title: Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries.
abstract: There is a need for techniques capable of identifying the antigenic epitopes targeted by polyclonal antibody responses during deliberate or natural immunization. Although successful, traditional phage library screening is laborious and can map only some of the epitopes. To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. This enabled us to precisely identify the regions of a model antigen, the meningococcal NadA virulence factor, targeted by serum antibodies in vaccinated individuals and to rank hundreds of antigenic fragments according to their immunoreactivity. We found that next generation sequencing can significantly empower the analysis of antigen-specific libraries by allowing simultaneous processing of dozens of library/serum combinations in less than two days, including the time required for antibody-mediated library selection. Moreover, compared with traditional plaque picking, the new technology (named Phage-based Representation OF Immuno-Ligand Epitope Repertoire or PROFILER) provides superior resolution in epitope identification. PROFILER seems ideally suited to streamline and guide rational antigen design, adjuvant selection, and quality control of newly produced vaccines. Furthermore, this method is also susceptible to find important applications in other fields covered by traditional quantitative serology.
authors.affiliation: SPGMB Department, University of Messina, Messina, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Domina
authors.affiliation: SPGMB Department, University of Messina, Messina, Italy.
authors.forename: Veronica
authors.initials: V
authors.lastname: Lanza Cariccio
authors.affiliation: SPGMB Department, University of Messina, Messina, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Benfatto
authors.affiliation: SPGMB Department, University of Messina, Messina, Italy.
authors.forename: Deborah
authors.initials: D
authors.lastname: D'Aliberti
authors.affiliation: SSMCSO Department, University of Messina, Messina, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Venza
authors.affiliation: Novartis Vaccines and Diagnostics, Siena, Italy.
authors.forename: Erica
authors.initials: E
authors.lastname: Borgogni
authors.affiliation: Novartis Vaccines and Diagnostics, Siena, Italy.
authors.forename: Flora
authors.initials: F
authors.lastname: Castellino
authors.affiliation: SPGMB Department, University of Messina, Messina, Italy.
authors.forename: Carmelo
authors.initials: C
authors.lastname: Biondo
authors.affiliation: Department of Physics, Sapienza University, Rome, Italy.
authors.forename: Daniel
authors.initials: D
authors.lastname: D'Andrea
authors.affiliation: Department of Physics, Sapienza University, Rome, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Grassi
authors.affiliation: Department of Physics, Sapienza University, Rome, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Tramontano
authors.affiliation: SPGMB Department, 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: SPGMB Department, University of Messina, Messina, Italy; Charybdis Vaccines Srl, Messina, Italy.
authors.forename: Concetta
authors.initials: C
authors.lastname: Beninati
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000097:blood / Q000235:genetics / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000939:Epitopes / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008585:Meningitis, Meningococcal / Q000097:blood / Q000276:immunology* / Q000382:microbiology; D009345:Neisseria meningitidis / Q000276:immunology / Q000472:pathogenicity; D019151:Peptide Library
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes; D019151:Peptide Library