Assessing Potential Latent Virus HIV-1 Viability using Next Generation Sequencing

使用下一代测序评估潜在的潜伏病毒 HIV-1 活力

• 批准号: 8892081
• 负责人: CHRISTOS J PETROPOULOS
• 金额: $ 10万
• 依托单位: MONOGRAM BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8892081
• 关键词: Address; Anti-Retroviral Agents; Apoptosis; Archives; Asses; Base Sequence; Basic Science; Biocompatible Materials; Bioinformatics; Biological Assay; CD4 Positive T Lymphocytes; Cell Line; Cells; Cessation of life; Clinical Trials; DNA; DNA Sequence; Data; Data Quality; Detection; Development; Drug resistance; Effectiveness; Funding Opportunities; Genes; Genome; Grant; HIV; HIV Genome; HIV Infections; HIV-1; Health; Histone Deacetylase Inhibitor; Immune system; Individual; Infection; Ions; Latent Virus; Length; Life; Measures; Memory; Methods; Modification; Mutate; Nanotechnology; Nucleic acid sequencing; Open Reading Frames; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Plasma; Polymerase; Population; Process; Production; Protocols documentation; Proviruses; RNA Sequences; Reading; Regimen; Research; Rest; Sampling; Sequence Analysis; Specific qualifier value; T memory cell; T-Lymphocyte; Technology; Time; Tissues; Variant; Viral; Viral Load result; Virion; Virus; Virus Replication; Work; chromatin remodeling; digital; experience; genomic RNA; immune activation; interest; latent infection; monocyte; next generation; next generation sequencing; prevent; resistance mutation; transcriptome sequencing; viral DNA

DESCRIPTION (provided by applicant): Strategies to eradicate HIV-1 infection in individuals with suppressed or undetectable viral loads are currently being explored in clinical trials. HIV+ individuals with suppressed replication are treated with agents that remodel chromatin, e.g. histone deacetylase inhibitors (HDACIs) or other T cell stimulators in efforts to reactivate expression of latent HIV resulting in de novo virus production, which subsequently results in the death of latent infected cells through a variety of postulated mechanisms, including programmed cell death (apoptosis) and/or immune activation. New virions produced by activated T cells are prevented from infecting new cells by the ongoing treatment of the individual with a fully suppressive regimen of anti-retroviral drugs. The virus elimination strategy is predicated on the assumption that multiple rounds of treatment with latency reversing drugs will decrease the reservoir over time leading to the eventual eradication of infection. The quantitative viral outgrowth assay (VOA) is considered the best method currently available to measure the level of latently infected cells. However the VOA is expensive, time consuming and labor intensive. And recent research shows that even the VOA may not be able to accurately quantitate the size of the latent reservoir as reactivation of latent functional viruses may involve poorly understood and/or stochastic mechanisms. By examining both the genomic RNA sequences of the reactivated viruses generated in the VOA and the archived proviral DNA sequences in the resting CD4+ T cells we should be able to obtain a greater understanding of the extent of latent infections. Developing a robust and sensitive method to amplify and sequence whole HIV-1 genomes found in early VOA supernatants would potentially save assay turnaround time and give access to a wealth of sequence information on these activated viruses. Additionally, the sensitive and reliable amplification of full-length HIV templates from enriched populations of CD4+ resting memory cells prior to activation, should enable direct comparisons of virus variants that appear after latent reservoir activation to those found in the cellular archive. Recently developed digital PCR platforms can be used to quantitate the number of copies of HIV DNA found in a sample and sequencing full length HIV proviral genomes would allow the estimation of what percentage of those copies appear to encode functional viruses. We propose developing robust and sensitive methods of amplifying HIV genomes from VOA supernatants as well as memory T cells and sequencing those templates using both conventional and next generation sequencing methods. This study proposal addresses several specific objectives of research interest as specified in the funding opportunity announcement (PA-12-162): (a) Development of new assays (including but not limited to development of new quantitative assays for sensitive detection of HIV-1 in tissue, a simple method for detecting replication-competent virus in latently infected cells, assays to measure diversification of viruses in reservoirs, assays to accurately discriminate and measure vDNA in integrated and unintegrated forms. (b) Technology advancement (including but not limited to methods to standardize isolation and quantification of replication competent vRNA and viral DNA (vDNA) from cells and tissues, and nanotechnology).

描述(由申请人提供)：目前正在临床试验中探索在病毒载量受到抑制或检测不到的个人中根除艾滋病毒-1感染的策略。复制被抑制的HIV+个体被用重塑染色质的药物治疗，例如组蛋白去乙酰化酶抑制剂(HDACIs)或其他T细胞刺激剂，以努力重新激活潜伏的HIV表达，导致从头病毒的产生，继而通过各种假设机制导致潜伏感染细胞死亡，包括程序性细胞死亡(细胞凋亡)和/或免疫激活。由激活的T细胞产生的新病毒粒子通过对个体进行全面抑制的抗逆转录病毒药物治疗来防止感染新细胞。病毒消除战略是基于这样的假设，即用潜伏期逆转药物进行多轮治疗将随着时间的推移减少病毒储存库，从而最终根除感染。定量病毒生长试验(VOA)被认为是目前测量潜伏感染细胞水平的最佳方法。然而，美国之音是昂贵的，耗时和劳动密集的。最近的研究表明，由于潜伏功能病毒的重新激活可能涉及知之甚少的和/或随机机制，即使是VOA也可能无法准确地量化潜伏病毒的大小。通过检查VOA中产生的重新激活的病毒的基因组RNA序列和静止的CD4+T细胞中存档的前病毒DNA序列，我们应该能够更好地了解潜伏感染的程度。开发一种可靠和灵敏的方法来扩增和测序在早期美国之音上清液中发现的HIV-1全基因组，将潜在地节省分析周转时间，并使人们能够获得关于这些激活病毒的丰富的序列信息。此外，在激活前灵敏而可靠地扩增来自丰富的CD4+静止记忆细胞群体的全长HIV模板，应该能够直接比较潜伏的储存库激活后出现的病毒变体与细胞档案中发现的病毒变体。最近开发的数字聚合酶链式反应平台可以用来定量在样本中发现的艾滋病毒DNA的拷贝数，并且对全长的艾滋病毒前病毒基因组进行测序将允许估计这些拷贝中似乎编码功能性病毒的百分比。我们建议开发稳健和敏感的方法，从美国之音上清液和记忆T细胞中扩增HIV基因组，并使用传统和下一代测序方法对这些模板进行测序。这项研究建议针对资助机会公告(PA-12-162)中规定的几项具体研究目标：(A)开发新的检测方法(包括但不限于开发用于灵敏地检测组织中的艾滋病毒-1的新的定量检测方法、检测潜伏感染细胞中具有复制能力的病毒的简单方法、测量宿主体内病毒多样性的检测方法、准确区分和测量综合和非整合形式的VDNA的检测方法)。(B)技术进步(包括但不限于标准化从细胞和组织中分离和量化具有复制能力的vRNA和病毒DNA(VDNA)的方法，以及纳米技术)。