Laboratory of Molecular Immunology, The Rockefeller University, New York, NY.
Laboratory of Molecular Immunology, The Rockefeller University, New York, NY
J Exp Med. 2019 Oct 7;216(10):2253-2264. doi: 10.1084/jem.20190896. Epub 2019 Jul 26.
HIV-1 infection requires lifelong therapy with antiretroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end, there are numerous ongoing studies to evaluate immunological approaches, including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here, we describe a relatively high-throughput combined quantitative PCR (qPCR) and next-generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (), polymerase (), and envelope () are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any two of the four probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.
由于存在转录沉默的整合前病毒潜伏库，HIV-1 感染需要终身接受抗逆转录病毒药物治疗。HIV-1 治愈研究的目标是消除或功能性沉默这个潜伏库。为此，有许多正在进行的研究评估免疫方法，包括单克隆抗体疗法。评估这些研究的结果需要对储库进行敏感和特异性的测量。在这里，我们描述了一种相对高通量的联合定量 PCR（qPCR）和下一代测序方法。四种不同的 qPCR 探针覆盖包装信号（PS）、群特异性抗原（）、聚合酶（）和包膜（），组合在单个多重反应中，以检测限制稀释样品中的 HIV-1 基因组，然后对任何两种探针组合呈阳性的单个反应进行序列验证（Q4PCR）。这种敏感和特异性的方法可以对 HIV-1 潜伏储库进行无偏倚的特征描述。
