Neutralizing Antibodies; Hookworm; Recombinant Vaccine; Hemoglobin Digestion
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 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 a new vaccine which is currently under pre-clinical development. This vaccine is an Alhydrogel® adjuvanted vaccine containing the mutant form of the Na-APR-1wt. Potent antibodies (IgG) will neutralizing Na-APR-1wt and 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.
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 in-vitro Cathepsin-D protease assay. Intra-Operator Precision and Ruggedness (inter-operator precision) of the assay were assessed by calculating the percent coefficient of variation (%CV). 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.
Five microgram of purified IgG neutralized 51.06% of the enzymatic activity of 250ng of Na-APR-1wt. This assay generated an excellent inter- and intra-operator precision with the %CVs < 20. 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).
These neutralizing antibody and potency assay results of Na-APR-1 M74 vaccine lay the foundation for a Phase 1 Clinical Trial in USA and Brazil. Subsequently, this vaccine will be combined with Necator americanus Glutathione transferase-1 (Na-GST-1) vaccine to form a multivalent human hookworm vaccine.
Jariwala, Amar R.; Chen, Xi; Pearson, Mark S.; Keegan, Brian; Brelsford, Jill B.; Plieskatt, Jordan L.; Zhan, Bin; Loukas, Alex; Hotez, Peter; and Bethony, Jeffrey M., "Vaccination of BALB/c Mice with the Na-APR-1 M74 Hookworm Vaccine Generates Neutralizing Antibodies and a Potent Immune Response" (2013). GW Research Days 2013. Paper 13.