. Parasitology 119, 396–405. Smith, W.D., 1993. Protection in lambs immunised with Haemonchus contortus gut membrane proteins. Res. Vet. Sci. 54, 94–101. Smith, T.S., Munn, E.A., 1990. Strategies for vaccination against gastro-intestinal nematodes. Rev. Sci. Tech. Off. Int. Epizooit. 9, 577–595. Smith, W.D., Smith, S.K., 1993. Evaluation of aspects of the protection afforded to sheep immunised with a gut membrane protein from Haemonchus contortus. Res. Vet. Sci. 55, 1–9. Smith, S.K., Smith, W.D., 1996. Immunisation of sheep with an integral membrane glycoprotein complex of Haemonchus contortus and its major polypeptide components. Res. Vet. Sci. 60, 1–6. Smith, T.S., Munn, E.A., Graham, M., Tavernor, A., Greenwood, C.A., 1993. Purification and evaluation of the integral membrane protein H11 as a protective antigen against Haemonchus contortus. Int. J. Parasitol. 23, 271–277. Smith, W.D., Smith, S.K., Murray, J.M., 1994. Protection studies with integral membrane fractions of Haemonchus contortus. Parasitol. Immunol. 16, 231–241. Smith, T.S., Graham, M., Munn, E.A., Newton, S.E., Knox, D.P., Coadwell, W.J., McMichael-Phillips, D., Smith, H., Smith, W.D., Oliver, J.J., 1997. Cloning and characterisation of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus. Biochim. Biophys. Acta 1338, 295–306. Smith, S.K., Pettit, D., Newlands, G.F.J., Redmond, D.L., Skuce, P.J., Knox, D.P., Smith, W.D., 1999. Further immunisation and biochemical studies with a protective antigen complex from the microvillar membrane of the intestine of Haemonchus contortus. Parasitol. Immunol. 21, 187–199. Smith, W.D., Angus, K.W., 1980. Haemonchus contortus: attempts to immunise lambs with irradiated larvae. Res. Vet. Sci. 29, 45–50. Smith, W.D., Smith, S.K., Pettit, D., 2000a. Evaluation of immunization with gut membrane glycoproteins of Ostertagia ostertagi against homologous challenge in calves and against Haemonchus contortus in sheep. Parasitol. Immunol. 22, 239–247. Smith, W.D., Smith, S.K., Pettit, D., Newlands, G.F., Skuce, P.J., 2000b. Relative protective properties of three membrane glycoprotein fractions from Haemonchus contortus. Parasitol. Immunol. 22, 63–71. Smith, W.D., Smith, S.K., Pettit, D., 2001a. Cross protection studies with gut membrane glycoprotein antigens from Haemonchus contortus and Teladorsagia circumcincta. Parasitol. Immunol. 23, 203–211. Smith, W.D., van Wyk, J.A., van Strijp, M.F., 2001b. Preliminary observations on the potential of gut membrane proteins of Haemonchus contortus as vaccine antigens in sheep grazing naturally infected pasture. Vet. Parasitol. 98, 285–297. Tavernor, A.S., Smith, T.S., Langford, C.F., Munn, E.A., Graham, M., 1992. Vaccination of Dorset lambs against haemochosis. Parasite Immunol. 14, 645–655. Urquhart, G.M., Jarrett, W.F.H., Jennings, F.N., MacIntyre, W.I.M., Mulligan, W., 1966. Immunity to Haemonchus contortus infection. Relationship between age and successful vaccination with irradiated larvae. Am. J. Vet. Res. 27, 1645–1648. Vervelde, L., 2000. Development of a vaccine against Haemonchus contortus in sheep. Res. Vet. Sci. 68 (Suppl. A), 24.
Vaccination against gastrointestinal nematode parasites of ruminants using gut-expressed antigens D.P. Knox∗ , W.D. Smith Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, Scotland EH26 0PZ, UK
Abstract To date, proteins isolated from the surface of the gut of gastrointestinal nematodes, particularly Haemonchus contortus, have generally proved to be useful protective antigens and several are being progressed towards recombinant protein-based vaccines. This paper describes the properties of some of the most promising antigens and summarises their performance in laboratory and field based trials. The antigens described include contortin, H11, H-gal-GP, GPI and cysteine proteinases. In addition, the discussion addresses the utility of selected antigens to protect against co-infecting nematode species such as Teladorsagia circumcincta and against related nematode infections such as Ostertagia ostertagi in cattle. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Vaccination; Gastrointestinal nematodes; Ruminants; Gut antigens
1. Introduction Anthelmintic resistance in nematode populations and concerns about the effects of drug residues on consumer health and the environment have focussed attention on the prospect of developing effective anti-nematode vaccines. These studies focus on characterising the host anti-parasite immune response, the nature of the protective components of the response, the parasite life-cycle stage targeted and, ultimately, with the aim of vaccine development, identifying the antigens responsible for protection. Vaccine development based on antigens that stimulate host immune responses during the course of infection is not straightforward. Partly so because no single component of this multifaceted response has been identified which mediates protective immunity and partly because, even if such a component were identified, the ability to induce it in the appropriate amount and location (i.e. the gastrointestinal mucosa) is currently lacking. An alternative approach is to ignore the natural host ∗ Corresponding author. Fax.: +44-131-4456111. E-mail address: [email protected] (D.P. Knox).
