Characterization of the intact and defective HIV reservoirs in myeloid cells in the brain

项目摘要/摘要 仅靠抗逆转录病毒疗法无法根除艾滋病毒-1。根除HIV-1的主要障碍是 整合、复制能力强的病毒DNA潜伏在细胞储存库中的能力。尽管存在着 关于HIV-1感染中枢神经系统和发展的重要和广泛的知识库 在HIV-1感染的病毒症患者中，对神经系统疾病的了解非常有限 病毒抑制后中枢神经系统持续存在的机制。尽管有一些数据支持CNS AS 在病毒抑制的个体中，HIV-1的潜在储备者，在我们的知识中仍然存在严重的差距 关于病毒在中枢神经系统持续存在的位置、频率和性质。这些数据对这两家公司都至关重要 制定旨在开发功能性和/或绝育疗法的战略。我们的目标是 建议描述中枢神经系统中艾滋病毒的持久储存库，特别是髓系储存库，以及 持久性病毒的复制能力。 这项建议的目标是，回应“髓系细胞在持续和根除艾滋病毒-1中的作用” 来自大脑的储藏物“RFA-MH-20-702将(I)使用基于高灵敏度的聚合酶链式反应来量化和表征 病毒抑制患者中枢神经系统中构成髓系储存库的完整和缺陷HIV，以及(Ii) 描述中枢神经系统髓系储存库的复制能力。在目标1中，我们将使用高度敏感的 成熟的聚合酶链式反应分析，包括完整的前病毒DNA分析(IPDA)与敏感性 免疫组织化学和改良的激光显微切割技术测定 病毒队列中可能完整的HIV病毒基因组CNS和非CNS组织的表型定位 被压抑的个体。我们将通过比较来确定中枢神经系统衍生的基因组的区划 非中枢神经系统组织。在目标2中，我们将全面描述前病毒复制能力的性质 来自髓系中枢神经系统储存库。简而言之，从猪的髓系细胞中分离出来的完整或缺陷前病毒的子集 病毒抑制个体的CNS(由IPDA确定)将由全长个体确认 前病毒测序(FLOPS)，并克隆到表达载体中，以鉴定其复制能力 巨噬细胞和T细胞。重要的是，有缺陷的前病毒继续产生RNA和/或 包括TAT和NEF在内的神经毒性蛋白将使用逆转录病毒载体系统或转染法进行测量。 我们根据初步数据推测，虽然髓系中枢神经系统储存库中的大多数前病毒 不会产生传染性病毒粒子，前病毒的子集将具有复制能力。此外，一个子集 有缺陷的前病毒会产生病毒蛋白。这些数据对艾滋病治疗的发展至关重要。 战略。

PROJECT SUMMARY/ABSTRACT HIV-1 cannot be eradicated by antiretroviral therapies alone. The major obstacle to eradicating HIV-1 is the ability of integrated, replication competent viral DNA to persist latently in cellular reservoirs. Despite there being a significant and extensive knowledge base regarding HIV-1 infection of the CNS and the development of neurological disorders in HIV-1 infected viremic individuals, there is only a very limited understanding of the mechanisms of persistence in the CNS following viral suppression. Despite some data supporting the CNS as a potential reservoir of HIV-1 in virally suppressed individuals, there remains critical gaps in our knowledge regarding the location, frequency and nature of viral persistence in the CNS. This data is critical to both developing strategies aimed at the development of both a functional and/or sterilising cure. The goal of our proposal is to characterise the persistent reservoir of HIV in the CNS, specifically the myeloid reservoir, and the replication competence of the persistent virus. The goals of this proposal, in response to the “Role of Myeloid Cells in Persistence and Eradication of HIV-1 Reservoirs from the Brain” RFA-MH-20-702 are to (i) use highly sensitive PCR based to quantify and characterise the intact and defective HIV composing the myeloid reservoir in the CNS of virally suppressed patients, and (ii) characterise the replication competence of the CNS myeloid reservoir. In Aim 1 we will use highly sensitive and well established PCR assays, including the intact proviral DNA assay (IPDA) in conjunction with sensitive immunohistochemistry and a modified laser microdissection technology to determine the quantity and phenotypical location of the potentially intact HIV viral genomes CNS and non-CNS tissue from a cohort virally suppressed individuals. We will determine the compartmentalisation of CNS derived genomes by comparison to non-CNS tissues. In Aim 2, we will comprehensively characterise the replication competent nature of proviruses from the myeloid CNS reservoir. Briefly, a subset of intact or defective proviruses isolated from myeloid cells of the CNS of virally suppressed individuals (as determined by IPDA) will be confirmed by full-length individual proviral sequencing (FLIPS) and cloned into expression vectors to characterise the replication competence in both macrophage and T cells. Importantly, the ability of defective proviruses to continue to produce RNA and/or neurotoxic proteins including tat and nef will be measured using retroviral vector systems or transfection. We hypothesise based on our preliminary data that whilst the majority of proviruses in the myeloid CNS reservoir will not generate infectious virions, a subset of proviruses will be replication competent. Furthermore, a subset of defective proviruses will produce viral proteins. These data are essential to the development of HIV cure strategies.