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

• 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
• 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

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 尽管抗逆转录病毒治疗（ART）已经成功了几十年，但持续感染HIV的静息CD 4 + T细胞仍然可以在体内存活。 仍然在组织储库中未被检测到。HIV复制在活化的CD 4 + T细胞中是致细胞病变的;然而， 的HIV感染的活化的CD 4 + T细胞在组织储库中回复到静止的G 0表型。这些持续 HIV感染的静息CD 4 + T细胞经历间歇性激活并产生完全感染性的病毒。艾滋病毒是 当患者停止ART时，血浆中总是检测到这种病毒，因为血浆病毒血症通过 组织储库中持续HIV感染的静息CD 4 + T细胞的间歇性激活。 针对RFA-AI-19-072：针对细胞内HIV靶点的新型治疗药物，我们提出了一种 创新的分析生物测定，以确定下一代单链抗体，维持艾滋病毒缓解。我们 研究的重点是热休克蛋白90（Hsp 90）在HIV复制中的重要作用，我们已经表明， 温和的热休克（39.5°C）加速了HIV的转录。在39.5°C下增加的Hsp 90活性对于 全长HIV RNA的合成宿主转录因子，我们已经表明，39.5°C重新激活潜伏的 ART抑制的HIV感染患者样本中HIV复制，分离的人静息CD 4 + T细胞 从完全抑制的人源化小鼠，并在三个不同的体外模型的HIV潜伏期。 潜伏的HIV的重新激活依赖于多种宿主蛋白与它们的HIV对应物的相互作用， 最近开发的HIV潜伏期的QUECEL（静止效应细胞潜伏期）模型是一种理想的体外系统， 鉴定破坏HIV-宿主蛋白相互作用的单链抗体。QUECEL携带一种改良的艾滋病毒 依赖于所有HIV-宿主蛋白质相互作用来重新激活潜伏的HIV RNA合成的前病毒。潜 QUECEL依赖于宿主蛋白质相互作用，用于HIV Tat介导的转录和HIV Rev介导的转录。 全长病毒基因组的出口，我们预计单链抗体将破坏这些关键的 HIV潜伏期再激活所需的HIV-宿主蛋白相互作用。我们有证据证明QUECEL可以 在39.5°C下重新活化，并设计了一个高复杂性的单链抗体库，以选择性地富集 防止QUECEL重新激活的候选项。随后将进一步丰富有前景的抗病毒候选药物 在从ART治疗的病毒血症患者中分离的静息CD 4 + T细胞中， 链抗体，破坏HIV-宿主蛋白质相互作用，使HIV从潜伏期重新激活。 我们的分析生物测定是创新的，因为1）温和的热休克激活在生理上类似于 间歇性细胞激活发生在持续的HIV感染组织水库和2）小尺寸的单个- 链抗体将靶向范围扩大到HIV蛋白的表面积，有效阻断关键的 宿主-蛋白质相互作用我们建议使用温和的热休克来鉴定有效的抗病毒单链抗体 1）选择性富集单链抗体，其防止潜伏性抗体的再活化。 QUECEL，和2）鉴定阻断HIV转录的有效单链抗体。