Metalloendopeptidases from the intestinal brush border of Haemonchus contortus as protective antigens for sheep

Abstract

Substantial protection against the economically important nematode Haemonchus contortus has been achieved by immunizing sheep with a glycoprotein fraction isolated from the intestinal membranes of this parasite. This fraction has been termed Haemonchus galactose-containing glycoprotein complex (H-gal-GP) since it was originally isolated through its selective binding to lectins with a specificity for N-acetylgalactosamine. A major component of this highly protective antigen complex is a family of four zinc metalloendopeptidases, designated MEPs 1-4. Various combinations of these MEPs were evaluated in immunization-challenge trials in sheep. In two experiments a combination of all four MEPs, separated from the rest of the complex by gel filtration in 8 m urea, significantly reduced H. contortus egg counts by 45 and 50%, an effect not significantly different from that conferred by 8 m urea treatment of H-gal-GP itself. Similarly, MEP3 alone or MEPs 1, 2 and 4 in combination, electroeluted from the complex following SDS gel electrophoresis, each reduced egg counts by some 33%. The MEPs are therefore protective components of H-gal-GP and from previously published findings, it appears that MEP3 is the most effective member of this metalloendopeptidase family. However, there was no significant protection when sheep were immunized with fully reduced and denatured H-gal-GP or with bacterially expressed recombinant forms of MEP 1 or the principal domains of MEP3, suggesting that conformational epitopes on the MEPs are required for immunity.

Proteases in Blood-Feeding Nematodes and Their Potential as Vaccine Candidates

“…MEP3 alone or MEPs 1, 2 and 4 in combination, electro-eluted from the complex following SDS gel electrophoresis, each reduced egg counts by about 30%. 32 These data indicated that the MEPs all contributed to the protection induced by H-gal-GP. Protection was lost when sheep were immunized with fully reduced and denatured H-gal-GP or with bacterially-expressed recombinant forms of MEP 1 or the principal domains of MEP3, suggesting that conformational epitopes on the MEPs are required for immunity.…”

“…Protection was lost when sheep were immunized with fully reduced and denatured H-gal-GP or with bacterially-expressed recombinant forms of MEP 1 or the principal domains of MEP3, suggesting that conformational epitopes on the MEPs are required for immunity. 32 Another component of H-gal-GP was shown to be an aspartyl protease with homology to mammalian pepsinogen sequences (HcPEP1), again expressed on the luminal surface of the gut in the blood-feeding stages. 25 The enzyme was shown to be almost exclusively expressed by the blood-feeding parasite stages.…”

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

“…The sheep barber's pole worm, Haemonchus contortus, expresses a family of metalloproteases in the intestine (43,44); although these enzymes have not been expressed in active form, it is thought that they are involved in Hb digestion. MEP-1 displayed a broad pH range when assessed in zymograms.…”

“…Haemonchus galactose-containing glycoprotein is a protein complex from the intestine of the sheep barber's pole worm, H. contortus. 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).…”

Comparative immunoprophylactic efficacy of Haemonchus contortus recombinant enolase (rHcENO) and Con A purified native glycoproteins in sheep

“…Recombinant DNA technology allows production of large amounts of parasite antigens in a heterologous system with high level of purity. Several attempts exploiting the recombinant versions of H11 (Reszka et al, 2007), H-gal-GP (Cachat et al, 2010;Smith et al, 2003) and Thiol separose binding proteins (TSBP) (Redmond and Knox, 2004) failed to induce a satisfactory immune response against H. contortus. This emphasizes the need to work for a more potential vaccine target against H. contortus.…”