A Novel Microfluidic HIV-1 Co-Culture Assay to Quantify Latent Reservoirs

描述（由申请人提供）：尽管抗逆转录病毒治疗长期抑制了循环病毒，但HIV-1储库仍以潜伏形式存在，实现HIV-1抗逆转录病毒（ART）缓解的主要挑战是这些潜伏病毒储库的持续存在。量化复制能力的病毒库对于理解HIV-1如何持续存在以及测量该库如何响应治疗策略而变化至关重要。已经开发了定量共培养试验，以通过在饲养细胞存在下活化来自患者的CD 4 + T细胞以进行病毒生长和HIV-1蛋白或遗传物质的测量来测量复制能力储库。然而，传统的定量共培养测定是耗时的、劳动密集型的，并且需要在大孔格式培养中进行平行反应。使共培养平台稳定化和增加样品通量的新方法具有显著改善HIV潜伏期研究的潜力，并且可用于简化和标准化新储库根除策略的测试。 我们建议适应和验证一种新的，基于微型芯片的微流控共培养分析，并比较其性能与传统的共培养分析。我们假设，与传统平台相比，这些检测试剂盒的重复使用将允许更高的通量，并且劳动强度更低。所提出的方法还将允许严格控制生长微环境，这将有助于提高灵敏度并使实验室之间的测试结果标准化。我们的目标是：（1）调整用于病毒共培养的基于芯片的微流体细胞培养/扩增方案，以将整合的有复制能力的前病毒定量为每百万个细胞的感染单位，和（2）与传统的实验室病毒生长测定相比，验证新的微流体共培养测定的性能。 这项为期两年的发展赠款将利用创新方法和现有微流控技术的调整，开发一种测定方法，以表征接受抗逆转录病毒治疗的患者体内的艾滋病毒储存库。我们的建议涉及具有不同但互补的研究背景和经验的主要研究人员，包括转化病毒学和生物工程/生物物理学。我们提出的检测有可能被用于各种应用，涉及感染的人细胞亚群和组织的分离和生长，我们最终计划使用该检测来量化各种细胞类型和接受异基因干细胞移植和细胞减灭化疗的患者的复制能力水库。

DESCRIPTION (provided by applicant): HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy, and a main challenge in achieving HIV-1 antiretroviral (ART)-free remission is the persistence of these latent viral reservoirs. Quantifying the replication competent viral reservoir is critical to understanding how HIV-1 persists and to measuring how this reservoir changes in response to therapeutic strategies. Quantitative co-culture assay have been developed to measure the replication competent reservoir by activating CD4+ T cells from patients in the presence of feeder cells for viral outgrowth and measurements of HIV-1 proteins or genetic material. However, traditional quantitative co-culture assays are time-consuming, labor-intensive and require parallel reactions in large-well format culture. Novel methods to miniaturize the co- culture platform and to increase sample throughput have the potential significantly to improve the study of HIV latency and could be used to streamline and standardize testing of novel reservoir eradication strategies. We propose to adapt and validate a novel, miniaturized chip-based microfluidic co-culture assay and compare its performance with the traditional co-culture assay. We hypothesize that miniaturizing these assays will allow for higher throughput and be less labor intensive than traditional platforms. The proposed method will also allow for tight control of the growth microenvironment which will help to improve sensitivity and standardize test results between laboratories. Our aims are: (1) adapt a chip-based microfluidic cell culture/expansion protocol for viral co-culture to quantify integrated, replication-competent provirus as infectious units per million cells, and, (2) validate the performance of the novel microfluidic co-culture assay compared with traditional laboratory viral outgrowth assays. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs in patients on antiretroviral therapy. Our proposal involves principal investigators with different but complimentary research backgrounds and experiences, including translational virology and bioengineering/biophysics. Our proposed assay has the potential to be used for a variety of applications involving the isolation and growth of infected human cell subsets and tissues, and we ultimately plan to use the assay to quantify replication competent reservoirs in various cell types and from patients undergoing allogeneic stem cell transplantation and cytoreductive chemotherapy.