title: Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries.
abstract: We explore here the potential of a newly described technology, which is named PROFILER and is 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 a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a 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 against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.
authors: Scylla Biotech Srl, Messina, Italy.
authors: Maria
authors: M
authors: Domina
authors: Scylla Biotech Srl, Messina, Italy.
authors: Veronica
authors: V
authors: Lanza Cariccio
authors: Department of Human Pathology, University of Messina, Messina, Italy.
authors: Salvatore
authors: S
authors: Benfatto
authors: Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy.
authors: Mario
authors: M
authors: Venza
authors: Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy.
authors: Isabella
authors: I
authors: Venza
authors: GSK Vaccines, Siena, Italy.
authors: Danilo
authors: D
authors: Donnarumma
authors: GSK Vaccines, Siena, Italy.
authors: Erika
authors: E
authors: Bartolini
authors: GSK Vaccines, Siena, Italy.
authors: Erica
authors: E
authors: Borgogni
authors: GSK Vaccines, Siena, Italy.|Department of Life Sciences, University of Siena, Siena, Italy.
authors: Marco
authors: M
authors: Bruttini
authors: GSK Vaccines, Siena, Italy.
authors: Laura
authors: L
authors: Santini
authors: Department of Human Pathology, University of Messina, Messina, Italy.
authors: Angelina
authors: A
authors: Midiri
authors: Department of Chemical, Biological and Pharmaceutical Sciences, University of Messina, Messina, Italy.
authors: Roberta
authors: R
authors: Galbo
authors: Department of Human Pathology, University of Messina, Messina, Italy.
authors: Letizia
authors: L
authors: Romeo
authors: Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy.
authors: Francesco
authors: F
authors: Patanè
authors: Department of Human Pathology, University of Messina, Messina, Italy.
authors: Carmelo
authors: C
authors: Biondo
authors: GSK Vaccines, Siena, Italy.
authors: Nathalie
authors: N
authors: Norais
authors: GSK Vaccines, Siena, Italy.
authors: Vega
authors: V
authors: Masignani
authors: Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy.|Charybdis Vaccines Srl, Messina, Italy.
authors: Giuseppe
authors: G
authors: Teti
authors: Department of Biosciences and Territory, University of Molise, Pesche, Isernia, Italy.
authors: Franco
authors: F
authors: Felici
authors: Scylla Biotech Srl, Messina, Italy.|Department of Human Pathology, University of Messina, Messina, Italy.
authors: Concetta
authors: C
authors: Beninati
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology*; D003429:Cross Reactions; D018604:Epitope Mapping / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D038541:Neisseria meningitidis, Serogroup B / Q000276:immunology; D019151:Peptide Library; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; C000620214:NHBA protein, Neisseria meningitidis; D019151:Peptide Library; D011994:Recombinant Proteins