Generation of Genetically Attenuated Rickettsiae

描述（由申请人提供）：立克次体病是一个很好的例子，它的重要性没有得到充分认识，尽管它们以地方性疫源地的形式在世界各地广泛分布，并伴有零星和经常季节性的暴发。立克次体感染以流行病的形式再次出现在人群中，在第一次世界大战期间和之后不久，虱子传播的斑疹伤寒的流行造成了3 000多万例病例，估计造成300万人死亡。尽管立克次体疾病在世界范围内分布，并且立克次体具有高致病性，但我们对立克次体发病的分子机制的理解仍有很大的差距。因此，本提案旨在产生缺乏功能毒力基因的普拉兹氏r、斑疹伤寒r和立克次体r突变体，并将其纳入开发用于广泛保护性立克次体疫苗的减毒无毒菌株中。为实现这一目标，提出了以下具体目标：(1)克隆和表征伤寒致病菌和立克次体致病菌推定毒力蛋白的基因。生物信息学方法将被用于根据先导序列的存在来识别编码假定分泌蛋白的基因；(2)产生靶向基因替代，培育出毒力减弱的立克次体突变体。将进行实验，破坏高致病性立克次体（在目标1中鉴定）的毒力蛋白编码基因，以产生缺乏毒力表型的减毒菌株，这将通过体外和体内重组菌株的功能表征进行评估；(3)表征重组立克次体（来自目标2）的免疫原性和在体内抵御致病性立克次体攻击的能力，并测试来自目标1的重组蛋白作为潜在候选疫苗的免疫原性。所测试的基本假设是，基因改变的重组菌株是否在哺乳动物体内保持其免疫原性，并在随后的毒力菌株攻击中提供交叉保护。

DESCRIPTION (provided by applicant): Rickettsioses are good examples of diseases whose importance are not adequately appreciated despite their wide distribution throughout the world in the form of endemic foci, with sporadic and often seasonal outbreaks. Rickettsial infections have re-emerged in epidemic form in human populations, the epidemics of louse-borne typhus were responsible for over 30 million cases during and immediately after World War I, causing an estimated three million deaths. Despite the worldwide distribution of rickettsial diseases and the highly pathogenic nature of rickettsiae, there is a substantial gap in our understanding of the molecular mechanisms underlying rickettsial pathogenesis. Thus, this proposal is submitted to generate R. prowazekii, R. typhi, and R. rickettsii mutants lacking functional virulence genes and their inclusion in developing attenuated non-virulent strains for a broad-based protective rickettsial vaccines. To achieve this goal the following Specific Aims are proposed: (1) To clone and characterize genes encoding putative virulence proteins from R. typhi and R. rickettsii. Bioinformatic approaches will be utilized to identify genes that encode putative secreted proteins based on presence of the leader sequence; (2) To generate targeted gene replacement to develop rickettsial mutants that are attenuated for virulence. Experiments will be performed to disrupt virulence protein encoding genes of highly pathogenic rickettsiae (identified in Aim 1) to generate attenuated strains lacking virulent phenotypes as will be assessed by functional characterization of recombinant strains in vitro and in vivo; and (3) To characterize the recombinant rickettsia (from aim 2) for their immunogenicity and ability to protect against virulent rickettsial challenge in vivo, and to test the immunogenicity of recombinant proteins from aim 1 as potential vaccine candidates. The underlying hypothesis tested is whether recombinant strains with altered genes maintain their immunogenicity within mammals and provide cross protection to subsequent challenge with virulent strains.