DEVELOPMENT OF A WEBTOOL FOR INTERACTOME­SEQUENCING DATA ANALYSIS AND IDENTIFICATION OF H. PYLORI EPITOPES RESPONSIBLE FOR HOST IMMUNO­RESPONSE MODULATION.

To elucidate the molecular mechanisms involved in persistency/latency of the H. pylori infection or in its progression towards serious diseases, it is necessary to analyse 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 aim of this work is the identification of H. pylori epitopes responsible for host immuno­response modulation through: a discovery­driven approach that couples “phage display” and deep sequencing (interactome-sequencing) and the development of a specific webtool for interactome-sequencing data analysis. We used this approach to identify novel antigens by screening gDNA libraries created from the pathogen’s genome, directly with sera from infected patients. Two genomic phage display libraries from 26695 and B128 H. pylori strains have been constructed by using ß­lactamase ORF selection vector. Genomic DNA was sonicated, fragments cloned into the filtering vector, after transformation libraries of 1x106 clones were obtained and sequenced by Illumina technology. More than 93% of Hp CDSs were represented in the phage genomic libraries therefore being representative of the whole H. pylori antigenic ORFeome. A webtool for interactome-sequencing data analysis was developed and used to identify the H. pylori antigens/epitopes which could be considered specific for infection progression towards three different pathological outcomes. Putative antigens were selected from libraries using sera from patients affected by: i) gastric adenocarcinoma; ii) autoimmune gastritis; iii) MALT lymphoma. The results, obtained thanks to the new interactome sequencing pipeline developed, show that the diversity of the libraries after selection is significantly reduced. Furthermore, individual ranks, for each infection condition, have been compared highlighting the pattern of putative antigens, shared by all the conditions, and some that can distinguish the different stages of infection. One of this new antigens, that seems to be specific for infection progression towards more serious diseases, has been successfully validated through ELISA assay on a wide number of sera from patients. Other more specific antigens identified by our approach and by the application of the new data analysis pipeline here described are in validation.