Background:
Lenacapavir (LEN) is a potent, first-in-class long-acting HIV capsid (CA) inhibitor, approved by the FDA as a twice-yearly treatment option for people living with multi-drug resistant HIV. Out of 258 people with HIV (PWH) who received LEN in clinical studies, CA mutations were observed in 14 participants (M66I, Q67H/K/N, K70H/N/R/S, N74D/H, A105T/S, and T107A/C/N/S). Phenotypic analyses of these mutants were successful in a single cycle (SC) assay; however, in the majority of capsid mutants, replication was impaired and too low for phenotyping by the multicycle (MC) MT-2 cytopathic assay. These mutants also had low replication capacity (RC) in the SC PhenoSense Gag-Pro assay (Monogram Biosciences). Here, we have developed and optimized a novel MC phenotyping assay using a Rev-dependent HIV reporter-controlled cell line, Rev-CEM-Luc/GFP (RevLucGFP), to characterize CA mutants with low infectivity.
Methods:
The HIV Gag-Protease fragments from plasma samples with CA resistance mutations (CAPELLA and CALIBRATE studies, n=21) and associated site-directed mutants (SDM, n=15) were cloned into pXXLAI HIV molecular clone followed by transfection into 293T cells. Replicative viral supernatants were evaluated in 2 different MC infection formats; MT-2 and RevLucGFP cell lines, with readouts of cell viability and viral replication, respectively. Patient isolates were also evaluated in the PhenoSense Gag-Pro assay. Outputs for antiviral assays included fold change (FC) in LEN susceptibility and RC (Gag-Pro assay).
Results:
We successfully phenotyped 11 mutants in RevLucGFP cells that were non-infectious in MT-2 assays, including clinical isolates containing M66I in various genetic contexts and combinations of LEN resistance associated mutations (RAMs) with FC ranging from 43.5 to >1000. Antiviral activity and susceptibility in the RevLucGFP MC assay aligned with the previously observed data in the SC PhenoSense Gag-Pro assay and MT-2 cells. Additionally, we observed that SDMs generated in a clinical isolate background containing common polymorphisms have a greater ability to be phenotyped, as compared to SDMs generated in WT lab isolate background. All CA mutants with resistance to LEN remained sensitive to other main HIV drug classes.
Conclusions:
Using a sensitive HIV-dependent reporter-based system, we evaluated phenotypic susceptibility of several viruses with low RC (0.6-24% of WT) that were previously uncharacterized, expanding our understanding of LEN resistance and interactions between CA RAMs.