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. 2016 Aug 17:6:31458.
doi: 10.1038/srep31458.

Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

Maria Domina  1 , Veronica Lanza Cariccio  1 , Salvatore Benfatto  2 , Mario Venza  3 , Isabella Venza  3 , Erica Borgogni  4 , Flora Castellino  4 , Angelina Midiri  2 , Roberta Galbo  5 , Letizia Romeo  2 , Carmelo Biondo  2 , Vega Masignani  4 , Giuseppe Teti  3   6 , Franco Felici  7 , Concetta Beninati  1   2
Affiliations

Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

Maria Domina et al. Sci Rep. .

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.

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Conflict of interest statement

M.D., V.L.C., C. Be. and G.T. hold shares in Scylla Biotech S.r.l., which has submitted a patent application for the PROFILER technology. E.B and V.M. are full-time employees of GSK Vaccines.

Figures

Figure 1
Figure 1
Upper panels: diversity of the GNA2091-fHbp lambda phage-displayed library before and after affinity selection with the 12C1 mAb. Shown is the abundance of unique “natural frame” sequences in the unselected (panel A) and affinity-selected (panel B) libraries. Each point represents the number of unique sequences (vertical axis) displaying the number of copies indicated in the horizontal axis. Lower panels: Enrichment of “natural frame” GNA2091-fHbp phage inserts after affinity selection with the 12C1 mAb. Each graph reports the cumulative occurrence, per single aminoacid position, of predicted “natural frame” sequences before (panel C) and after (panel D) affinity selection with the 12C1 mAb. The horizontal axis reports the aa sequence corresponding to the GNA2091-fHbp fusion gene used to engineer the library. The occurrence of each “natural frame” sequence was normalized over the total number of sequences. A schematic representation of the GNA2091-fHbp fusion protein is reported below the horizontal axis.
Figure 2
Figure 2. Enrichment of fHbp fragments after affinity-selection of phage-displayed libraries and immunoreactivity of selected antigenic fragments with the 12C1 mAb.
Panel A shows the 30 most enriched fragments in the GNA2091-fHbp lambda library after selection, as identified by next generation sequencing. The small spots along the horizontal axis indicate the 23 contact points between mAb 12C1 and fHbp var 1, as previously determined by X-ray spectroscopy and the arrow identifies the shortest fragment. Panel B shows the most enriched fragments identified by Sanger sequencing of immunoreactive clones after immunoblotting in the same lambda library. Panel C shows the fragments identified by NGS sequencing in the filamentous phage library after affinity selection with mAb 12C1. Fragments with the same frequency were artificially separated for clarity of representation and are indicated by the curly brackets. A schematic representation of the GNA2091-fHbp fusion protein is reported below the horizontal axis. Panel D shows the results of an ELISA assay in which recombinant fHbp or its fragments, fused to GST, were immobilized on plastic wells followed by the addition of mAb 12C1 and enzyme-conjugated anti-mouse Ig. The ELISA assay was performed in triplicate and results are from one experiment representative of three producing similar data.
Figure 3
Figure 3. Competitive inhibition of binding of mAb 12C1 to fHbp by recombinant fHbp fragments.
Plates were coated with the GST-fHbp recombinant protein and reacted with limiting amounts of 12C1 mAb in the presence of increasing concentrations of the indicated fragments (GST was used as a negative control). Data are from one experiment representative of three producing similar results.
Figure 4
Figure 4. Cumulative “enrichment factor” values for each fHbp aa residue.
Enrichment factor was calculated as the ratio between the occurrence of the residue in the affinity-selected phage population and its occurrence in the unselected library. The horizontal axis reports the sequence of the GNA2091-fHbp protein. Red spots on the horizontal axis indicate the 23 contact points between mAb 12C1 and fHbp var 1, as previously determined by X-ray spectroscopy. The arrows indicate points of sudden increase in enrichment factor values. A schematic representation of the GNA2091-fHbp fusion protein is reported below the horizontal axis.
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