Abstract
We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete “hot spots” with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.
Highlights
Phage vectors that can tolerate expression of large protein domains[10,11]
The ability of this system to express a wide variety of protein domains spanning several hundred residues, as well as oligopeptides[10,14], renders it ideally suited for epitope mapping
We have recently combined the efficiency of this antigen display system with the power of generation sequencing into a platform allowing the characterization of antibody repertoires in polyclonal antibody mixtures such as serum samples from vaccinated individuals
Summary
Construction and characterization of an antigen-specific lambda phage-displayed library. GST-FrN produced slight inhibition, which is compatible with the known presence of 3 contact points between fHbp and mAb 12C1 in this fragment[16] These data suggested that the long fragments identified in the lambda display library, such as FrC, might better reconstitute the complex conformational epitope recognized by mAb12C1, relative to the considerably shorter fragments (e.g. FrS) identified in a previous study[16]. Our data suggest that, by virtue of its ability to detect overrepresented residues in affinity-selected libraries, PROFILER may provide useful clues for the functional characterization of the epitope This stems from the ability of generation sequencing to identify thousands of different inserts, thereby empowering phage display-based methods. The PROFILER technology, by virtue of its ability to identify large numbers of unique antigen fragments, can provide insights into the structural and functional properties of mAb epitopes and may, be useful in simplifying further structural analysis and in guiding the construction of mutagenized, second-generation libraries
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