Giandhari Jennifer, Basson Adriaan E, Sutherland Katherine, Parry Chris M, Cane Patricia A, Coovadia Ashraf, Kuhn Louise, Hunt Gillian, Morris Lynn
Centre for HIV and STIs, National Institute for Communicable Diseases (NICD), Johannesburg, South Africa Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa.
Department of Infection, University College London, London, United Kingdom.
Antimicrob Agents Chemother. 2016 Mar 25;60(4):2248-56. doi: 10.1128/AAC.02682-15. Print 2016 Apr.
Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicativein vitroassay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of thegaggene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.
蛋白酶抑制剂（PIs）被用作HIV-1感染儿童的一线治疗方案。在此，我们研究了在接受PI治疗失败的儿科患者中，Gag和蛋白酶的氨基酸变化对洛匹那韦（LPV）和利托那韦（RTV）敏感性的表型影响。使用非复制性体外试验对来自20例接受基于LPV和/或RTV方案治疗失败的HIV-1 C亚型感染儿科患者的分离株的Gag-蛋白酶进行表型分析。计算相对于匹配的基线（治疗前）样本对LPV和RTV的敏感性变化。通过定点诱变在参考克隆中产生与PI治疗失败相关的Gag和蛋白酶氨基酸替代，并进行评估。使用斯坦福耐药性算法确定预测的表型。在10例（50%）患者的分离株中观察到对RTV和/或LPV的表型耐药或敏感性降低，所有这些患者均接受过RTV治疗。在大多数情况下，这与蛋白酶耐药性突变相关，但也检测到Gag裂解位点和非裂解位点的替代。在3例药物敏感性降低但蛋白酶为野生型的患者的分离株中也发现了Gag氨基酸替代。定点诱变证实，Gag中的一些氨基酸变化导致了PI耐药性，但仅在存在主要蛋白酶耐药相关替代的情况下。所有仅接受LPV治疗的患者的分离株在表型上均敏感。来自20例患者的基线分离株在LPV的50%有效浓度方面显示出较大（47倍）的范围，这解释了实验确定的表型与预测表型之间出现的大部分不一致。总体而言，纳入gag基因和使用匹配的基线样本能够更全面地评估PI诱导的氨基酸变化对PI耐药性的影响。对LPV缺乏表型耐药性支持在儿科患者中继续使用该药物。
