Reverse vaccinology of enterotoxigenic E. coli

描述(由申请人提供)：建议研究的意义。由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。在当前的军事行动和生物恐怖主义中，腹泻病原体仍然是一个重要的健康问题。尽管ETEC对一般人类健康的威胁很大，而且严重抑制了军事表现，但我们仍然没有有效的疫苗，我们也不完全了解导致肠道定植和随后的腹泻病的毒力决定因素。需要基础研究来确定新的治疗和疫苗靶点。这项应用的目的是使用定量蛋白质组分析来发现广泛保守的抗原，从而开发出有效对抗ETEC的疫苗。推动这项研究目标的主要需要是，尽管使用ETEC粘附素的疫苗试验花费了大量的精力和费用，但这些疫苗在调节交叉保护免疫方面都没有特别有效，一些试验甚至存在重大的健康风险。需要替代疫苗目标和策略。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型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暴露在表面的ETEC蛋白来做到这一点。我们在初步研究中表明，所提出的方法快速且经济地识别保守的、暴露在表面的ETEC蛋白，而不依赖于菌株特定的CFs，这些蛋白可以被纯化并用于ETEC毒力的强大动物模型的保护分析。其具体目的是：1)用定量蛋白质组学方法鉴定ETEC外膜蛋白质组；2)制备重组ETEC蛋白和抗血清；3)用重组ETEC抗原免疫小鼠，并量化它们抵御ETEC致死攻击的能力。这项拟议的研究具有重大意义和创新性，因为它为一种被忽视的病原体提供了一种新的保护性抗原发现策略，这种病原体是造成巨大的全球腹泻疾病负担的罪魁祸首。事实上，我们已经发现了17种表面暴露的抗原，它们是一种新的ETEC疫苗配方的潜在成分。此外，这些实验将优化确定其他肠道病原体候选疫苗所需的蛋白质组策略。

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.