Microfluidic PCR Method to Identify and Characterize HIV-Infected Single Cells

描述(申请人提供)：尽管长期使用抗逆转录病毒疗法抑制循环病毒，HIV-1储存库仍然以潜伏的形式存在。实现治愈艾滋病毒-1感染的主要挑战是这些潜伏的病毒库的持久性。化验 对潜伏感染的单个细胞进行鉴定、鉴定和分离，以便进行下游基因组测序，以有效和充分地描述HIV-1宿主的特征是必要的。然而，现有的分析潜伏感染细胞的分析方法需要繁琐和昂贵的连续细胞稀释。其他提出的识别和分析艾滋病毒感染细胞的方法需要在昂贵的设备和试剂上进行大量投资，而且还没有被调整用于潜在水库的下游特征。我们建议开发和验证一种使用微流控方法和聚合酶链式反应的创新和新的检测方法，用于从潜伏感染的人类细胞中识别、计数、分离和下游鉴定病毒基因组。我们方法的应用将特别有助于对联合抗逆转录病毒治疗患者的样本进行分析，和/或在研究新的消灭宿主模式方面。具体目标包括：1)建立和测试所提出的方法的有效性，使用微流控方法和聚合酶链式反应来识别和计数潜伏感染的人PBMCs、组织来源的巨噬细胞和其他原代人类细胞，以及2)验证我们的方法来分离带有整合的HIV-1 DNA的单细胞液滴，用于下游测序和次级靶基因定量。这笔为期两年的开发赠款将利用现有微流控技术的创新方法和改编，开发一种分析方法，在单细胞水平上表征接受抗逆转录病毒治疗的患者的艾滋病毒宿主。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，包括翻译病毒学和生物工程/生物物理学。我们以前在检测和量化极低水平的HIV-1遗传物质以及开发用于病毒和免疫表征的微流控设备方面的经验将对 开发新的检测方法，在单细胞水平上识别和表征艾滋病毒感染。所提出的方法有可能适用于广泛的多学科研究，如病毒和细胞内的单细胞表征。 病原体或干细胞和/或恶性组织的分析。

DESCRIPTION (provided by applicant): HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy. The main challenge in achieving a cure for HIV-1 infection is the persistence of these latent viral reservoirs. Assays that allow for identification, characterization, and isolation of latently infected single cells fo downstream genomic sequencing are needed to efficiently and fully characterize HIV-1 reservoirs. However, existing assays that analyze latently-infected cells require burdensome and costly serial cell dilutions. Other proposed methods to identify and analyze HIV-infected cells require significant investment in costly equipment and reagents and have not been adapted for downstream characterization of latent reservoirs. We propose to develop and validate an innovative and novel assay using microfluidic methods with PCR for identification, enumeration, and isolation and downstream characterization of viral genomes from latently-infected human cells. The application of our approach will be particular useful in the analysis of samples from patients on combination antiretroviral therapy and/or in studies of novel modalities of reservoir eradication. Specific aims include: 1) develop and test the efficacy of the proposed method to identify and enumerate latently-infected human PBMCs, tissue derived macrophages, and other primary human cells using microfluidic methods and PCR, and, 2) validate our assay to isolate single-cell droplets with integrated HIV-1 DNA for downstream sequencing and secondary target gene quantification. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs on the single-cell level 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 prior experiences in the detection and quantification of very low-levels of HIV-1 genetic material and in the development of microfluidic devices for viral and immune characterization will be crucial to the development of novel assays to identify and characterize HIV-infection at the single-cell level. The proposed method has the potential to be adapted for a wide variety of multidisciplinary research studies, such as single-cell characterization of viral and intracellular pathogens or analysis of stem cells and/or malignant tissues.