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

Background 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 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”. Objectives The aim of this work is the identification of H. pylori epitopes responsible for host immuno­response modulation though a discovery­driven approach that couples “phage display” and deep sequencing. Methods We used an approach 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 (HP­26695, HP­B128, HP­SS1) have been constructed by using ß­lactamase ORF selection vectors (Di Niro et al., 2010). Genomic DNA was sonicated, fragments cloned into the filtering vector, after transformation libraries of 1x106 clones were obtained and sequenced by 454 technology. Conclusions More than 93% of HP CDSs were represented in the phage genomic library therefore being representative of the whole H. pylori antigenic ORFeome. 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. The results show that the diversity of the libraries obtained 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.