New tools for NTD vaccines: A case study of quality control assays for product development of the human hookworm vaccine Na-APR-1_M74

Abstract

Na-APR-1_M74 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-1_wt) in the gut of the hookworm, thereby depriving the worm of its ability to digest its blood meal. Previously, canines immunized with Na-APR-1_M74 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-1_wt in vitro, thereby providing proof of concept of Na-APR-1_M74 as a vaccine antigen. The mutated version, Na-APR-1_M74, 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-1_M74 IgG. As little as 0.99 μg of recombinant Na-APR-1_M74 could induce anti Na-APR-1_M74 IgG in mice that were capable of inhibiting Na-APR-1_wt-mediated digestion of a peptide substrate by 89%. In the absence of enzymatic activity of Na-APR-1_M74 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-1_wt was demonstrated by inhibition of enzymatic activity, and competitive binding with unlabelled pepstatin A. BDP also bound to Na-APR-1_M74 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-1_M74 as it proceeds through the clinical development as part of the Human Hookworm Vaccine pipeline.

Keywords:

• aspartic protease
• fluorescence polarization
• hookworm
• Necator americanus
• vaccine

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This research was funded by the Sabin Vaccine Institute with a grant awarded by the Bill and Melinda Gates Foundation. MSP was supported by an NHMRC Early Career Fellowship. AL is an NHMRC Senior Principal Research Fellow.