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cDNAs encoding 2 Ancylostoma-secreted proteins (ASPs), Ancylostoma ceylanicum (Ay)-ASP-1 and Ay-ASP2, were cloned from infective third-stage larvae (L3) of the hookworm A. ceylanicum and were expressed as soluble recombinant fusion proteins secreted by the yeast Pichia pastoris. The recombinant fusion proteins were purified, adjuvant formulated, and injected intramuscularly into hamsters. Hamsters vaccinated either by oral vaccination with irradiated L3 (irL3) or by injections of the adjuvants alone served as positive and negative controls, respectively. Anti-ASP-1 and anti-ASP-2 antibody titers exceeded 1:100,000. Each vaccinated hamster was challenged orally with 100 L3. Two groups of vaccinated hamsters (i.e., those vaccinated with either irL3 or ASP-2 formulated with Quil A) exhibited significant reductions in adult hookworm burdens, compared with control hamsters. The hookworms recovered from the hamsters vaccinated with ASP-2 plus Quil A were reduced in length. Splenomegaly, which was observed in control hamsters, was not seen in hamsters vaccinated with either irL3 or ASP-2 formulated with Quil A. These results indicate that ASP-2 is a promising molecule for the development of a hookworm vaccine. [ABSTRACT FROM AUTHOR]

Hookworms are voracious blood-feeders. The cloning and functional expression of an aspartic protease, Na-APR-2, from the human hookworm Necator americanus are described here. Na-APR-2 is more similar to a family of nematode-specific, aspartic proteases than it is to cathepsin D or pepsin, and the term "nemepsins" for members of this family of nematode-specific hydrolases is proposed. Na-apr-2 mRNA was detected in blood-feeding, developmental stages only of N. americanus, and the protease was expressed in the intestinal lumen, amphids, and excretory glands. Recombinant Na-APR-2 cleaved human hemoglobin (Hb) and serum proteins almost twice as efficiently as the orthologous substrates from the nonpermissive dog host. Moreover, only 25% of the Na-APR-2 cleavage sites within human Hb were shared with those generated by the related N. americanus cathepsin D, Na-APR-1. Antiserum against Na-APR-2 inhibited migration of 50% of third-stage N. americanus larvae through skin, which suggests that aspartic proteases might be effective vaccines against human hookworm disease. [ABSTRACT FROM AUTHOR]