Neutralizing Antibodies; Hookworm; Recombinant Vaccine; Hemoglobin Digestion
Backgound: Human Hookworm Infection, a neglected tropical disease infects more than 600 million people around the world. Hookworms ingest hemoglobin containing erythrocytes and Necator americanus Aspartic Protease-1 wild type (Na-APR-1wt) a hemoglobinase cleaves hemoglobin to form Heme and Globin. Globin is further digested by other gut enzymes and the nutritional end products are absorbed by the hookworm’s gut wall. Also, Heme which is toxic to hookworm is detoxified by the Necator americanus Glutathione Transferase-1 (Na-GST-1) a detoxification enzyme secreted by the gut of the hookworm. Necator americanus Aspartic Protease-1 M74 (Na-APR-1 M74) is the new vaccine for the Human Hookworm Infection which is currently under pre-clinical development. Na-APR-1 M74 vaccine is an Alhydrogel® adjuvanted vaccine containing the mutant form of the Na-APR-1wt. Neutralizing Na-APR-1wt by potent antibodies (IgG) in the vaccinees will block the initiation of the hemoglobin digestion cascade and starve the hookworms from essential nutrition, leading to their death. Here, we report the results of the neutralizing capacity of antibodies and potency (immunogenicity) of Na-APR-1 M74 vaccine in BALB/c mice.
Methods: Serum for IgG was generated by vaccinating BALB/c mice twice subcutaneously with Na-APR-1 M74 an enzymatically inactive mutant form of Na-APR-1wt formulated with Alhydrogel®. Assessment of neutralizing capacity of IgG was performed using the standard Cathepsin-D protease assay using MOCAc substrate. Dose response (% Inhibition vs Dose) was assessed using linear regression analysis. Potency testing of the Na-APR-1M74 clinical drug product was performed by standard bioassay. Median Effective Dose 50 (ED50) with the 95% fiducial limits (95%FL) was estimated using Probit Analysis (SAS® 9.3). Also, Relative Potency (RP) was estimated by the methods described in European Pharmacopeia's Chapter 5.3.
Results: Five microgram of IgG neutralized 51.06% of the enzymatic activity of 250ng of Na-APR-1wt. An excellent dose response was also observed. ED50 of 14.15μg (95%FL = 10.47μg -- 18.93μg) and 11.46μg (95%FL = 4.86μg --27.42μg) was estimated for time 1 and 7 month post manufacture respectively. RP at 7 months was found to be 1.23 (95%FL = 0.792--1.917).
Conclusion: These preclinical results of the Na-APR-1 M74 vaccine lay the foundation for a Phase 1 Clinical Trial in USA and Brazil. This Na-APR-1 M74 vaccine will be subsequently combined with Necator americanus Glutathione transferase-1 (Na-GST-1) vaccine to form a multivalent human hookworm vaccine.