Trials with the Haemonchus vaccine, Barbervax®, in ewes and lambs in a tropical environment: Nutrient supplementation improves protection in periparturient ewes.

Haemonchus contortus is an economic problem in sheep farms worldwide, mainly in the tropics and subtropics. A vaccine against haemonchosis, called Barbervax®, was evaluated in ewes under two nutritional status, naturally infected with gastrointestinal nematodes. Ewes were divided into four groups: Supplemented Diet - Vaccine; Supplemented Diet - No vaccine; Basal Diet - Vaccine and Basal Diet - No vaccine. Their lambs were divided in Vaccinated and No vaccine. Ewes were immunised six times starting about 1 month of pregnancy with the first three doses at 3 week intervals and the last three shots at 4 week intervals. Supplemented ewes had higher body weight, body score and packed cell volume compared with those fed a basal diet. Both groups of vaccinated ewes showed a similar response in circulating anti-vaccine antibodies but the vaccine had no discernible effect on either body weight, body score and packed cell volume. There was a marked group difference in the number of ewes that received precautionary treatments with anthelmintic. All 14 Basal Diet - No vaccine ewes required treatment. In contrast only 7 ewes, in the Supplemented Diet - Vaccine group required anthelmintic treatment. In the Basal Diet - Vaccine and in the Supplemented Diet - No Vaccine groups, 12 and 13 ewes needed anthelmintic treatment, respectively. Vaccinated lambs showed much higher antibody titres resulting in 80% less Haemonchus spp. egg counts comparing with no vaccine lambs. Taken together these results clearly suggest that in pregnant and lactating ewes a combined protective effect between vaccination and improved nutrition resulted in fewer precautionary anthelmintic treatments. Thus, it was possible to achieve a more sustainable level of control of the haemonchosis, less dependent on anthelmintic drugs.

Attempts to vaccinate ewes and their lambs against natural infection with Haemonchus contortus in a tropical environment.

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of grazing sheep each containing 13 ewes and their 16 lambs naturally infected with gastrointestinal nematodes. Two groups were vaccinated with either 5 or 50µg of the antigen per immunisation, while the third, the control group, received adjuvant alone. The sheep were immunised six times at 3week intervals, partly because the vaccine antigens are hidden and thus no immunological boost would be delivered by subsequent infection and partly because the level of Haemonchus spp. challenge was expected to be high. The vaccinated ewes, first immunised approximately 1month before lambing, showed a circulating antibody response but no signs of reduced anaemia or Haemonchus spp. egg counts, compared with control ewes. Several ewes with severe haemonchosis in all three groups had to be given precautionary treatment with anthelmintic drugs. In contrast, vaccinating their lambs with either 5 or 50µg of the antigen per immunisation resulted in 10 fold higher antibody titres. In the case of the lower antigen dose this was associated with significantly less anaemia, 72% reduction in the overall number of Haemonchus spp. eggs produced and significantly fewer worms compared with control lambs. It is hypothesised that the heavily pregnant or lactating ewes did not have sufficient physiological reserves to mount a protective response following vaccination in the tropical weather and high challenge conditions that prevailed. Nevertheless, the vaccine could afford useful protection for lambs against H. contortus.

Vaccination of grazing calves with antigens from the intestinal membranes of Haemonchus contortus: effects against natural challenge with Haemonchus placei and Haemonchus similis.

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of eight 5 months old grazing calves, naturally infected by Haemonchus similis, Haemonchus placei and other gastrointestinal nematodes. Vaccinated calves received 5 or 50µg of the antigen and 1mg of saponin adjuvant, while the controls received adjuvant alone, initially three times, 3 weeks apart and then four more times at 6 weeks intervals. Three weeks after the last immunisation all of the calves were euthanised for worm counts. Immunisation stimulated high titre antibodies against the vaccine antigens, reduced the egg output of Haemonchus spp. by 85% and the numbers of H. placei and H. similis by 63% and 32%, respectively, compared with control calves. It was concluded that vaccination with intestinal membrane glycoproteins from H. contortus could substantially reduce the transmission of H. placei and H. similis, thus providing protective benefit downstream. This appears to be the first known successful demonstration of a vaccine protective for cattle naturally exposed to infection with any gastrointestinal nematode parasite.

