Stage-specific gene expression in Teladorsagia circumcincta (Nematoda: Strongylida) infective larvae and early parasitic stages.

Suppression subtractive hybridisation was used to enrich genes expressed in a stage-specific manner in infective, exsheathed L3s (xL3) versus early L4s of the ovine nematode, Teladorsagia circumcincta prior to gene expression profiling by microarray. The 769 cDNA sequences obtained from the xL3-enriched library contained 361 unique sequences, with 292 expressed sequence tags (ESTs) being represented once ("singletons") and 69 sequences which were represented more than once (overlapping and non-overlapping "contigs"). The L4-enriched EST dataset contained 472 unique sequences, with 314 singletons and 158 contigs. Of these 833 sequences, 85% of the xL3 sequences and 86% of the L4 sequences exhibited homology to known genes or ESTs derived from other species of nematode. Quantitative differential expression (P<0.05) was demonstrated for 563 (68%) of the ESTs by microarray. Within the L3-specific dataset, more than 30% of the transcripts represented the enzyme, guanosine-5'-triphosphate (GTP)-cyclohydrolase, which is the first and rate-limiting enzyme of the tetrahydrobiopterin synthesis pathway and may be involved in critical elements of larval development. In L4s, proteolytic enzymes were highly up-regulated, as were collagens and a number of previously characterised secretory proteins, reflecting the rapid growth of these larvae in abomasal glands. Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under accession numbers AM 743198-AM 744942.

Evaluation of Caenorhabditis elegans glycoproteins as protective immunogens against Haemonchus contortus challenge in sheep.

High levels of protection can be attained against Haemonchus contortus challenge infection in sheep using native antigens isolated from the gut of the adult parasite. However, vaccination with recombinant forms of these antigens, or components thereof, has disappointingly failed to generate similar levels of protection, suggesting that appropriate nematode glycosylation may be a prerequisite for protection. The free-living nematode, Caenorhabditis elegans is closely related to H. contortus and has been shown to share similar glycan moieties. In order to investigate the potentially protective role of these glycan moieties, a complex set of glycoproteins was isolated from C. elegans using ConA-lectin chromatography and their efficacy as immunogens against H. contortus challenge infection evaluated in sheep. Despite the generation of a high titre systemic IgG antibody response to the C. elegans glycoproteins and the ability of these antibodies to bind to the microvillar surface of the gut of H. contortus, no protection against challenge infection was observed. Serum antibodies to the C. elegans glycoproteins cross-reacted with the H. contortus host-protective antigen, H-gal-GP, by ELISA, although the level of cross-reactivity was not of a magnitude considered protective. Qualitative differences were also determined between the glycan epitopes of the C. elegans ConA-binding proteins and those of H-gal-GP, suggesting the presence of H. contortus-specific patterns of glycosylation.

Protection studies in sheep using affinity-purified and recombinant cysteine proteinases of adult Haemonchus contortus.

Vaccination with a membrane-bound thiol Sepharose-binding fraction (TSBP) of adult Haemonchus contortus has been shown to confer significant levels of protection against homologous challenge in sheep. This fraction is greatly enriched for cysteine proteinase activity. Following fractionation of TSBP by anion-exchange chromatography on MonoQ, protection was found to partition with those fractions further enriched for cysteine proteinase activity. In this study, the cysteine proteinases of adult H. contortus TSBP were specifically purified by affinity chromatography using recombinant H. contortus cystatin, a potent cysteine proteinase inhibitor. Although only 1-1.5% of total TSBP bound to cystatin-Sepharose, this fraction contained 100% of the cysteine proteinase activity, as determined by gelatin substrate gel analysis. When used to immunise sheep, less than 3microg per dose of this cysteine proteinase fraction was found to confer a substantial and repeatable level of protection against homologous challenge infection, reducing faecal egg counts by 48 and 28% and worm burdens by 44 and 46% over two trials. Host serum immunoglobulin levels and abomasal mast cell and eosinophil numbers were evaluated, although no correlation with protection was observed. Three cathepsin B-like cysteine proteinases present in TSBP (hmcp1, 4 and 6) have been identified previously by cDNA library immunoscreening. The predicted mature forms of these three cysteine proteinases were expressed in bacteria as insoluble, GST-fusion proteins. Following solubilisation in urea/DTT, the protective capacity of a cocktail of recombinant proteins was evaluated in sheep. Although no reduction in faecal egg counts was observed, sheep vaccinated with recombinant cysteine proteinases showed a highly significant 38% reduction (P <0.01) in worm burdens.

The nature and prospects for gut membrane proteins as vaccine candidates for Haemonchus contortus and other ruminant trichostrongyloids.

Substantial progress has been made in the last decade in identifying several antigens from Haemonchus contortus which, in their native form, stimulate useful levels of protective immunity (70-95% reductions in faecal egg output) in the ovine host. Much work has focussed on proteins/protein complexes expressed on the surface of the worm gut which are exposed to the blood meal, and, hence, antibody ingested with it. The antigens generally, but not in all cases, show protease activity and antibody is thought to mediate protective immunity by blocking the activity of enzymes involved in digestion within the worm. This review summarises the protective efficacy, as well as the biochemical and molecular properties, of the principal candidate antigens which are expressed in the gut of these parasites. Of course, such antigens will have to be expressed as recombinant proteins to be sufficiently cost-effective for use in a commercial vaccine and the current status of recombinant antigen expression is discussed with particular reference to conformation and glycosylation. There is a need for continued antigen definition even in the confines of gut antigens and potential targets can be selected from the rapidly expanding genome/EST datasets on the basis of predicted functional homology. Gene knockout technologies such as RNA interference have the potential to provide high throughput, rapid and inexpensive methods to define whether the protein product of a particular gene would be a suitable vaccine candidate.