Effect of Vaccination with a Recombinant Fusion Protein Encoding an Astacinlike Metalloprotease (MTP-1) Secreted by Host-Stimulated Ancylostoma caninum Third-Stage Infective Larvae

Abstract

Laboratory dogs were vaccinated intramuscularly with a recombinant fusion protein (expressed and isolated from Escherichia coli) formulated with the Glaxo SmithKline Adjuvant System 02 (AS02). The fusion protein encoded Ac-MTP-1, a developmentally regulated astacinlike metalloprotease secreted by host-stimulated Ancylostoma caninum third-stage larvae (L3). Control dogs were injected intramuscularly with an equivalent amount of AS02 adjuvant alone. The vaccinated and control dogs were then challenged by s.c. injection of 500 L3 of the canine hookworm A. caninum. The vaccinated dogs developed prechallenge immunoglobulin G2 (IgG2) antibody responses specific to anti-Ac-MTP-1-fusion protein with titers ranging between 1:40,000 and 1:364,000, whereas they developed antigen-specific immunoglobulin E antibody responses with titers ranging between 1:500 and 1:1,500. By immunoblotting, canine sera obtained from the vaccinated dogs recognized a protein of the estimated apparent molecular weight of Ac-MTP-1 in activated L3 secretory products. Spearman rank order correlations between the canine intestinal adult hookworm burden and quantitative egg counts at necropsy and anti-Ac-MTP-1 IgG2 antibody titers revealed a statistically significant inverse association (r = -0.89; P = 0.04), suggesting that this molecule offers promise as a recombinant vaccine.

Ancylostoma caninum MTP-1, an Astacin-Like Metalloprotease Secreted by Infective Hookworm Larvae, Is Involved in Tissue Migration

“…A similar inverse association was noted between IgG2 antibody titers and median quantitative egg counts (13). More recently, Ay-MTP-1, the orthologue of Ac-MTP-1 from the related hookworm Ancylostoma ceylanicum, was shown to provide significant reductions in worm burdens and egg counts when used to vaccinate hamsters in a challenge model of hookworm infection (21).…”

“…In a previous study, the vaccination of dogs with recombinant Ac-MTP-1 expressed in E. coli in an insoluble form resulted in an inverse association between antibody titers and worm burdens after larval challenge (13). A similar inverse association was noted between IgG2 antibody titers and median quantitative egg counts (13).…”

“…Notably, MTP-1 is not found in the cuticles of developmentally arrested L3 (data not shown). Previously, it was also shown that MTP-1 is not secreted by developmentally arrested L3 in the soil but is rapidly released when worms are given a host-like stimulus (serum) (8,13,28). A family of pathogenesis-related proteins called the Ancylostoma secreted proteins (ASPs) are also secreted upon host-like activation of A. caninum larvae (9, 10).…”

Secreted Proteomes of Different Developmental Stages of the Gastrointestinal Nematode Nippostrongylus brasiliensis

“…In eukaryotes, astacins adopt a range of functions, including the activation of growth factors, degradation of polypeptides, and the processing of extracellular proteins (69). Astacins have been found in other free-living and parasitic nematodes (50, 70 -72) including the canine hookworm A. caninum (73); A. caninum L3 secrete an astacin which digests connective tissues upon invasion of the host skin, and the protein has shown potential as a hookworm vaccine (73,74). S. stercoralis strongylastacin is only expressed by L3, and it is thought to be involved in the invasion process (75).…”

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

“…76 The dogs mounted a strong antibody response to the protein and the numbers of adult worms retrieved from small intestine was reduced compared to controls. Moreover, worms appeared to be displaced from their predilection site into the colon, the effect being particularly pronounced for female worms and correlated with antibody titre.…”