Identification of H. pylori epitopes responsible for host immuno-response modulation through ORF-filtered phage display libraries and Interactome-Sequencing.

To elucidate the main molecular mechanisms involved in persistency/latency of the H. pylori infection or in its progression towards more serious diseases, it is necessary to analyze the host pathogen interaction in vivo. The circulating antibody repertoire represents an important source of diagnostic information, serving as biomarker to provide a “disease signature”. The so called epitome analysis can lead to the identification of H. pylori epitopes responsible for host immuno-response modulation. Here we propose an unbiased discovery-driven approach that couples emerging technologies “phage display” and deep sequencing. This approach provides a simple procedure for identifying novel antigens, by screening gDNA libraries, created from the pathogen genome, directly with sera from infected patients. Three phage display libraries from three H. pylori strains (HP-26695, HP-B128 and HP-SS1) have been constructed by using ß-lactamase ORF selection vectors (Di Niro et al. NAR 2010). Genomic DNA was sonicated, fragments cloned into the ORF filtering vector and after transformation libraries of 1x106 clones were obtained. Libraries were then sequenced by 454 technology showing that 93% of the CDSs were represented for each strain therefore being representative of the whole H. pylori antigenic ORFeome. Finally putative antigens were selected from libraries using sera from patients affected by H. pylori presenting increasing degrees of infection: i) autoimmune gastritis and pernicious anemia; ii) gastric adenocarcinoma; iii) MALT lymphoma. Two cycles of selection have been performed and mini-libraries of selected phages have been collected and sequenced. The results show that the diversity of the libraries obtained after selection on patients’ sera is significantly reduced. Furthermore, individual ranks, for each infection condition, have been compared to each other highlighting the pattern of putative antigens shared by all the conditions and potentially some that can distinguish the different stages of infection.