A, Cytotoxicity profile of SIL in TZM-bl cells. Cells were infected with LAI, a CXCR4-using virus, or BAL, a CCR5-using virus, at an MOI of 0.05 in the presence of the indicated concentrations of SIL and ATP was measured using the ATPlite kit 48 hours later. The data are representative of 2 (BAL) and 3 (LAI) independent technical repeats. B, Antiviral profile of SIL in TZM-bl cells. Serial dilutions of SIL were tested for inhibition of infection in TZM cells. Following addition of compounds and virus, cells were incubated for 48 hours before luciferase activity was measured. Percent inhibition refers to percent reduction in luciferase activity of SIL versus untreated cultures. Error bars represent standard deviation of 3 independent technical repeats. C, SIL inhibits pseudovirus replication in TZM-bl cells. TZM-bl cells were infected with the indicated viruses in the presence of the indicated concentrations of SIL and luciferase activity was measured 48 hours post-infection. The D013M12 psuedovirus contains a subtype D envelope sequence, while the D769 psuedovirus contains a subtype A envelope sequence. Error bars represent standard deviations of triplicate wells per condition.

A, SIL inhibits LAI and BAL infection of PBMCs derived from 5 different donors. PBMC were activated with PHA and 3 days later, cells were washed to remove PHA and resuspended in media containing IL-2. The cells were plated in 96 well plates in the presence or absence of 243 µM SIL. Cells were then infected with 3-fold serial dilutions of LAI and BAL virus stocks until the end point dilution was reached. Cultures were incubated 24 hours, input virus removed, and cultures were fed with media containing IL-2 and SIL. Supernatants were harvested 6 days later and assayed for p24 levels using HIV p24 Antigen Capture ELISA. B, Dose response of LAI inhibition by SIL. The five PBMC cultures were treated as described above and infected with LAI in the presence of the indicated concentrations of SIL. p24 ELISA was performed at 7 days post-infection. C, SIL inhibits HIV-1 infection of PBMCs and CEM cells. PBMCs were treated as described above. CEM cells were infected with LAI (MOI = 0.001) in the presence of the indicated amounts of SIL and p24 antigen was measured in culture supernatants at 4–7 days post-infection. The data represent pooled data from individual experiments of PBMCs infected with LAI (N = 10) or BAL (N = 6) and CEM infected with LAI (N = 8). D, SIL's anti-HIV effects are durable. PBMCs were activated and infected with LAI in the presence of 243 µM of SIL and p24 antigen was measured at the indicated times (days post-infection). Cells were fed every 3–4 days with medium containing fresh SIL. Virus control refers to cells that only received virus and no SIL.

A, SIL suppresses growth of PBMC and CEM cells. Cells were treated with SIL as described in the Materials and Methods and cells were counted 24 hours later by hemacytometer and trypan blue. B, Suppression of PHA-stimulated PBMC cell growth. After 24 hours of PHA stimulation, PBMC were placed in culture for 24 or 96 hours and the concentration of live cells per µL of sample was measured by flow cytometry. C, Suppression of CD4+ T cell proliferation measured by CFSE staining. PBMCs were labeled with CFSE and stimulated with PHA (2 µg/ml) for 24 hours prior to incubation with SIL for 4 days. D, SIL suppresses growth of T Cells and B Cells. Three day PHA-stimulated PBMC were treated with or without SIL for 24 hours and analyzed by flow cytometry following staining of T (CD4 or CD8) and B (CD20) cells and labeling of DNA using the Click-iT® EdU labeling kit. Peaks on the left of each graph represent EdU-, non-proliferating cells, while peaks on the right represent EdU+ proliferating cells. Top two rows depict unstimulated PBMC with and without SIL. Bottom two rows depict 72 h PHA and 24 h IL-2 stimulated PBMC with and without SIL. E, the number of EdU+ proliferating cells is inhibited by SIL. Cells were treated as described for panel C following exposure to SIL. The cell concentration is expressed per µl of cell suspension and is determined using counting beads as for panel B. F, G, SIL is not cytotoxic to PBMCs. PBMCs were stimulated with PHA for 3 days and treated with IL-2 in the presence of varying doses of SIL and 24 hours later, stained for CD4+, CD8+ and CD19+ (B cell) immune cell subsets and dead cells (F) as well as Annexin V+ apoptotic cells (G) were quantified.

PBMCs were stimulated with PHA for 3 days prior to exposure to IL-2 and the indicated concentrations of SIL. Twenty-four hours later, cells were stained for CD4, CXCR4, and CCR5 and analyzed by flow cytometry. A, Y-axis represents the concentration of the indicated cell type. The cell concentration is expressed per µl of cell suspension and was determined using counting beads. Panel B shows the percentage of total CD4+ T cells that express one, both, or neither co-receptor.

PBMCs were activated with either SEB (0.5 µg/ml) or PHA (2 µg/ml) for 24 hours, and treated with the indicated concentrations of SIL for 12 hours. Representative flow cytometry dot plots showing expression of HLA-DR (A), CD38 (B), Ki67 (C), and CCR5 (D). Data are representative of 3 HIV-seronegative individuals tested for each marker.