BC Centre for Excellence in HIV/AIDS, 603-1081 Burrard St., Vancouver, BC, Canada.
J Clin Microbiol. 2011 Jan;49(1):201-8. doi: 10.1128/JCM.01868-10. Epub 2010 Nov 17.
Initial in vitro studies of bevirimat resistance failed to observe mutations in the clinically significant QVT motif in SP1 of HIV-1 gag. This study presents a novel screening method involving mixed, clinically derived gag-protease recombinant HIV-1 samples to more accurately mimic the selection of resistance seen in vivo. Bevirimat resistance was investigated via population-based sequencing performed with a large, initially antiretroviral-naïve cohort before (n = 805) and after (n = 355) standard HIV therapy (without bevirimat). The prevalence of any polymorphism in the motif comprising Q, V, and T was ∼ 6%, 29%, and 12%, respectively, and did not change appreciably over the course of therapy. From these samples, three groups of 10 samples whose bulk sequences were wild type at the QVT motif were used to generate gag-protease recombinant viruses that captured the existing diversity. Groups were mixed and passaged with various bevirimat concentrations for 9 weeks. gag variations were assessed by amplicon-based "deep" sequencing using a GS FLX sequencer (Roche). Unscreened mutations were present in all groups, and a V370A minority not originally detected by bulk sequencing was present in one group. V370A, occurring together with another preexisting, unscreened resistance mutation, was selected in all groups in the presence of a bevirimat concentration above 0.1 μM. For the two groups with V370A levels below consistent detectability by deep sequencing, the initial selection of V370A required 3 to 4 weeks of exposure to a narrow range of bevirimat concentrations, whereas for the group with the V370A minority, selection occurred immediately. This approach provides quasispecies diversity that facilitates the selection of mutations observed in clinical trials and, coupled with deep sequencing, could represent an efficient in vitro screening method for detecting resistance mutations.
最初针对贝伐单抗耐药性的体外研究未能观察到 HIV-1 gag 中 SP1 中临床显著的 QVT 基序发生突变。本研究提出了一种新的筛选方法，涉及混合的、源自临床的 gag-蛋白酶重组 HIV-1 样本，以更准确地模拟体内观察到的耐药性选择。通过对大量初始未接受抗逆转录病毒治疗的队列（n=805）和接受标准 HIV 治疗（无贝伐单抗）后的队列（n=355）进行基于群体的测序，研究了贝伐单抗耐药性。在该 motif 中由 Q、V 和 T 组成的任何多态性的流行率分别约为 6%、29%和 12%，并且在治疗过程中没有明显变化。从这些样本中，选择了三个 10 个样本组，其 bulk 序列在 QVT 基序处为野生型，用于生成捕获现有多样性的 gag-蛋白酶重组病毒。将这些组混合并在不同的贝伐单抗浓度下传代 9 周。通过基于扩增子的“深度”测序（使用 GS FLX 测序仪（Roche））评估 gag 变异。所有组中都存在未筛选的突变，并且一个最初未通过 bulk 测序检测到的 V370A 少数突变存在于一个组中。在贝伐单抗浓度高于 0.1 μM 的情况下，所有组中都选择了 V370A 与另一个预先存在的未筛选的耐药突变一起发生。对于两个 V370A 水平低于深度测序一致可检测性的组，V370A 的初始选择需要 3 到 4 周的时间暴露在窄范围的贝伐单抗浓度下，而对于 V370A 少数的组，选择立即发生。这种方法提供了有助于选择临床试验中观察到的突变的准种多样性，并且与深度测序相结合，可能代表一种用于检测耐药性突变的有效体外筛选方法。
