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

潜伏感染HIV-1的长寿命细胞形成了一个不能被当前病毒消除的病毒库 抗逆转录病毒治疗方案，即使这些疗法是在感染者的一生中应用的。一个 全面了解基于细胞和组织的储集层将有助于开发新的 根除这种持久的病毒库的方法。 我们假设大脑和免疫组织的巨噬细胞系细胞构成了一个重要的HIV-1 在遗传和表型上与CD4T细胞储存库不同的储存库。高度灵敏的方法 将用于量化脑、脑脊液和免疫中巨噬细胞系细胞中存在的HIV-1核酸 并确定星形胶质细胞是否对非T细胞储存库有贡献。一个关键问题是 巨噬细胞系细胞实际上被感染了，或者可检测到的HIV-1核酸是否代表被摄取 或污染T细胞。使用高纯度的巨噬细胞，我们将确定它们是否携带全长， 完整的前病毒，主动表达HIV-1mRNA，并能产生传染性病毒。我们还将建立 巨噬细胞前体能否被潜伏期反转剂(LRAs)体外激活。 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HIV-1在脑、脑脊液和免疫组织中的系统发育关系和区划 以及特定类型的细胞，即纯化的巨噬细胞、星形胶质细胞和T细胞。我们提出了以下三个目标： 目的1.测定和鉴定脑和免疫组织巨噬细胞中的HIV-1储存库。HIV-1 前病毒的DNA和mRNA将在巨噬细胞系细胞和从脑、脑脊液、 和免疫组织，以更好地了解感染和表达的细胞的数量和分布 HIV-1mRNA。 目的2.评价脑和免疫来源的巨噬细胞中是否存在具有复制能力的HIV-1 组织。聚合酶链式反应和培养方案将评估完整的、整合的、具有复制能力的前病毒 存在于巨噬细胞中，并可被LRA激活。 目的3.探讨巨噬细胞中HIV-1与脑内T细胞和免疫系统发育的关系 组织。将使用env和pol基因的单基因组聚合酶链式反应和PacBio深度测序来研究 巨噬细胞中发现的HIV-1与脑、脑脊液和免疫组织中T细胞的关系 我们的建议旨在(1)确定免疫组织中的巨噬细胞是否感染艾滋病毒-1和 形成病毒库，(2)建立量化单核/巨噬细胞中传染性HIV-1病毒的方法 以及(3)建立HIV-1种群之间的系统发育关系 存在于纯化的巨噬细胞中，存在于不同组织中的T细胞。研究结果将对 告知策略，以实现缓解和潜在的治愈，在艾滋病毒-1感染者。

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.