A Microengraving Technology for the Study of Latently HIV-infected Primary Cells

描述(由申请人提供)：高效抗逆转录病毒疗法(HAART)有效地减少了艾滋病毒-1的复制，但早期对通过长期使用这些药物治疗来根除病毒的乐观情绪并未得到满足。这在很大程度上是因为HIV-1可以引起宿主细胞的潜伏感染，当被感染、激活的细胞恢复到静止状态时，宿主细胞就会发展。这些潜伏感染的细胞是HIV-1感染的转录沉默的储存库，对目前可用的抗逆转录病毒药物和HIV-1特异性免疫反应具有耐药性。潜伏感染细胞的半衰期延长(约44个月)，加上它们在停止HAART后迅速启动病毒学反弹的能力，是导致病毒长期存在的主要因素。这些特征与裂解感染细胞形成鲜明对比，裂解感染细胞代表着高度激活、短暂的细胞群体，具有高病毒产量，可以被抗逆转录病毒药物和HIV-1特异性CD8+T细胞有效抑制。了解控制潜伏和裂解感染细胞的维持、存活、周转和HIV-1基因表达的分子机制将为开发针对持续病毒复制或潜伏感染的细胞的特定治疗方案开辟新的前景，因此是当前HIV-1研究的首要课题之一。要充分详细地确定潜伏感染细胞的特征，主要的技术障碍是我们目前无法有效和准确地识别和物理分离这些细胞。在这项提案中，我们通过提出一种新的微雕技术来解决这一关键问题，该技术允许对单个原代细胞的感染状态进行前所未有的高通量测试。为此，我们将使用一种独特的生物芯片，允许将单个单个核细胞物理分离到超过105-106个亚纳升体积的孔中。从每个单个细胞的培养上清液打印的蛋白质微阵列，以及随后与gp120抗体的询问，将被用于在单个细胞水平上检测具有活跃病毒产生的细胞。通过选择性地对体外激活后的原代激活细胞或静止细胞进行这种检测，我们将能够识别、物理分离和扩增裂解和潜伏的HIV-1感染细胞，并研究它们的关键生物学特性，包括它们的TCR谱系和克隆型组成，它们的表型特征，以及决定它们生长、增殖和长期维持的分子机制。此外，将这种新颖的多重技术应用于分析不同HIV-1疾病进展速度的人的外周血和组织样本中的单个核细胞，将使我们能够确定与HIV-1感染的不同临床结果相关的裂解或潜伏的HIV-1储存库的特定数量或质量特征。该项目是拥有单细胞生物芯片检测工程专业知识的Love实验室(MIT)和拥有HIV免疫学和病毒学专业知识的Yu实验室(Ragon Institute/MGH)合作完成的。这种跨学科的方法解决了HIV-1研究中最重要的问题之一，有可能导致针对潜伏感染细胞的新策略，从而完全治愈HIV-1。 公共卫生相关性：潜伏着HIV-1感染的静止的CD4T细胞是病毒持续存在和我们无法治愈HIV-1感染的主要原因。在这项提案中，我们将使用一种新颖的、多路生物芯片分析系统，该系统将允许识别、分离和表征这些潜伏感染的细胞，并为开发针对它们的特定治疗策略做出贡献。

DESCRIPTION (provided by applicant): Highly active antiretroviral therapy (HAART) effectively reduces HIV-1 replication, but the early optimism for viral eradication through prolonged treatment with these drugs has not been met. This is largely due to the fact that HIV-1 can cause a latent infection of host cells that develops when infected, activated cells revert back to a quiescent state. These latently infected cells represent a transcriptionally silent reservoir for HIV-1 infection that is resistant to currently available antiretroviral drugs and HIV-1-specific immune responses. The prolonged half life of latently infected cells (approximately 44 months), combined with their ability to rapidly initiate virological rebound after discontinuation of HAART, are the main factors responsible for viral long-term persistence. These characteristics are in sharp contrast to lytically infected cells, which represent a highly activated, short-lived cell population with high viral production that can be effectively suppressed by antiretroviral drugs and HIV-1-specific CD8+ T cells. Understanding the molecular mechanisms that govern the maintenance, survival, turnover and HIV-1 gene expression in latently and lytically infected cells would open up novel perspectives for developing specific therapeutic options to target cells with persistent viral replication or latent HIV-1 infection, and therefore represent one of the highest priority topics in current HIV-1 research. The major technological obstacle to characterizing latently infected cells in sufficient detail is our current inability to identify and physically isolate these cells in an efficient and accurate manner. In this proposal, we address this key issue by proposing a novel microengraving technology that allows unprecedented high-throughput testing of the infection status of individual primary cells. For this purpose, we will use a unique biochip that allows for the physical separation of individual mononuclear cells into more than ~105-106 wells with subnanoliter volumes. Protein microarrays printed from the supernatant of each individual cell and their subsequent interrogation with gp120 antibodies will then be used to detect cells with active viral production on a single cell level. By selectively subjecting primary activated cells or quiescent cells following ex vivo activation to this assay, we will be able to identify, physically isolate, and expand lytically and latently HIV-1 infected cells, and to study their key biological properties, including their TCR repertoire and clonotypic composition, their phenotypic characteristics, and the molecular mechanisms that determine their growth, proliferation and long-term maintenance. Moreover, applying this novel, multiplexed technology to the analysis of mononuclear cells from peripheral blood and tissue specimens in individuals with different rates of HIV-1 disease progression will allow us to determine specific quantitative or qualitative characteristics of the lytic or latent HIV-1 reservoir associated with distinct clinical outcomes of HIV-1 infection. This project is a collaboration between the Love lab (MIT) with expertise in engineering of single-cell biochip assays and the Yu lab (Ragon Institute/MGH) with expertise in the immunology and virology of HIV. This interdisciplinary approach to one of the most significant problems in HIV-1 research has the potential to result in novel strategies to target latently infected cells, and thus to cure HIV-1 entirely. PUBLIC HEALTH RELEVANCE: Quiescent CD4 T cells with latent HIV-1 infection represent the main reason for viral persistence and our inability to cure HIV-1 infection. In this proposal, we will use a novel, multiplexed bio-chip analysis system that will allow to identify, isolate and characterize these latently infected cells, and to contribute to the development of specific therapeutic strategies to target them.