From a technical standpoint, our data confirm previous studies showing that next generation sequencing can improve the resolution of methods using phage or ribosome display libraries[19],[20],[21],[22],[23],[24]. generation sequencing can significantly empower the analysis of antigen-specific libraries by permitting 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 selecting, the new technology (namedPhage-basedRepresentationOF Immuno-LigandEpitopeRepertoire or PROFILER) provides superior resolution in epitope recognition. 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 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 indication 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 induced by any immunizing antigen are not equally directed against the various portions of the antigen molecule[2]. Often, within an antigen, you will find 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 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 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 Rabbit polyclonal to ABHD12B 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 Lasmiditan of vaccine-induced antibody repertoires would be useful to lead rational antigen design and selection of appropriate adjuvants. Indeed, the ability of certain adjuvants to broaden the antibody repertoire and to provide extended protection of antigen regions targeted by polyclonal responses is becoming progressively obvious[5],[6]. Moreover, because the spectrum of antibody specificities varies with age and physiology, repertoire profiles may be useful to specifically tailor vaccine formulations 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 circulation 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]. Lasmiditan These techniques are, however, labor-intensive, time consuming and can identify only a limited quantity 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, that we named PROFILER (forPhage-basedRepresentationOF ImmunoLigandEpitopeRepertoire), Lasmiditan generates a high-resolution profile of antigen-specific antibody repertoires in less than two days. As a model antigen we used here NadA[17], one of the 4 components of the 4CMenB anti-meningococcal vaccine (Bexsero). We first generated a lambda phage displayed library in which individual phage particles display on their surface different NadA fragments of various lengths, followed by selection with sera from Bexsero-immunized volunteers and analysis through the Illumina MiSeq platform. == Results == == Library sequencing == The gene-specific library that was used in this study is composed of phage particles transporting, as recombinant inserts, fragments of thenadAgene of group BN. meningiditis(Fig. 1). Since the sequence of this gene is known, the entire sequence of the inserts can be inferred from your relatively short (150 bp) reads obtained at each end of insert-containing amplicons using Illumina MiSeq paired end sequencing. After quality control of the sequencing output and demultiplexing of the data, FASTQ raw Lasmiditan files were generated by the Illumina MiSeq sequencer.