TRYPANOSOMA CRUZI:GENE DISCOVERY USING CDNA MICROARRAYS

南美锥虫病仍然是中美洲和南美洲发病率和死亡率的重要原因，估计有1亿人处于T。克氏感染 目前的南美锥虫病控制措施使用的是几十年前的技术。 由于对自身免疫在恰加斯病病理学中的重要性存在分歧，以及基因发现方法效率低下，疫苗的开发尤其缓慢。这项拟议中的研究将利用三个领域的最新进展来加速T。cruzi疫苗的开发。 这些发现表明，CD 8 + T细胞介导的免疫应答对T细胞免疫的结果至关重要。cruzi感染，基因疫苗可以增强T. cruzi感染的动物，DNA微阵列可用于全基因组表达分析和基因发现。 本项目的长期目标是确定新的T。克氏锥虫病多组分基因疫苗的候选疫苗。 具体地，申请人提出从来自T. cruzi基因组表达文库。 使用双色荧光方案，将用在锥鞭毛体至无鞭毛体分化期间的不同时间点从寄生虫制备的标记的cDNA探针的混合物探测微阵列，以使得能够同时成对比较阵列中代表的基因的相对表达水平。 将使用阵列扫描仪测定阵列上每个点的每个荧光标记产生的信号强度。 将选择代表在锥鞭毛体至无鞭毛体分化早期上调的基因的克隆进行测序。基于序列分析的结果，将测试上调基因的子集在小鼠中引发保护性免疫应答的能力。 预计该项目将产生至少20-30种新型候选疫苗。

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.