背景:细胞相关的HIV-1DNA,HIV-12-LTR圆圈,和HIV-1未剪接RNA(usRNA)是监测HIV-1持久性和潜伏HIV-1激活的重要病毒学参数。未来的临床试验需要通过CLIA和/或GCLP标准完全验证的测定,以寻求评估针对HIV-1治愈的治疗方法。
目的:为了确定敏感,中等吞吐量,细胞相关HIV-1DNA的数字液滴PCR(ddPCR)分析,HIV-12-LTR圆圈,和HIV-1usRNA,可以检测广泛的HIV-1M组亚型。
方法:要评估线性度,检测限,精度,和每个分析的准确性,在未感染的PBMC背景下分析人为标本。通过引物和探针组的计算机模拟分析和从体外感染各种HIV-1亚型的PBMC中收获的材料分析来评估检测宽度。分析了一组来自病毒血症和病毒学抑制个体的临床标本,以证明其对临床研究的适用性。
结果:根据经验确定的这些检测方法的检测极限为HIV-1DNA的每百万PBMC中29、7和60个拷贝,HIV-12-LTR圆圈,和HIV-1usRNA,分别。该测定法检测广泛范围的HIV-IM组亚型。最后,临床标本的分析表明,这些检测可以检测低水平的细胞相关的HIV-1DNA,HIV-1usRNA,和HIV-12-LTR循环,并与未经治疗和抗逆转录病毒治疗的个体的临床病史和病毒载量相关。
结论:我们报告了三种HIV-1储库检测方法的临床验证,这些方法具有广泛的HIV-1覆盖率,可用于未来的治愈研究。
Cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 unspliced RNA (usRNA) are important virological parameters for monitoring HIV-1 persistence and activation of latent HIV-1. Assays fully validated by CLIA and/or GCLP standards are needed for future clinical trials that seek to evaluate treatments directed towards HIV-1 cure.
To determine performance characteristics of sensitive, moderate-throughput, digital droplet PCR (ddPCR) assays for cell-associated HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA that can detect a broad range of HIV-1 M-group subtypes.
To evaluate linearity, limit of detection, precision, and accuracy of each assay, contrived specimens were analyzed in a background of uninfected PBMC. Detection breadth was evaluated by in silico analysis of primer and probes sets and analysis of material harvested from PBMC infected in vitro with various HIV-1 subtypes. A cohort of clinical specimens from viremic and virologically suppressed individuals was analyzed to demonstrate applicability to clinical research.
The empirically determined limit of detection of these assays was 29, 7, and 60 copies per million PBMC for HIV-1 DNA, HIV-1 2-LTR circle, and HIV-1 usRNA, respectively. The assays detect a broad range of HIV-1 M-group subtypes. Finally, analysis of clinical specimens demonstrate that these assays can detect low levels of cell-associated HIV-1 DNA, HIV-1 usRNA, and HIV-1 2-LTR circle and correlate with clinical histories and viral loads of untreated and antiretroviral treated individuals.
We report the clinical validation of three HIV reservoir assays with broad HIV-1 coverage for future cure studies.