Assessment of serotype-independent immunity elicited by Shigella T3SS proteins.

说明(申请人提供)：志贺氏菌病是一种对全球公共卫生具有重要意义的胃肠道疾病，目前还没有获得许可的疫苗。本课题组开发了一种基于志贺氏菌3型分泌系统(T3SS)的两种蛋白的非血清型疫苗。这些蛋白在致病过程中具有重要作用，并在志贺氏菌强毒株中保守。我们产生了一个融合蛋白(DB融合)，它由T3SS末端蛋白IPAB和iPad组成。该疫苗已被证明在小鼠肺模型中具有保护作用。我们建议使用T3SS疫苗作为一个模型来识别对志贺氏菌感染具有保护作用的宿主免疫反应。我们假设，通过使用肠外和粘膜接种途径、两种不同的洗涤剂以及Balb/c和基因敲除小鼠的致死性肺部和IP模型对DB Fusion接种后的免疫反应进行彻底检查，我们将确定可以在未来使用该疫苗进行的临床试验中测量的保护相关性。为此，本研究的具体目的是：1.确定DB融合疫苗在小鼠模型中的保护机制。我们将通过为不同免疫反应不足的动物接种疫苗来确定参与保护的免疫类型。B细胞和几种细胞因子缺陷的动物将接种DB融合蛋白。之后，小鼠将被挑战感染福氏志贺氏菌。利用这些实验鉴定的免疫反应将通过与适当的细胞或细胞因子进行转移实验来进一步分析，以确定保护机制；2.确定抗原提呈的最佳途径。我们将用IPAB、iPad及其组合在dmLT存在下刺激树突状细胞，并检测细胞因子的释放和激活标志物的上调。这种上调的机制将通过识别树突状细胞中负责这一反应的受体分子来进一步表征；以及3.优化疫苗配方，以确定洗涤剂在传递保护性免疫原性方面的重要性。将通过分析存在两种不同洗涤剂的DB Fusion来评估疫苗配方中存在的洗涤剂的作用。特别是，我们将分析融合在每种洗涤剂中的聚集状态和一般二级结构稳定性。我们还将表征从接种DB Fusion的小鼠中提取的免疫细胞的特征，以及每种洗涤剂的存在，以将生物物理特征与保护效果联系起来。我们已经组建了一个具有免疫学和蛋白质生物化学专业知识的研究小组，以探索这种新型疫苗能够传递对志贺氏菌病的保护作用的机制。通过将保护相关因素的鉴定与使用生物物理方法优化疫苗配方相结合，我们将为评估这种疫苗在保护人类感染病毒方面的可能效力奠定基础。 志贺氏菌病。

DESCRIPTION (provided by applicant): Shigellosis is a gastrointestinal disease of worldwide public health importance for which there is no licensed vaccine. Our group has developed a serotype-independent vaccine based on two proteins of the Shigella type three secretion system (T3SS). These proteins have important roles in pathogenesis and are conserved among virulent Shigella strains. We generated a fusion protein (DB fusion) that comprises the T3SS tip proteins IpaB and IpaD. This vaccine has been shown to be protective in the mouse pulmonary model. We propose to use the T3SS vaccine as a model to identify the host immune responses that confer protection against Shigella infection. We hypothesize that by through a thorough examination of the immune response after vaccination with the DB Fusion using parenteral and mucosal vaccination routes, two different detergents, and the lethal pulmonary and IP models in Balb/c and knockout mice, we will identify correlates of protection that can be measured in future clinical trials with this vaccine. Toward this end, the Specific Aims of this study are: 1. Determine the protective mechanism of the DB fusion vaccine in a mouse models. We will determine the type of immunity involved in protection by vaccinating animals deficient in different immune responses. B cell and several cytokine deficient animals will be vaccinated with the DB Fusion protein. Afterwards, mice will be challenged IP with S. flexneri. The immune responses identified using these experiments will be further analyzed by making transfer experiments with the appropriate cells or cytokines in order to confirm the protective mechanism; 2. Determine the optimal pathway for antigen presentation. We will stimulate dendritic cells with IpaB, IpaD and the combination in the presence of dmLT and measure cytokine release and up-regulation of activation markers. The mechanism of this up-regulation will be further characterized by identifying receptor molecules in dendritic cells responsible for this response; and 3. Optimize the vaccine formulation to identify the importance of detergent in conveying protective immunogenicity. The role of detergent present in the vaccine formulation will be evaluated by analyzing the DB Fusion in presence of two different detergents. In particular we will analyze the aggregation state and general secondary structure stability of the fusion in each detergent. We will also characterize immune cells extracted from mice vaccinated with the DB Fusion with each detergent present to correlate biophysical characteristics with protective efficacy. We have assembled a team of investigators with expertise in immunology and protein biochemistry to explore the mechanism by which this novel vaccine is able to convey protection against shigellosis. In combining an identification of the correlates of protection with the use of biophysical methods for optimizing vaccine formulation, we will establish the basis for evaluating the likely efficacy of this vaccine in protecting humans against shigellosis.