Figure 2
Distinct efficacy of entry inhibitors to block cell-free HIV-1 infection and cell-to-cell virus transmission. Activated human PBMCs (105) were incubated for 30 min with the different entry inhibitors at various concentrations and then infected with HIV-1 Ba-L (R5), A204 (R5X4), LAI (X4) or KH025 (X4) for 1 h at 37°C in the presence of drugs at varying doses. After 3 washes, cells were resuspended in RPMI1640 containing IL-2 and the same drug they were pre-incubated with. HIV-1 infected PBMCs were then left in culture for 3 days, when the culture supernatants were replaced with RPMI1640-IL-2 medium without drug. The cultures were continued for 4 more days and HIV-1 p24 antigen was assayed in the supernatants. For the inhibition of HIV-1 passage by entry inhibitors, BeWo cells (CD4 negative, white bars or CD4 positive, black bars) were pre-incubated for 30 min at 37°C in the presence of varying doses of TAK779, SCH-350581, or AMD3100. If T20 or C34 were used as inhibitors, PBMCs infected with different HIV-1 isolates were pre-incubated with varying doses of T20 or C34 for 30 minutes before the contact with BeWo cells. At this point, HIV-1 Ba-L, HIV-1 A204, HIV-1 LAI or HIV-1 KH025 infected PBMCs were added and both cell types were left in contact for 3 h at 37°C. Extensive washes were then performed and the Transwell® inserts transferred on new plates containing activated HIV negative indicator PBMCs, without drugs. The inserts were removed 5 days later and the culture continued 3 more days, when the HIV-1 p24 core antigen was quantified in the supernatants. The IC50s were determined graphically from the titration curves. Figure 2A: comparison of IC50s during the inhibition of cell-free HIV-1 Ba-L infection of PBMC (grey bars), HIV-1 Ba-L/CD4 negative BeWo cells (white bars) and HIV-1 Ba-L PBMC/CD4 positive BeWo cells (black bars). Figure 2B: the PBMCs were infected with HIV-1 A204. Figure 2C: the PBMCs were infected with HIV-1 LAI or HIV-1 KH025 and the inhibition was achieved by AMD3100.