Phenotypic Characterization of Replication-Impaired Lenacapavir-Resistant HIV Clinical Isolates

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.