Utility of the Trypanosoma cruzi sequence database for identification of potential vaccine candidates by in silico and in vitro screening

Glycosylphosphatidylinositol (GPI)-anchored proteins are abundantly expressed in the infective and intracellular stages of Trypanosoma cruzi and are recognized as antigenic targets by both the humoral and cellular arms of the immune system. Previously, we demonstrated the efficacy of genes encoding GPI-anchored proteins in eliciting partially protective immunity to T. cruzi infection and disease, suggesting their utility as vaccine candidates. For the identification of additional vaccine targets, in this study we screened the T. cruzi expressed sequence tag (EST) and genomic sequence survey (GSS) databases. By applying a variety of web-based genomemining tools to the analysis of similar to2,500 sequences, we identified 348 (37.6%) EST and 260 (17.4%) GSS sequences encoding novel parasite-specific proteins. Of these, 19 sequences exhibited the characteristics of secreted and/or membrane-associated GPI proteins. Eight of the selected sequences were amplified to obtain genes TcG1, TcG2, TcG3, TcG4, TcG5, TcG6, TcG7, and TcG8 (TcG1-TcG8) which are expressed in different developmental stages of the parasite and conserved in the genome of a variety of T. cruzi strains. Flow cytometry confirmed the expression of the antigens encoded by the cloned genes as surface proteins in trypomastigote and/or amastigote stages of T. cruzi. When delivered as a DNA vaccine, genes TcG1-TcG6 elicited a parasite-specific antibody response in mice. Except for TcG5, antisera to genes TcG1-TcG6 exhibited trypanolytic activity against the trypomastigote forms of T. cruzi, a property known to correlate with the immune control of T. cruzi. Taken together, our results validate the applicability of bioinformatics in genome mining, resulting in the identification of T. cruzi membrane-associated proteins that are potential vaccine candidates.