Pearson, Mark S., Jariwala, Amar R., Abbenante, Giovanni, Plieskatt, Jordan, Wilson, David, Bottazzi, Maria Elena, Hotez, Peter J., Keegan, Brian, Bethony, Jeffrey M., and Loukas, Alex (2015) New tools for NTD vaccines: a case study of quality control assays for product development of the human hookworm vaccine Na-APR-1M74. Human Vaccines & Immunotherapeutics, 11 (5). pp. 1251-1257.

PDF (Published Version) - Published Version Restricted to Repository staff only

 

13

3

Abstract

Na-APR-1M74 is an aspartic protease that is rendered enzymatically inactive by site-directed mutagenesis and is a candidate antigen component in the Human Hookworm Vaccine. The mutant protease exerts vaccine efficacy by inducing antibodies that neutralize the enzymatic activity of wild type enzyme (Na-APR-1wt) in the gut of the hookworm, thereby depriving the worm of its ability to digest its blood meal. Previously, canines immunized with Na-APR-1M74 and challenged with Ancylostoma caninum were partially protected against hookworm challenge infection, especially from the loss in hemoglobin observed in control canines and canine immunoglobulin (Ig) G raised against Na-APR-1 was shown to inhibit the enzymatic activity of Na-APR-1wt in vitro, thereby providing proof of concept of Na-APR-1M74 as a vaccine antigen. The mutated version, Na-APR-1M74, was then expressed at the cGMP level using a Nicotiana benthamiana expression system (Fraunhofer, CMB, Delaware, MD), formulated with Alhydrogel®, and used to immunize mice in a dose-ranging study to explore the enzyme-neutralizing capacity of the resulting anti- Na-APR-1M74 IgG. As little as 0.99 μg of recombinant Na-APR-1M74 could induce anti Na-APR-1M74 IgG in mice that were capable of inhibiting Na-APR-1wt-mediated digestion of a peptide substrate by 89%. In the absence of enzymatic activity of Na-APR-1M74 as a surrogate marker of protein functionality, we developed an assay based on the binding of a quenched fluorescence-labeled inhibitor of aspartic proteases, BODIPY-FL pepstatin A (BDP). Binding of BDP in the active site of Na-APR-1wt was demonstrated by inhibition of enzymatic activity, and competitive binding with unlabelled pepstatin A. BDP also bound to Na-APR-1M74 which was assessed by fluorescence polarization, but with an ~50-fold reduction in the dissociation constant. Taken together, these assays comprise a "toolbox" that could be useful for the analyses of Na-APR-1M74 as it proceeds through the clinical development as part of the Human Hookworm Vaccine pipeline.

Actions (Repository Staff Only)

Item Control Page