Generation of Genetically Attenuated Rickettsiae

遗传减毒立克次体的产生

• 批准号: 7171592
• 负责人: Abdu F Azad
• 金额: $ 41.17万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-15 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171592
• 关键词: Attenuated; Bioinformatics; Biological Products; Cessation of life; Condition; Crowding; Disasters; Disease; Disease Outbreaks; Disruption; Dysentery; Effectiveness; Epidemic; Epidemic Louse-Borne Typhus; Generations; Genes; Goals; Human; In Vitro; Infection; Infectious Diseases Research; Influenza; Japanese Population; Lice; Mammals; Molds; Molecular; Nature; Numbers; Pathogenesis; Pathway interactions; Phenotype; Plague; Plaque Assay; Population; Proteins; Reaction; Recombinant Proteins; Recombinants; Recording of previous events; Rickettsia; Rickettsia Infections; Rickettsia prowazekii; Rickettsial Vaccines; Testing; Typhus; Vaccines; Virulence; Virulent; War; World War I; base; biodefense; gene replacement; immunogenicity; in vivo; mutant; research study; response

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.

说明(申请人提供)：立克次体病是一种很好的疾病，其重要性没有得到充分认识，尽管它们在世界各地以地方性疫源地的形式广泛分布，有零星的和往往是季节性的暴发。立克次体感染在人类中以流行的形式重新出现，在第一次世界大战期间和之后不久，由虱子传播的斑疹伤寒的流行造成了3000多万病例，估计造成300万人死亡。尽管立克次体疾病在世界范围内广泛分布，且具有高度致病性，但我们对立克次体致病的分子机制的认识还存在很大差距。因此，这项建议是为了产生缺乏功能性毒力基因的普罗瓦泽克、伤寒和立克次体突变株，并将它们包括在开发广泛基础保护性立克次体疫苗的无毒减毒株中。为实现这一目标，提出了以下具体目标：(1)克隆和鉴定伤寒杆菌和立克次体可能的毒力蛋白编码基因。生物信息学方法将被用于根据前导序列的存在来识别编码假定的分泌蛋白的基因；(2)产生有针对性的基因替换，以开发毒力减弱的立克次体突变体。将进行实验以干扰(在Aim 1中鉴定的)高致病性立克次体的毒力蛋白编码基因，以产生缺乏毒力表型的减毒菌株，该弱毒株将通过体外和体内重组菌株的功能鉴定来评估；以及(3)鉴定(来自Aim 2的)重组立克次体的免疫原性和在体内抵抗强毒力立克次体攻击的能力，并测试来自Aim 1的重组蛋白作为潜在疫苗候选的免疫原性。检验的基本假设是，具有改变基因的重组菌株是否在哺乳动物体内保持其免疫原性，并为随后与毒力菌株的攻击提供交叉保护。