The overall goal of this project is to use DNA microarraying technology as a gene discovery tools for the identification of vaccine candidates in Trypanosoma cruzi. DNA microarrays provide the means to monitor the expression of many, or all genes in a genome, under different growth conditions, during different development stages, or in different tissues. Our specific application of this technology will be to identify upon the reasoning that the protein products of such genes would be among the first to reach the class I MHC processing and presentation pathway and thus would serve as early indicators to the immune system of the infection status of host cells. The genomic libraries generated for gene in genetic immunization project described in Project 1 will serve as the source material for generating a random, genomic DNA array from T. cruzi. The initial microarray will consist of a minimal 1X representation of the T. cruzi genome numbering approximately 20,000 clones. This array will be probed with cDNAs isolated from trypomastigotes prior to infection of host cells, with trypomastigotes in the process of converting to amastigotes, and fully differentiated and dividing amastigotes in host cells. The genes shown to be significantly up-regulated during the conversion to amastigotes will then be identified from the array and end- sequenced to attempt to determine the identity or function of the encoded protein product. Novel genes of unknown function or with properties which might be consistent with serve as good vaccine will be tested individually for the ability to confer resistance to infection in a murine model of T. cruzi infection. Genes which provide a significant degree of protection will then be used for further vaccine trials and optimization in the mouse (Project 3) and in Project 4 to determine the response pattern of humans with chronic infection to the proteins encoded by these genes.
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