Phage-display library biopanning as a novel approach to identifying nematode vaccine antigens.

Infections with parasitic nematodes are of significant welfare and economic importance worldwide, and because of the emergence of anthelmintic resistance, this has lead to alternative methods of parasite control being required. Vaccination offers a feasible alternative control, and the majority of research has focused on the production of recombinant versions of native antigens previously identified as protective in vaccinated animals. Attempts at the production of protective recombinant subunit vaccines have been hindered, however, as these antigens have invariably failed to replicate the same level of protective immune response as seen with the native versions. It has been proposed that these failures are owing to the fact that the recombinant proteins do not contain the appropriate post-translational modifications to retain the protective capacity of the native molecules. In this review, we discuss a novel approach to vaccine antigen identification through the application of random peptide phage-display libraries and their use to identify peptide sequences that potentially mimic the structure(s) of antigenic epitopes. This area of research is still relatively novel with respect to parasites, and the current state of the art will be discussed here.

Protection of calves against Haemonchus placei and Haemonchus contortus after immunization with gut membrane proteins from H. contortus.

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in four groups of nine worm-free calves challenged with either 8000 H. contortus or Haemonchus placei infective larvae. Vaccinates received 50 µg of the antigen and 1 mg QuilA adjuvant three times 21 days apart, while the controls got adjuvant alone. The calves were challenged 7 days after the last immunization and killed for worm counts 43 days later. Immunization resulted in high titre antibodies against the vaccine antigens and significant reduction in egg output and worm numbers of both challenge species, compared with control calves. It was concluded that vaccination of calves with native parasite gut membrane glycoproteins obtained from H. contortus conferred protection against both H. placei and H. contortus.

Attempts to immunize sheep against Haemonchus contortus using a cocktail of recombinant proteases derived from the protective antigen, H-gal-GP.

The objective of this study was to determine whether an antigen cocktail containing recombinantly expressed versions of most of the protective proteases of H-gal-GP, a known protective antigen from Haemonchus contortus, would confer any protection to lambs in a vaccine-challenge trial. Haemonchus contortus metalloendopeptidases, MEP1, MEP3 and MEP4, were expressed as soluble recombinant proteins in insect cells, but attempts to express the H. contortus aspartyl proteases, PEP1 and PEP2, by the same techniques were not successful. Recombinant H. contortus PEP1 was therefore expressed in Escherichia coli and refolded. Groups of sheep were immunized thrice with either native H-gal-GP, a cocktail of recombinantly expressed proteins (rMEP1, rMEP3, rMEP4 and rPep1), or adjuvant only (QuilA in PBS). All sheep were challenged with 5000 infective larvae 1 week after the final vaccination. High levels of serum antibodies that recognized H-gal-GP were detected in both the native antigen and recombinant cocktail-immunized groups by the time of challenge, but protective immunity was only observed in the group immunized with native H-gal-GP.

Molecular characterization of a family of metalloendopeptidases from the intestinal brush border of Haemonchus contortus.

