IMMUNITY TO INTRACELLULAR PATHOGENS

对细胞内病原体的免疫力

基本信息

批准号：
8663168
负责人：
EMIL Raphael UNANUE
金额：
$ 37.62万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-12-01 至 2015-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8663168
关键词：
Adjuvant Adjuvanticity Amino Acids Antigen Presentation Antigen Presentation Pathway Antigen-Presenting Cells Antigens Apoptosis Autophagocytosis Avidity Binding Biochemical Biochemistry Biological Biological Assay Biology CD8B1 gene Catabolism Cell Death Induction Cell surface Cells Cellular biology Cholesterol Cytolysins Family Generations Goals Haplotypes Histocompatibility Immunity In Vitro Infection Inflammatory Investigation Knowledge Listeria monocytogenes Listeria monocytogenes hlyA protein Membrane Membrane Protein Traffic Modification Molecular Mutagenesis Natural Immunity Pathway interactions Process Production Property Proteins Role Series Staging T cell response T-Lymphocyte Testing Toxic effect Toxin Vaccines Virulence base cell type cytokine immunogenic immunogenicity in vivo insight interest member microbial mutant pathogen perforin response trafficking uptake

项目摘要

DESCRIPTION (provided by applicant): The goals are to explain, at the biochemical and cellular levels, the high immunogenicity of the Listeria monocytogenes (LM) pore-forming toxin listeriolysin O (LLO). LLO provides dominant antigens for both CD4+ and CD8+ T cell responses on multiple MHC haplotypes. The LLO molecule has two notable dual functions, first, it is a strongly stimulatory protein (i.e., it has an "adjuvant" effect), inducing cytokine production and cell activation on the antigen presenting cells (APC). Second, LLO is an exceptional donor of antigens to the major histocompatibility class II molecules (MHC-II), eliciting strong T cell responses when given as a purified protein. Our hypothesis is that the strong avidity for cholesterol-rich membranes makes LLO a highly efficient immunogen. We want to take advantage of this feature to develop new knowledge on antigen processing and presentation biochemistry and to determine the relationship of LLO toxicity with its immunogenicity. We have chosen to focus on examining LLO as a purified protein, separate from the LM that normally expresses it. The benefits of this strategy are twofold: first, we can examine the immunogenicity of LLO in the absence of any other bacterial product that could change the biology of the APC, and, or induce an inflammatory effect; and second, we can take advantage of the known biochemistry of LLO. Examining the immunogenicity of LLO is important in order to: i) understand the natural infection and how LM antigens are presented; ii) explain LLO as an immunogen and a carrier molecule for ectopic antigens; and iii) provide us with unique clues on the basic mechanisms of antigen presentation. In Aim 1, we describe the generation of an extensive series of LLO mutants that will be used to examine their immunogenicity both in vitro and in vivo. Mutagenesis of key amino acid residues on LLO will be informative in that it will allow us to probe the biochemical and biological features that makes LLO such a strong immunogen; such as binding to membranes, trafficking pathways, possible association with other molecules involved in presentation, capacity to activate the APC, etc. Of course, the information will be of interest to those using LLO as a vaccine vehicle and to those who study cholesterol dependent cytolysins. The various LLOs will be tested in the context of basic presentation assays and used in all subsequent aims. In Aim 2, we describe approaches for determining the localization and catabolism of LLO inside APC. The subcellular localization of LLO will be followed by different approaches including developing fluorescently-tagged LLOs. We also plan to examine the relationship between the induction of cell death and autophagic processes by LLO and its immunogenicity. In Aim 3, we examine the adjuvanticity of LLO in three contexts: i) activation of APC, ii) priming of naive T cells, and iii) generation of protective immunity to LM.

描述（由申请人提供）：目标是在生化和细胞水平上解释单核细胞增生李斯特菌（LM）孔形成的毛孔形成毒素listeriolysin o（LLO）的高免疫原性。 LLO为多种MHC单倍型上的CD4+和CD8+ T细胞反应提供了显性抗原。 LLO分子具有两个显着的双重功能，首先，它是一种强刺激蛋白（即，它具有“辅助”效应），可诱导细胞因子的产生和细胞激活抗原存在细胞（APC）。其次，LLO是对主要组织相容性II类分子（MHC-II）的抗原的杰出供体，当给出作为纯化蛋白时，会引起强T细胞反应。我们的假设是，富含胆固醇的膜的强烈亲和力使LLO成为高效的免疫原。我们希望利用这一特征来发展有关抗原加工和表现生物化学的新知识，并确定LLO毒性与其免疫原性的关系。我们选择专注于将LLO作为一种纯化的蛋白质，与通常表达它的LM分开。该策略的好处是双重的：首先，我们可以在没有任何其他可能改变APC生物学的细菌产品的情况下检查LLO的免疫原性，或者诱导炎症作用；其次，我们可以利用已知的LLO生物化学。检查LLO的免疫原性对于：i）了解自然感染以及如何呈现LM抗原； ii）将LLO解释为异位抗原的免疫原和载体分子； iii）为我们提供有关抗原表现基本机制的独特线索。在AIM 1中，我们描述了一系列广泛的LLO突变体的产生，这些突变体将用于检查其在体外和体内的免疫原性。 LLO上关键氨基酸残基的诱变将是有益的，因为它将允许我们探测使LLO使LLO如此强大的免疫原的生化和生物学特征。例如与膜，运输途径的结合，可能与涉及表现的其他分子相关，激活APC的能力等。当然，使用LLO作为疫苗的人以及研究胆固醇依赖的细胞蛋白酶的人都会感兴趣的信息。各种LLO将在基本演示分析的背景下进行测试，并在所有后续目标中使用。在AIM 2中，我们描述了确定LLO在APC中的定位和分解代谢的方法。 LLO的亚细胞定位将采用不同的方法，包括开发荧光标记的LLOS。我们还计划检查LLO诱导细胞死亡与自噬过程之间的关系及其免疫原性。在AIM 3中，我们在三种情况下检查了LLO的辅助性：i）APC的激活，ii）启动幼稚T细胞，ii）对LM产生了保护性免疫。