The Dual Reporter Sensor Cell (DRSC) Assay: An Enhanced Tool for Measuring the Viral Reservoir

项目总结 潜伏的病毒库是根除艾滋病毒感染的主要障碍。不幸的是，可用的工具非常少 量化储存库并监测旨在减少储存库的前瞻性治疗的有效性。海流 金标准分析，定量病毒生长分析(QVOA)，使具有里程碑意义的科学研究成为可能， 但缺乏大规模临床实施所需的简单性和健壮性。其他选项包括 一些基于核酸的分析，但这些被认为高估了潜在的艾滋病毒的数量，即 实际上是复制能力强的。因此，存在对下一代储集层分析的未得到满足的需求 能够以直截了当和准确的方式分析艾滋病毒前病毒宿主的技术。 我们提出的检测方法的基石是一种新型的“双报告”传感器细胞系(DrSc)，它包含两个 独立的艾滋病毒感染相关报告系统，必须触发这两个系统才能指定为“真阳性” 结果，从而增强了特异性。像QVOA一样，我们的DrSc要求艾滋病毒感染在 多个细胞触发阳性信号，从而消除了许多非实时事件中的假阳性事件 细胞分析(例如，在许多基于聚合酶链式反应的储存库分析中，有缺陷的艾滋病毒DNA会被错误地量化)。 重要的是，初步数据表明，DrSc的速度要快得多(2天比7天)，所需时间也更少 与QVOA相比的样本量。 在拟议的项目中，我们通过开发两个关键概念来进一步优化DrSc：第一，我们将 评估和实施“低对流”培养环境，这将加速检测动力学(并改善 敏感性)，通过消除对流稀释的分泌因子(包括病毒粒子)，导致 生产细胞附近的因子浓度。第二，我们将继续提高我们的自动化程度 成像和图像分析技术(包括实时成像过程)，促进进一步 优化化验性能(例如，通过更好地选择化验时间)并简化最终的 将这项技术转化为更广泛的研究和临床角色(例如，通过整合基于图像的 基因分型“进入我们的读数)。这两个关键的发展实现了许多有利的特征， 包括：1)减少总分析时间；2)DRSCs的空间分辨率与扩散相结合- 占主导地位的流体力学消除了限制稀释终点的需要(与QVOA一样，这需要 多个T细胞稀释的多次复制以获得单个终点)，大大减少了 所需样本量；3)与QVOA相比，总体降低了分析的复杂性。重要的是，在 这项研究的结论是，我们将进行一项初步研究，将患者样本与预期的最高和最低进行比较 同时使用DrSc分析和QVOA评估储集层价值，从而为未来更大规模的研究奠定了基础。

PROJECT SUMMARY The latent viral reservoir is a major barrier to eradicating HIV infection. Unfortunately, very few tools are available to quantify the reservoir and monitor the effectiveness of prospective therapies that aim to reduce it. The current gold standard assay, the quantitative viral outgrowth assay (QVOA), has enabled landmark scientific studies, but lacks the simplicity and robustness required for large-scale clinical implementation. Other options include a number of nucleic acid-based assays, but these are thought to overestimate the quantity of latent HIV that is actually replication-competent. Thus, there exists an unmet need for next-generation reservoir assay technologies that are capable of analyzing the HIV proviral reservoir in a straightforward and precise manner. The cornerstone of our proposed assay is a novel “dual reporter” sensor cell (DRSC) line that contains two independent HIV infection-linked reporter systems, both of which must be triggered to designate a “true positive” result, thereby enhancing specificity. Like QVOA, our DRSC requires successful spread of HIV infection between multiple cells to trigger a positive signal, thereby eliminating many of the false positive events seen with non-live cell assays (e.g., defective HIV DNA will be erroneously quantified in many PCR-based reservoir assay). Importantly, preliminary data suggests that the DRSC is significantly faster (2 vs. 7 days) and requires less sample volume when compared with the QVOA. In the proposed project, we further optimize the DRSC via development of two key concepts: First, we will evaluate and implement “low convection” culture environments, which will accelerate assay kinetics (and improve sensitivity) via elimination of the convective dilution of secreted factors (including virions), resulting in the concentration of factors within the vicinity of producing cells. Second, we will continue to improve our automated imaging and image analysis techniques (including real-time imaging processes), facilitating the further optimization of assay performance (e.g., through better selection of assay timing) and streamlining the ultimate translation of this technology into broader research and clinical roles (e.g., via integration of “image-based genotyping” into our readout). These two key developments enable a number of advantageous features, including: 1) Reduction in total assay time; 2) The spatial resolution of the DRSCs combined with diffusion- dominant fluid mechanics eliminates the need for limiting dilution endpoints (as with QVOA, which requires multiple replicates of multiple T cell dilutions to obtain a single endpoint), substantially reducing the amount of sample volume required; 3) an overall reduction in assay complexity, relative to the QVOA. Importantly, at the conclusion of this study, we will perform a pilot study comparing patient samples with expected high and low reservoir values using both the DRSC assay and QVOA, thus laying the foundation for larger future studies.