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.