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

CNS 和免疫组织骨髓 HIV-1 储存库的测量和表征

• 批准号: 10319583
• 负责人: Katherine F Luzuriaga
• 金额: $ 65.87万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10319583
• 关键词: Address; Astrocytes; Biological; Blood; Brain; CD4 Positive T Lymphocytes; Cell Lineage; Cells; Clinical; DNA; Data; Development; Disease remission; Genetic; Genome; HIV; HIV-1; Immune; Individual; Infection; Ingestion; Length; Longevity; Measurement; Measures; Messenger RNA; Methods; Microglia; Minority; Myelogenous; Neurologic; Neurological observations; Nucleic Acids; Persons; Phenotype; Phylogenetic Analysis; Population; Property; Protocols documentation; Proviruses; Regimen; Reporting; T-Lymphocyte; Tissues; Type I Epithelial Receptor Cell; Viral; Viral reservoir; Virus; Work; antiretroviral therapy; base; brain tissue; deep sequencing; env Genes; genome analysis; improved; macrophage; monocyte; nervous system disorder; next generation sequencing; novel strategies; pol genes; side effect

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.

潜伏感染HIV-1的长寿细胞形成了一个病毒库，无法通过电流消除 抗逆转录病毒治疗方案，即使这些治疗是在感染者的生命周期中应用。一 全面了解细胞和组织为基础的水库将促进新的发展， 根除这种持久性病毒储存库的方法。 我们假设，脑和免疫组织巨噬细胞系细胞构成了重要的HIV-1 CD 4 + T细胞库在遗传和表型上与CD 4 + T细胞库不同。高灵敏度方法 将用于定量存在于脑、CSF和免疫系统中巨噬细胞谱系细胞中的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， 巨噬细胞系细胞是否真的被感染，或者是否可检测到HIV-1核酸代表摄入 或者污染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， 完整前病毒，活性表达HIV-1 mRNA，并可产生感染性病毒。我们还将建立 巨噬细胞前病毒是否可以被潜伏期逆转剂（LRA）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）， 研究脑、CSF和免疫组织中HIV-1的系统发育关系和区室化 以及在特定细胞类型即纯化的巨噬细胞、星形胶质细胞和T细胞中。我们提出以下三个目标： 目标1。测量和表征来自脑和免疫组织的巨噬细胞中的HIV-1储库。HIV-1 在从脑，CSF， 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。 目标二。为了评估在脑和免疫组织中的巨噬细胞中是否存在有复制能力的HIV-1， 组织. PCR和培养方案将评估完整的、整合的、有复制能力的前病毒是否被 存在于巨噬细胞中，并可被LRA激活。 目标3.目的探讨HIV-1在脑巨噬细胞和T细胞中的系统发育关系， 组织.将使用env和pol基因的单基因组PCR和PacBio深度测序来研究 在巨噬细胞中发现的HIV-1与脑、CSF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， 形成病毒库，（2）建立定量单核细胞/巨噬细胞中感染性HIV-1前病毒的方法 在脑、CSF和免疫组织中的分布;（3）建立HIV-1群体之间的系统发育关系 存在于纯化的巨噬细胞和存在于不同组织中的T细胞中。研究结果对于以下方面很重要： 为HIV-1感染者实现缓解和潜在治愈的策略提供信息。