Quantitative Viral Outgrowth Assays with Improved Throughput and Performance

描述（由申请人提供）：临床试验正在积极探索在病毒复制受到抑制或检测不到的个体中根除HIV-1感染的策略。用重塑染色质的试剂，例如组蛋白脱乙酰酶抑制剂（HDACI）或其他T细胞刺激剂治疗病毒复制受抑制的HIV+个体，以努力重新激活潜伏HIV的表达，导致从头病毒产生，这随后将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（凋亡）和/或免疫激活。通过用完全抑制性抗逆转录病毒药物方案对个体进行持续治疗，防止由活化的T细胞产生的新病毒体感染新细胞。这样的病毒清除策略 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逐渐减少潜伏的小瓶储库，导致感染的最终根除。在临床试验中评价根除方案的有效性的主要挑战是缺乏用于检测潜伏病毒消除的标准化测定法作为储库根除的量度。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代静息T细胞的有限稀释液。这些测定需要大量的细胞，并且需要数天，如果不是数周的话来完成。使用包括ELISA和PCR的各种方法从细胞或细胞培养基级分定量病毒产生。使用各种化验和检测方法导致各实验室的结果不一致，妨碍了实验室间的比较。以前，引入标准化的中和抗体检测方法用于评价HIV候选疫苗，从而在评价不同免疫原和疫苗接种策略时能够对有效性进行精细区分。同样地，可靠的标准化Q-VOA测定的实施应该提供在根除研究期间辨别潜在储库大小的细微差异的能力。Monogram Biosciences（www.monogrambio.com）拥有超过15年的开发高通量基于细胞的病毒检测的经验，这些检测用于评估HIV-1治疗。我们打算利用我们现有的专业知识来追求和完成拟议的研究目标。Monogram将探索不同的Q-VOA方法，以选择和开发一种方法，该方法可以生成一种稳健，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。本研究提案涉及资助机会公告中指定的几个具体目标（PA-12-162）：（a）开发新的化验方法（包括但不限于开发新的定量化验方法，用于灵敏地检测组织中的HIV-1，检测潜伏感染细胞中有复制能力的病毒的简单方法，测量储存库中病毒多样性的化验方法，用于准确区分和测量整合和未整合形式的vDNA的测定。(b)技术进步（包括但不限于标准化从细胞和组织中分离和定量可复制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).