Neutralizing Antibodies; Hookworm; Recombinant Vaccine; Hemoglobin Digestion
Necator Americanus, a human hookworm causes approximately 85% of the global hookworm infections. Hookworm ingest hemoglobin containing erythrocytes. Hemoglobin is further digested to Heme and Globin by hookworm's gut enzymes. Iron-containing Heme is a potent enzyme inhibitor and generates toxic reactive oxygen species which is toxic to hookworms. Hookworm's gut enzyme Na-GST-1 (Necator Americanus Glutathione S-Transferase-1) has been hypothesized to detoxify Heme. Na-GST-1 adjuvanted with Alhydrogel® is a new vaccine which is currently under clinical development. Na-GST-1 has two active sites, the ligand binding or Heme detoxification site (H-site) and the catalytic active glutathione binding site (G-site). We have developed in-vitro assays to assess the neutralizing capacity of antibodies against the functional activity of these two active sites. The antibodies used in this assay were purified from serum of BALB/c mice vaccinated with Na-GST-1 vaccine. Here, we report the development and results of these two novel in-vitro assays.
Heme detoxification function (H-site) of Na-GST-1 was evaluated by oxidizing and degrading hematin to release iron using a potent oxidizer, hydrogen peroxide. Free iron released from hematin was measured by iron detection reagent called ferrozine. BALB/c mice were vaccinated with Na-GST-1 vaccine and polyclonal IgG from the mice sera was purified using immuno-precipitation. The neutralizing capacity of IgG against the catalytic activity (G-site) of Na-GST-1 was assessed using the CDNB assay. Similarly, the neutralizing capacity of IgG against the H-site was assessed by the ferrozine iron releasing assay after incubating Na-GST-1 with Hematin and polyclonal IgG.
Na-GST-1 significantly inhibited free-iron release from hematin when incubated with hydrogen peroxide. No statistically significant reduction in iron release was found when other iron containing molecules like cytochrome C, ferredoxin or myoglobin were incubated with Na-GST-1 and H2O2. Four microgram of polyclonal IgG reversed 56.9% of the free-iron release from Hematin. Moreover, 4µg monoclonal Sj-GST IgG completely reversed the inhibition of the release of free-iron caused by Na-GST-1. The neutralizing antibody assay against G-site of Na-GST-1 generated a dose response (%Inhibition vs Negative-IgG) when 20µg (25.87%), 15µg (19.61%), 10µg (13.78%) and 5µg (6.32%) of the purified IgG was incubated with 500ng of Na-GST-1.
In-vitro assay showed that Na-GST-1 could prevent release of free-iron from hematin. Antibodies purified from Na-GST-1 vaccinated mice neutralized both the heme-detoxification activity (H-site) and catalytic activity (G-site) of Na-GST-1. These neutralizing antibody assay results of Na-GST-1 vaccine lay the foundation for a Phase 2 Clinical Trial in Brazil.
Chen, Xi; Keegan, Brian; Hotez, Peter J.; Bethony, Jeffrey M.; and Jariwala, Amar R., "Novel Neutralizing Antibody Assays for Recombinant Human Hookworm Na-GST-1 Vaccine" (2013). GW Research Days 2013. Paper 14.