How to cite this article: Vet. Res. (2009) 40:41
DOI: 10.1051/vetres/2009025

An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

Erik De Vries¹, Nicole Bakker¹, Jeroen Krijgsveld², Dave P. Knox³, Albert J.R. Heck² and Ana Patricia Yatsuda<sup>4

1</sup>  Division of Parasitology and Tropical Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, P.O. Box 80165, 3508 TD, Utrecht, The Netherlands
²  Department of Biomolecular Mass Spectrometry, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands
³  Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 OPZ, United Kingdom
⁴  Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av do Café, sn/n, 14040-903, Ribeirão Preto, SP, Brazil

Received 24 July 2008; accepted 24 April 2009; published online 30 April 2009

Abstract - The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.

Key words: Haemonchus contortus / excretory-secretory products / cysteine protease / vaccination / AC-5

Corresponding author: ayatsuda@fcfrp.usp.br

© INRA, EDP Sciences 2009