Harnessing Transcriptomics to Identify and Test Novel Hookworm Vaccine Targets

利用转录组学来识别和测试新型钩虫疫苗靶标

• 批准号: 9063524
• 负责人: RAFFI V AROIAN
• 金额: $ 24.36万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-05 至 2017-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9063524
• 关键词: Adult; Agriculture; Albendazole; Ancylostoma (genus); Anthelmintics; Binding; Binding Proteins; Bioinformatics; Biology; Blood; Candidate Disease Gene; Cathepsins B; Complementary DNA; Data; Digestion; Disease; Experimental Models; Family; Gene Targeting; Genes; Genomics; Goat; Haemonchus; Hamsters; Health; Homologous Gene; Hookworms; Human; Humoral Immunities; Immune system; Immunity; Immunocompetent; Immunocompromised Host; Individual; Infection; Injection of therapeutic agent; Integral Membrane Protein; Intestinal parasite; Intestines; Larva; Legal patent; Life; Livestock; Mammals; Membrane; Mesocricetus auratus; Methods; Multi-Drug Resistance; Necator americanus; Nematoda; Old World Hookworm; Organism; Parasite Control; Parasites; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacotherapy; Pichia; Population; Protease Inhibitor; Proteins; RNA; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Sheep; Staging; T cell response; T-Lymphocyte; Testing; Tissues; Translating; Vaccinated; Vaccination; Vaccines; Yeasts; comparative genomics; cost; feeding; gene product; genome sequencing; genome-wide; killings; member; next generation; novel; novel vaccines; parasite genome; preference; protein expression; response; success; transcriptome; transcriptome sequencing; transcriptomics; vaccine candidate; vaccine development; vaccine trial; vaccinology

 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.

 描述（由适用提供）：我们将使用RNA-seq和比较基因组学对钩虫开发疫苗，以识别感染必不可少的靶标。钩虫（Ancylostoma duodeNale和necator Americanus感染）等钩虫感染超过4亿人，使它们发育迟缓和贫穷。现有药物仅部分有效，并且没有可靠的针对钩虫的有效疫苗。人畜共患钩虫ceylanicum很容易感染人类和其他哺乳动物（例如金仓鼠）。因此，这是一个有用的实验模型，在其中测试可能治疗钩虫疾病。我们的第一个假设是，通过使用肝曲霉和相关寄生虫Haemonchus contortus感染期间更新的基因的转录分析，我们已经确定了一组新型的分泌蛋白酶和蛋白酶抑制剂，这些蛋白酶和蛋白酶抑制剂对于寄生虫生存至关重要，并且是疫苗靶标的。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一组更深的疫苗靶标，这些疫苗靶标由钩虫否定宿主免疫系统所需的基因编码，并且可以通过疫苗接种。最近，我们在从第三阶段幼虫到完全成熟的成年人的锡兰曲霉感染过程中生成并分析了含有约27,000个基因的313 MB的锡兰曲霉基因组序列，以及RNA-Seq数据。这已经为我们提供了顶级 优先蛋白和蛋白抑制剂疫苗的候选物。我们的第一个目的是通过组织特异性表达来优先考虑这些候选者，将它们合成在酵母菌Pichia Pastoris中，然后将其作为对针对肝曲霉的疫苗进行测试。尽管先前在寄生虫中被杀死或弱化的线虫蛋白的粗糙混合物的疫苗的努力已经取得了希望的结果，但来自单个寄生虫基因的单个蛋白质的疫苗通常失败了。我们怀疑可以集体表达并产生以产生有效的免疫组织化学免疫力，在感染过程中表达的多个基因组。 T细胞对成功疫苗配方的反应将被表征。我们的第二个目的是将免疫呼吸肠钩虫蛋白鉴定为进一步的疫苗候选物。我们建议对肝洲感染的肝曲霉和抑制免疫系统的肝曲霉的金仓鼠进行额外的RNA-SEQ，并在解剖的肠道和非智能组织上执行。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，在感染期间，它们要么与宿主的免疫系统相互作用和/或在寄生虫的肠道中进行稳健翻译。这些是寄生虫用于免疫系统神经化的蛋白质的好候选者。我们将通过人类钩虫尸体在Americanus和H. totortus中的保存来进一步将候选者分泌和膜结合的基因产物优先考虑。疫苗接种将从最高的一两个候选人开始，将在随后的R01中扩展。