TRYPANOSOMA CRUZI:GENE DISCOVERY USING CDNA MICROARRAYS

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Chagas' disease remains a significant cause of morbidity and mortality in Central and South America where an estimated 100 million people are at risk for T. cruzi infection. Current control measures for Chagas' disease utilize decades-old technologies. Vaccine development in particular has been slowed by disagreement about the significance of autoimmunity in Chagas' disease pathology and by inefficient methods of gene discovery. The proposed study will utilize recent advances in three areas to accelerate gene discovery efforts for T. cruzi vaccine development. These are the findings that CD8+ T cell-mediated immune responses are critical to the outcome of T. cruzi infections, that genetic vaccination can potentiate protective CTL responses in T. cruzi infected animals, and that DNA microarrays can be used for genome-wide expression analyses and gene discovery. The long-term objective of this project is to identify new T. cruzi vaccine candidates for use in a multi- component genetic vaccine for Chagas' disease. Specifically, the applicants propose to create multiple, high density microarrays of DNAs from 10,000 random, ORF-containing clones from a T. cruzi genomic expression library. Using a two color fluorescence scheme the microarrays will be probed with mixtures of labeled cDNA probes prepared from parasites at different time points during trypomastigote to amastigote differentiation to enable simultaneous, pairwise comparisons of the relative expression levels for the genes represented in the arrays. The signal intensities produced by each fluor tag for each spot on the arrays will be determined using an array scanner. Clones representing genes upregulated early in trypomastigote to amastigote differentiation will be selected for sequencing. Based on the results of the sequence analyses a subset of the upregulated genes will be tested for their ability to elicit protective immune responses in mice. It is anticipated that this project will yield a minimum of 20-30 novel vaccine candidates.