Harnessing Transcriptomics to Identify and Test Novel Hookworm Vaccine Targets

 描述（由申请人提供）：我们将使用RNA-seq和比较基因组学开发针对钩虫的疫苗，以确定感染所必需的靶标。钩虫如钩虫和美洲钩虫感染了4亿多人，使他们发育迟缓和死亡。现有的药物只是部分有效，没有可靠有效的疫苗对抗钩虫。人畜共患钩虫（Ancylostoma ceylanicum）很容易感染人类和其他哺乳动物（如金黄仓鼠）。因此，这是一个有用的实验模型，其中测试可能的治疗钩虫病。我们的第一个假设是，通过对在感染A. ceylanicum和相关的寄生虫捻转血矛线虫，我们已经鉴定了一组新的分泌蛋白酶和蛋白酶抑制剂，它们对于寄生虫存活是必需的，并且是有希望的疫苗靶标。我们的第二个假设是，我们可以使用RNA-seq和组织特异性表达来识别更深层次的疫苗靶点，这些靶点由钩虫所需的基因编码，以否定宿主免疫系统，并且可以通过疫苗接种获得。我们最近生成并分析了一个A。ceylanicum基因组序列的313 Mb，包含约27，000个基因，沿着A.从第三期幼虫到完全成熟的成虫的锡兰虫感染。这已经为我们提供了 优先蛋白酶和蛋白酶抑制剂疫苗候选物。我们的第一个目标是通过组织特异性表达来优先考虑这些候选物，在酵母毕赤酵母中合成它们，然后在混合物中测试它们作为抗A.锡兰尽管先前用来自杀死或弱化的寄生虫的线虫蛋白质的粗混合物进行疫苗接种的努力已经给出了有希望的结果，但是用来自单个寄生虫基因的单一蛋白质进行疫苗接种通常失败。我们怀疑，多个基因组在感染过程中表达的一个单一的寄生虫基因组可以集体表达和共注射，以引起有效的免疫。将表征对成功疫苗制剂的T细胞应答。我们的第二个目标将是确定免疫应答肠道钩虫蛋白作为进一步的疫苗候选者。我们建议对来自A.感染免疫系统正常或受抑制的金黄仓鼠。我们将使用生物信息学来寻找编码分泌或膜结合蛋白的基因，这些蛋白在感染期间与宿主的免疫系统相互作用和/或在寄生虫的肠道中强烈翻译。这些是寄生虫用于中和免疫系统的蛋白质的良好候选物。我们将进一步优先考虑候选分泌和膜结合的基因产物，他们在人类钩虫美洲钩虫和H。扭曲疫苗接种将开始于前一两名候选人，并在随后的R 01中扩大。

 DESCRIPTION (provided by applicant): We will develop vaccines against hookworms using RNA-seq and comparative genomics to identify targets essential for infection. Hookworms such as Ancylostoma duodenale and Necator americanus infect over 400 million human beings, stunting and impoverishing them. Existing drugs are only partially effective, and no reliably effective vaccines against hookworms exist. The zoonotic hookworm Ancylostoma ceylanicum easily infects both humans and other mammals (such as golden hamsters). It is therefore a useful experimental model in which to test possible treatments for hookworm disease. Our first hypothesis is that by using transcriptional analysis of genes upregulated during infection by both A. ceylanicum and the related parasite Haemonchus contortus, we have identified a novel set of secreted proteases and protease inhibitors that are essential for parasite survival and that are promising vaccine targets. Our second hypothesis is that we can use RNA-seq and tissue-specific expression to identify a deeper set of vaccine targets, encoded by genes whose products are required for hookworms to negate the host immune system and that are accessible via vaccination. We have recently generated and analyzed an A. ceylanicum genomic sequence of 313 Mb containing ~27,000 genes, along with RNA-seq data during an A. ceylanicum infection from third-stage larvae to fully mature adults. This has already provided us with our top priority protease and protease inhibitor vaccine candidates. Our first aim will be to prioritize these candidates by tissue-specific expression, synthesize them in the yeast Pichia pastoris, and then test them in mixtures as vaccines against A. ceylanicum. Although previous efforts at vaccination with crude mixtures of nematode proteins from killed or weakened parasites have given promising results, vaccinations with single proteins from individual parasite genes have generally failed. We suspect that multiple gene groups expressed during infection by a single parasite genome can be collectively expressed and coinjected to elicit effective immunity. T cell responses to successful vaccine formulations will be characterized. Our second aim will be to identify immunoresponsive intestinal hookworm proteins as further vaccine candidates. We propose to perform additional RNA-seq on dissected intestines and non-intestinal tissues, from A. ceylanicum infecting golden hamsters with either normal or suppressed immune systems. We will use bioinformatics to look for genes encoding secreted or membrane-bound proteins, which, during infection, are either interacting with the host's immune system and/or robustly translated in the parasite's intestines. These are good candidates for proteins used by the parasite for neutralization of the immune system. We will further prioritize candidate secreted and membrane-bound gene products by their conservation in the human hookworm Necator americanus and in H. contortus. Vaccination will begin with the top one or two candidates, to be expanded upon in a subsequent R01.