0304-4017/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 4 8 0 - 0
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immune response and instead direct a response against parasite proteins which are essential to parasite survival and which are accessible to antibody such as excretory/secretory, cuticular or, in the case of blood-feeding species, gut-expressed proteins. This paper outlines the progress on vaccination against nematode parasites based on proteins isolated from the microvillar surface of the parasite enterocyte.
2. The nematode gut The nematode gut comprises, from anterior to posterior, a buccal capsule, a muscular pharynx, a fairly straight intestine and, finally, a rectum or cloaca. The pharynx and cloaca are, in part, lined with cuticle. The intestine is a syncytial or oligocytous structure with two cells in any circumference containing a lumen lined with a brush-border of millions of microvilli (Bird, 1971; Munn and Greenwood, 1984). In trichostrongylid nematodes, there is a fibrous sub-microvillar layer, known as the endotube, to which the filamentous cores of the microvilli attach (Munn, 1977) and this layer, together with the microvilli, can be dissected from the remainder of the intestine in most species (Munn and Greenwood, 1984).
3. Brush-border associated proteins from the intestinal cells of Haemonchus contortus Several proteins have now been characterised from the microvillar surface of Haemonchus. Since this parasite is a voracious blood feeder, these proteins are exposed to host immunoglobulin in the blood meal. Most, but not all, of these proteins have been found to be protective when used to immunise sheep against challenge infection with the parasite. What follows is an outline of the salient characteristics of these proteins together with a description of their protective antigen capability. 3.1. Contortin The microvillar surface is coated with a layer of electron dense amorphous material (glycocalyx). In Haemonchus contortus there are helical filaments, composed of contortin associated with this layer, which fill the spaces between the microvilli. Contortin is not attached to the plasma membrane and some lies free in the lumen of the intestine and is also present in large amounts in the pharynx although this may not be the case in vivo (Munn, 1977). Contortin, which can be purified from phosphate-buffered saline extracts of adult parasites by ultra-centrifugation, is composed of subunits with an apparent molecular weight of 60 kDa as judged by SDS-PAGE. Lambs vaccinated with a contortin-enriched preparation and subsequently challenged with Haemonchus infective larvae showed a mean reduction in worm burden of 78% (Munn et al., 1987). This result was particularly significant because it was the first to show that proteins expressed on the surface of the gut of a blood-feeding nematode, could induce high levels of protective immunity when used as an immunogen. Intestinal brush-border proteins are not normally accessible to the host immune system, even following repeated infection (Smith,
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1993), hence they are termed hidden or concealed antigens and the immunity conferred by them is described as artificial immunity. 3.2. H11 Contortin-enriched preparations contained a 110 kDa contaminant as judged by Western blotting despite only faint staining being evident in protein gels (Smith and Munn, 1990). This protein was purified using concanavalin A lectin-affinity chromatography combined with, on some occasions, Mono Q anion-exchange chromatography and it migrated as a doublet at 110 kDa as judged by SDS-PAGE, hence the short-hand designation, H11. It is the most effective immunogen isolated from a parasitic nematode to date, inducing high levels of protection (>90% reductions in worm burdens) which closely correlate with serum antibody titre (Smith et al., 1993). The immune response in sheep primed by immunisation with H11 can be boosted following challenge, presumably by H11 released from dead or dying parasites (Andrews et al., 1997), suggesting that this glycoprotein may not in fact be a completely hidden antigen. H11 is an integral membrane glycoprotein only expressed on the intestinal microvilli of the parasitic stages and homologues have been identified in Teladorsagia circumcincta and Ostertagia ostertagi (McMichael-Philips et al., 1995; Smith et al., 2000a, 2001). It shows microsomal aminopeptidase A and M activities and is encoded by at least three genes (Graham et al., 1993; McMichael-Philips et al., 1995; Smith et al., 1997). H11 has the predicted structure of a type II integral membrane protein with a short N-terminal cytoplasmic tail, a transmembrane region and an extracellular region organised into four domains (Smith et al., 1997; Newton and Munn, 1999). Enzyme activity is localised exclusively to the intestinal brush-border and is inhibited by H11 antisera in vitro (Smith et al., 1997), the level of inhibition observed being correlated to protection (Munn et al., 1997). 3.3. H-gal-GP Smith et al. (1994) used several different techniques, including lectin screening of worm sections and radiolabelling, to target integral membrane glycoprotein on the luminal surface of H. contortus intestinal cells. These proteins were then purified from detergent extracts of whole worms and evaluated in protection trials. One fraction, which selectively bound to lectins with specificity for N-acetylgalactosamine, reduced mean challenge worm burdens by up to 72% and mean faecal egg counts by up to 93%. This fraction was termed Haemonchus galactose-containing glycoprotein complex (H-gal-GP). The microvillar surface of the intestinal cells of worms retrieved from vaccinated lambs was coated with sheep immunoglobulin and protection was correlated with systemic antibody titre (Smith et al., 1999). H-gal-GP can be visualised as a single diffusely staining band after Blue Native PAGE with an estimated molecular weight of about 1000 kDa and resolves as four major protein zones, designated A, B, C and D, at ∼230, 170, 45 and <35 kDa, respectively, under non-reducing SDS-PAGE (Smith et al., 1999). Each zone resolved as more than one band after reduction. Biochemical analyses indicated that H-gal-GP exhibited aspartyl-,
