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

根治艾滋病毒需要从包括中枢神经系统(CNS)在内的所有组织中消除病毒，最重要的是彻底了解含有完整和/或复制能力病毒并在治疗中断时引起或支持反弹病毒血症的艾滋病毒储存库。作为长期存活的中枢神经系统髓系细胞，即小胶质细胞和常驻巨噬细胞是HIV的储存库。虽然HIV在原发病毒血症期间进入CNS，并且感染后不久分离的主要HIV变异株显示出T细胞趋向性，但在慢性感染过程中，感染表面CD4密度较低的细胞(如小胶质细胞)的HIV变异株被发现，这是由从血管周围和CNS髓系细胞中分离的HIV病毒DNA支持的，并在人源化髓系小鼠中独立于T细胞在巨噬细胞中持续复制HIV。在PLWH抑制ART的脑脊液(CSF)中也检测到来自T细胞的HIV。在脑脊液中检测到的HIV变异很可能是在中枢神经系统中以CD4+T和/或髓系细胞分隔的HIV独立复制的结果，以及HIV通过T细胞或单核细胞或作为游离病毒从外周转运的结果。技术和解剖学的限制限制了对(I)在中枢神经系统中参与艾滋病毒复制的细胞和病毒因素以及(Ii)在正确的细胞环境中引起反弹病毒的选择压力，特别是在小胶质细胞中的全面检查。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，尽管如此，它们在体内很快就失去了关键的特征，以及缺乏成功概括关键的小胶质细胞特征的体外模型。我们将使用从成人成纤维细胞分化出的人诱导多能干细胞来源的小胶质细胞(IMG)作为一个易于处理的系统来检测控制CNS髓系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，这是目前可用的模型无法实现的。关于反弹病毒的两个最新发展导致了这一提议的概念。首先，在最近一项对接受分析治疗中断(ATI)的患者的关键病毒表型进行的研究中，我们的合作者Bar博士发现，在整个患者的纵向随访过程中，在从血液中分离出的病毒中，在ATI后反弹的病毒显示出比任何其他病毒更高的干扰素-1耐药性，包括传播型方正(TF)病毒和蓄水池病毒。这些独一无二的干扰素-1耐药反跳病毒的来源尚不清楚。尽管ART受到抑制，但PLWH的中枢神经系统中干扰素-1信号的持续上调增加了这样一种可能性，即选择压力可能导致在中枢神经系统髓系细胞中建立高效的干扰素-1耐药病毒储存库。第二，我们的初步数据表明，Bar博士发现的一些反弹病毒的感染性分子克隆(IMCs)在IMG中的复制可能比TF病毒的IMCs更好。在这个方案中，我们将使用IMG和一组特征良好的TF、蓄积物和反弹病毒的IMC来验证我们的假设，即中枢神经系统髓系细胞可能是复制干扰素-1耐药反弹病毒的场所。

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.