An enzymatically inactivated hemoglobinase fromNecator americanusinduces neutralizing antibodies against multiple hookworm species and protects dogs against heterologous hookworm infection

Abstract

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-1wt) and mutant (Na-APR-1mut—mutagenesis of the catalytic aspartic acids) forms of Na-APR-1 from the human hookworm, Necator americanus. Refolded Na-APR-1wt was catalytically active, and Na-APR-1mut was catalytically inactive but still bound substrates. Vaccination of canines with Na-APR-1mut 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-1mut induced antibodies that bound the native enzyme in the parasite gut and neutralized enzymatic activity of Na-APR-1wt and APR-1 orthologues from three other hookworm species that infect humans. IgG1 against Na-APR-1mut 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-1mut 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.

Vaccinomics for the Major Blood Feeding Helminths of Humans

“…It is not known whether the orthologous hookworm proteases form a similar complex to H-gal-GP, but, with the development of techniques such as LMM Xu, 2010) and tissue profiling via mass spectrometry, the characterization of proteins in microscopic tissues, such as a hookworm gut, is now a possibility. Indeed, one of the lead hookworm vaccine antigens is the intestinal haemoglobinase, Na-APR-1 (a cathepsin D-like aspartic protease), where the protective effect appears to establish neutralizing antibodies that inhibits substrate proteolysis (Loukas et al, 2005;Pearson et al, 2009). …”

Expression, purification, and characterization of theNecator americanusaspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine

“…The Na-APR-1 protein is expressed in the gut of the adult N. americanus parasites where it functions in concert with the cysteine protease Na-CP-3 and the metalloprotease Na-MEP-1 to digest the hemoglobin tetramer. [7][8][9] Due to its role in hemoglobin digestion, Na-APR-1 was selected as a candidate for the development of a hookworm vaccine since neutralization of this crucial enzyme would result in starvation of the parasite and concomitantly could lead to parasite death and the prevention of blood loss in the infected host. In support of this hypothesis, vaccination of dogs with the canine homolog of the wild type Na-APR-1 (Ancylostoma caninum-APR-1) resulted in the production of neutralizing antibodies against the hookworm hemoglobinase and thus, higher level of hemoglobin in the blood of vaccinated dogs compared to the non-vaccinated control group, as well as a reduction in both egg counts and adult parasite burden in the gut of the infected host.…”

“…Vaccination of canines with Na-APR-1 (M74) followed by a heterologous challenge with the canine hookworm Ancylostoma caninum resulted in a significant reduction in both parasite egg burden and weight loss. 9 In addition, vaccination with Na-APR-1 (M74) induced antibodies that not only bound to the native enzyme in the parasite gut, but also neutralized enzymatic activity of both wild type Na-APR-1 as well as APR-1 orthologues from 3 other hookworm species that infect humans. 9 The production of Na-APR-1 (M74) protein at high yields and with adequate stability, however, has been challenging due to the very low expression levels observed in P. pastoris and the lack of solubility of the Escherichia.…”

Lipid core peptide targeting the cathepsin D hemoglobinase ofSchistosoma mansonias a component of a schistosomiasis vaccine

“…14 While it is possible to generate recombinant Sm-CatD for small-scale vaccine testing, high-yield expression of eukaryotic proteases is problematic. 15,16 Moreover, the most efficacious vaccines for helminth infections are likely to require multiple antigens or fragments thereof, and must induce high titer antibody responses that are long-lasting. Currently, most adjuvants approved for use in subunit vaccines for human use are those based on aluminum salts.…”