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metallo- and, on occasion, cysteine protease activities. Initial N-terminal sequence (Smith et al., 1999) and cDNA analysis showed that the complex contained an aspartyl protease with homology to pepsin (Longbottom et al., 1997) and a metalloprotease (MEP, Redmond et al., 1997). Further molecular analysis has now shown that H-gal-GP contains at least 4 distinct MEPs with 38–54% similarity at the amino acid level to each other, these proteases being associated with the prominent 230 (MEP 1, 2 & 4) and 170 kDa (MEP 3) protein zones (Smith et al., 1999). It also contains a thrombospondin homologue, which migrates between the 170 and 230 kDa zones, this protein being related to a variety of “adhesin-type” proteins (Skuce et al., unpublished). The pepsin is the major component of the 45 kDa zone, a galectin is associated with the 35 kDa zone (Newlands et al., 2001) and a low molecular weight (∼13 kDa) inhibitor of Haemonchus cysteine proteases, cystatin has also been identified (Newlands et al., in press). H-gal-GP has proved resistant to fractionation using a variety of chromatographic techniques under native conditions. However, a closely related, somewhat simpler complex has been isolated using jacalin lectin. Unlike peanut lectin, jacalin can bind glycoproteins with sialylated forms of  1–3 N-acetylgalactosamine; a feature that resulted in this newer complex being designated Haemonchus sialylated galactose-containing glycoprotein complex (H-sialgal-GP). H-sialgal-GP is just as protective against Haemonchus as H-gal-GP (Smith et al., 2000a). The complexes appear identical except that the 230 kDa zone is missing in H-sialgal-GP, suggesting that MEPs 1, 2 and 4 are not essential for protection (Smith et al., 2000a). The precise function of these complexes remains undefined, but strong haemoglobinase activity associated with the aspartyl protease suggests that they play a role in digestion of the blood meal.
3.4. p46 and p52 Jacalin lectin, in combination with ion exchange and gel filtration chromatography, has also been used to isolate a pair of protective glycoproteins of 46 and 52 kDa from Haemonchus membranes. In combination these proteins reduced the egg output and worm count of immunised lambs by 78 and 33%, respectively (Smith et al., 2000b). The p46 molecule had been described earlier as a 45 kDa glycoprotein that protected guinea pigs against Haemonchus (Sharp et al., 1992). N-terminal sequence analysis of p46 revealed significant homology with that predicted from a cDNA fragment which encoded the 45 kDa protein of Sharp et al. (1992). Furthermore, the N-terminal data from p52 was almost identical to the predicted product of a second, closely related cDNA fragment (Sharp et al., 1992), now known to be part of a recently isolated full length gene, designated Hc40 (Rehman and Jasmer, 1998). Hc40 would be predicted to encode a protein of 51 kDa, in near agreement with the estimated mass of p52. The sequence of Hc40 suggests that the N and C terminal halves of p52 are closely related, with many motifs repeated in each half (Rehman and Jasmer, 1998). Intriguingly, many of these are also common to ES 24, a natural antigen discovered in Haemonchus excretory–secretory products which, in combination with a 15 kDa molecule, is also highly protective when used to vaccinate sheep (Schallig et al., 1997).
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3.5. Cysteine protease enriched extracts Extracts from adult H. contortus and excretory/secretory harvested by in vitro maintenance of adult parasites contain several strong cysteine protease activities with the capacity to degrade blood proteins such as haemoglobin and albumin, major constituents of the blood meal (Knox et al., 1993; Rhoads and Fetterer, 1995, 1997). Water-soluble extracts of adult H. contortus were shown to contain a 35 kDa cysteine protease which degraded fibrinogen and increased clotting time in sheep plasma in vitro (Cox et al., 1990a). Lambs immunised with extracts from the adult parasite enriched for this activity were claimed to be protected against challenge infection (Boisvenue et al., 1992) despite unconvincing raw data from two trials involving small numbers of sheep (Cox et al., 1990b). The cDNA (AC1) encoding this protease was isolated and showed 42% amino acid sequence identity to human cathepsin B. A cysteine protease enriched fraction (TSBP) was prepared from membrane extracts of adult H. contortus by passage over a Thiol-Sepharose affinity column and the proteins obtained were clearly localised to the microvillar surface of the intestinal cells (Knox et al., 1995, 1999). Lambs immunised with TSBP were substantially protected against a single challenge infection with H. contortus with reductions in daily faecal egg outputs of 77% and final worm burdens of 47% over three trials. Again, the microvillar surface of worms surviving in vaccinated lambs was coated with sheep immunoglobulin and recent experiments in our laboratory show that antibody harvested from vaccinated lambs functionally inhibits the cysteine protease components of TSBP (Knox, unpublished). Interestingly, TSBPs prepared in the same way from water-soluble parasite extracts were ineffective immunogens. TSBP comprised a prominent 60 kDa protein and several minor bands between 35 and 45 kDa and 97–120 kDa. Protease activity was mostly attributable to cysteine proteases although serine/metallo proteases were also identified (Knox et al., 1999). Lectinbinding studies showed that most of the TSBP proteins were glycosylated. Expression library immunoscreening revealed that the prominent 60 kDa component of TSBP is a glutamate