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

描述（由申请人提供）：目前正在临床试验中探索在病毒载量被抑制或检测不到的个体中根除HIV-1感染的策略。复制受到抑制的HIV+个体用重塑染色质的药物治疗，例如组蛋白去乙酰化酶抑制剂（hdac）或其他T细胞刺激剂，努力重新激活潜伏HIV的表达，导致新病毒的产生，随后通过各种假定的机制导致潜伏感染细胞死亡，包括程序性细胞死亡（凋亡）和/或免疫激活。被激活的T细胞产生的新病毒粒子可以通过持续使用完全抑制的抗逆转录病毒药物治疗来防止感染新细胞。病毒消除策略是基于这样的假设，即多轮治疗的潜伏期逆转药物会随着时间的推移减少病毒库，从而最终根除感染。定量病毒生长测定（VOA）被认为是目前可用来测量潜伏感染细胞水平的最佳方法。然而，VOA是昂贵的，耗时和劳动密集型的。最近的研究表明，即使VOA也可能无法准确地量化潜伏病毒库的大小，因为潜伏功能病毒的再激活可能涉及人们知之甚少和/或随机机制。通过检查VOA中产生的再激活病毒的基因组RNA序列和静止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。开发一种强大而灵敏的方法来扩增和测序早期VOA上清中发现的整个HIV-1基因组，可能会节省分析周转时间，并提供这些活化病毒的丰富序列信息。此外，从激活前富集的CD4+静息记忆细胞群体中灵敏可靠地扩增全长HIV模板，应该能够直接比较潜伏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PCR平台可用于定量样品中发现的艾滋病毒DNA拷贝数，对全长艾滋病毒原基因组进行测序将允许估计这些拷贝中编码功能病毒的百分比。我们建议开发稳健和敏感的方法，从VOA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。本研究提案涉及资助机会公告（PA-12-162）中规定的几个研究兴趣的具体目标：(a)开发新的测定方法(包括但不限于开发新的定量测定方法，以灵敏地检测组织中的艾滋病毒-1，在潜伏感染细胞中检测具有复制能力的病毒的简单方法，测量病毒库中病毒多样化的测定方法，准确区分和测量整合和非整合形式的vDNA的测定方法。(b)技术进步（包括但不限于从细胞和组织中分离和定量复制能力vRNA和病毒DNA （vDNA）的标准化方法，以及纳米技术）。

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).