Single-chain antibodies to block HIV transcription and prevent reactivation from latently infected resting CD4+ T cells

单链抗体可阻断 HIV 转录并防止潜伏感染的静息 CD4 T 细胞重新激活

基本信息

批准号：
10308728
负责人：
Cheryl Stoddart
金额：
$ 20.19万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10308728
关键词：
Adopted Affinity Aftercare Animals Antibodies Area Binding Biogenesis Biological Assay Blocking Antibodies CD4 Positive T Lymphocytes Cell Nucleus Cell division Cells Clinical Data Development Disease remission Dissociation Effector Cell Epitopes Genetic Transcription Genome Goals Grant HIV Heat-Shock Proteins 90 Heat-Shock Response Human Immune Immunization In Vitro Infection prevention Integration Host Factors Length Longevity Mediating Modeling Molecular Conformation Mus Nuclear Envelope Nuclear Export Nucleic Acids Oral Patients Pharmaceutical Preparations Phenotype Physiological Plasma Process Protein Export Pathway Proteins Proviruses RNA RNA Splicing RNA chemical synthesis Recruitment Activity Reporter Reporter Genes Reporting Research Resistance Rest Role Sampling Specificity Spleen Surface System Therapeutic Tissues Transcript Viral Genome Viremia Virus Withholding Treatment antibody libraries antiretroviral therapy base clinically relevant cytotoxicity design humanized mouse in vitro Model inhibitor innovation next generation novel novel therapeutics prevent promoter prospective reactivation from latency response rev-Responsive Elements side effect stem therapeutic candidate transcription factor translation factor viral RNA viral genomics virtual

项目摘要

PROJECT SUMMARY Despite decades of successful antiretroviral therapy (ART), persistent HIV-infected resting CD4+ T cells can remain undetected in tissue reservoirs. HIV replication is cytopathic in activated CD4+ T cells; however, a fraction of HIV-infected activated CD4+ T cells revert to the resting G0 phenotype in tissue reservoirs. These persistent HIV-infected resting CD4+ T cells undergo intermittent activation and produce fully infectious virus. HIV is invariably detected in the plasma when patients discontinue ART because plasma viremia is reseeded by intermittent activation of persistent HIV-infected resting CD4+ T cells in the tissue reservoirs. In response to RFA-AI-19-072: Novel Therapeutics Directed to Intracellular HIV Targets, we propose an innovative analytic bioassay to identify next-generation single-chain antibodies that sustain HIV remission. Our research is focused on the essential role of heat shock protein 90 (Hsp90) in HIV replication, and we have shown that mild heat shock (39.5°C) accelerates HIV transcription. Increased Hsp90 activity at 39.5°C was critical for full-length HIV RNA synthesis by host transcription factors, and we have shown that 39.5°C reactivates latent HIV replication in ART-suppressed aviremic HIV-infected patient samples, in human resting CD4+ T cells isolated from fully suppressed humanized mice, and in three distinct in vitro models of HIV latency. Reactivation of latent HIV depends on multiple host proteins interacting with their HIV counterpart, and the recently developed QUECEL (quiescent effector cell latency) model of HIV latency is an ideal in vitro system to identify single-chain antibodies that disrupt HIV-host protein interactions. The QUECEL carries a modified HIV provirus that depends on all the HIV-host protein interactions to reactivate latent HIV RNA synthesis. The latent QUECEL depends on host protein interaction for both the HIV Tat-mediated transcription and HIV Rev-mediated export of the full-length viral genome, and we anticipate that single-chain antibodies will disrupt these critical HIV-host protein interactions required for reactivation of HIV latency. We have proof that the QUECEL can be reactivated at 39.5°C and designed a high-complexity single-chain antibody library to selectively enrich for candidates that prevent QUECEL reactivation. The prospective antiviral candidates will then be further enriched in resting CD4+ T cells isolated from ART-treated aviremic patients, and we anticipate identifying potent single- chain antibodies that disrupt the HIV-host protein interactions required to reactivate HIV from latency. Our analytic bioassay is innovative because 1) mild heat shock activation is physiologically similar to the intermittent cellular activation occurring in persistent HIV-infected tissue reservoirs and 2) the small size of single- chain antibodies will increase the target range to the surface area of HIV proteins, effectively blocking critical host-protein interactions. We propose to use mild heat shock to identify potent antiviral single-chain antibodies in the following Specific Aims: 1) Selectively enrich for single-chain antibodies that prevent reactivation of latent QUECEL, and 2) Identify potent single-chain antibodies that block HIV transcription.

项目摘要尽管数十年成功的抗逆转录病毒疗法（ART），但持续的HIV感染的静止CD4+ T细胞可以在组织储层中保持未发现。 HIV复制在活化的CD4+ T细胞中是细胞的。但是，一小部分 HIV感染的活化的CD4+ T细胞恢复为组织储层中的静息G0表型。这些持久 HIV感染的静息CD4+ T细胞经过间歇性激活并产生完全感染性病毒。艾滋病毒是当患者中断ART时，在血浆中总是检测到，因为血浆病毒血症已通过组织储层中持续的HIV感染的静息CD4+ T细胞的间歇性激活。为了响应RFA-AI-19-072：针对细胞内HIV靶标的新型治疗剂，我们提出了一个创新的分析生物测定，以识别维持HIV缓解的下一代单链抗体。我们的研究的重点是热休克蛋白90（HSP90）在HIV复制中的基本作用，我们已经显示轻度热休克（39.5°C）加速了HIV转录。 HSP90在39.5°C时的活性增加至关重要宿主转录因子全长HIV RNA RNA合成，我们已经表明39.5°C重新激活潜在在人类静止的CD4+ T细胞中，在抑制ART抑制的Art抑制的Avirememic HIV感染患者样品中的HIV复制来自完全抑制的人源性小鼠，以及三种不同的艾滋病毒潜伏期体外模型。潜在艾滋病毒的重新激活取决于多种宿主蛋白与艾滋病毒的相互作用，并且 HIV潜伏期的最近开发的Quecel（静态效应细胞潜伏）模型是一个理想的体外系统确定破坏HIV宿主蛋白相互作用的单链抗体。 Quecel带有改良的HIV 取决于所有HIV宿主蛋白相互作用以重新激活潜在的HIV RNA合成的病毒。潜在 Quecel取决于HIV TAT介导的转录和HIV Rev介导的宿主蛋白相互作用全长病毒基因组的导出，我们预计单链抗体会破坏这些关键 HIV蛋白相互作用需要重新激活HIV潜伏期。我们有证据表明Quecel可以是在39.5°C下重新激活，并设计了一个高复杂性单链抗体库，以选择性地丰富预防Quecel重新激活的候选者。然后将进一步丰富预期的抗病毒药候选人在静止从ART治疗的无毒患者中分离出的CD4+ T细胞时，我们预计会发现有效的单个破坏了从潜伏期重新激活HIV所需的HIV宿主蛋白相互作用的链抗体。我们的分析生物测定具有创新性，因为1）轻度热激活在物理上与间歇性细胞活化发生在持续的HIV感染组织储层中，2）链抗体将增加目标范围对HIV蛋白的表面积，从而有效阻断临界宿主蛋白质相互作用。我们建议使用轻度热冲击来鉴定潜在的抗病毒单链抗体在以下特定目的中：1）选择性地富集了防止潜在重新激活的单链抗体 Quecel和2）确定阻断HIV转录的潜在单链抗体。