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

大脑髓细胞中完整和有缺陷的 HIV 储存库的表征

• 批准号: 10491333
• 负责人: Thomas A Angelovich
• 金额: $ 13.44万
• 依托单位: ROYAL MELBOURNE INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10491333
• 关键词: Activities of Daily Living; Anatomy; Antiviral Therapy; Autopsy; Biological Assay; Brain; Cell Lineage; Cells; Central Nervous System Infections; Competence; DNA; Data; Development; Exhibits; Frequencies; Genome; Genomic DNA; Genotype; Goals; HIV; HIV-1; HIV-associated neurocognitive disorder; Immune system; Immunohistochemistry; Individual; Infection; Knowledge; Lasers; Length; Location; Measurement; Measures; Microdissection; Microglia; Modeling; Myelogenous; Myeloid Cells; Nature; Neuraxis; Neurodevelopmental Disorder; Patients; Peripheral; Persons; Pharmaceutical Preparations; Phenotype; Phylogenetic Analysis; Proteins; Proviruses; RNA; Retroviral Vector; Role; SIV; Sequence Analysis; Signal Transduction; Site; System; T-Lymphocyte; Technology; Testing; Tissues; Transfection; Viral; Viral Genome; Viral Proteins; Viral reservoir; Virion; Virus; antiretroviral therapy; base; cohort; comorbidity; expression vector; immunogenic; knowledge base; laser capture microdissection; lymph nodes; macrophage; nervous system development; neurotoxic; nonhuman primate; novel; peripheral blood; response; success; viral DNA; viral RNA

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.

项目总结/摘要 单靠抗逆转录病毒疗法无法根除艾滋病毒1型。根除HIV-1的主要障碍是 整合的、有复制能力的病毒DNA在细胞库中潜伏存在的能力。尽管有 关于CNS的HIV-1感染和发展的重要和广泛的知识基础 在HIV-1感染的病毒血症个体的神经系统疾病，只有一个非常有限的了解， 病毒抑制后在CNS中持续存在的机制。尽管有一些数据支持CNS， HIV-1在病毒抑制个体中的潜在储存库，我们的知识仍然存在重大空白 关于CNS中病毒持续存在的位置、频率和性质。这些数据对双方都至关重要。 制定旨在开发功能性和/或灭菌治疗的战略。我们的目标 建议是消除CNS中HIV的持续储存库，特别是骨髓储存库， 持续性病毒的复制能力。 该提案的目标是响应“骨髓细胞在HIV-1持续存在和根除中的作用”， 来自大脑的储库”RFA-MH-20-702将（i）使用基于高灵敏度PCR的定量和分析方法， 完整和有缺陷的HIV构成病毒抑制患者CNS中的骨髓储库，和（ii） 抑制CNS髓系储库的复制能力。在目标1中，我们将使用高度敏感和 完善的PCR检测，包括完整前病毒DNA检测（IPDA）结合敏感的 免疫组织化学和改良的激光显微切割技术，以确定数量和 来自病毒感染队列的潜在完整HIV病毒基因组CNS和非CNS组织的表型定位 被压制的人。我们将通过比较CNS衍生基因组的区室化， 非CNS组织。在目标2中，我们将全面阐述前病毒的复制能力 从髓系中枢神经系统储库中取出简而言之，从骨髓细胞分离的完整或有缺陷的前病毒的亚组， 病毒抑制个体的CNS（由IPDA测定）将通过全长个体进行确认 前病毒测序（FLIPS）并克隆到表达载体中以鉴定在 巨噬细胞和T细胞。重要的是，缺陷型前病毒继续产生RNA和/或 使用逆转录病毒载体系统或转染来测量包括达特和Nef的神经毒性蛋白。 基于我们的初步数据，我们假设虽然髓系CNS储库中的大多数前病毒 将不产生感染性病毒体，前病毒的子集将具有复制能力。此外，一个子集 会产生病毒蛋白这些数据对艾滋病治疗的发展至关重要 战略布局