Purification and molecular cloning of and immunization with Ancylostoma ceylanicum excretory-secretory protein 2, an immunoreactive protein produced by adult hookworms

Hookworms remain major agents of global morbidity, and vaccination against these bloodfeeding parasites may be an attractive complement to conventional control methods. Here we describe the cloning of Ancylostoma ceylanicum excretdry-secretory protein 2 (AceES-2), a novel immunoreactive protein produced by adult worms. Native AceES-2 was purified from excretory-secretory (ES) products by reverse-phase high-pressure liquid chromatography, subjected to amino-terminal sequencing, and cloned from adult worm RNA by using reverse transcription-PCR. The translated AceES-2 cDNA predicts that the mature protein consists of 102 amino acids and has a molecular mass of 11.66 kDa. Western immunoblot and enzyme-linked immunosorbent assay analyses demonstrated that recombinant AceES-2 (rAceES-2) reacted strongly with antibodies from A. ceylanicum-infected hamsters. Immunization of hamsters with native ES products adsorbed to alum induced antibodies that recognized rAceES-2, while rAceES-2-alum vaccination resulted in antibodies that reacted with a single protein band in ES products that closely approximated the size predicted for the native molecule. Infected hamsters that were passively immunized with hyperimmune rabbit anti-rAceES-2 serum exhibited more rapid and complete recovery from anemia than controls that received normal serum. Oral immunization with rAceES-2 was associated with significantly reduced anemia upon challenge, an outcome similar to the outcome observed in hamsters that were orally vaccinated with soluble hookworm extract (the latter animals were also resistant to weight loss). These data suggest that AceES-2 plays an important role in the host-parasite interaction and that vaccination against this protein may represent a useful strategy for controlling hookworm anemia.