To accelerate and improve epitope identification, we have employed massive sequencing of phage-displayed antigen-specific libraries using the Illumina MiSeq platform. 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 guideline 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. Introduction Measuring the total concentration of antigen-specific serum antibodies is usually a fundamental step in the diagnosis of infectious and PX-866 (Sonolisib) autoimmune diseases and is used to monitor the efficacy of vaccination, which is the most powerful tool to preserve human health and to reduce the costs of medical care. However, a purely quantitative analysis of serum antibodies is usually a poor indicator of the complexity of the antibody response, which involves the activation of thousands of different B cell clones and the secretion of a wide variety of antibodies, each directed against a different region of the immunizing antigen(s) [1]. For reasons that are only partially understood, the antibodies PX-866 (Sonolisib) induced by any immunizing antigen are not equally directed against the various portions of the antigen molecule [2]. Often, within an antigen, there are regions that are strongly reactive with antibodies (i.e. immunodominant regions) flanked by domains that seem to be partially or completely ignored by the immune system. Anti-microbial vaccination induces the production DFNB39 of a great variety of antigen-specific antibodies, only a minority of which possesses the ability to protect against target infections [2], [3]. In other words, only PX-866 (Sonolisib) certain antibodies – those directed against specific warm spots of the antigen molecule – have immunoprotective activities. Therefore, it has been proposed that pathogens sometimes adopt the strategy of incorporating, in the context of their virulence factors, immunodominant regions that function as decoys by preventing the immune system from targeting the hot spots [3]. Although the exact nature and function of such decoy epitopes are still ill-defined, it is well established that selective removal of immunodominant, non-protective regions can boost the ability of the antigen to protect against contamination after immunization [4]. In view of these considerations it would be helpful, particularly in the course of preclinical studies and clinical trials involving vaccines, to establish whether the immune response is usually optimally targeted against the antigenic residues crucial for immune-mediated protection. To this end, a method capable of providing a detailed analysis of the fine specificity of vaccine-induced antibody repertoires would be useful to guide rational antigen design and selection of appropriate adjuvants. Indeed, the ability of certain adjuvants to broaden the antibody repertoire and to provide extended coverage of antigen regions targeted by polyclonal responses is becoming increasingly clear [5], [6]. Moreover, because the spectrum of antibody specificities varies with age and physiology, repertoire profiles may be useful to specifically tailor vaccine formulations PX-866 (Sonolisib) for different age groups and for high-risk populations [7], [8], [9]. The recent development of high-throughput methods for repertoire data collection – from single cell mass spectroscopy and multicolor flow cytometry to massively parallel sequencing of immunoglobulin transcripts – offers today an opportunity to analyze large samples of lymphocyte repertoires [10], [11], [12]. Although these methods provide extensive information regarding the diversity of clonotypes and immunoglobulin gene usage, they have limited usefulness, by their nature, in sampling the antibody repertoire in terms of epitope specificity. Libraries consisting of phage particles or cells expressing on their surface peptides of various lengths have been widely used in epitope mapping [13], [14], [15], [16]. These techniques are, however, labor-intensive, time consuming and can identify only a limited number of epitopes. We describe here a novel approach, based on the combined use of phage-displayed antigen-specific libraries and massive parallel sequencing of the entire populace of affinity-selected phages. This approach,.