Generation of Genetically Attenuated Rickettsiae

描述（由申请人提供）：立克次体病是一种重要性尚未得到充分认识的疾病，尽管其在世界各地以地方病疫源地的形式广泛分布，并有零星和经常季节性的爆发。立克次体感染在人群中以流行病的形式重新出现，虱传斑疹伤寒的流行在第一次世界大战期间和之后立即造成了超过3000万例病例，估计造成300万人死亡。尽管立克次体病在世界范围内分布，立克次体具有高致病性，但我们对立克次体发病机制的理解仍存在很大差距。因此，提出了生成R.普劳瓦泽基河typhi和R.缺乏功能性毒力基因的立克次氏体突变体，以及将它们包括在开发用于广泛保护性立克次氏体疫苗的减毒无毒力菌株中。为实现这一目标，本研究提出了以下具体目标：（1）克隆和鉴定红曲霉毒力蛋白编码基因; typhi和R.立克次氏体生物信息学方法将用于基于前导序列的存在来鉴定编码推定的分泌蛋白的基因;（2）产生靶向基因置换以开发毒力减毒的立克次体突变体。将进行破坏高致病性立克次体毒力蛋白编码基因的实验（在目的1中鉴定）以产生缺乏毒力表型的减毒菌株，如将通过体外和体内重组菌株的功能表征来评估的;（3）鉴定重组立克次体（来自AIM2）的免疫原性和在体内保护免受毒性立克次体攻击的能力，并测试来自AIM1的重组蛋白作为潜在疫苗候选物的免疫原性。检验的基本假设是，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，并对随后的强毒株攻击提供交叉保护。

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.