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.
Moredun Research Institute, International Research Centre, Pentlands Science Park, Penicuik, Midlothian EH260PZ, Scotland, UK. redmd@mri.sari.ac.uk
Diane L
DL
Redmond
David P
DP
Knox
D000818:Animals; D000947:Antigens, Helminth / Q000276:immunology; D015153:Blotting, Western; D002846:Chromatography, Affinity; D015891:Cystatins / Q000737:chemistry / Q000276:immunology; D003546:Cysteine Endopeptidases / Q000276:immunology* / Q000302:isolation & purification; D018509:DNA, Helminth / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000469:parasitology; D005982:Glutathione Transferase / Q000276:immunology; D006188:Haemonchiasis / Q000276:immunology* / Q000517:prevention & control / Q000662:veterinary*; D006189:Haemonchus / Q000737:chemistry / Q000276:immunology*; D007070:Immunoglobulin A / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008566:Membranes / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012685:Sepharose / Q000737:chemistry; D012756:Sheep; D012757:Sheep Diseases / Q000276:immunology* / Q000517:prevention & control*; D014614:Vaccines, Synthetic / Q000276:immunology*
D000947:Antigens, Helminth; D015891:Cystatins; D018509:DNA, Helminth; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D007073:Immunoglobulin E; D012685:Sepharose; D005982:Glutathione Transferase; D003546:Cysteine Endopeptidases