title: Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform.
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.
authors.affiliation: Scylla Biotech Srl, Messina, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Domina
authors.affiliation: Scylla Biotech Srl, Messina, Italy.
authors.forename: Veronica
authors.initials: V
authors.lastname: Lanza Cariccio
authors.affiliation: Department of Human Pathology, University of Messina, Messina, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Benfatto
authors.affiliation: Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Venza
authors.affiliation: Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Venza
authors.affiliation: GSK Vaccines, Siena, Italy.
authors.forename: Erica
authors.initials: E
authors.lastname: Borgogni
authors.affiliation: GSK Vaccines, Siena, Italy.
authors.forename: Flora
authors.initials: F
authors.lastname: Castellino
authors.affiliation: Department of Human Pathology, University of Messina, Messina, Italy.
authors.forename: Angelina
authors.initials: A
authors.lastname: Midiri
authors.affiliation: Department of Biological, Chemical and Environmental Sciences, University of Messina, Messina, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Galbo
authors.affiliation: Department of Human Pathology, University of Messina, Messina, Italy.
authors.forename: Letizia
authors.initials: L
authors.lastname: Romeo
authors.affiliation: Department of Human Pathology, University of Messina, Messina, Italy.
authors.forename: Carmelo
authors.initials: C
authors.lastname: Biondo
authors.affiliation: GSK Vaccines, Siena, Italy.
authors.forename: Vega
authors.initials: V
authors.lastname: Masignani
authors.affiliation: Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy.|Charybdis Vaccines Srl, Messina, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Teti
authors.affiliation: Department of Biosciences and Territory, University of Molise, Pesche, Isernia, Italy.
authors.forename: Franco
authors.initials: F
authors.lastname: Felici
authors.affiliation: Scylla Biotech Srl, Messina, Italy.|Department of Human Pathology, University of Messina, Messina, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Beninati
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D010582:Bacteriophage lambda / Q000235:genetics*; D000939:Epitopes / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D051379:Mice; D019151:Peptide Library
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D000939:Epitopes; D019151:Peptide Library