dehydrogenase (GDH) homologue (Skuce et al., 1999a) and that the cysteine protease activity can be attributed to the protein products of three distinct genes, cDNAs derived from which have been designated hmcp1, 4 and 6 (Skuce et al., 1999b). Like AC1, these cDNAs showed closest homology to mammalian cathepsin B cysteine proteases. The expression of the GDH and cysteine protease encoding genes coincides with the onset of blood-feeding and immunolocalisation studies showed that the former was expressed in the cytoplasm of the intestinal cells (Skuce et al., 1999a) while the latter was expressed on the microvillar surface (Skuce et al., 1999b). This would argue against the GDH being the protective component of TSBP as it would be inaccessible to antibody and this, is indeed the case (Skuce et al., 1999a). TSBP can be further fractionated by anion exchange chromatography into a fraction which contains the bulk of the GDH protein and activity, fractions which contain the cysteine proteases and very little GDH and a protein fraction which does not bind to the column (Knox et al., 1995). Lambs immunised with the GDH fraction, or the unbound protein fraction, were completely unprotected against
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challenge whilst lambs immunised with the pooled protease-containing fractions were protected against challenge to the same degree as positive controls which received unfractionated TSBP (Knox et al., 1995), supporting the view that protection is attributable to the cysteine proteases. 3.6. GA1 and P1 antigens Monoclonal antibodies were used to identify and purify a group of proteins, Mr 46, 52 and 100 kDa, which, collectively, induced reductions of 60 and 50% in worm and faecal egg outputs, respectively, in immunised goats (Jasmer et al., 1993). N-terminal protein sequence analyses of the three proteins, termed p46GA1 , p52GA1 and p100GA1 , and cDNA library immunoscreening showed that all three were encoded by the same GA1 gene and are initially expressed as a polyprotein (p100GA1 , Jasmer et al., 1996). Western blot analysis indicated that the GA1 proteins were expressed in adult worms but not in infective larvae, a result essentially confirmed by Northern blot analysis. The GA1 gene product showed closest homology with bacterial Tolb proteins that are associated with the bacterial membrane and periplasm and may be involved in transport. GA1 derived proteins were detected in ES products from adult worms and host abomasal mucus indicative of release from the microvillar surface. Following on from this, protective immunity stimulated by immunisation with these proteins may involve anamnestic and mucosal immune responses. This suggestion was supported in a later study (Karanu et al., 1997) which provided evidence for a contribution from CD4+ lymphocytes to gut antigen-induced immunity. Smith et al. (1993) identified a group of three peptides (P1) which were separated from H11 by ion-exchange chromatography. The constituent peptides (45, 49 and 53 kDa) showed some similarities to the GA1 proteins and induced mean reductions of 69% in faecal egg output and 38 and 20% reductions in female and male worm numbers, respectively (Smith et al., 1993). The proteins, designated p45, p49 and p53, form a complex (p150) which is a ubiquitous constituent of the microvillar membrane of the intestinal cells. p53 has a membrane anchor and associates non-covalently with a disulphide bridged dimer of p45 and p49. All three components share peptide epitopes (Rocha and Munn, 1997).
4. Protective antigen capacity of intestinal brush-border proteins for other helminth species 4.1. Cross-protective capacity of Haemonchus proteins Compared to Haemonchus, much less is known about the gut membrane proteins of the other important helminth parasites of livestock. Although, T. circumcincta possesses antigenically cross-reactive homologues of H11 and H-gal-GP (McMichael-Philips et al., 1995; Smith et al., 2001a), immunisation of sheep with a mixture of H11 and H-gal-GP did not provide any cross-protection (Smith et al., 2001a). Nor was any cross-protection recorded against Trichostrongylus axei or Cooperia oncophora (Smith et al., 2001a), endorsing
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earlier findings with Nematodirus battus, when detergent extracts of Haemonchus intestines were employed as antigen (Smith, 1993). 4.2. Protective capacity of homologous gut membrane proteins Such trials have provided mixed results. The most promising was from Fasciola hepatica, where 89% fewer flukes were recently reported from sheep immunised with aminopeptidases purified from the parasite (Piacenza et al., 1999), a finding which needs to be repeated and extended. An earlier report stated that vaccination with homologous aminopeptidases induced protection in pigs challenged with Ascaris suum (50% reduction in larval counts, Ferguson et al., 1969). Results from only two of the ruminant trichostrongyle species have been published, namely O. ostertagi and T. circumcincta. Calves immunised with a combination of H11 and H-gal-GP homologues from O. ostertagi showed 30–50% reduction in egg counts, but there was no effect on worm numbers and neither antigen protected when administered on its own (Smith et al., 2000a). With T. circumcincta, lambs vaccinated with homologous TSBP were highly protected (71 and 65% reduction in eggs and worms, respectively; Knox et al. (1995)), but this could not be reproduced in subsequent trials. Furthermore, lambs immunised with fractions of T. circumcincta containing a mixture of its H11 and H-gal-GP homologues were not protected at all (Smith et al., 2001a). Despite this, the same T. circumcincta and O. ostertagi preparations did cross-protect sheep against a Haemonchus challenge (Smith et al., 2000a, 2001), indicating that neither immunogen was defective, and suggesting that these species do not ingest sufficient antibody for the gut antigen approach to vaccination to be appropriate.
