| description |
In this work, we explored the potential of the newly described PROFILER technology, based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used mAb 31E10/E7, a novel antibody directed against Neisserial Heparin-Binding Antigen (NBHA), an important component of the anti-group B meningococcus Bexsero® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7 followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule in ELISA-inhibition experiments using mAb 31E10/E7. The binding properties of mAb 31E10/E7were also tested against a panel of overlapping recombinant fragments derived from the pep2 vaccine variant, and against a set of chemically synthetized peptides covering the 10 most frequent variants of NHBA. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of NHBA-mAb31E10/E7 complex was performed. All the approaches used were compatible with PROFILER-based epitope identification. Collectively, these results indicate that PROFILER technology can reliably identify epitope-containing antigenic fragments and requires less work, time and reagents compared with the immunoscreening/colony picking method. |