Quantitative Viral Outgrowth Assays with Improved Throughput and Performance

描述(由申请人提供)：在病毒复制受到抑制或检测不到的个人中，根除艾滋病毒-1感染的战略正在临床试验中积极探索。病毒复制受到抑制的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的表达，导致新病毒的产生，这将随后通过各种假定的机制导致潜伏感染细胞的死亡，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完全抑制的抗逆转录病毒药物方案的持续治疗来防止感染新细胞。这样的病毒清除策略 预测是基于这样的假设，即用潜伏期逆转药物进行多轮治疗将随着时间的推移逐渐减少潜在的小瓶储存库，从而最终根除感染。在临床TRILS中评估根除方案的有效性的一个主要挑战是缺乏一种标准化的检测潜伏病毒耗竭的方法来衡量宿主的根除。目前公认的金标准试验(Q-VOA)是基于病毒生长的定量测量，使用暴露于候选治疗性T细胞刺激剂和有效对照化合物后分离的原始静止T细胞的有限稀释。这些检测需要大量的细胞，需要数天，甚至数周才能完成。使用各种方法，包括酶联免疫吸附试验和聚合酶链式反应，对细胞或细胞培养上清液中的病毒产量进行定量。各种化验和检测方法的使用导致不同执行实验室的结果不一致，并阻碍了实验室间的比较。以前，在评估不同的免疫原和疫苗接种策略时，采用标准化的中和抗体测试来评估艾滋病毒候选疫苗，可以在疗效上产生细微的差异。以大致相同的方式，实施可靠、标准化的Q-VOA分析应该能够在根除研究期间辨别潜在储存库大小的细微差异。Mongraph Biosciences(www.monogramBio.com)拥有超过15年的开发高通量细胞病毒分析的经验，用于评估HIV-1疗法。我们打算利用我们已有的专业知识来追求和完成拟议的研究目标。Mongraph将探索不同的Q-VOA方法，以选择和开发一种方法，产生一种健壮、可重复、快速和负担得起的方法，以准确测量临床样本中的HIV-1潜伏库。这项研究建议涉及资助机会公告(PA-12-162)中规定的几个具体目标：(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病毒DNA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)。

DESCRIPTION (provided by applicant): Strategies to eradicate HIV-1 infection in individuals with suppressed or undetectable viral replication are being actively explored in clinical trials. HIV+ individuals with suppressed viral 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 will 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 anti-retroviral drug regimen. Such virus elimination strategies are predicated on the assumption that multiple rounds of treatment with latency reversing drugs will incrementally diminish the latent vial reservoir over time leading to an eventual eradication of infection. A major challenge in evaluating the efficacy of eradication protocols in clinical trils is the lack of a standardized assay for detecting latent virus depletion as a measure of reservoir eradication. The current recognized gold-standard assay (Q-VOA) is based on quantitative measures of viral outgrowth using limit dilutions of primary resting T cells isolated following exposure to candidate therapeutic T cell stimulators and potent control compounds. These assays require large cell numbers and require days, if not weeks to complete. Virus production is quantitated from cell or cell culture media fractions using various methods that include ELISA and PCR. The use of various assays and detection methods has led to inconsistent results from the various performing laboratories and hinders inter-laboratory comparisons. Previously, the introduction of standardized neutralizing antibody assays for the evaluation of HIV vaccine candidates enabled fine distinctions in efficacy when evaluating different immunogens and vaccination strategies. In much the same way, the implementation of a reliable, standardized Q-VOA assay should provide the ability to discern subtle differences in latent reservoir size during eradication studies. Monogram Biosciences (www.monogrambio.com) has more than 15 years of experience developing high throughput cell based viral assays that evaluate HIV-1 therapeutics. We intend to leverage our established expertise to pursue and complete the proposed study objectives. Monogram will explore different Q-VOA approaches to select and develop a method that generates a robust, reproducible, rapid and affordable method to accurately measure the HIV-1 latent reservoir in clinical samples. This study proposal addresses several specific objectives 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 viruse 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).