Vaccination of Dogs with a Recombinant Cysteine Protease from the Intestine of Canine Hookworms Diminishes the Fecundity and Growth of Worms

Abstract

We expressed a catalytically active cysteine protease, Ac-CP-2, from the blood-feeding stage of the canine hookworm Ancylostoma caninum and vaccinated dogs with the purified protease. Dogs acquired high-titer, antigen-specific antibody responses, and adult hookworms recovered from the intestines of vaccinated dogs were significantly smaller than hookworms from control dogs. There was also a marked decrease in fecal egg counts and the number of female hookworms in vaccinated dogs. Ac-CP-2 is expressed by the parasite in the brush-border membrane of its alimentary canal, and anti-Ac-CP-2 antibodies were bound to the gut of hookworms from vaccinated dogs, which suggests that these antibodies were ingested by the parasites with their blood meal. IgG from vaccinated dogs decreased proteolytic activity against a peptide substrate by 73%, which implies that neutralizing antibodies were induced by vaccination. These results indicate that cysteine proteases involved in parasite nutrition are promising candidates as vaccines against hookworm disease.

A Multi-enzyme Cascade of Hemoglobin Proteolysis in the Intestine of Blood-feeding Hookworms

“…AF273084). Expression and purification of recombinant APR-1 in baculovirus (20) and CP-2 in the yeast Pichia pastoris (21) have been reported previously. To express MEP-1 in baculovirus, the entire open reading frame minus the predicted signal peptide was amplified by PCR using a MEP-1 pBluescript plasmid as the template.…”

“…Recombinant Ac-CP-2 cleaved the substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin with an optimal pH range of 5.0 -7.0 as described previously (21). Optimal assay conditions for Ac-MEP-1 were determined using pre-cast gelatin gels (Invitrogen) and the Novex® zymogram gels developing system per the manufacturer's guidelines (Invitrogen).…”

“…Vaccination of sheep with H-gal-GP confers high levels of protection against parasite challenge; the complex is predominantly made up of aspartic, cysteine, and metalloproteases (57), and recent evidence suggests that the purified enzymes from Haemonchus galactose-containing glycoprotein are responsible for the vaccine efficacy (44,58,59). We recently described the efficacy of Ac-CP-2 as an anti-hookworm vaccine in dogs experimentally challenged with A. caninum (21). Vaccinated animals produced antibodies that neutralized the proteolytic activity of the recombinant enzyme and resulted in decreased growth and fecundity of female worms.…”

“…We have suggested that hookworms likely employ a cascade of hemoglobinolysis utilizing multiple proteases of distinct mechanistic classes (18). A cathepsin B-like cysteine protease, Ac-CP-2 (24), was recently shown to be expressed in the intestinal brush border of adult Ancylostoma caninum hookworms, and immunization of dogs with the recombinant protease resulted in an anti-fecundity effect on female hookworms (21). In addition, a neprilysin-like metalloendopeptidase was identified from the intestinal brush border of adult A. caninum (25); however, the recombinant molecule was not expressed in catalytically active form, precluding the determination of a functional role for this enzyme.…”

Assessment of Caenorhabditis elegans as a model in Haemonchus contortus vaccine research

“…C. elegans was used to investigate the function of intestinal cathepsin B-like proteinases in nematodes [5]. These gut expressed enzymes have proven to be effective targets for vaccination against Haemonchus contortus and Ancylostoma caninum, resulting in lower faecal egg output and worm burdens [6,7]. Surprisingly, simultaneous RNAi of the predominant intestinal cathepsin B enzymes in adult C. elegans did not result in any visible phenotype despite the complete abolishment of all cysteine proteinase activity, detectable by substrate gel analysis of worm extracts.…”