Substantial protection against the economically important parasitic nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of the worm (H-gal-GP). Previous studies showed that one of the major components of H-gal-GP is a family of at least 4 zinc metalloendopeptidases, designated MEPs 1-4. This paper describes aspects of the molecular architecture of this protease family, including the proteomic analysis of the MEP fraction of the H-gal-GP complex. These enzymes belong to the M13 zinc metalloendopeptidase family (EC 3.4.24.11), also known as neutral endopeptidases or neprilysins. The sequences of MEPs 1 and 3 suggested a typical Type II integral membrane protein structure, whilst MEPs 2 and 4 had putative cleavable signal peptides, typical of secreted proteins. Proteomic analysis of H-gal-GP indicated that the extracellular domain of all 4 MEPs had been cleaved close to the transmembrane region/signal peptide with additional cleavage sites mid-way along the polypeptide. MEP3 was present as a homo-dimer in H-gal-GP, whereas MEP1 or MEP2 formed hetero-dimers with MEP4. It was found that expression of MEP3 was confined to developing 4th-stage larvae and to adult worms, the stages of Haemonchus which feed on blood. MEP-like activity was detected in the H-gal-GP complex over a broad pH range (5-9). Since all 4 MEPs must share a similar microenvironment in the complex, this suggests that each might have a different substrate specificity.

Presence of the LDNF glycan on the host-protective H-gal-GP fraction from Haemonchus contortus.

Immunization of sheep with the gut membrane-associated protein complex H-gal-GP of adult Haemonchus contortus induces high levels of protection against a homologous challenge infection. Protection is correlated with a systemic IgG response against the antigen. Analysis of the antibody response showed that the majority of the antigen-specific IgG was of the IgG2 isotype. A substantial proportion (74%) of this response was directed against the glycan component of H-gal-GP. The high immunogenicity of the H-gal-GP glycans may be due to the presence of the fucosylated LacdiNAc (LDNF) antigen. 2D electrophoresis, Western blotting and mass spectrometry analysis of H-gal-GP showed that this glycan epitope was specifically located on a metalloendopeptidase, MEP3. MEP3 is the most abundant protein in H-gal-GP and has been identified as one of the most likely protective components of the complex. Here, we present evidence that the LDNF glycan does not contribute to the protective capacity of H-gal-GP. Animals vaccinated with reduced and denatured H-gal-GP are not protected against subsequent infection, although the antibody response against the LDNF glycan is very similar to that of animals vaccinated with the native H-gal-GP. In addition, an alternative version of H-gal-GP, H-sialgal-GP, which is equally protective, but isolated by affinity chromatography on jacalin lectin rather than peanut lectin, contains a MEP3 component which has no detectable LDNF glycan.

Metalloendopeptidases from the intestinal brush border of Haemonchus contortus as protective antigens for sheep.

Substantial protection against the economically important nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of this parasite. This fraction has been termed Haemonchus galactose-containing glycoprotein complex (H-gal-GP) since it was originally isolated through its selective binding to lectins with a specificity for N-acetylgalactosamine. A major component of this highly protective antigen complex is a family of four zinc metalloendopeptidases, designated MEPs 1-4. Various combinations of these MEPs were evaluated in immunization-challenge trials in sheep. In two experiments a combination of all four MEPs, separated from the rest of the complex by gel filtration in 8 m urea, significantly reduced H. contortus egg counts by 45 and 50%, an effect not significantly different from that conferred by 8 m urea treatment of H-gal-GP itself. Similarly, MEP3 alone or MEPs 1, 2 and 4 in combination, electroeluted from the complex following SDS gel electrophoresis, each reduced egg counts by some 33%. The MEPs are therefore protective components of H-gal-GP and from previously published findings, it appears that MEP3 is the most effective member of this metalloendopeptidase family. However, there was no significant protection when sheep were immunized with fully reduced and denatured H-gal-GP or with bacterially expressed recombinant forms of MEP 1 or the principal domains of MEP3, suggesting that conformational epitopes on the MEPs are required for immunity.

Aspartyl proteases from the intestinal brush border of Haemonchus contortus as protective antigens for sheep.

