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: 8790309
• 负责人: CHRISTOS J PETROPOULOS
• 金额: $ 17.5万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8790309
• 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; Genomics; Grant; HIV; HIV Genome; HIV Infections; HIV-1; 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; immune activation; interest; latent infection; monocyte; next generation; next generation sequencing; prevent; public health relevance; resistance mutation; 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抑制或无法检测到的病毒负荷患者中HIV-1感染的策略。 HIV+被抑制复制的个体用重塑染色质的药物治疗，例如组蛋白脱乙酰基酶抑制剂（HDACIS）或其他T细胞刺激剂在重新激活潜在HIV的表达，从而导致从头病毒产生，这随后通过包括各种假设的机制导致潜在感染细胞死亡，包括程序性细胞死亡（凋亡）和/或免疫激活。活化的T细胞产生的新病毒体可以通过持续治疗抗逆转录病毒药物的治疗方法来阻止新细胞感染新细胞。该病毒消除策略的假设是，多发药物逆转药物的治疗将随着时间的流逝而减少储层，从而导致最终消除感染。定量病毒生长测定（VOA）被认为是目前可用于测量潜在感染细胞水平的最佳方法。但是，VOA昂贵，耗时和劳动量很大。最近的研究表明，即使VOA也可能无法准确量化潜在储层的大小，因为潜在功能病毒的重新激活可能涉及较知的理解和/或随机机制。通过检查VOA中产生的重新激活病毒的基因组RNA序列和静息CD4+ T细胞中存档的病毒DNA序列，我们应该能够更了解潜在感染程度。开发一种强大而敏感的方法来放大和序列，在早期VOA上清液中发现的整个HIV-1基因组可能会节省测定的周转时间，并访问有关这些激活病毒的大量序列信息。此外，在激活之前，从富集的CD4+静息记忆细胞中对全长HIV模板的敏感和可靠的扩增，应能够直接比较潜在储层激活后与细胞档案中发现的病毒变体进行直接比较。最近开发的数字PCR平台可用于量化样品中发现的HIV DNA副本数量，并测序全长HIV病毒基因组将允许估计这些副本似乎是哪些百分比似乎编码功能病毒。我们提出了开发可靠和敏感的方法，以使用常规和下一代测序方法同时使用VOA上清液以及记忆T细胞的HIV基因组以及记忆T细胞进行测序。 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以综合和不整合的形式（b）技术进步（包括但不限于标准化的方法，可以从细胞和组织中隔离和量化复制的VRNA和病毒DNA（VDNA），以及纳米技术）。