Measurement and characterization of CNS and immune tissue myeloid HIV-1 reservoirs

潜伏感染 HIV-1 的长寿命细胞形成病毒库，目前无法消除 抗逆转录病毒治疗方案，即使这些治疗方法适用于感染者的整个生命周期。一个 对基于细胞和组织的储库的全面了解将有助于开发新的 根除这种持久病毒库的方法。 我们假设大脑和免疫组织巨噬细胞谱系细胞构成了重要的 HIV-1 其在遗传和表型上与 CD4 T 细胞储库不同。高灵敏度方法 将用于量化大脑、脑脊液和免疫系统中巨噬细胞谱系细胞中存在的 HIV-1 核酸 组织并确定星形胶质细胞是否有助于非 T 细胞库。一个关键问题是是否 巨噬细胞谱系细胞实际上已被感染，或者可检测到的 HIV-1 核酸是否代表被摄入 或污染T细胞。使用高度纯化的巨噬细胞，我们将确定它们是否携带全长， 完整的原病毒，主动表达HIV-1 mRNA，并能产生感染性病毒。我们还将设立 巨噬细胞原病毒是否可以被潜伏期逆转剂（LRAs）离体激活。 最后，我们将使用 PacBio 下一代测序（NGS）和单基因组分析（SGA）来 研究大脑、脑脊液和免疫组织中 HIV-1 的系统发育关系和区室划分 以及特定的细胞类型，即纯化的巨噬细胞、星形胶质细胞和 T 细胞。我们提出以下三个目标： 目标 1. 测量和表征大脑和免疫组织巨噬细胞中的 HIV-1 储存库。 HIV-1 将从脑、脑脊液、脑脊液中纯化的巨噬细胞系细胞和 T 细胞中的前病毒 DNA 和 mRNA 进行定量 和免疫组织，以更好地了解被感染和表达的细胞的数量和分布 HIV-1 mRNA。 目标 2. 评估大脑和免疫巨噬细胞中是否存在具有复制能力的 HIV-1 组织。 PCR 和培养方案将评估完整、整合、具有复制能力的原病毒是否是完整的、整合的、具有复制能力的原病毒。 存在于巨噬细胞中，可以被 LRA 激活。 目标 3. 研究大脑和免疫系统中巨噬细胞和 T 细胞中 HIV-1 的系统发育关系 组织。单基因组 PCR 和 env 和 pol 基因的 PacBio 深度测序将用于研究 巨噬细胞中发现的 HIV-1 与大脑、脑脊液和免疫组织中的 T 细胞之间的关系。 我们的建议旨在（1）确定免疫组织中的巨噬细胞是否感染了 HIV-1 以及 形成病毒库，(2) 建立量化单核细胞/巨噬细胞中感染性 HIV-1 原病毒的方法 在大脑、脑脊液和免疫组织中，(3) 建立 HIV-1 群体之间的系统发育关系 存在于不同组织中的纯化巨噬细胞和 T 细胞中。研究结果将很重要 为 HIV-1 感染者提供缓解和潜在治愈的策略。

Long-lived cells latently infected with HIV-1 form a viral reservoir that cannot be eliminated by current anti-retroviral therapy regimens, even when those therapies are applied over an infected person's lifespan. A comprehensive understanding of cellular and tissue-based reservoirs would facilitate the development of new approaches to eradicate this persistent viral reservoir. We hypothesize that brain and immune tissue macrophage-lineage cells constitute a significant HIV-1 reservoir that is genetically and phenotypically distinct from the CD4+ T cell reservoir. Highly sensitive methods will be used to quantify HIV-1 nucleic acids present in macrophage lineage cells in brain, CSF, and immune tissue and to also determine whether astroglia contribute to a non-T cell reservoir. A key issue is whether macrophage-lineage cells are actually infected, or whether detectable HIV-1 nucleic acids represent ingested or contaminating T cells. Using highly purified macrophages, we will ascertain whether they carry full length, intact proviruses, actively express HIV-1 mRNA, and can produce infectious virus. We will also establish whether macrophage proviruses can be activated ex vivo by latency reversing agents (LRAs). Finally, we will use PacBio next generation sequencing (NGS) and single genome analyses (SGA) to investigate phylogenetic relationships and compartmentalization of HIV-1 in brain, CSF, and immune tissues and in specific cell types i.e. purified macrophages, astroglia and T cells. We propose the following three aims: Aim 1. To measure and characterize the HIV-1 reservoir in macrophages from brain and immune tissue. HIV-1 proviral DNA and mRNA will be quantified in macrophage-lineage cells and T cells purified from brain, CSF, and immune tissue to better understand the number and distribution of cells that are infected and expressing HIV-1 mRNA. Aim 2. To evaluate the presence of replication competent HIV-1 in macrophages from brain and immune tissue. PCR and culture protocols will evaluate whether intact, integrated, replication-competent proviruses are present in macrophages and can be activated with LRAs. Aim 3. To investigate the phylogenetic relationships of HIV-1 in macrophages and T cells in brain and immune tissue. Single genome PCR and PacBio deep sequencing of env and pol genes will be used to investigate the relationships between HIV-1 found in macrophages and T cells in brain, CSF, and immune tissue. Our proposal aims to (1) determine whether macrophages in immune tissue are infected with HIV-1 and form a viral reservoir, (2) set up approaches to quantify infectious HIV-1 proviruses in monocyte/ macrophages in brain, CSF and immune tissue and (3) establish phylogenetic relationships between HIV-1 populations present in purified macrophages and T-cells present in different tissues. Study results will be important for informing strategies to achieve remission, and potential cure, in HIV-1 infected individuals.