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.