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.

项目总结 尽管几十年的抗逆转录病毒治疗(ART)取得了成功，但持续感染艾滋病毒的静息CD4+T细胞可以 在组织储藏室中仍未被检测到。HIV复制是激活的CD4+T细胞的细胞病变；然而，一小部分 在组织储存库中，HIV感染的活化的CD4+T细胞恢复到静止的G0表型。这些坚持不懈 HIV感染的静止的CD4+T细胞经历间歇性激活，并产生完全具有感染性的病毒。艾滋病毒是 当患者停止抗逆转录病毒治疗时，总是在血浆中检测到，因为血浆病毒血症是通过 组织储存库中持续感染艾滋病毒的静息CD4+T细胞的间歇性激活。 为了回应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，以确定维持艾滋病毒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复制中的重要作用，我们已经证明 温和的热休克(39.5摄氏度)加速了艾滋病毒的转录。在39.5摄氏度时增加Hsp90活性对 宿主转录因子合成全长HIV RNA，我们发现39.5℃重新激活潜伏 ART抑制的无核型HIV感染者样本中HIV的复制，分离的人类静息CD4+T细胞 来自完全受抑制的人源化小鼠，并在三个不同的体外模型中潜伏着艾滋病毒。 潜伏的HIV的重新激活依赖于多个宿主蛋白与其对应的HIV蛋白相互作用，而 最近开发的HIV潜伏期的QUECEL(静态效应细胞潜伏期)模型是一种理想的体外系统 识别破坏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 前病毒依赖于所有HIV-宿主蛋白的相互作用来重新激活潜在的HIV RNA合成。潜伏者 QUECEL依赖于HIV Tat介导的转录和HIV Rev介导的宿主蛋白相互作用 输出全长病毒基因组，我们预计单链抗体将破坏这些关键的 HIV-宿主蛋白相互作用是重新激活HIV潜伏期所必需的。我们有证据证明QUECEL可以 在39.5℃下重新激活，并设计了一个高复杂性的单链抗体库，用于选择性地富集 阻止QUECEL重新激活的候选人。然后，未来的抗病毒候选药物将进一步丰富 在静息状态下，从接受抗逆转录病毒治疗的无氧血症患者中分离出CD4+T细胞，我们预计将鉴定出有效的单一... 破坏HIV-宿主蛋白相互作用的链抗体，这些相互作用是从潜伏期重新激活HIV所需的。 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 在持续感染HIV的组织储存库中发生间歇性细胞激活，以及2)单个小尺寸的 链抗体会将靶向范围扩大到HIV蛋白的表面积，有效地阻断关键的 宿主与蛋白质的相互作用。我们建议使用轻度热休克来鉴定有效的抗病毒单链抗体。 在以下特定目的中：1)选择性地富含单链抗体，以防止潜伏期重新激活 QUECEL，以及2)识别阻止HIV转录的有效单链抗体。