A novel pepsin-like aspartyl protease was identified as a component of Haemonchus galactose-containing glycoprotein (H-gal-GP), which is an integral membrane glycoprotein complex located on the intestinal cells of Haemonchus contortus, and a highly protective antigen for sheep. This molecule, designated HcPEP2, showed 50% sequence identity with a previously described aspartyl protease from H-gal-GP known as HcPEP1. Fractions of H-gal-GP, either containing both HcPEP1 and 2 or other lower molecular weight components of the complex, were evaluated as protective antigens in immunization - challenge trials in sheep. When separated from the rest of the complex by gel filtration in 8 m urea, the HcPEP1 and 2 fraction significantly reduced H. contortus egg counts by 48% and worm numbers by 36%, but the lower molecular weight components were not significantly protective. However, the HcPEP1 and 2 fraction did not protect if electro-eluted from SDS-dissociated H-gal-GP, nor did bacterially expressed recombinant HcPEP1, suggesting that conformational epitopes are important for inducing immunity.

The contribution of molecular biology to the development of vaccines against nematode and trematode parasites of domestic ruminants.

Rapid developments in molecular biology have had an enormous impact on the prospects for the development of vaccines to control the major nematode and trematode infestations of livestock. Vaccine candidates are purified using conventional protein chemistry techniques but the limitations imposed by the scarcity of parasite material provide an insurmountable barrier for commercial vaccine production by this means. The ability to purify mRNA from different parasite life-cycle stages and to prepare cDNA expression libraries from it has proven central to the identification of immunogenic parasite proteins. Potentially, protective parasite antigens can now be produced in recombinant form in a variety of vectors and this represents a key breakthrough on the road to commercial vaccine production. The contribution of molecular biology to this process is discussed using several examples, particularly in vaccine development against the pathogenic abomasal nematode of sheep and goats, Haemonchus contortus, and the liver fluke of sheep and cattle, Fasciola hepatica. The difficulties of producing recombinant proteins in the correct form, with appropriate post-translational modification and conformation, are discussed as well as emerging means of antigen delivery including DNA vaccination. The opportunities offered by genome and expressed sequence tag analyses programmes for antigen targeting are discussed in association with developing microarray and proteomics technologies which offer the prospect of large scale, rapid antigen screening and identification.

Cloning and expression of cystatin, a potent cysteine protease inhibitor from the gut of Haemonchus contortus.

A cDNA encoding a cysteine protease inhibitor (cystatin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a protective membrane protein complex (H-gal-GP) derived from the gut of the parasite. The cDNA sequence, designated Cys-1, showed significant levels of similarity with cystatins from several species of nematode as well as with human cystatin. Recombinant H. contortus cystatin was expressed in Escherichia coli in a soluble and functionally active form, which proved to be a potent inhibitor of both mammalian cathepsin B and native H. contortus cysteine proteases. Immunolocalization studies using antisera raised against recombinant H. contortus cystatin showed that the inhibitor was predominantly expressed in the cytoplasm of intestinal cells. To determine whether H. contortus had any protective capacity against infection, lambs were vaccinated with the recombinant molecule and subsequently given a single challenge infection. Although vaccination did not confer any protection against infection with H. contortus, as judged by faecal egg output or worm counts, cystatin will be a valuable tool in the analysis of the function of the cysteine proteases which are the subject of on-going study as potential vaccine components.

Relative protective properties of three membrane glycoprotein fractions from Haemonchus contortus.

Jacalin lectin was used as a ligand to isolate a fraction containing two distinct protective antigens from detergent extracts of membranes from Haemonchus contortus. The first antigen was identified as a complex which appeared very similar to Haemonchus galactose-containing glycoprotein (H-gal-GP), which is a previously described protective protease complex, except that it was substantially depleted of one of the main H-gal-GP components, a 230 kDa metallopeptidase-containing band. The new complex was termed Haemonchus sialylated galactose-containing glycoprotein (H-sialgal-GP), because it bound to jacalin but not to peanut lectin and only jacalin will bind the sialylated form of galactosyl (beta-1, 3) N-acetylgalactosamine. Two protection trials with sheep showed that H-sialgal-GP and H-gal-GP were equally efficacious, reducing numbers of Haemonchus eggs by between 86% and 93% and worms by between 52% and 75%, respectively. The second jacalin-binding protective antigen fraction was separated from H-sialgal-GP by ion exchange and gel filtration chromatography. It was greatly enriched for two proteins termed p46 and p52 according to their apparent molecular weights. Immunization of sheep with these proteins gave protection values of 78% for eggs and 33% for worms, which are significantly lower than those obtained with either H-gal-GP or H-sialgal-GP. N-terminal amino acid sequence data from p46 and p52 showed that both proteins were closely related to a previously described 45 kDa Haemonchus membrane protein, which had conferred protection against Haemonchus in guinea-pigs.

