Reverse vaccinology of enterotoxigenic E. coli

描述（由申请人提供）：拟研究的意义。肠道细菌引起的腹泻病是一种重要的地方性健康威胁，也是食源性疾病的主要来源。腹泻病原体在当前的军事行动和生物恐怖主义中仍然是一个重要的健康问题。尽管人们认识到ETEC对一般人类健康的威胁和对军事表现的严重抑制的严重性，但我们仍然没有有效的疫苗，我们也没有充分了解导致肠道定植和随后的腹泻病的毒力决定因素。需要进行基础研究以确定新的治疗和疫苗靶点。该应用程序的目的是使用定量蛋白质组学蛋白质分析来发现广泛保守的抗原，从而允许开发有效对抗ETEC的疫苗。激发这一研究目标的主要需求是，尽管在使用ETEC粘连素的疫苗试验中付出了巨大的努力和费用，但这些疫苗在介导交叉保护性免疫方面都没有特别有效，有些试验甚至存在重大的健康风险。需要替代疫苗目标和战略。我们将利用我们在ETEC微生物学和定量蛋白质组学方面的成熟经验来表征新的ETEC疫苗抗原。我们将通过鉴定可能被宿主免疫系统识别的新的表面暴露的ETEC蛋白来做到这一点。我们在初步研究中表明，所提出的方法可以快速、经济地识别出保守的、表面暴露的ETEC蛋白，而不依赖于菌株特异性的CFs，这些蛋白可以被纯化并用于ETEC毒力动物模型的保护试验。具体目标是：1)使用定量蛋白质组学分析表征ETEC外膜蛋白质组，2)生产重组ETEC蛋白质和抗血清，以及3)用重组ETEC抗原免疫小鼠并量化其抵抗ETEC致命挑战的能力。提出的研究是重要的和创新的，因为它引入了一种新的策略来发现保护性抗原的一个被忽视的病原体负责造成巨大的全球腹泻疾病负担。事实上，我们已经发现了17种表面暴露抗原，它们是一种新的ETEC疫苗配方的潜在成分。此外，这些实验将优化表征其他肠道病原体候选疫苗所需的蛋白质组学策略。

DESCRIPTION (provided by applicant): Significance of the proposed research. Diarrheal disease caused by enteric bacteria is an important endemic health threat and a major source of food-borne disease. Diarrheal pathogens also remain an important health concern in current military operations and in bioterrorism. In spite of the appreciated magnitude of the threat to both general human health and the severe inhibition of military performance caused by ETEC, we still do not have an effective vaccine, nor do we fully understand the virulence determinants responsible for intestinal colonization and subsequent diarrheal disease. Basic research is needed to identify new therapeutic and vaccine targets. The objective of this application is to use quantitative proteomic protein profiling to discover broadly conserved antigens that will permit development of a vaccine efficacious against ETEC. The primary need that motivates this research objective is that despite the significant effort and expense that has been employed in vaccine trials using ETEC adhesins, none of these vaccines have been especially effective in mediating cross- protective immunity, and some trials have even had significant health risks. Alternative vaccine targets and strategies are demanded. We will utilize our proven experience in ETEC microbiology and quantitative proteomics to characterize novel ETEC vaccine antigens. We will do this by identifying novel surface-exposed ETEC proteins likely to be recognized by the host immune system. We show in preliminary studies that the proposed approach rapidly and economically identifies conserved, surface-exposed ETEC proteins, independent of strain-specific CFs, which can be purified and used in protection assays in a robust animal model of ETEC virulence. The specific aims are to: 1) Characterize ETEC outer membrane proteomes using quantitative proteomic profiling, 2) Produce recombinant ETEC proteins and antisera, and 3) Immunize mice with recombinant ETEC antigens and quantify their ability to protect against an otherwise lethal challenge with ETEC. The proposed research is significant and innovative because it introduces a novel strategy for protective antigen discovery for a neglected pathogen responsible for causing an enormous global diarrheal disease burden. Indeed, we have already discovered seventeen surface-exposed antigens that are potential components of a new ETEC vaccine formulation. Additionally, these experiments will optimize proteomic strategies needed to characterize vaccine candidates in other enteric pathogens.