Harnessing Transcriptomics to Identify and Test Novel Hookworm Vaccine Targets

 描述(由申请人提供)：我们将利用RNA-SEQ和比较基因组学来开发针对钩虫的疫苗，以确定感染的关键靶点。钩虫，如十二指肠钩虫和美洲钩虫，感染了超过4亿人，使他们发育迟缓和贫困。现有的药物只有部分有效，也没有针对钩虫的可靠有效疫苗。人畜共患钩虫很容易感染人类和其他哺乳动物(如金黄仓鼠)。因此，这是一种有用的实验模型，可以用来测试钩虫疾病的可能治疗方法。我们的第一个假设是，通过对头孢曲霉和相关寄生虫螺旋体血吸虫感染期间上调的基因的转录分析，我们已经鉴定出一组新的分泌型蛋白水解酶和蛋白水解酶抑制剂，它们对寄生虫的生存是必不可少的，并且有望成为疫苗靶标。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，这些基因的产物是钩虫否定宿主免疫系统所需的，并且可以通过接种疫苗获得。我们最近已经产生并分析了一个313 Mb的天牛基因组序列，包含大约27,000个基因，以及从第三期幼虫到完全成熟的成虫感染天牛的RNA-SEQ数据。这已经为我们提供了我们最好的 优先考虑的蛋白水解酶和蛋白水解酶抑制剂候选疫苗。我们的第一个目标是根据组织特异性表达对这些候选基因进行优先排序，在毕赤酵母中合成它们，然后在混合物中测试它们作为对抗头孢曲霉的疫苗。尽管以前用灭活或弱化寄生虫的线虫蛋白粗混合物接种疫苗的努力取得了令人振奋的结果，但用单个寄生虫基因的单一蛋白接种通常都是失败的。我们怀疑，在单个寄生虫基因组感染期间表达的多个基因组可以集体表达并共同注射，以诱导有效的免疫。将描述T细胞对成功的疫苗配方的反应。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候选疫苗。我们建议对分离的肠道和非肠道组织进行额外的RNA-SEQ，这些组织来自感染免疫系统正常或受抑制的金黄地鼠的头孢曲霉。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要么与宿主的免疫系统相互作用，要么在寄生虫的肠道中强有力地翻译。这些都是寄生虫用来中和免疫系统的蛋白质的很好候选者。我们将进一步优先考虑候选的分泌和膜结合基因产物，因为它们在人钩虫、美洲钩虫和螺旋钩虫中具有保守性。疫苗接种将从前一到两名候选疫苗开始，并在随后的R01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.