title: New tools for NTD vaccines: A case study of quality control assays for product development of the human hookworm vaccine Na-APR-1M74.
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-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-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-1w t-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.
authors.affiliation: a Centre for Biodiscovery and Molecular Development of Therapeutics; Australian Institute of Tropical Health and Medicine; James Cook University; Cairns , Queensland , Australia.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Pearson
authors.forename: Amar R
authors.initials: AR
authors.lastname: Jariwala
authors.forename: Giovanni
authors.initials: G
authors.lastname: Abbenante
authors.forename: Jordan
authors.initials: J
authors.lastname: Plieskatt
authors.forename: David
authors.initials: D
authors.lastname: Wilson
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Bottazzi
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hotez
authors.forename: Brian
authors.initials: B
authors.lastname: Keegan
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Bethony
authors.forename: Alex
authors.initials: A
authors.lastname: Loukas
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage; D000536:Aluminum Hydroxide / Q000008:administration & dosage; D000722:Ancylostoma / Q000201:enzymology* / Q000276:immunology; D000724:Ancylostomiasis / Q000517:prevention & control*; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood; D057134:Antibodies, Neutralizing / Q000097:blood; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology*; D057055:Aspartic Acid Proteases / Q000235:genetics / Q000276:immunology*; D055808:Drug Discovery / Q000379:methods; D005260:Female; D006725:Hookworm Infections / Q000517:prevention & control*; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D000909:Antibodies, Helminth; D057134:Antibodies, Neutralizing; D000947:Antigens, Helminth; D050505:Mutant Proteins; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D000536:Aluminum Hydroxide; D057055:Aspartic Acid Proteases
keywords: Necator americanus; aspartic protease; fluorescence polarization; hookworm; vaccine