Phenotypic impacts of treatment-selected mutations in HIV-2 protease on darunavir and lopinavir susceptibility: Evaluating genotypic HIV-2 drug resistance tools

Compared to HIV-1, HIV-2 infection is characterized by lower viral loads and slower decline in CD4 cells, however the majority of people living with HIV-2 (PLWH2) progress to AIDS and will benefit from antiretroviral therapy. Mutations leading to protease inhibitor (PI) resistance in HIV-2 are poorly characterized, but have important implications for second-line therapy. We evaluated the phenotypic drug susceptibility impacts of HIV-2 protease changes which are identified in genotypic resistance tools. We generated a library of 54 full length HIV-2ROD9 clones that included 21 individual protease mutations, alone or in various combinations. We generated eight additional clones containing combinations of changes observed in PI-treated PLWH2. We tested the clones in a single-cycle PI assay to determine darunavir (DRV) and lopinavir (LPV) EC50, and calculated fold change resistance relative to wild-type HIV-2ROD9. Four of the 21 amino acid changes tested alone conferred PI resistance: V47A and T56V conferred 4.1 and 2.9-fold resistance, respectively, to LPV, I50V conferred 4.6-fold resistance to DRV, and I54M conferred 5.5-fold resistance to DRV and 2.2-fold resistance to LPV. Other changes either lowered the EC50 or caused no change. Some combinations including V47A, I50V, I54M, or T56V also conferred resistance, with EC50 values 4.4 to 17-fold higher than wild-type. Six of eight PLWH2-derived strains were replication-competent: five exhibited resistance to LPV (>8.8-fold resistance), and three exhibited resistance to DRV (>4.7-fold). HIV-1 and HIV-2 are not equivalently susceptible to all antiretroviral agents and do not utilize identical pathways to resistance. We provide phenotypic evidence supporting the resistance role of changes in HIV-2 protease which do not have HIV-1 analogues, as well as evidence that analogues of "major" resistance changes in HIV-1 may have no resistance impacts in HIV-2, despite apparent treatment selection. These results should inform the HIV-2 genotypic resistance tools and help improve treatment for PLWH2.