Qualification of Assays to Measure Human T-cell Responses Against Mycobacterial Lipid Antigens

项目概要（摘要） 结核分枝杆菌（M.tb）在2013年造成150多万人死亡， 一种导致全球死亡的主要传染病目前，牛分枝杆菌卡介苗（BCG） 是唯一获得许可的结核病疫苗。卡介苗提供保护，防止传播形式的 但在预防成人肺结核的发展方面并不一致。以来 成人肺结核具有高度传染性，用卡介苗无法控制流行， 急需新的疫苗。目前有多种疫苗正在开发中，包括 重组BCG和减毒结核分枝杆菌菌株。对结核分枝杆菌的成功免疫应答关键取决于T- 细胞，其通常被结合到主要组织相容性复合物的细菌肽抗原激活 (MHC)分子。与卡介苗一样，候选全细胞疫苗是多抗原性的，既含有肽，又含有 被人类T细胞识别的非肽抗原。分枝杆菌细胞壁脂质决定性地 当与抗原呈递细胞上的CD 1蛋白结合时，显示出激活人T细胞。CD1 系统在哺乳动物中是保守的，在其他脊椎动物中几乎没有，这表明它进化到 执行与MHC系统非冗余的重要功能。然而，没有经过验证的 测定人T细胞对非肽抗原如脂质的应答的测定。我们有 与脂质抗原发现、流式细胞术、疫苗和计算机科学领域的专家建立了伙伴关系， 在过去的五年里，生物学解决了这一技术挑战。我们最近开发了可溶性CD 1 四聚体和基于活化的T细胞谱分析作为促进基于群体的T细胞研究的工具 对分枝杆菌脂质的反应。我们还开发了无偏的计算方法， 高维流式细胞术数据分析。在目标1中，我们将使用脂质- 加载的CD 1四聚体，以研究静息时脂质特异性T细胞的记忆表型和活化状态。在 目标2，我们将优化并鉴定一种互补的基于激活的测定方法来研究T的效应子功能 由脂质抗原激活的细胞。与MHC蛋白不同，CD 1蛋白实际上是非多态性的，因此这些 可以独立于遗传背景应用测定。在目标3中，我们将使用这两种测定来研究 使用BCG作为模型系统，分枝杆菌疫苗接种对人类脂质特异性T细胞应答的影响。 这项建议的主要合作者是艾滋病毒疫苗试验网络（HVTN）和南非艾滋病毒研究所。 结核病疫苗倡议（SATVI），在开发基于免疫的检测方面拥有丰富的经验 分别用于艾滋病毒和结核病候选疫苗试验。在融资期结束时，我们将 鉴定了一套检测方法，这些方法将补充现有的方法，以确定保护性 全细胞分枝杆菌疫苗有效性研究中的免疫力。

PROJECT SUMMARY (ABSTRACT)   Mycobacterium tuberculosis (M.tb) was responsible for more than 1.5 million deaths in 2013, making it a leading infectious cause of death worldwide. Currently, Mycobacterium bovis bacille Calmette-Guérin (BCG) is the only licensed vaccine for tuberculosis. BCG provides protection against disseminated forms of the disease in children but is inconsistent in preventing the development of pulmonary tuberculosis in adults. Since adults with pulmonary tuberculosis are highly infectious, control of the epidemic will not be achieved with BCG, and new vaccines are urgently needed. There are a number of vaccines under development, including recombinant BCG and attenuated M.tb strains. A successful immune response to M.tb depends critically on T- cells, which are typically activated by bacterial peptide antigens bound to major histocompatibility complex (MHC) molecules. Like BCG, candidate whole cell vaccines are poly-antigenic and contain both peptide and non-peptide antigens that are recognized by human T cells. Mycobacterial cell wall lipids have conclusively been shown to activate human T cells when bound to CD1 proteins on antigen-presenting cells. The CD1 system is conserved among mammals and mostly absent from other vertebrates, suggesting it evolved to perform an important function that is non-redundant with the MHC system. However, there are no validated assays to measure human T-cell responses against non-peptide antigens, such as lipids. We have established partnerships with experts in lipid antigen discovery, flow cytometry, vaccines, and computational biology to tackle this technical challenge over the last five years. We recently developed soluble CD1 tetramers and an activation-based T-cell profiling assay as tools to facilitate population-based studies of T-cell responses to mycobacterial lipids. We have also developed unbiased computational approaches to the analysis of high-dimensional flow cytometry data. In Aim 1, we will optimize and qualify an assay using lipid- loaded CD1 tetramers to study the memory phenotype and activation status of lipid-specific T cells at rest. In Aim 2, we will optimize and qualify a complementary activation-based assay to study the effector functions of T cells activated by lipid antigens. Unlike MHC proteins, CD1 proteins are virtually non-polymorphic so these assays can be applied independent of genetic background. In Aim 3, we will use both assays to study the effect of mycobacterial vaccination on lipid-specific T-cell responses in humans using BCG as a model system. Major collaborators on this proposal are the HIV Vaccine Trials Network (HVTN) and the South African Tuberculosis Vaccine Initiative (SATVI), which have extensive experience in developing immune-based assays for candidate HIV and tuberculosis vaccine trials, respectively. By the end of the funding period, we will have qualified a suite of assays that will supplement existing approaches to identifying correlates of protective immunity in efficacy studies of whole cell mycobacterial vaccines.