Viral dynamics of rebound and reservoir HIV species in IPSC-derived myeloid cells

IPSC 衍生骨髓细胞中反弹和储存 HIV 物种的病毒动态

• 批准号: 10684814
• 负责人: Cagla Akay Espinoza
• 金额: $ 20.31万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10684814
• 关键词: Address; Adherence; Adult; Anatomy; Binding; Blood; Blood Vessels; Cell Lineage; Cells; Central Nervous System; Cerebrospinal Fluid; Characteristics; Conceptions; Data; Development; Exhibits; Fibroblasts; HIV; Human; Infection; Interferons; Interruption; Knowledge; Macrophage; Microglia; Modeling; Molecular Cloning; Mus; Myelogenous; Myeloid Cells; Patients; Phenotype; Productivity; Resistance; Role; Signal Transduction; Site; Source; Surface; System; T-Lymphocyte; Technology; Testing; Tissues; Toxic effect; Tropism; Up-Regulation; Variant; Viral; Viral reservoir; Viremia; Virus; Virus Replication; antiretroviral therapy; chronic infection; density; follow-up; in vitro Model; in vivo; induced pluripotent stem cell; lymph nodes; monocyte; novel strategies; pressure; trafficking; transmission process; variant detection; viral DNA; viral rebound; virus identification

Eradicative HIV cure requires elimination of virus from all tissues, including the central nervous system (CNS), and a thorough understanding of HIV reservoirs that harbor intact and/or replication-competent virus and give rise or support rebound viremia upon treatment interruption is paramount. As long-lived CNS myeloid cells, namely microglia and resident macrophages are an HIV reservoir. While HIV enters the CNS during primary viremia and the predominant HIV variants isolated soon after infection exhibit T-cell tropism, HIV variants that infect cells with lower surface CD4 density, such as microglia, are found later during chronic infection, supported by HIV viral DNA isolated from perivascular and CNS myeloid cells and sustained HIV replication in macrophages independently of T cells in humanized myeloid-only mice. HIV from a T-cell source has also been detected in the cerebrospinal fluid (CSF) of PLWH on suppressive ART. HIV variants detected in the CSF are likely a result of a combination of independent replication of HIV compartmentalized in the CNS in CD4+ T and/or myeloid­ lineage cells and HIV trafficking via T cells or monocytes or as free virus from the periphery. Technical and anatomical constraints limit the comprehensive examination of (i) cellular and viral factors involved in HIV replication in the CNS and (ii) selection pressures that give rise to rebound viruses in the correct cellular context, especially in microglia. Among the reasons are limited availability of freshly isolated primary human microglia, which nonetheless rapidly lose key in vivo features, and the paucity of in vitro models that successfully recapitulate key microglial characteristics. We will use human induced pluripotent stem cell-derived microglia (iMg) differentiated from adult human fibroblasts as a tractable system to examine host and viral determinants governing rebound virus replication in CNS myeloid cells with detailed analyses that are not possible with currently available models. Two recent developments regarding rebound viruses has led to the conception of this proposal. First, in a recent study examining key viral phenotypes in patients undergoing analytical treatment interruption (ATI), our collaborator Dr. Bar found that viruses that rebounded after ATI exhibited higher IFN-1 resistance than any other viruses, including transmitted founder (TF) viruses and reservoir viruses, among viruses isolated from the blood throughout the longitudinal follow-up of patients. The origins of these uniquely IFN-1-resistant rebound viruses remain unclear. Persistent upregulation of IFN-1 signaling in the CNS of PLWH despite suppressive ART raises the possibility that selection pressures might lead to the establishment of a productive reservoir of IFN-1-resistant virus within CNS myeloid-lineage cells. Second, our preliminary data suggest that infectious molecular clones (IMCs) of some rebound viruses identified by Dr. Bar may replicate better than the IMCs of TF viruses in iMg. In this proposal, we will use iMg with a panel of well-characterized IMCs of TF, reservoir, and rebound viruses to test our hypothesis that CNS myeloid cells may be a site for replication of IFN-1-resistant rebound viruses.

根除性HIV治疗需要从所有组织（包括中枢神经系统（CNS））中消除病毒，并且彻底了解HIV储库，这些储库含有完整和/或复制能力的病毒，并在治疗中断时引起或支持反弹病毒血症。由于长寿命的CNS髓样细胞，即小胶质细胞和常驻巨噬细胞是HIV储库。虽然HIV在原发性病毒血症期间进入CNS，并且感染后不久分离的主要HIV变体表现出T细胞嗜性，但感染具有较低表面CD 4密度的细胞（如小胶质细胞）的HIV变体在慢性感染期间较晚发现，由从血管周围和CNS髓样细胞分离的HIV病毒DNA支持，并且在人源化仅髓样小鼠中不依赖于T细胞而在巨噬细胞中持续HIV复制。在接受抑制性ART治疗的PLWH的脑脊液（CSF）中也检测到来自T细胞来源的HIV。CSF中检测到的HIV变体可能是CNS中CD 4 + T和/或髓系造血细胞中区室化的HIV独立复制与HIV通过T细胞或单核细胞或作为游离病毒从外周转运的组合的结果。技术和解剖学上的限制限制了对（i）CNS中HIV复制所涉及的细胞和病毒因素以及（ii）在正确的细胞环境中（特别是在小胶质细胞中）引起病毒反弹的选择压力的全面检查。原因之一是新鲜分离的原代人类小胶质细胞的可用性有限，但很快就会失去关键的体内特征，以及缺乏成功重现关键小胶质细胞特征的体外模型。我们将使用人诱导多能干细胞衍生的小胶质细胞（iMg）分化成人成纤维细胞作为一个易于处理的系统，检查主机和病毒决定因素管理反弹病毒复制中枢神经系统骨髓细胞与详细的分析，是不可能与目前可用的模型。关于反弹病毒的两个最新进展导致了这一建议的概念。首先，在最近的一项研究中，我们的合作者Bar博士检查了接受分析性治疗中断（ATI）的患者的关键病毒表型，发现ATI后反弹的病毒表现出比任何其他病毒更高的IFN-1抗性，包括传播的创始人（TF）病毒和储库病毒，在整个患者纵向随访期间从血液中分离的病毒中。这些独特的抗IFN-1反弹病毒的起源仍不清楚。持续上调IFN-1信号在中枢神经系统的PLWH，尽管抑制性ART提出的可能性，选择压力可能会导致建立一个生产水库的IFN-1耐药病毒在中枢神经系统的骨髓系细胞。其次，我们的初步数据表明，由Bar博士鉴定的一些反弹病毒的感染性分子克隆（IMC）可能比TF病毒的IMC在iMg中复制得更好。在这个提议中，我们将使用iMg与一组充分表征的TF、储库和反弹病毒的IMC来测试我们的假设，即CNS髓样细胞可能是IFN-1抗性反弹病毒复制的位点。