Characterize replication competent myeloid reservoirs in the central nervous system

摘要 根除艾滋病毒-1(HIV)感染必须针对感染持续存在的所有组织。净空 中枢神经系统(CNS)的感染不是单靠抗逆转录病毒疗法(ART)就能实现的，就像在 外围。CNS中潜在的HIV宿主可能会在ART停止后允许病毒反弹，因为HIV 可以从大脑进入外周血液。然而，需要研究来阐明这些贡献 潜伏感染的中枢神经系统细胞亚群与中枢神经系统中持续存在的艾滋病毒有关。循环中的T细胞已经 具有外周血液中主要的艾滋病毒宿主的特征，并可能循环到中枢神经系统， 有助于艾滋病毒在大脑中的持续存在。然而，T细胞是否是唯一的 中枢神经系统的主要病毒库。 髓系细胞是大脑中感染艾滋病毒的免疫系统的主要细胞隔间。在……里面 活体成像和免疫染色研究表明，脑髓系细胞(BMC)含有HIV DNA和 生产艾滋病毒核糖核酸和蛋白质。然而，目前还不清楚骨髓细胞，特别是长期存活的小胶质细胞是否 潜伏感染，如果它们是真正的艾滋病毒宿主，编码具有复制能力的艾滋病毒，尽管耐用， 成功的艺术。我们在北卡罗来纳大学艾滋病毒治疗中心的团队最近建立了一项协议，将高度分离 来自SIV感染猕猴大脑的纯髓系细胞，其中ART被中断。这些SIV 含有BMCs的细胞可在体外培养多代。该技术在其他领域的进一步应用 完全抑制ART的动物发现，骨髓细胞含有SIV前病毒DNA，SIV RNA可以有效地 潜伏期反转剂诱导。这个在体外研究髓系细胞的平台允许严格的 脑髓系细胞的特征，以解决它们作为真正的艾滋病毒储存库的角色，可以产生复制- 有能力的病毒。我们的初步数据强调了表观遗传调控，如组蛋白去乙酰化 和组蛋白甲基化，可能在调节HIV在中枢神经系统中的潜伏期中发挥重要作用。我们 假设骨髓细胞潜伏感染艾滋病毒，并携带有复制能力的艾滋病毒。 在这项提议中，我们将从大脑的不同区域分离高度纯净的脑髓细胞 艾滋病病毒携带者(PLWH)登记在“最后的礼物”队列中，并完全抑制SIV感染的恒河猴 猕猴。我们将使用完整的前病毒检查BMC是否具有复制能力的HIV或SIV DNA测定法(IPDA)和病毒生长定量测定法(QVOA)(Aim1)。我们将检查潜伏的HIV病毒 对目前可用的与表观遗传调控有关的潜伏期反转剂的反应 HIV潜伏期(目标2)，这可能为大脑中允许HIV潜伏期的机制提供线索。我们的研究 将解决知识方面的关键差距，以更好地了解骨髓基质细胞是否真的是艾滋病毒宿主和 HIV是如何在这些中枢神经系统细胞中持续存在的。

SUMMARY The eradication of HIV-1(HIV) infection must address all tissues where infection persists. Clearance of infection in the central nervous system (CNS) is not achieved by antiretroviral therapy (ART) alone, just as in the periphery. Latent HIV reservoirs in the CNS may allow viral rebound upon discontinuation of ART, as HIV can egress from the brain into the peripheral blood. However, studies are needed to elucidate the contributions of subsets of latently infected CNS cells to ongoing HIV persistence in the CNS. Circulating T cells have been well characterized as the major HIV reservoirs in the peripheral blood, and may circulate into the CNS, contributing to HIV persistence in the brain. Nevertheless, it is still not known whether T cells are the only major viral reservoir in the CNS. Myeloid cells are a major cellular compartment of the immune system infected by HIV in the brain. In vivo imaging and immunostaining studies have revealed that brain myeloid cells (BMCs) harbor HIV DNA and produce HIV RNA and proteins. However, it is not clear whether BMCs, and especially long-lived microglia, are latently infected, and if they are true HIV reservoirs encoding replication-competent HIV despite durable, successful ART. Our team at the UNC HIV Cure Center has recently established a protocol to isolate highly pure myeloid cells from the brain of SIV-infected macaques, in whom ART was interrupted. These SIV containing BMCs can be cultured for many generations ex vivo. Further application of this technology to other fully ART-suppressed animals found that BMCs contain proviral SIV DNA, and that SIV RNA can be effectively induced by latency reversal agents. This platform to study myeloid cells ex vivo allows rigorous characterization of brain myeloid cells to address their role as true HIV reservoirs that can produce replication- competent viruses. Our preliminary data highlights that epigenetic regulation, such as histone deacetylation and histone methylation, may play an essential role in the modulation of HIV latency in the CNS. We hypothesize that BMCs are latently infected by HIV and harbor replication competent HIV. In this proposal, we will isolate highly pure brain myeloid cells from different regions of the brain in both people living with HIV (PLWH) enrolled in the “Last Gift” cohort and fully ART-suppressed SIV-infected rhesus macaques. We will examine whether BMCs harbor replication-competent HIV or SIV using the intact proviral DNA assay (IPDA) and quantitative viral outgrowth assay (QVOA) (Aim1). We will examine whether latent HIV in myeloid cells responds to the currently available latency reversal agents related to epigenetic regulation of HIV latency (Aim 2), which may provide clues to the mechanisms that allow HIV latency in the brain. Our study will address critical gaps in knowledge to better understand whether BMCs serve as true HIV reservoirs and how HIV persists in these CNS cells.