5. Glycosylation, conformation and protective efficacy of Haemonchus brush-border proteins With the possible exception of contortin, all of the proteins described above are known to be, or predicted from gene sequence data to be, glycosylated. Because several (H11, H-gal-GP, P1, p46/42 and GA1) can be separated from each other by lectin-affinity chromatography, their sugar residues must differ. The nature of the N-linked oligosaccharides of glycoproteins in detergent extracts of adult Haemonchus and of H11 specifically has been investigated in detail (Haslam et al., 1996). These experiments identified a core fucosylation of a type not previously observed in eukaryotic glycoproteins. The major N-linked glycans had up to three fucose residues attached to chitobiose cores. These were found at the 3- and/or 6-positions of the proximal N-acetylglucosamine (GlcNAc) and at the 3-position of the distal GlcNAc. The latter substitution was stated to be unique in N-glycans (Haslam et al., 1996). It was proposed that these multifucosylated core structures could be highly immunogenic. Protection is reduced when either H11 or H-gal-GP is progressively denatured by treatment with SDS alone or SDS+ dithiothreitol (Munn et al., 1997; Smith and Smith, 1996), suggesting that conformational epitopes are essential for the full expression of protective immunity.
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6. Expression of Haemonchus protective antigens by recombinant DNA technology The cDNAs encoding H11 as well as most of the components of H-gal-GP and TSBP have all been expressed in E. coli and, to date, none of these recombinant proteins have been reported to be protective. These findings tend to confirm the need for conformational epitopes suggested by the dissociation and reduction experiments described above. However, enzymatically active recombinant protein versions of H11 and a cysteine protease component of TSBP have been expressed in the baculovirus-Sf9 insect cell system (Smith et al., 1997) and the yeast Pichia pastoris, respectively (Skuce et al., unpublished). The fact that these proteins show enzymatic activity suggests that they are folded correctly and the outcome of protection trials is awaited. Moreover, recent work has demonstrated the feasibility of expressing heterologous nematode gut-expressed genes in the free-living nematode Caenorhabditis elegans in a tissue-specific manner, this work being conducted with the gene encoding the pepsin component of H-gal-GP (Redmond et al., in press). In theory, the post-translational processing in C. elegans should be very close to that found in parasitic nematodes. Given that C. elegans can be cultivated in large-scale cultures, this may provide the best method for the large-scale production of nematode glycoprotein antigens.
7. Practical considerations of vaccination against Haemonchus with gut membrane proteins 7.1. Laboratory trials Many trials using worm-free, housed sheep have shown that H11 and H-gal-GP are effective antigens, stimulating high levels of protection single a challenge infection of Haemonchus after administration of two or three injections with QuilA, a commercially acceptable adjuvant (Newton and Munn, 1999; Smith et al., 2000a,b). Both were also effective in young lambs (Tavernor et al., 1992; Smith et al., 1994), demonstrating that the hidden antigen approach to vaccination was not restricted by the age-related unresponsiveness phenomenon. Such unresponsiveness prevented the development of irradiated larval vaccines for Haemonchus (Urquhart et al., 1966; Smith and Angus, 1980) and could also prove to be problematic with the natural antigen approach to vaccination (Vervelde, 2000). Worms begin to be shed from immunised lambs between 7 and 14 days after challenge soon after the onset of blood-feeding (Smith and Smith, 1993). A consistent trend after vaccination with H11, H-gal-GP or TSBP is that the female worms are more susceptible than the males, possibly reflecting higher metabolic activity associated with egg production. Protection afforded by vaccination with H11 in Freunds adjuvant persisted for at least 23 weeks after vaccination (Andrews et al., 1997) and did not interfere with the development of acquired immunity (Smith and Smith, 1993). Immunised lambs given a trickle infection were largely protected against the anaemia normally associated with infection and grew as fast and as efficiently as uninfected lambs (Smith and Smith, 1993). Vaccination with H11 reduced worm egg output (98% reduction) from pregnant ewes challenged with 10,000 H. contortus L3 during the third trimester (Andrews et al., 1995), almost eliminating the increase in
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egg output normally associated with the peri-parturient relaxation of immunity. In addition, lambs born and reared on vaccinated ewes had high antibody titres to H11 as a result of colostral transfer and this antibody conferred moderate protection against a single challenge infection of 3000 infective larvae (Andrews et al., 1995). This latter result indicated that protection was antibody mediated, a conclusion supported by serum transfer experiments (Smith, 1993) and observations that antisera from vaccinated lambs inhibited the microsomal aminopeptidase activity and that the degree of inhibition was highly correlated to the level of protection obtained (Munn et al., 1997). 7.2. Field trials Sterner, more realistic tests of potential vaccine efficacy can only be obtained from field trials where sheep grazing Haemonchus contaminated pasture may be continuously or sporadically exposed to variable dose rates of infective larvae depending on the prevailing climate. Several trials have recently been completed using a combination of native H11 and H-gal-GP as antigen formulated with QuilA. Most of these experiments were conducted in Louisiana, USA in the summer, a fairly extreme test because the warm humid conditions were highly favourable to the development of Haemonchus (Bahirathan et al., 1996), providing a heavy and continuous larval exposure. One trial, was complicated by the fact a proportion of the ewes had already acquired natural immunity, nevertheless, immunisation did reduce the egg counts of those deemed to be susceptible by 65% (Kabagambe et al., 2000). However, further trials with lambs indicated that these antigens were unable to control the infection before the animals succumbed to potentially fatal anaemia (Miller and Smith, unpublished data). Another 11-month long experiment was conducted in yearling sheep, near Pretoria, South Africa where the relatively dry climate with sporadic, seasonal rain ensured a discontinuous challenge of parasites more normal to conditions encountered by commercial sheep farmers in the tropics and sub-tropics. Here, vaccination reduced egg output by more than 82% on average during one 4-month period of the trial and simultaneously reduced the degree of anaemia and deaths due to haemonchosis (Smith et al., 2001b). Although vaccine immunity was not sufficiently long lasting to prevent a surge in egg output which occurred after the onset of a period of irrigation, re-vaccinating the sheep at this point cleared their newly acquired infection and rapidly restored protection to approximately the level observed beforehand. It was clear that in this situation a vaccine based on parasite gut membrane proteins could offer substantial benefits in the control of natural haemonchosis.