Cloning and characterization of a beta-galactoside-binding protein (galectin) from the gut of the gastrointestinal nematode parasite Haemonchus contortus.

A cDNA encoding a beta-galactoside-binding lectin (galectin) was identified by immunoscreening a Haemonchus contortus cDNA library with antisera from lambs vaccinated with a membrane protein complex (H-gal-GP) derived from the parasites' gut. The cDNA sequence, exhibiting a tandem repeat structure and designated Hco-gal-2, showed significant levels of similarity with galectins from several species of nematode as well as mammalian galectin type 4. Native galectin was preferentially extracted from the H-gal-GP complex and also from an insoluble membrane fraction prepared from adult worms using lactose-agarose affinity chromatography. The affinity-purified material had apparent molecular mass of around 35 kDa with 3 distinct bands visible on SDS-PAGE. All 3 bands were identified as galectins by reaction with antiserum raised to recombinant Hco-GAL-2 on Western blot. To determine whether H. contortus galectins have any protective capacity against infection lambs were vaccinated with affinity-purified galectin and subsequently given a single challenge infection. Vaccination did not confer any protection against infection with H. contortus as judged by faecal egg output or worm counts.

Molecular cloning and characterization of gut-derived cysteine proteinases associated with a host protective extract from Haemonchus contortus.

Cysteine proteinases have been implicated in the protection conferred by vaccination with detergent-soluble extracts of Haemonchus contortus. In the present study, antisera from sheep refractory to Haemonchus challenge following vaccination with a 'proteinase-enriched' Haemonchus gut membrane extract, were employed to screen a cDNA expression library of the adult parasite. This resulted in the isolation of 3 cDNAs (designated hmcp1, 4 and 6) encoding cathepsin B-like cysteine proteinases. Immunocytochemical studies specifically localized the products of these genes to the microvillar surface of the parasite's gut and RT-PCR experiments revealed that these were developmentally regulated, being expressed exclusively during the blood-feeding parasitic stages. In addition, a generic PCR approach was adopted in order to identify the predominant cysteine proteinases in a UK strain of Haemonchus. A panel of 5 cDNAs, including hmcp1 and 4, was amplified in this way. Genomic Southern blot analysis indicated that some of these enzymes were encoded by single-copy genes, whereas others were encoded by multi-copy genes. Subsequent sequence analysis revealed that the proteases identified in this study were distinct from those previously reported in USA strains of the parasite.

Further immunization and biochemical studies with a protective antigen complex from the microvillar membrane of the intestine of Haemonchus contortus.

Immunization of sheep with a protective antigen complex from the intestinal cells of Haemonchus contortus in Freund's adjuvant stimulated individually variable antibody responses but still conferred significant protection against parasite infection. Correlation between antibody concentration and degree of protection was suggestive of antibody being the effector mechanism. The antigen is known as Haemonchus galactose-containing glycoprotein complex (H-gal-GP) because it binds to lectins with a specificity for N-acetyl-galactosamine. Polypeptide composition analysis by polyacrylamide gel electrophoresis indicated an apparent molecular weight of about 1000 kDa and SDS gels revealed four major polypeptides, containing between 2 and 5 disulphide linked subunits, nearly all being glycosylated. N-terminal amino acid sequence was obtained from 12 subunits, ten showing homologies with cDNAs from Haemonchus encoding either pepsin, metalloprotease or cysteine protease-like enzymes. pH optima, inhibitor and various substrate studies confirmed that the native complex possessed proteolytic activities in agreement with the sequence data. Although the cDNAs predicted water soluble enzymes, little of the complex was solubilized from worm membranes without the use of a detergent, such as Triton X-100. It is hypothesized that H-gal-GP is a gut membrane associated multiprotease complex which is involved in the digestion of the blood meal and which can be neutralized by specific antibodies with drastic consequences for the parasite.

