BECAS FULBRIGHT / FUNDACIÓN BUNGE Y BORN / FUNDACIÓN WILLIAMS
Trypanosoma cruzi novel antigen discovery by positional scanning of synthetic peptide libraries with pathogen-specific T-cell lines
Gonzalo Raúl Acevedo
Torrey Pines Institute for Molecular Studies
Chagas disease, caused by infection with the hemiflagellate protozoan Trypanosoma cruzi, is a major health issue in its endemic area, which comprises South, Central and southern North America. In the last few years, these burdens have been expanded due to global migration patterns, raising concern in Europe, Japan and the USA. The disease has an early acute infection phase, with little or no symptoms, followed by a chronic stage that can remain asymptomatic for decades, or develop into cardiac or digestive pathologies, which affects drastically the patient’s life quality and leads ultimately to death. Mechanisms proposed to explain pathogenesis have varied in time, but it is broadly accepted now that both persistence of the infection and an imbalanced inflammatory response generated against the parasite, but which also present an important self-reactive component, are the main causes for the symptoms observed. Although a unicellular organism, T. cruzi is of an enormous complexity and a very particular -and even sometimes, cryptic- biology, hindering the search of potentially protective and/or therapeutic vaccine candidates by most traditional approaches. Despite research in the field is profuse and active, efforts towards the discovery of T. cruzi antigens useful for vaccine design have been, thus far, of very limited success, for the reasons previously discussed. Our line of investigation proposes a novel approach that may bring us closer to that goal, by using information gathered from infected patients’ immune memory, by means of a high-throughput strategy. Briefly, samples of peripheral blood mononuclear cells will be used to generate T-cell lines specifically reactive against T. cruzi. Amplification and selection of such cells is accomplished by an in vitro stimulation, expansion and challenge protocol designed and adjusted by our research team. T. cruzi-specific cell pools generated this way will be cloned by limiting dilution and further expanded. The resulting specific T-cell clonal lines will be then used to scan combinatorial synthetic peptide libraries, consisting of 200 rationally ordered decapeptide mixes, encompassing all the possible decamer peptidic sequences (over ten trillions). A biometrical algorithm will be applied to deconvolute the resulting output of peptide mixes that activate T-cell response, allowing us to “read” individual reactive peptide sequences out of this group. Finally, individual decamers will be tested for T-cell reactivity using the same previously generated T-cell lines to confirm their antigenicity. It is important to note that antigens obtained by this method are, by definition, immunogenic, given they can stimulate a response from specific T-cells generated in vivo, by the patients’ own, natural mechanisms of immunity. Also, the high-throughput nature of this approach helps overcoming the difficulty presented by a vast, complex proteome such as the one of T. cruzi. This strategy has previously been applied with successful results to other pathogens by Drs. Pinilla’s and Judkowski’s research team, and their laboratories in Torrey Pines Institute for Molecular Studies count with exceptional facilities for the development of this project. Dr. Pinilla has vast experience in the field of combinatorial chemistry and its application to the discovery of epitopes and drugs. Dr. Judkowski specializes in antigen discovery and immune modulation, and is currently involved in projects regarding T. cruzi antigens identification and characterization. Collaboration between our group at INGEBI and theirs at TPIMS has been on for years now, and Drs. Pinilla and Judkowski have provided most valuable expertise for the design and optimization of cell culture protocols to be employed. They have also agreed to provide the forementioned decapeptide libraries, and the required equipment and facilities necessary for the purpose of this investigation.