Typhoid Pathogenesis and Immunity

 描述（由申请人提供）：人类病原体伤寒沙门氏菌（STy）每年导致超过2000万例感染和20万例死亡。我们的实验室已经开发出一种新的伤寒模型，该模型使用从脐带血中获得的人造血干细胞移植的人源化小鼠。这是第一个小动物模型， 急性STy感染的特征性病理和致死性。我们还获得了证据表明STy通过促进替代活化途径而在人巨噬细胞中持续存在，并显示人源化小鼠可以用活减毒STy疫苗株免疫以产生保护性适应性免疫应答。本申请建立在我们的初步观察结果的基础上，以测试中心假设，即伤寒沙门氏菌通过促进替代性巨噬细胞活化来逃避人类先天免疫，并需要获得性免疫来清除。申请的具体目的是：1.确定允许STy在人巨噬细胞中持续存在的机制。我们已经发现STy持续存在于培养的人巨噬细胞中，而STm导致快速细胞死亡。这是了解伤寒和非伤寒沙门氏菌感染发病机制差异的重要线索。将在人巨噬细胞中测定与巨噬细胞极化和凋亡相关的细胞表面标志物、信号传导途径和细胞因子/趋化因子，以了解沙门氏菌与人巨噬细胞相互作用中的血清型特异性差异。2.分析人源化小鼠中毒力所需的STy遗传基因座。一个高复杂性转座子突变体库已经鉴定了人源化小鼠中STy增殖所需的细菌基因。筛选鉴定了已知的和新的毒力决定因子，包括参与Vi荚膜多糖产生和输出、转录调节、中枢代谢、O-抗原修饰和IV型菌毛生物合成的基因。深入分析将确定反沉默转录调节因子，一种新的通透酶复合物，铁吸收系统和IV型皮利在伤寒发病机制中的作用。3.表征人源化小鼠中对STy疫苗的保护性适应性免疫应答。我们已经表明，人源化小鼠在用活减毒STy接种后产生保护性应答。详细的免疫学表征将分析对人源化小鼠疫苗接种的适应性免疫应答，并确定疫苗引发的保护机制。相关性：这些研究将加深我们对伤寒发病机制的理解，并为伤寒候选疫苗的评价建立新的研究平台。

 DESCRIPTION (provided by applicant): The human pathogen Salmonella Typhi (STy) causes more than 20 million infections and 200,000 deaths each year. Our laboratory has developed a novel model of typhoid fever that uses humanized mice engrafted with human hematopoietic stem cells obtained from umbilical cord blood. This is the first small animal model to recapitulate the characteristic pathology and lethality of acute STy infection. We have also obtained evidence to suggest that STy persists in human macrophages by promoting an alternative pathway of activation and shown that humanized mice can be immunized with a live attenuated STy vaccine strain to engender protective adaptive immune responses. This application builds upon our preliminary observations to test the central hypothesis that Salmonella Typhi evades human innate immunity by promoting alternative macrophage activation and requires adaptive immunity for clearance. The specific aims of the application are to: 1. Determine the mechanisms that allow STy to persist in human macrophages. We have found that STy persists in cultured human macrophages whereas STm causes rapid cell death. This is an important clue to understanding differences in the pathogenesis of typhoidal and non-typhoidal Salmonella infections. Cell surface markers, signaling pathways, and cytokines/chemokines associated with macrophage polarization and apoptosis will be assayed in human macrophages to understand serovar-specific differences in Salmonella interaction with human macrophages. 2. Analyze STy genetic loci required for virulence in humanized mice. A high-complexity transposon mutant library has identified bacterial genes required for STy proliferation in humanized mice. The screen has identified both known and novel virulence determinants including genes involved in Vi capsular polysaccharide production and export, transcriptional regulation, central metabolism, O-antigen modification, and type IV pilus biosynthesis. In-depth analysis will determine the roles of a counter-silencing transcriptional regulator, a novel permease complex, iron uptake systems and type IV pili in typhoid pathogenesis. 3. Characterize protective adaptive immune responses to STy vaccines in humanized mice. We have shown that humanized mice develop protective responses following vaccination with live attenuated STy. Detailed immunological characterization will analyze adaptive immune responses to vaccination of humanized mice and determine the mechanism of vaccine-elicited protection. Relevance: These studies will advance our understanding of typhoid pathogenesis and establish a new research platform for the evaluation of candidate typhoid vaccines.