TARGETING VIRAL PROTEINS INTO THE CLASS I PATHWAY

将病毒蛋白靶向 I 类途径

• 批准号: 6099904
• 负责人: ROBERT F SILICIANO
• 金额: --
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6099904
• 关键词: MHC class I antigen; cell mediated lymphocytolysis test; clone cells; cytotoxic T lymphocyte; disease /disorder model; drug design /synthesis /production; influenza vaccines; interleukin 2; laboratory mouse; nucleic acid sequence; ubiquitin; vaccine development; viral vaccines; virus antigen; virus protein

This project will explore a novel vaccine strategy designed to enhance the induction of virus-specific CD8+ cytolytic T lymphocytes (CTL). The strategy involves the use of viral vectors or other delivery systems to introduce into host cells viral genes modified to contain sequences that will target the relevant viral protein for rapid degradation in the cytoplasm. Because cytoplasmic degradation of viral proteins provides the substrate for the class I antigen processing pathway, we hypothesize that these targeting strategies will facilitate the induction of CD8+ CTL responses specific for the relevant viral protein. We will directly test the hypotheses that such targeting strategies will result in increased loading of class I MHC molecules with viral peptides and enhanced induction of CTL responses. Several independent approaches will be taken to defining transferable sequence elements that will target viral proteins for rapid degradation in the cytoplasm of human cells. Modified viral proteins designed to undergo ubiquitin (Ub)-dependent degradation by the N-end rule pathway or by the related second codon rule pathway will be expressed in human cell lines. Kinetics of degradation will be measured biochemically, and the Ub dependence of degradation will be evaluated using cell lines with temperature sensitive defects in the E1 Ub activating enzyme. Constructs designed to undergo rapid cytoplasmic degradation by other two other mechanisms will also be analyzed. Based on preliminary studies of processing of viral envelope proteins for class I-restricted recognition by a TAP 1/2-dependent pathway, it is proposed that insertion that an "out of context" hydrophobic domain in a cytoplasmic protein will trigger extremely rapid degradation, resulting in efficient generation of peptides that can be presented with class I molecules. in addition, the C-terminal degradation-targeting domain of the short lived RAG-2 protein will be used to force the rapid degradation of test viral proteins. For each degradation targeting strategy, quantitative analysis of naturally processed peptides will be carried out to determine whether targeting results in increased loading of class I MHC molecules with the relevant viral peptides. In addition and IL-2-independent clonal expansion of antigen-specific CTL will be compared. Five different approaches will be taken to measuring the immunogenicity of cells expressing degradation- targeted viral proteins. These include in vitro studies of antigen- specific proliferative and cytolytic responses of human T cell clones, and studies of secondary in vitro CTL responses using responding cells from infected human donors. In addition, in vivo studies in the mouse model will be used to evaluate the generation of CTL responses from naive T cell populations and to evaluate this vaccine strategy in a influenza virus immunization/challenge model. Together, these studies would provide a foundation for the development of optimized CTL-based vaccine strategies.

该项目将探索一种新颖的疫苗策略，旨在增强 诱导病毒特异性CD8+细胞溶解淋巴细胞（CTL）的诱导。 这 策略涉及使用病毒向量或其他输送系统的使用 引入被修饰的宿主细胞病毒基因，以包含序列 将针对相关病毒蛋白以快速降解 细胞质。 因为病毒蛋白的细胞质降解提供了 I类抗原处理途径的底物，我们假设 这些目标策略将有助于CD8+ CTL的诱导 针对相关病毒蛋白的反应。 我们将直接测试 这种定位策略将导致增加的假设 用病毒肽和增强诱导的I类MHC分子加载 CTL响应。 将采用几种独立的方法来定义可转移 将靶向病毒蛋白的序列元素以快速降解 人类细胞的细胞质。 改良的病毒蛋白旨在进行 泛素（UB）依赖性降低n-end规则途径或通过 相关的第二密码子规则途径将在人类细胞系中表达。 降解的动力学将以生化测量，UB 降解的依赖性将使用带有的细胞系进行评估 E1 UB激活酶的温度敏感缺陷。 构造 设计用于经历其他两个人的快速细胞质降解 机制也将进行分析。 基于对 处理病毒包膜蛋白，用于I类限制识别 TAP 1/2依赖性途径，提议插入“ 上下文“细胞质蛋白中的疏水结构域将极度触发 快速降解，导致有效产生的肽可以 用I类分子表示。 另外，C端 将使用短寿命RAG-2蛋白的降解靶向域 迫使测试病毒蛋白的快速降解。 对于每个降解策略，自然的定量分析 将进行加工的肽以确定是否针对 导致与相关的I类MHC分子的负载增加 病毒肽。 另外以及与IL-2无关的克隆膨胀 将比较抗原特异性的CTL。 将是五种不同的方法 测量表达降解的细胞的免疫原性 靶向病毒蛋白。 这些包括抗原的体外研究 人类T细胞克隆的特定增生和细胞溶解反应，以及 使用反应细胞的次级体外CTL反应的研究 感染的人类捐助者。 另外，小鼠模型中的体内研究 将用于评估天真T细胞的CTL响应的产生 种群并评估流感病毒中的疫苗策略 免疫/挑战模型。 这些研究在一起将提供 开发基于CTL的疫苗策略的基础。