Rapid profiling of the antigen regions recognized by serum antibodies using massively parallel sequencing of antigen-specific libraries

PLoS One. 2014 Dec 4;9(12):e114159. doi: 10.1371/journal.pone.0114159. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / blood
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology
  • Antigens, Bacterial / genetics*
  • Antigens, Bacterial / immunology
  • Antigens, Bacterial / isolation & purification
  • Epitopes / genetics*
  • Epitopes / immunology
  • Epitopes / isolation & purification
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • Meningitis, Meningococcal / blood
  • Meningitis, Meningococcal / immunology*
  • Meningitis, Meningococcal / microbiology
  • Neisseria meningitidis / immunology
  • Neisseria meningitidis / pathogenicity
  • Peptide Library

Substances

  • Antibodies, Monoclonal
  • Antigens, Bacterial
  • Epitopes
  • Peptide Library

Grants and funding

Funding for this work came from CLUSTER MEDINTECH (Project CTN01_00177_962865), PANLab (PON a3_00166), King Abdullah University of Science and Technology (Award KUK I1-012-43), Progetti di Ricerca di Rilevante Interesse Nazionale (20108XYHJS), and the Flagship EPIGEN. All funding for this project is gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.