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

用于研究潜在 HIV 感染原代细胞的微雕刻技术

• 批准号: 8312490
• 负责人: Xu Yu
• 金额: $ 40.56万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-15 至 2014-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312490
• 关键词: Address; Affect; Anti-Retroviral Agents; Antibodies; Back; Basic Science; Biologic Characteristic; Biological; Biological Assay; Biological Markers; Biomedical Engineering; CD4 Positive T Lymphocytes; CD8B1 gene; Cells; Characteristics; Clinical; Collaborations; Development; Disease; Disease Progression; Engineering; Evaluation; Gene Expression; Genes; Genome; Growth; HIV; HIV Envelope Protein gp120; HIV-1; Half-Life; Highly Active Antiretroviral Therapy; Human; Immune response; Immunologist; Immunology; Individual; Infection; Institutes; Investigation; Life; Love; Lytic; Lytic Phase; Maintenance; Memory; Molecular; Mononuclear; NIH Program Announcements; Outcome; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Population; Printing; Production; Property; Protein Microchips; Research; Resistance; Resources; Rest; Sampling; Solutions; Source; Specimen; System; Systems Analysis; T-Lymphocyte; Technology; Testing; Therapeutic; Therapeutic Intervention; Tissues; Viral; Viremia; Virion; Virus; antiretroviral therapy; base; biochip; cohort; innovation; interdisciplinary approach; latent infection; meetings; novel; novel strategies; optimism; physical separation; public health relevance; stem; therapeutic development; tool; virology

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.

描述（由申请人提供）：高效抗逆转录病毒疗法（HAART）可有效降低HIV-1复制，但通过长期治疗这些药物根除病毒的早期乐观情绪尚未得到满足。这在很大程度上是由于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感染的转录沉默储存库，其对目前可用的抗逆转录病毒药物和HIV-1特异性免疫应答具有抗性。潜伏感染细胞的半衰期延长（约44个月），加上它们在HAART停药后迅速启动病毒学反弹的能力，是病毒长期持续存在的主要因素。这些特征与裂解性感染细胞形成鲜明对比，裂解性感染细胞代表具有高病毒产量的高度活化的短寿命细胞群，其可以被抗逆转录病毒药物和HIV-1特异性CD 8 + T细胞有效抑制。了解的分子机制，管理的维持，生存，营业额和HIV-1的基因表达在潜伏和裂解感染的细胞将开辟新的前景，开发特定的治疗方案，以靶细胞与持续的病毒复制或潜伏的HIV-1感染，因此代表在当前的HIV-1研究的最高优先级的主题之一。足够详细地表征潜伏感染细胞的主要技术障碍是我们目前无法以有效和准确的方式鉴定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刻技术来解决这个关键问题，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，该芯片允许将单个单核细胞物理分离到超过约105-106个威尔斯的亚纳升体积的孔中。从每个单个细胞的上清液打印的蛋白质微阵列和随后用gp 120抗体进行的询问将用于检测在单细胞水平上具有活性病毒产生的细胞。通过选择性地使原代活化细胞或离体活化后的静止细胞经受该测定，我们将能够鉴定、物理分离和扩增溶胞性和潜伏性HIV-1感染的细胞，并研究它们的关键生物学特性，包括它们的TCR库和克隆型组成、它们的表型特征以及决定它们的生长、增殖和长期维持的分子机制。此外，将这种新型的多路复用技术应用于分析具有不同HIV-1疾病进展率的个体的外周血和组织标本中的单核细胞，将使我们能够确定与HIV-1感染的不同临床结果相关的溶解性或潜伏性HIV-1储库的特定定量或定性特征。该项目是爱实验室（麻省理工学院）与单细胞生物芯片检测工程专业知识和Yu实验室（Ragon研究所/MGH）在艾滋病毒免疫学和病毒学方面的专业知识之间的合作。这种针对HIV-1研究中最重要问题之一的跨学科方法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HIV-1感染的静止CD 4 T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，我们将使用一种新型的多路复用生物芯片分析系统，该系统将允许识别，分离和表征这些潜伏感染的细胞，并有助于开发针对它们的特定治疗策略。