Mucosal mast cell responses and release of mast cell protease-I in infections of mice with Hymenolepis diminuta and H. microstoma: modulation by cyclosporin A.

The dynamics of intestinal mucosal mast cells and the major mucosal mast cell protease were followed during the course of laboratory infections of mice with Hymenolepis diminuta and H. microstoma. The effects of the drug cyclosporin A (CsA), which is both immunosuppressive and selectively anthelmintic depending upon dose regime, were determined. In H. diminuta infections worm expulsion occurred around day 9 and coincided with peak mastocytosis and peak mMCP-I concentrations in tissues and serum. Immunosuppressive treatment with CsA prevented worm expulsion, permitting some individuals to reach maturity, and abrogated mast cell proliferation and mMCP-I production and release. By contrast, H. microstoma infections persisted for 64 days in spite of a considerable mastocyosis in both intestine and bile duct tissues accompanied by a high level of mMCP-I in tissues and serum. A subimmunosuppressive regime of CsA had only limited effects on worms and mast cell numbers and activity. Together these data shed light on the variable mast cell response to gastrointestinal infections and on the potential significance of parasite location in evasion of mast cell action. Use of CsA reveals the contributions of both T cell-dependent mechanisms, including mast cell proliferation and activation, and T cell-independent events in regulating intestinal helminth infections.

Mast cell heterogeneity in the gastrointestinal tract: variable expression of mouse mast cell protease-1 (mMCP-1) in intraepithelial mucosal mast cells in nematode-infected and normal BALB/c mice.

Soluble granule chymases in rodent intestinal mucosal mast cells (IMMCs) may play an important role in altering epithelial permeability during immediate hypersensitivity reactions. Using a monoclonal antibody against the chymase mouse mast cell protease-1 (mMCP-1), we have shown that it is constitutively expressed in < or = 20% of esterase-positive (esterase+) IMMCs but not in esterase+ gastric mucosal mast cells (GMMCs) in normal BALB/c mice. Intestinal infection with mouse- or rat-adapted strains of Nippostrongylus brasiliensis resulted in IMMC hyperplasia with 100% of esterase+ IMMCs expressing mMCP-1. In contrast, there was a variable response in terms of numbers of GMMCs and of the proportion expressing mMCP-1. Esterase+ mast cells in the gastric submucosa, muscularis, ear pinna, lung parenchyma, major airway submucosa, and peritoneal cavity did not express mMCP-1. The few airway esterase+ mast cells expressing mMCP-1 were, like the great majority of IMMCs and GMMCs, located intraepithelially. In conclusion, mMCP-1 is predominantly expressed by intraepithelial mucosal mast cells but not in all sites; the immunological stimulus associated with intestinal nematodiasis substantially up-regulates mMCP-1 expression by mast cells in the jejunum but not in the stomach; IMMCs and GMMCs in BALB/c mice are phenotypically and possibly functionally distinct.

Mast cell and mast cell granule phenotypes in normal and Nippostrongylus-infected rats. A qualitative laser confocal microscopic study.

In the rat, the individual mast cell secretory granules may be divided into three subpopulations based on the presence of the specific proteases RMCP-1, RMCP-2, or a variable combination of these two proteases. Mast cells in the tongue only express RMCP-1, both in normal and infected animals, whereas in the other tissue locations studied (lung, intestinal mucosa and submucosa, tracheal epithelium and submucosa) the mast cells contain all three granule subtypes in a wide variation of combinations. These studies demonstrate that there is wide heterogeneity in protease expression in rat mast cells, which may be influenced by local stimulation with environmental tissue factors.