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HIV-1的复制，但通过这些药物的长期治疗来根除病毒的早期乐观情绪尚未得到满足。这主要是由于HIV-1可以引起宿主细胞的潜伏感染，当被感染时，激活的细胞会恢复到静止状态。这些潜伏感染的细胞代表了HIV-1感染的转录沉默库，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），再加上它们在停止HAART治疗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。这些特征与溶性感染细胞形成鲜明对比，溶性感染细胞是一种高度活化、寿命短的细胞群，具有高病毒产量，可被抗逆转录病毒药物和hiv -1特异性CD8+ T细胞有效抑制。了解潜伏和裂解感染细胞中控制维持、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HIV-1感染的细胞的特定治疗方案开辟新的视角，因此是当前HIV-1研究中最优先考虑的课题之一。对潜伏感染细胞进行充分详细描述的主要技术障碍是我们目前无法以有效和准确的方式识别和物理分离这些细胞。在本提案中，我们通过提出一种新的微雕刻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，可以将单个核细胞物理分离到约105-106个亚纳升体积的孔中。从每个细胞的上清液中打印的蛋白质微阵列以及随后的gp120抗体将用于检测单细胞水平上产生活跃病毒的细胞。通过选择性地将原代活化细胞或离体激活后的静止细胞置于该试验中，我们将能够识别、物理分离和扩增裂解和潜伏HIV-1感染细胞，并研究其关键生物学特性，包括其TCR库和克隆型组成、表型特征以及决定其生长、增殖和长期维持的分子机制。此外，将这种新颖的多路复用技术应用于不同HIV-1疾病进展率个体外周血和组织标本中的单个核细胞分析，将使我们能够确定与HIV-1感染的不同临床结果相关的溶解性或潜伏性HIV-1储存库的特定定量或定性特征。该项目是Love实验室（MIT）与Yu实验室（Ragon研究所/MGH）在HIV免疫学和病毒学方面的合作项目，Love实验室拥有单细胞生物芯片分析的工程专业知识。这种跨学科的方法解决了HIV-1研究中最重要的问题之一，有可能产生针对潜伏感染细胞的新策略，从而完全治愈HIV-1。

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.