ena-STUDY-UNIVERSITY OF MESSINA-24-09-2014-15:51:33:086-513
Rapid profiling of the Neisserial NadA antigen regions  recognized by serum antibodies using massively parallel  sequencing of antigen-specific libraries
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 antigen32 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.
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 antigen32 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.