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

评估志贺氏菌 T3SS 蛋白引起的血清型无关免疫。

• 批准号: 9107330
• 负责人: Wendy L Picking
• 金额: $ 37.5万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9107330
• 关键词: Adjuvant; Animal Model; Animals; Antibody Response; Antigen Presentation Pathway; Antigens; Apoptosis; B-Lymphocytes; Cell Extracts; Cells; Characteristics; Chimeric Proteins; Clinical Trials; Colon; Dendritic Cells; Detergents; Dysentery; Epithelium; Flow Cytometry; Formulation; Foundations; Future; Gastrointestinal Diseases; Health; Human; Immune; Immune Cell Activation; Immune response; Immune system; Immunity; Immunology; Infection; Inflammatory Response; Interferons; Interleukin-17; Intestines; Investigation; Knockout Mice; Licensing; Lung; Measures; Modeling; Mus; Organ; Pathogenesis; Population; Protein Biochemistry; Proteins; Public Health; Research Personnel; Role; Route; Serotyping; Shigella; Shigella Infections; Shigella dysenteriae; Shigella flexneri; Shigella sonnei; Solubility; Structure; System; Traveler' s diarrhea; Type III Secretion System Pathway; Up-Regulation; Vaccinated; Vaccination; Vaccine Adjuvant; Vaccine Production; Vaccines; Virulent; base; biophysical properties; biophysical techniques; cost; cytokine; dodecyldimethylamine oxide; human disease; immunogenicity; intraperitoneal; macrophage; mouse model; mucosal vaccination; novel; novel vaccines; pathogen; protective efficacy; receptor; research study; response; vaccine development; vaccine efficacy

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.

描述（由申请人提供）：Shigellosis是全球公共健康重要性的胃肠道疾病，没有许可疫苗。我们的小组基于Shigella型三型分泌系统（T3SS）的两种蛋白质开发了一种独立于血清型的疫苗。这些蛋白质在发病机理中具有重要作用，并且在有毒的志贺氏菌菌株中保守。我们生成了一个融合蛋白（DB融合），该蛋白包括T3SS尖端蛋白IPAB和iPad。该疫苗已显示在小鼠肺模型中具有保护性。我们建议使用T3SS疫苗作为模型，以识别宿主免疫反应，从而赋予志贺氏菌感染的保护。我们假设，通过使用肠胃外和粘膜疫苗接种途径疫苗接种DB融合后的免疫反应，两种不同的洗涤剂以及BALB/C和敲除小鼠中致命的肺和IP模型，我们将在未来与该疫苗的临床试验中测量的保护相关性。为此，这项研究的具体目的是：1。确定小鼠模型中DB融合疫苗的保护机制。我们将通过疫苗接种不同免疫反应的动物来确定保护涉及的免疫力的类型。 B细胞和几种细胞因子缺乏动物将通过DB融合蛋白接种。之后，将小鼠与S. flexneri挑战IP。使用这些实验确定的免疫反应将通过使用适当的细胞或细胞因子进行转移实验来进一步分析，以确认保护机制； 2。确定抗原呈递的最佳途径。我们将使用IPAB，iPad刺激树突状细胞以及在存在DMLT的情况下的组合，并测量细胞因子释放和激活标记的上调。这种上调的机制将进一步特征，以鉴定负责这种反应的树突状细胞中的受体分子。 3。优化疫苗配方，以确定洗涤剂在传达保护性免疫原性中的重要性。洗涤剂在疫苗配方中的作用将通过在存在两个不同的洗涤剂的情况下分析DB融合来评估。特别是我们将分析每个洗涤剂中融合的聚集状态和一般二级结构稳定性。我们还将表征从用DB融合疫苗的小鼠中提取的免疫细胞，每种洗涤剂都存在，以将生物物理特性与保护效果相关联。我们已经组建了一个研究人员，具有免疫学和蛋白质生物化学方面的专业知识，以探索这种新型疫苗能够传达对志尿病的保护的机制。在将保护的相关性鉴定与使用生物物理方法优化疫苗配方的使用时，我们将为评估该疫苗保护人类的可能疗效的基础建立基础 志智利病。