8. Concluding remarks Obviously the biggest single barrier to the commercialisation of a vaccine against haemonchosis is the production of recombinant proteins which approach the efficacy of the best native antigens. To date, recombinant proteins expressed in E. coli have not induced significant protective immunity but work is progressing in a variety of expression systems to overcome this problem. It will be disappointing if these difficulties cannot be overcome in the short-term.
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In addition, further work is required to determine whether other adjuvant formulations can give longer lasting responses than QuilA. More field trials to optimise when and how often such a vaccine would be administered are needed, bearing in mind the age and reproductive status of the sheep as well as the likely level of parasite exposure, before the potential benefits and limitations of a vaccine can be defined. Gut antigen-based vaccines (based on the antigens described above, at least) and mediated by high systemic antibody responses, are unlikely to be useful against non-blood-feeding nematodes. Different antigens may be required and may need to be delivered in such a way as to stimulate specific local mucosal immune responses. This will require continued efforts to define the key protective immune responses, to define the parasite antigens to which these responses are directed and to develop the means to deliver antigen to the host immune system in a manner to stimulate the correct response. New technologies will play a part. Already, the possibility of DNA vaccination is being explored, a topic which has recently been the subject of a comprehensive review (Alarcon et al., 1999). However, such an approach is quite likely to be met with consumer hostility towards the consumption of meat products contaminated with foreign DNA. We will be in a position to exploit genome-sequencing initiatives that should enable micro-array analysis of gene expression in parasite and host. These analyses should permit a very detailed analysis of the host–parasite interaction at the molecular level and allow us to target parasite gene-products associated with key events in the life-cycle such as the switch to the next developmental stage and moulting. In the future, it may be possible to generate a virulent mutant worms by targeted gene knock-out. The latter is already being applied extensively in C. elegans and veterinary parasitologists have much to learn from these experiments. References Alarcon, J.B., Waine, G.W., McManus, D.P., 1999. DNA vaccines: technology and application as anti-parasite and anti-microbial agents. Adv. Parasitol. 42, 344–410. Andrews, S.J., Hole, N.J.K., Munn, E.A., Rolph, T.P., 1995. Vaccination of sheep against haemonchosis with H11-prevention of the periparturient rise and colostral transfer of protective immunity. Int. J. Parasitol. 25, 839–846. Andrews, S.J., Rolph, T.P., Munn, E.A., 1997. Duration of protective immunity against ovine haemonchosis following vaccination with the nematode gut membrane antigen H11. Res. Vet. Sci. 62, 223–227. Bahirathan, M., Miller, J.E., Barras, S.R., Kearney, M.T., 1996. Susceptibility of Suffolk and Gulf Coast Native suckling lambs to naturally acquired strongylate nematode infection. Vet. Parasitol. 65, 259–268. Bird, A.F., 1971. The Structure of Nematodes. Academic Press, London. Boisvenue, R.J., Stiff, M.I., Tonkinson, L.V., Cox, G.N., Hageman, R., 1992. Fibrinogen-degrading proteins from Haemonchus contortus used to vaccinate sheep. Am. J. Vet. Res. 53, 1263–1265. Cox, G.N., Pratt, D., Hageman, R., Boisvenue, R.J., 1990a. Molecular cloning and sequencing of a cysteine protease expressed by Haemonchus contortus adult worms. Mol. Biochem. Parasitol. 41, 25–34. Cox, G.N., Pratt, D., Hageman, R., 1990b. Anticoagulant and anthelmintic proteins and methods for the production and use of the same. European Patent Application no. 90117879.8 Ferguson, D.L., Rhodes, M.B., Marsh, C.L., Payne, L.C., 1969. Resistance of immunised animals to infection by the larvae of the large roundworm of swine (Ascaris suum). Fed. Proc. 28, 497. Graham, M., Smith, T.S., Munn, E.A., Newton, S.E., 1993. Recombinant DNA molecules encoding aminopeptidase enzymes and their use in the preparation of vaccines against helminth infections. Patent no. WO 93/23542. Haslam, S.M., Coles, G.C., Munn, E.A., Smith, T.S., Smith, H.F., Morris, H.R., Dell, A., 1996. Haemonchus contortus glycoproteins contain oligosaccharides with novel highly fucosylated core structures. J. Biol. Chem. 271, 30561–30570.
