Vaccination with Recombinant Aspartic Hemoglobinase Reduces Parasite Load and Blood Loss after Hookworm Infection in Dogs

BACKGROUND: Hookworms infect 730 million people in developing countries where they are a leading cause of intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adult hookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasites subsequently digest hemoglobin in their intestines using a cascade of proteolysis that begins with the Ancylostoma caninum aspartic protease 1, APR-1. METHODS AND FINDINGS: We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellular responses and resulted in significantly reduced hookworm burdens (p = 0.056) and fecal egg counts (p = 0.018) in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p = 0.049) and most did not develop anemia, the major pathologic sequela of hookworm disease. IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interferes with the parasite's ability to digest blood. CONCLUSION: To the best of our knowledge, this is the first report of a recombinant vaccine from a hematophagous parasite that significantly reduces both parasite load and blood loss, and it supports the development of APR-1 as a human hookworm vaccine

Progress in the development of a recombinant vaccine for human hookworm disease: The Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia, and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunizing laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titers. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunizing large impoverished populations living in the developing nations of the tropics

The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus

We have previously reported the successful adaptation of human hookworm Necator americanus in the golden hamster, Mesocricetus auratus. This animal model was used to test a battery of hookworm (N. americanus and Ancylostoma caninum) recombinant antigens as potential vaccine antigens. Hamsters immunized a leading vaccine candidate N. americanus–Ancylostoma secreted protein 2 (Na-ASP-2) and challenged with N. americanus infective larvae (L3), resulted in 30–46.2% worm reduction over the course of three vaccine trials, relative to adjuvant controls. In addition, significant reduction of worm burdens was also observed in the hamsters immunized with adult hookworm antigens A. caninum aspartic protease 1 (Ac-APR-1); A. caninum-glutathione-S transferase 1 (Ac-GST-1) and Necator cysteine proteases 2 (Na-CP-2) (44.4%, 50.6%, and 29.3%, respectively). Our data on the worm burden reductions afforded by these hookworm antigens approximate the level of protection reported previously from dogs challenged with A. caninum L3, and provide additional evidence to support these hookworm antigens as vaccine candidates for human hookworm infection. The hamster model of N. americanus provides useful information for the selection of antigens to be tested in downstream vaccine development

Vaccination with recombinant aspartic hemoglobinase reduces parasitic load and blood loss after hookworm infection in dogs

Background Hookworms infect 730 million people in developing countries where they are a leading cause of intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adult hookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasites subsequently digest hemoglobin in their intestines using a cascade of proteolysis that begins with the Ancylostoma caninum aspartic protease 1, APR-1. Methods and Findings We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellular responses and resulted in significantly reduced hookworm burdens (p = 0.056) and fecal egg counts (p = 0.018) in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p = 0.049) and most did not develop anemia, the major pathologic sequela of hookworm disease. IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interferes with the parasite\u27s ability to digest blood. Conclusion To the best of our knowledge, this is the first report of a recombinant vaccine from a hematophagous parasite that significantly reduces both parasite load and blood loss, and it supports the development of APR-1 as a human hookworm vaccine

Vaccination of Dogs with APR-1 Reduces Blood Loss and Protects against Anemia

<p>Hb concentrations of vaccinated dogs were significantly (<i>p</i> = 0.049) greater than those of control dogs when blood was drawn after larval challenge (0 and 7 d before necropsy [post]) but not when blood was drawn 5 d before larval challenge (pre).</p

Vaccination with APR-1 Reduces Adult Worm Burdens of Dogs after Challenge Infection with Hookworms

<p>Statistically significant reduction at the <i>p</i> < 0.1 level (<i>p</i> = 0.065) in median adult worm (both sexes) burdens of dogs vaccinated with APR-1 compared to dogs that received adjuvant alone (A). Reductions are also shown when only male (B) (<i>p</i> = 0.111) and only female (C) (<i>p</i> = 0.1905) worms were considered; however, statistically significant reductions were not achieved for single sex analyses. Bars represent the median value for each group.</p

Vaccination with APR-1 Reduces Fecal Egg Counts of Dogs after Challenge Infection with Hookworms

<div><p>Statistically significant reduction (<i>p</i> = 0.018) in median fecal egg counts sampled on days 21, 23, and 26 of dogs vaccinated with APR-1 compared to dogs that received adjuvant alone.</p> <p>(A). Geometric mean values of fecal egg counts from vaccinated and control dogs between challenge infection and necropsy.</p> <p>(B). Error bars refer to the standard error of the mean.</p></div

P. pastoris Secrete <i>Ac-</i>APR-1 Zymogen that Autoactivates at Low pH and Degrades Canine Hb

<div><p>SDS-PAGE gel stained with Coomassie Brilliant Blue showing purification of recombinant APR-1 zymogen from <i>P. pastoris</i> culture supernatant.</p> <p>(A) Lane 1, molecular weight markers; lane 2, concentrated culture supernatant; lane 3, flow-through from a nickel-IDA column; lane 4, 5 mM imidazole wash; lane 5, 20 mM imidazole column eluate; lane 6, 60 mM imidazole eluate; and lane 7, 1 M imidazole eluate. Purified recombinant APR-1 zymogen was activated by buffer exchange into 0.1 M sodium formate/0.1 M NaCl (pH 3.6).</p> <p>(B) Lane 1, molecular weight markers; lane 2, 5.0 μg of canine Hb (pH 3.6); and lane 3, 5.0 μg of canine Hb (pH 3.6) incubated with 0.2 μg of recombinant APR-1.</p></div

Antibodies Bind In Situ to the Intestines of Hookworms that Feed on Vaccinated Dogs

<p>Detection of antibodies that bound to the gut of worms recovered from vaccinated dogs (A and B) but not control dogs (C and D) by immunofluorescence. Binding was detected using Cy3-conjugated rabbit anti-dog IgG, allowing only detection of antibodies that had bound in situ while parasites were feeding on blood from vaccinated or control dogs. ic. intestinal contents; in, hookworm intestine; mv, intestinal microvillar surface; ro, reproductive organs.</p