TRYPANOSOMA CRUZI:GENE DISCOVERY USING CDNA MICROARRAYS

恰加斯病仍然是中美洲和南美洲发病和死亡的一个重要原因，估计有1亿人面临感染克氏锥虫的风险。目前对恰加斯病的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中的重要性存在分歧，以及基因发现方法效率低下，疫苗开发尤其受到阻碍。拟议的研究将利用三个领域的最新进展来加速克氏锥虫疫苗开发的基因发现工作。这些研究结果表明，CD8+ T细胞介导的免疫反应对克氏锥虫感染的结果至关重要，遗传疫苗可增强克氏锥虫感染动物的保护性CTL反应，DNA微阵列可用于全基因组表达分析和基因发现。该项目的长期目标是确定新的克氏锥虫候选疫苗，用于恰加斯病的多组分遗传疫苗。具体来说，申请人建议从克氏T. crozi基因组表达文库中随机提取10,000个含有orf的克隆，创建多个高密度dna微阵列。使用双色荧光方案，将从寄生虫中提取的标记cDNA探针的混合物用于微阵列的探测，这些探针在锥马鞭毛向无尾鞭毛分化的不同时间点制备，以便同时两两比较阵列中所代表基因的相对表达水平。每个氟标签对阵列上的每个点产生的信号强度将使用阵列扫描仪确定。克隆代表基因上调在早期的密集mastigote向无尾轴分化将被选择测序。基于序列分析的结果，将测试上调基因子集在小鼠中引发保护性免疫反应的能力。预计该项目将产生至少20-30种新的候选疫苗。

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.