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Jasmer, D.P., Perryman, L.P., Conder, G.A., Crow, S., McGuire, T.C., 1993. Protective immunity to Haemonchus contortus induced by immunoaffinity isolated antigens that share a phylogenetically conserved carbohydrate gut surface epitope. J. Immunol. 151, 5450–5460. Jasmer, D.P., Perryman, L.P., McGuire, T.C., 1996. Haemonchus contortus GA1 antigens: related phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection. Proc. Natl. Acad. Sci. U.S.A. 93, 8642–8647. Kabagambe, E.K., Barras, S.R., Li, Y., Pena, M.T., Smith, W.D., Miller, J.E., 2000. Attempts to control haemonchosis in grazing ewes by vaccination with gut membrane proteins of the parasite. Vet. Parasitol. 92, 15–23. Karanu, F.N., McGuire, T.C., Davis, W.C., Besser, T.E., Jasmer, D.P., 1997. CD4+ T lymphocytes contribute to protective immunity induced in sheep and goats by Haemonchus contortus gut antigens. Parasitol. Immunol. 19, 435–445. Knox, D.P., Redmond, D.L., Jones, D.G., 1993. Characterisation of proteases in extracts of adult Haemonchus contortus, the ovine abomasal nematode. Parasitology 106, 395–404. Knox, D.P., Smith, S.K., Smith, W.D., Redmond, D.L., Murray, J.M., 1995. Thiol Binding Proteins. Patent Application no. PCT/GB95/00665. Knox, D.P., Smith, S.K., Smith, W.D., 1999. Immunization with an affinity purified protein extract from the adult parasite protects lambs against Haemonchus contortus. Parasitol. Immunol. 21, 201–210. Longbottom, D., Redmond, D.L., Russell, M., Liddell, S., Smith, W.D., Knox, D.P., 1997. Molecular cloning and characterisation of an aspartate protease associated with a highly protective gut membrane protein complex from adult Haemonchus contortus. Mol. Biochem. Parasitol. 88, 63–72. McMichael-Philips, D., Munn, E.A., Graham, M., 1995. Helminth parasite antigen with aminopeptidase-like activity. International patent application. WO 95/12671. Munn, E.A., 1977. A helical, polymeric extracellular protein associated with the luminal surface of Haemonchus contortus intestinal cells. Tissue Cell 9, 23–34. Munn, E.A., Greenwood, C.A., 1984. The occurrence of submicrovillar endotube (modified terminal web) and associated cytoskeletal structures in the inestinal epithelia of nematodes. Phil. Trans. R. Soc. London B 306, 1–18. Munn, E.A., Graham, M., Coadwell, W.J., 1987. Vaccination of young lambs by means of a protein fraction extracted from adult Haemonchus contortus. Parasitology 94, 385–397. Munn, E.A., Smith, T.S., Smith, H., Smith, F., Andrews, S.J., 1997. Vaccination against Haemonchus contortus with denatured forms of the protective antigen H11. Parasitol. Immunol. 19, 243–248. Newlands, G.F.J., Skuce, P.J., Knox, D.P., Smith, S.K., Smith, W.D., 1999. Cloning and characterisation of a -galactoside-binding protein (galectin) from the gastrointestinal nematode Haemonchus contortus. Parasitology 119, 483–490. Newlands, G.F.J., Skuce, P., Knox, D.P., Smith, W.D., 2001. Cloning and expression of cystatin, a potent cysteine protease inhibitor from the gut of Haemonchus contortus. Parasitology 122, 371–378. Newton, S.E., Munn, E.A., 1999. The development of vaccines against gastrointestinal nematodes, particularly Haemonchus contortus. Parasitol. Today 15, 116–122. Piacenza, L., Acosta, D., Basmadjan, I., Dalton, J.P., Carmona, C., 1999. Vaccination with cathepsin L proteases and with leucine aminopeptidase induces high levels of protection against fascioliasis in sheep. Infect. Immunnol. 67, 1954–1961. Redmond, D.L., Knox, D.P., Newlands, G.F.J., Smith, W.D., 1997. Molecular cloning of a developmentally regulated putative metallo-peptidase present in a host protective extract of Haemonchus contortus. Mol. Biochem. Parasitol. 85, 77–87. Redmond, D.L., Clucas, C., Johnstone, I., Knox, D.P., 2001. Expression of Haemonchus contortus pepsinogen in Caenorhabditis elegans. Mol. Biochem. Parasitol. 112, 125–131. Rehman, A., Jasmer, D.P., 1998. A tissue specific approach for analysis of membrane and secreted protein antigens from Haemonchus contortus gut and its application to diverse nematode species. Mol. Biochem. Parasitol. 97, 55–68. Rhoads, M.L., Fetterer, R.H., 1995. Developmentally regulated secretion of cathepsin L-like proteases by Haemonchus contortus. J. Parasitol. 81, 505–512. Rhoads, M.L., Fetterer, R.H., 1997. Extracellular matrix: a toll for defining extracorporeal digestion of parasite proteases. Parasitol. Today 13, 119–122.
