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: 10548292
• 负责人: Cagla Akay Espinoza
• 金额: $ 22.81万
• 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/10548292
• 关键词: Address; Adherence; Adult; Anatomy; Blood; Cell Lineage; Cells; Cerebrospinal Fluid; Characteristics; Conceptions; Data; Development; Exhibits; Fibroblasts; HIV; Human; Infection; Interferons; Interruption; Knowledge; Lead; Microglia; Modeling; Molecular Cloning; Mus; Myelogenous; Myeloid Cells; Neuraxis; Patients; Phenotype; 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; macrophage; monocyte; novel strategies; pressure; trafficking; viral DNA; viral rebound

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HIV需要清除包括中枢神经系统（CNS）在内的所有组织中的病毒，并且彻底了解HIV储存库，这些储存库包含完整和/或具有复制能力的病毒，并在治疗中断时引起或支持反弹病毒血症，这一点至关重要。作为长寿命的中枢神经系统髓细胞，即小胶质细胞和巨噬细胞是HIV病毒的储存库。虽然HIV在原发性病毒血症期间进入中枢神经系统，并且在感染后不久分离的主要HIV变体表现出T细胞的亲和性，但在慢性感染期间发现感染表面CD4密度较低的细胞（如小胶质细胞）的HIV变体，这得到了从血管周围和中枢神经系统髓细胞中分离的HIV病毒DNA的支持，并且在人源化的纯髓细胞小鼠中巨噬细胞中独立于T细胞的持续HIV复制。在接受抑制性抗逆转录病毒治疗的患者脑脊液（CSF）中也检测到t细胞来源的HIV。在脑脊液中检测到的HIV变异可能是CD4+ T细胞和/或髓系细胞在中枢神经系统中区隔的HIV独立复制和通过T细胞或单核细胞或作为游离病毒从外周运输HIV的结合结果。技术和解剖学上的限制限制了对以下方面的全面研究：(1)参与HIV在中枢神经系统复制的细胞和病毒因素；(2)在正确的细胞环境下，特别是在小胶质细胞中，产生反弹病毒的选择压力。其中一个原因是，新分离的初级人类小胶质细胞的可用性有限，尽管如此，它们很快就会失去体内的关键特征，而且缺乏成功概括小胶质细胞关键特征的体外模型。我们将使用从成人成纤维细胞分化而来的人类诱导多能干细胞衍生的小胶质细胞（iMg）作为一种可处理的系统来检查控制中枢神经系统髓细胞中反弹病毒复制的宿主和病毒决定因素，并进行详细的分析，这是目前可用模型无法实现的。关于回弹病毒的两个最新发展导致了这一建议的构想。首先，在最近的一项研究中，研究了接受分析性治疗中断（ATI）的患者的关键病毒表型，我们的合作者Bar博士发现，在患者的纵向随访中，从血液中分离的病毒中，ATI后反弹的病毒比任何其他病毒（包括传播型创始人（TF）病毒和库病毒）表现出更高的IFN-1耐药性。这些独特的ifn -1抗性反弹病毒的起源尚不清楚。尽管抗逆转录病毒疗法具有抑制性，但PLWH的中枢神经系统中IFN-1信号的持续上调提出了一种可能性，即选择压力可能导致中枢神经系统髓系细胞内产生IFN-1抗性病毒库。其次，我们的初步数据表明，Bar博士发现的一些反跳病毒的传染性分子克隆（IMCs）在iMg中的复制可能比TF病毒的IMCs更好。在本提案中，我们将使用iMg与一组具有良好特征的TF、库病毒和反弹病毒的imc来验证我们的假设，即中枢神经系统髓细胞可能是ifn -1抗性反弹病毒复制的一个位点。