An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection

Hookworms digest hemoglobin from erythrocytes via a proteolytic cascade that begins with the aspartic protease, APR‐1. Ac ‐APR‐1 from the dog hookworm, Ancylostoma caninum , protects dogs against hookworm infection via antibodies that neutralize enzymatic activity and interrupt blood‐feeding. Toward developing a human hookworm vaccine, we expressed both wild‐type ( Na ‐APR‐1 wt ) and mutant ( Na ‐APR‐1 mut —mutagenesis of the catalytic aspartic acids) forms of Na ‐APR‐1 from the human hookworm, Necator americanus. Refolded Na ‐APR‐1 wt was catalytically active, and Na ‐APR‐1 mut was catalytically inactive but still bound substrates. Vaccination of canines with Na ‐APR‐1 mut and heterologous challenge with A. caninum resulted in significantly reduced parasite egg burdens ( P =0.034) and weight loss ( P =0.022). Vaccinated dogs also had less gut pathology, fewer adult worms, and reduced blood loss compared to controls but these did not reach statistical significance. Vaccination with Na ‐APR‐1 mut induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na ‐APR‐1 wt and APR‐1 orthologues from three other hookworm species that infect humans. IgG1 against Na ‐APR‐1 mut was the most prominently detected antibody in sera from people resident in high‐transmission areas for N. americanus , indicating that natural boosting may occur in exposed humans. Na ‐APR‐1 mut is now a lead antigen for the development of an antihematophagy vaccine for human hookworm disease.—Pearson, M. S., Bethony, J. M., Pickering, D. A., de Oliveira, L. M., Jariwala, A., Santiago, H., Miles, A. P., Zhan, B., Jiang, D., Ranjit, N., Mulvenna, J., Tribolet, L., Plieskatt, J., Smith, T., Bottazzi, M. E., Jones, K., Keegan, B., Hotez, P. J., Loukas, A. An enzymatically inactivated hemoglobinase from Necator americanus induces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection. FASEB J. 23, 3007–3019 (2009). www.fasebj.org