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Rocha, J., Munn, E.A., 1997. p150, a protective glycoprotein complex of the microvillar membrane of Haemonchus contortus. Conference Abstract from Parasitic Helminths — from Genomes to Vaccines, Edinburgh, UK, 6–9 September 1997. Schallig, H.D.F., Van Leeuwen, M.A., Verstrepen, B.E., Cornelissen, A.W., 1997. Molecular characterization and expression of two putative protective excretory secretory proteins of Haemonchus contortus. Mol. Biochem. Parasitol. 88, 202–213. Sharp, P.J., Wagland, B.M., Cobon, G.S., 1992. Nematode vaccine. International Patent WO 92/13889. Skuce, P.J., Stewart, E.M., Smith, W.D., Knox, D.P., 1999a. Cloning and characterisation of glutamate dehydrogenase (GDH) from the gut of Haemonchus contortus. Parasitology 118, 297–304. Skuce, P.J., Redmond, D.L., Liddell, S., Stewart, E.M., Newlands, G.F.J., Smith, W.D., Knox, D.P., 1999b. Molecular cloning and characterisation of gut-derived cysteine proteases associated with a host-protective extract from Haemonchus contortus. Parasitology 119, 396–405. Smith, W.D., 1993. Protection in lambs immunised with Haemonchus contortus gut membrane proteins. Res. Vet. Sci. 54, 94–101. Smith, T.S., Munn, E.A., 1990. Strategies for vaccination against gastro-intestinal nematodes. Rev. Sci. Tech. Off. Int. Epizooit. 9, 577–595. Smith, W.D., Smith, S.K., 1993. Evaluation of aspects of the protection afforded to sheep immunised with a gut membrane protein from Haemonchus contortus. Res. Vet. Sci. 55, 1–9. Smith, S.K., Smith, W.D., 1996. Immunisation of sheep with an integral membrane glycoprotein complex of Haemonchus contortus and its major polypeptide components. Res. Vet. Sci. 60, 1–6. Smith, T.S., Munn, E.A., Graham, M., Tavernor, A., Greenwood, C.A., 1993. Purification and evaluation of the integral membrane protein H11 as a protective antigen against Haemonchus contortus. Int. J. Parasitol. 23, 271–277. Smith, W.D., Smith, S.K., Murray, J.M., 1994. Protection studies with integral membrane fractions of Haemonchus contortus. Parasitol. Immunol. 16, 231–241. Smith, T.S., Graham, M., Munn, E.A., Newton, S.E., Knox, D.P., Coadwell, W.J., McMichael-Phillips, D., Smith, H., Smith, W.D., Oliver, J.J., 1997. Cloning and characterisation of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus. Biochim. Biophys. Acta 1338, 295–306. Smith, S.K., Pettit, D., Newlands, G.F.J., Redmond, D.L., Skuce, P.J., Knox, D.P., Smith, W.D., 1999. Further immunisation and biochemical studies with a protective antigen complex from the microvillar membrane of the intestine of Haemonchus contortus. Parasitol. Immunol. 21, 187–199. Smith, W.D., Angus, K.W., 1980. Haemonchus contortus: attempts to immunise lambs with irradiated larvae. Res. Vet. Sci. 29, 45–50. Smith, W.D., Smith, S.K., Pettit, D., 2000a. Evaluation of immunization with gut membrane glycoproteins of Ostertagia ostertagi against homologous challenge in calves and against Haemonchus contortus in sheep. Parasitol. Immunol. 22, 239–247. Smith, W.D., Smith, S.K., Pettit, D., Newlands, G.F., Skuce, P.J., 2000b. Relative protective properties of three membrane glycoprotein fractions from Haemonchus contortus. Parasitol. Immunol. 22, 63–71. Smith, W.D., Smith, S.K., Pettit, D., 2001a. Cross protection studies with gut membrane glycoprotein antigens from Haemonchus contortus and Teladorsagia circumcincta. Parasitol. Immunol. 23, 203–211. Smith, W.D., van Wyk, J.A., van Strijp, M.F., 2001b. Preliminary observations on the potential of gut membrane proteins of Haemonchus contortus as vaccine antigens in sheep grazing naturally infected pasture. Vet. Parasitol. 98, 285–297. Tavernor, A.S., Smith, T.S., Langford, C.F., Munn, E.A., Graham, M., 1992. Vaccination of Dorset lambs against haemochosis. Parasite Immunol. 14, 645–655. Urquhart, G.M., Jarrett, W.F.H., Jennings, F.N., MacIntyre, W.I.M., Mulligan, W., 1966. Immunity to Haemonchus contortus infection. Relationship between age and successful vaccination with irradiated larvae. Am. J. Vet. Res. 27, 1645–1648. Vervelde, L., 2000. Development of a vaccine against Haemonchus contortus in sheep. Res. Vet. Sci. 68 (Suppl. A), 24.