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）成孔毒素李斯特菌溶血素 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“佐剂”作用），诱导抗原呈递细胞（APC）产生细胞因子和细胞活化。其次，LLO 是主要组织相容性 II 类分子 (MHC-II) 的特殊抗原供体，当以纯化蛋白形式给予时，会引发强烈的 T 细胞反应。我们的假设是，对富含胆固醇的膜的强烈亲合力使 LLO 成为高效的免疫原。我们希望利用这一特性来开发有关抗原加工和呈递生物化学的新知识，并确定 LLO 毒性与其免疫原性的关系。我们选择将重点放在检查 LLO 作为纯化蛋白的情况，与通常表达它的 LM 分开。这种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 生物学和/或诱导炎症效应的细菌产物的情况下检查 LLO 的免疫原性；其次，我们可以利用 LLO 已知的生物化学特性。检查 LLO 的免疫原性对于以下方面非常重要： i) 了解自然感染以及 LM 抗原如何呈递； ii) 解释脂质连接寡糖作为一种免疫原和异位抗原的载体分子； iii) 为我们提供有关抗原呈递基本机制的独特线索。在目标 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它们的体外和体内免疫原性。 LLO 上关键氨基酸残基的诱变将提供丰富的信息，因为它将使我们能够探究使 LLO 成为如此强大的免疫原的生化和生物学特征；例如与膜的结合、运输途径、与参与呈递的其他分子的可能关联、激活 APC 的能力等。当然，这些信息将引起那些使用 LLO 作为疫苗载体的人和研究胆固醇依赖性的人的兴趣。溶细胞素。各种 LLO 将在基本呈现分析的背景下进行测试，并用于所有后续目标。在目标 2 中，我们描述了确定 LLO 在 APC 内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 LLO。我们还计划研究 LLO 诱导细胞死亡和自噬过程及其免疫原性之间的关系。在目标 3 中，我们检查了 LLO 在三种情况下的佐剂作用：i）APC 的激活，ii）初始 T 细胞的启动，以及 iii）对 LM 的保护性免疫的产生。