Plague vaccine candidates by a functional genomic screen

描述(由申请人提供)：我们工作的长期目标是生产一套全面的疫苗，即使是基因工程病原体也能有效地保护大量人口。这里描述的提案的直接目标是筛选鼠疫耶尔森氏菌的整个基因组，以寻找最有效的候选疫苗。至少出于两个原因，鼠疫是一个重要的目标。1)这是一种极其致命的疾病，自古以来就一直困扰着人类。2)迫切需要生产一种广泛有效、安全和廉价的疫苗来对抗生物恐怖主义的威胁。目前正在研究的一种亚单位疫苗的免疫原性简单，可能会限制其保护多样化人类群体的能力，并可能使其对转基因病原体无效。我们的第一阶段的具体目标是：i)建立所需的信息学，以支持对整个鼠疫杆菌基因组进行技术要求高、功能强大的保护性抗原筛选；ii)确定生产完整的鼠疫菌基因库的方案；iii)通过在免疫和保护分析中测试鼠疫杆菌基因，为该基因组筛选确定最佳的基因剂量和设计。我们的实验方法结合了我们的三项技术：基因免疫、表达文库筛选和线性基因表达元件。在第二阶段，我们将应用这个系统来评估每个鼠疫耶尔森氏菌基因作为阵列矩阵中的PCR产物，以保护小鼠免受感染。积极得分的基因将构成一系列抗原性选择，以保护广大人口免受自然和非自然的鼠疫杆菌感染。除了识别未来疫苗的成分外，这些保护性抗原还将揭示保护的免疫标记，从而促进这些疫苗以及其他潜在鼠疫疫苗的临床验证。

DESCRIPTION (provided by applicant): The long-term objective of our work is to produce a comprehensive set of vaccines that would effectively protect a large population against even genetically-engineered pathogens. The immediate objective of the proposal described here is to screen the whole genome of Yersinia pestis for the most potent vaccine candidates. Plague is a significant target for at least two reasons. 1) It is an extremely virulent, often lethal disease that has been suffered by humans since antiquity. 2) There is an immediate need to produce a broadly effective, safe, and inexpensive vaccine to counter the threat of bioterrorism. The immunogenic simplicity of a subunit vaccine currently under study may limit its ability to protect the diverse human population, and may render it ineffectual against a genetically-modified pathogen. Our phase I specific aims are to i) establish the informatics required to support a technically demanding, functional screen of the entire Y. pestis genome for protective antigens, ii) identify protocols for producing a complete Y. pestis gene library iii) define the optimal gene dose and design for this genome screen by testing Y. pestis genes in immune and protection assays. Our experimental approach combines three of our technologies: genetic immunization, expression library screening, and linear gene expression elements. In phase II we will apply this system to evaluate every Y. pestis gene as a PCR product in an arrayed matrix for protection of mice against infection. Positively scoring genes will constitute a portfolio of antigenic options to protect a broad population against natural and unnatural Y. pestis infections. In addition to identifying the components for a future vaccine, these protective antigens will reveal immune markers of protection, and thereby facilitate clinical validation of these as well as other potential plague vaccines.