Division of Biotechnology Review and Research II, Food and Drug Administration, Silver Spring, Maryland, USA.
Division of Biotechnology Review and Research III, Food and Drug Administration, Silver Spring, Maryland, USA.
J Virol. 2023 Jul 27;97(7):e0015423. doi: 10.1128/jvi.00154-23. Epub 2023 Jul 5.
Baloxavir marboxil (baloxavir) is a recently FDA-approved influenza virus polymerase acidic (PA) endonuclease inhibitor. Several PA substitutions have been demonstrated to confer reduced susceptibility to baloxavir; however, their impacts on measurements of antiviral drug susceptibility and replication capacity when present as a fraction of the viral population have not been established. We generated recombinant A/California/04/09 (H1N1)-like viruses (IAV) with PA I38L, I38T, or E199D substitutions and B/Victoria/504/2000-like virus (IBV) with PA I38T. These substitutions reduced baloxavir susceptibility by 15.3-, 72.3-, 5.4-, and 54.5-fold, respectively, when tested in normal human bronchial epithelial (NHBE) cells. We then assessed the replication kinetics, polymerase activity, and baloxavir susceptibility of the wild-type:mutant (WT:MUT) virus mixtures in NHBE cells. The percentage of MUT relative to WT virus necessary to detect reduced baloxavir susceptibility in phenotypic assays ranged from 10% (IBV I38T) to 92% (IAV E199D). While I38T did not alter IAV replication kinetics or polymerase activity, IAV PA I38L and E199D MUTs and the IBV PA I38T MUT exhibited reduced replication levels and significantly altered polymerase activity. Differences in replication were detectable when the MUTs comprised ≥90%, ≥90%, or ≥75% of the population, respectively. Droplet digital PCR (ddPCR) and next-generation sequencing (NGS) analyses showed that WT viruses generally outcompeted the respective MUTs after multiple replication cycles and serial passaging in NHBE cells when initial mixtures contained ≥50% of the WT viruses; however, we also identified potential compensatory substitutions (IAV PA D394N and IBV PA E329G) that emerged and appeared to improve the replication capacity of baloxavir-resistant virus in cell culture. Baloxavir marboxil, an influenza virus polymerase acidic endonuclease inhibitor, represents a recently approved new class of influenza antivirals. Treatment-emergent resistance to baloxavir has been observed in clinical trials, and the potential spread of resistant variants could diminish baloxavir effectiveness. Here, we report the impact of the proportion of drug-resistant subpopulations on the ability to detect resistance in clinical isolates and the impact of substitutions on viral replication of mixtures containing both drug-sensitive and drug-resistant variants. We also show that ddPCR and NGS methods can be successfully used for detection of resistant subpopulations in clinical isolates and to quantify their relative abundance. Taken together, our data shed light on the potential impact of baloxavir-resistant I38T/L and E199D substitutions on baloxavir susceptibility and other biological properties of influenza virus and the ability to detect resistance in phenotypic and genotypic assays.
巴洛沙韦马索利（baloxavir marboxil）是一种最近获得美国食品药品监督管理局（FDA）批准的流感病毒聚合酶酸性（PA）内切酶抑制剂。已经证实几种 PA 取代可降低对巴洛沙韦的敏感性；然而，当它们以病毒群体的一部分存在时，对测定抗病毒药物敏感性和复制能力的影响尚未确定。我们生成了具有 PA I38L、I38T 或 E199D 取代的重组 A/加利福尼亚/04/09（H1N1）样病毒（IAV）和具有 PA I38T 的 B/维多利亚/504/2000 样病毒（IBV）。在正常人支气管上皮细胞（NHBE）中进行测试时，这些取代分别使巴洛沙韦的敏感性降低了 15.3 倍、72.3 倍、5.4 倍和 54.5 倍。然后，我们评估了野生型：突变（WT:MUT）病毒混合物在 NHBE 细胞中的复制动力学、聚合酶活性和巴洛沙韦敏感性。在表型测定中，需要 MUT 相对于 WT 病毒的百分比来检测到降低的巴洛沙韦敏感性，范围从 10%（IBV I38T）到 92%（IAV E199D）。虽然 I38T 没有改变 IAV 复制动力学或聚合酶活性，但 IAV PA I38L 和 E199D MUT 和 IBV PA I38T MUT 表现出降低的复制水平和显著改变的聚合酶活性。当 MUT 分别占群体的≥90%、≥90%或≥75%时，可检测到复制差异。液滴数字 PCR（ddPCR）和下一代测序（NGS）分析表明，当初始混合物含有≥50%的 WT 病毒时，WT 病毒通常在 NHBE 细胞中多次复制循环和连续传代后优于各自的 MUT；然而，我们还发现了一些潜在的补偿性突变（IAV PA D394N 和 IBV PA E329G），这些突变出现并似乎提高了细胞培养中耐巴洛沙韦病毒的复制能力。巴洛沙韦马索利是一种流感病毒聚合酶酸性内切酶抑制剂，代表了最近批准的一类新的流感抗病毒药物。在临床试验中已经观察到治疗出现的对巴洛沙韦的耐药性，耐药变体的潜在传播可能会降低巴洛沙韦的有效性。在这里，我们报告了耐药亚群比例对在临床分离物中检测耐药性的能力的影响，以及取代对含有敏感和耐药变体的混合物中病毒复制的影响。我们还表明，ddPCR 和 NGS 方法可成功用于检测临床分离物中的耐药亚群并定量其相对丰度。总之，我们的数据阐明了耐巴洛沙韦的 I38T/L 和 E199D 取代对巴洛沙韦敏感性和流感病毒其他生物学特性的潜在影响，以及在表型和基因型测定中检测耐药性的能力。
