EFdA (4´-ethynyl-2-fluoro-2´-deoxyadenosine; MK-8591; Merck & Co.) is an investigational NRTI that blocks HIV-1 replication in culture with 50% effective concentrations (EC50) in the low-nanomolar to picomolar range. EFdA is highly active against HIV-1 and SIV in humanized mice and rhesus macaques, respectively, and a single 10-mg dose of EFdA demonstrated potent antiviral activity for 10 days in a phase 1b proof-of-concept clinical trial. However, studies evaluating the activity of the EFdA against HIV-2 are lacking, and the ability of the drug to inhibit NRTI-resistant mutants of HIV-2 is unknown.
HIV-1 and HIV-2 isolates from antiretroviral-naïve individuals were tested against EFdA in single-cycle infections of MAGIC-5A cells. Site-directed mutants of HIV-2 reverse transcriptase (RT) were generated in a full-length plasmid clone (pROD9) and were evaluated for EFdA resistance in the single-cycle assay. 50% cytotoxic concentrations (CC50) for EFdA were determined using a CellTiter-Glo® luminescence kit.
EFdA inhibited HIV-2 infection of MAGIC-5A cells with mean EC50 values (± SD) of 0.58 ± 0.13 nM for 6 group A isolates and 0.55 ± 0.16 nM for 6 group B isolates (range = 0.34–0.83 nM for all 12 HIV-2 strains tested). In contrast, the mean EC50 for 6 HIV-1 isolates, including group M subtype A, B, C and D strains and the group O isolate MVP5180-91, was 2.0 ± 0.43 nM (range = 1.29–2.54 nM; p < 0.0001 for HIV-1 vs. HIV-2, Mann-Whitney test). In spreading infections of CEMss cells, EC50 values for HIV-2 ROD9 and HIV-1 NL4-3 were 38 and 120 pM, respectively. EFdA was fully active against HIV-2 RT mutants K65R and Q151M (EC50 = 0.17 ± 0.04 nM and 0.31 ± 0.05 nM, respectively), whereas the M184V variant was 10-fold resistant to the drug. Similar levels of resistance (12–16-fold) were seen for HIV-2 mutants that harbored M184V plus one or more additional NRTI resistance-associated changes in RT, including a patient-derived clone encoding K65R+N69S+V111I+Q151M+M184V. The CC50 for EFdA in MAGIC-5A cells was >100 nM.
EFdA is the most potent inhibitor of HIV-2 replication described to date and is more active against HIV-2 than against HIV-1 in culture. EFdA also inhibits multi-NRTI–resistant HIV-2 mutants with single-cycle EC50 values ≤ 10 nM. These data indicate that EFdA should be evaluated in clinical studies involving HIV-2–infected individuals.