Defining the HLA ligandome of HIV-1 latently infected CD4 + T cells

项目摘要 抗逆转录病毒疗法未能治愈HIV-1感染，因为潜伏感染的细胞库恢复病毒复制， 治疗中断时的阳离子。这激发了治疗方法的发展， HIV-1感染。那些引起针对病毒表位的免疫应答的需要先前的病毒再活化 与不能有效诱导病毒抗原表达的潜伏期逆转剂。 近年来，已经开发了技术来鉴定天然加工和呈递的表位， I类和II类HLA分子（HLA配体组），以引发CD 4 + T辅助细胞和CD 8 + CTL特异性效应， 和记忆反应。这一策略已被应用于癌症领域，从而允许设计治疗- 寻求引发针对肿瘤相关肽的基于细胞的免疫的疫苗接种策略。这 目前正在临床环境中评估新的方法来治疗人类癌症，例如成胶质细胞瘤， MA、多发性骨髓瘤、急性髓性白血病、肝细胞癌、卵巢癌和肾细胞癌。一 对HIV-1潜伏感染细胞上独特表达的表位的全面分析从未被... 诱惑然而，这可能为开发基于免疫的治疗性AP提供基础。 在没有病毒再活化的情况下，杀死携带潜伏HIV-1的细胞。 本申请的主要目标是定义潜伏性HLA-IgM抗体的HLA“配体组”（病毒和宿主表位）。 与未感染的细胞相比。这种独特的肽子集的鉴定可以导致 设计治疗性疫苗接种策略，在没有病毒的情况下靶向并消除潜伏感染的细胞， 重新激活为了实现这一目标，我们提出了两个具体目标。具体目标1旨在确定独特的 在MHC-I和II分子的背景下，在潜伏感染细胞的表面上表达的配体组。 我们将获得在潜伏感染和未感染细胞上表达的MHC-I和II相关肽， 用我们实验室开发的体外模型，我们将通过质谱法鉴定它们。肽 在潜伏感染的中央记忆CD 4 + T细胞上独特表达的CD 4 + T细胞将针对大量数据进行筛选， 在正常健康的人类细胞表面表达的肽的基础。这将定义病毒和宿主 HIV-1潜伏感染的CD 4 + T细胞的独特特征。第2章评价 对HIV-1潜伏感染细胞配体组中鉴定的特异性肽的免疫应答， cART治疗的HIV-1感染受试者。我们将长期评估HIV-1中T细胞应答的频率， 感染的患者针对选定的表位。将通过细胞内IFNγ和IL-4评估特异性应答。 细胞因子染色、IFNγ和IL-4 ELISPOT测定和四聚体结合测定。结果将提供相关数据- ta，以确定HIV-1潜伏感染患者对所鉴定表位的应答程度。 本申请中提出的研究将与Luigi Buonaguro博士（国家 癌症研究所，意大利）、Maureen Kane（马里兰州大学药学院）和Mathias Lichterfeld (BWH/MGH，波士顿），每个人都贡献了完成项目所需的一套独特的专业知识。

PROJECT SUMMARY Antiretroviral therapy fails to cure HIV-1 infection due to a pool of latently infected cells that resume viral repli- cation upon treatment interruption. This has sparked the development of therapeutic approaches toward a cure for HIV-1 infection. Those entailing immune responses against viral epitopes require previous viral reactivation with latency reversing agents, which do not efficiently induce the expression of viral antigens. In recent years, techniques have been developed to identify epitopes naturally processed and presented by class I and class II HLA molecules (HLA ligandome) to elicit both CD4+ T helper and CD8+ CTL specific effec- tor and memory responses. This strategy has been applied in the cancer field, thus allowing the design of ther- apeutic vaccination strategies seeking to elicit cell-based immunity against tumor-associated peptides. This novel approach is currently being evaluated in the clinical setting to treat human cancers, such as glioblasto- ma, multiple myeloma, acute myeloid leukemia, hepatocellular carcinoma, ovarian and renal cell cancer. A comprehensive analysis of epitopes uniquely expressed on HIV-1 latently infected cells has never been at- tempted. Nevertheless, this might provide the basis for the development of immune-based therapeutic ap- proaches aimed at killing cells harboring latent HIV-1 in the absence of viral reactivation. The main goal of this application is to define the HLA “ligandome” (viral and host epitopes) of latently infected compared to uninfected cells. The identification of such unique subset of peptides could lead to devise therapeutic vaccination strategies to target and eliminate latently infected cells in the absence of viral reactivation. To achieve this goal, we propose two specific aims. Specific Aim 1 seeks to identify the unique ligandome expressed on the surface of latently infected cells in the context of MHC-I and II molecules. We will obtain MHC-I and II-associated peptides expressed on latently infected and uninfected cells generated with an in vitro model developed in our laboratory, and we will identify them by mass spectrometry. Peptides uniquely expressed on latently infected central memory CD4+ T cells will be screened against a large data- base of peptides expressed on the surface of normal, healthy human cells. This will define the viral and host ligandome that uniquely characterizes CD4+ T cells latently infected with HIV-1. Specific Aim 2 will evaluate immune responses to specific peptides identified in the ligandome of HIV-1-latently infected cells in cART-treated HIV-1 infected subjects. We will assess the frequency of T-cell responses in HIV-1 chronically infected patients against selected epitopes. Specific responses will be assessed via IFNγ and IL-4 intracellular cytokine staining, IFNγ and IL-4 ELISPOT assay, and tetramer binding assay. Results will provide relevant da- ta in order to determine the extent of responses to the identified epitopes in HIV-1 latently infected patients. The studies proposed in this application will be conducted in collaboration with Drs. Luigi Buonaguro (National Cancer Institute, Italy), Maureen Kane (University of Maryland School of Pharmacy), and Mathias Lichterfeld (BWH/MGH, Boston), each contributing a unique set of expertise needed for the completion of the project.