Demirdjian Sally, Naik Vidula, Margot Nicolas, Falkard Brie, Callebaut Christian
HIV Clinical Virology, Gilead Sciences Inc., Foster City, California, USA.
J Med Virol. 2025 Apr;97(4):e70340. doi: 10.1002/jmv.70340.
Lenacapavir (LEN) is a potent, first-in-class long-acting HIV-1 capsid (CA) inhibitor, approved for treatment of people with multi-drug resistant HIV in combination with other antiretrovirals. In clinical studies with LEN, the CA resistance-associated mutations (RAMs) M66I, Q67H/K/N, K70H/N/R/S, N74D/H/K, A105S/T, and T107A/C/N/S were observed. Phenotypic analyses of these CA mutants were possible in single cycle (SC) PhenoSense Gag-Pro assays; however, most CA mutants exhibited severely impaired replication capacity (RC), rendering phenotyping with multicycle (MC) MT-2 cytopathic assays unsuccessful for many. Here, we developed and optimized a novel MC phenotyping assay using a Rev-dependent HIV reporter cell line, Rev-CEM-Luc/GFP (RevLun assay), to further characterize these replication-impaired LEN-resistant HIV CA mutants. HIV Gag-Protease fragments from patients' plasma samples with CA RAMs and associated site-directed mutants were cloned into the pXXLAI HIV molecular clone, and replicative viral supernatants were evaluated in MT-2 and RevLun MC assays, with readouts of cytopathic effect and reporter gene expression, respectively. Viruses were also evaluated in the SC assay. We successfully phenotyped CA mutants in RevLun that were noninfectious in MT-2 assay, including clinical isolates containing M66I in various genetic contexts and combinations of LEN RAMs. LEN susceptibility in the RevLun MC assay aligned with data in the SC PhenoSense Gag-Pro assay and MC MT-2 assay when available. All viruses with LEN RAMs remained sensitive to other HIV drug classes. Using a sensitive HIV-dependent reporter system enhanced our ability to assess the phenotypes of viruses with low RC, allowing for further investigation into LEN resistance and CA RAMs.
来那卡帕韦（LEN）是一种强效的、同类首创的长效HIV-1衣壳（CA）抑制剂，已被批准与其他抗逆转录病毒药物联合用于治疗多重耐药HIV感染者。在LEN的临床研究中，观察到了与CA耐药相关的突变（RAMs）M66I、Q67H/K/N、K70H/N/R/S、N74D/H/K、A105S/T和T107A/C/N/S。在单循环（SC）PhenoSense Gag-Pro试验中可以对这些CA突变体进行表型分析；然而，大多数CA突变体的复制能力（RC）严重受损，使得许多突变体无法通过多循环（MC）MT-2细胞病变试验进行表型分析。在此，我们开发并优化了一种使用依赖Rev的HIV报告细胞系Rev-CEM-Luc/GFP的新型MC表型分析方法（RevLun分析），以进一步表征这些复制受损的LEN耐药HIV CA突变体。将来自具有CA RAMs的患者血浆样本和相关定点突变体的HIV Gag-蛋白酶片段克隆到pXXLAI HIV分子克隆中，并分别在MT-2和RevLun MC试验中评估复制性病毒上清液，通过细胞病变效应和报告基因表达进行读数。还在SC试验中对病毒进行了评估。我们成功地对在MT-2试验中无感染性的RevLun中的CA突变体进行了表型分析，包括在各种基因背景下含有M66I的临床分离株以及LEN RAMs的组合。当有数据时，RevLun MC试验中的LEN敏感性与SC PhenoSense Gag-Pro试验和MC MT-2试验中的数据一致。所有具有LEN RAMs的病毒对其他HIV药物类别仍敏感。使用敏感的HIV依赖报告系统增强了我们评估低RC病毒表型的能力，有助于进一步研究LEN耐药性和CA RAMs。
