SAMMA inhibits HIV transmission by DCs. (a) MDDCs (1.25–2×10⁵ per well) were exposed to replication-competent R5 virus HIV_BaL at MOI of 0.002 in the presence or absence of SAMMA at 100 μg/ml at 37°C for 2 hr. Cells were washed and co-cultured with autologus activated primary CD4⁺ T cells (1×10⁶) in complete media in the presence or absence of SAMMA at 100 μg/ml. (b) HIV_BaL was pre-incubated without (control) or with SAMMA at 100 μg/ml at 37°C for 1 hr followed by addition to DCs (2×10⁵) for 2 hr in the absence or presence of the inhibitor. After washing four times with media, DCs (1.25 × 10⁵) were incubated with activated primary CD4⁺ T cells (1 × 10⁶) in the absence of SAMMA. HIV released into media was monitored by measuring the level of HIV p24 antigen at day 0, 5, 7, 9, 14 after infection. Data in Fig. 1a are mean ± SD of duplicated samples and represent two independent experiments. In the wash-off setting (panel b), results from two different donors are shown.

SAMMA does not cause cytotoxicity in DCs and primary CD4⁺ T cells. DCs or activated primary CD4⁺ T cells (5×10⁴) were treated with SAMMA at 10, 100, 500 μg/ml for three days at 37°C. Cytotoxicity was determined by measuring the number of viable cells by the MTS assay (Promega). MTS substrate was added to cells for 1 hr at 37°C. Data are mean ± SD of triplicated samples and represent two independent experiments.

SAMMA inhibits HIV glycoprotein-mediated cell-cell fusion. HeLa-Tat cells were transfected HIV_JR-FL envelope plasmid for 48 hr. Cells were then treated with a fusion inhibitor T-20 or SAMMA at 37°C for 1 hr before addition to TZM cells, indicator cells expressing CD4, CXCR4 and CCR5 and containing a luciferase reporter gene driven by HIV LTR. After incubation at 37°C for 24 hr, luciferase activity was measured. Data are mean ± SD of duplicated samples and represent three independent experiments. (b) Viability of TZM-bl cells in the presence of SAMMA at various concentrations for 48 hr was determined by MTS assay as described in Fig. 3.

SAMMA inhibits HIV infection in trans by DCs in a single-round infection assay. (a) MDDCs (1×10⁶) were incubated with SAMMA (10 and 100 μg/ml), mannan (0.1 and 5 mg/ml) or anti-DC-SIGN antibody (20 μg/ml, mAb 612 from R&D systems) for 1 hr at 37°C and then exposed to pseudotyped HIV_JR-FL luciferase reporter virus for 2 hr at 37°C. Cells were washed four times before co-culturing with HeLa-CD4.CCR5 cells for 48 hr. DCs were removed by washing and luciferase activity in relative light unit (R.L.U.) in HeLa cells were measured. (b). DCs were incubated with SAMMA at 100 μg/ml, anti-CD4 antibody (10 μg/ml, Leu3a from BD Biosciences) or an isotype control antibody (mouse IgG₁, κ) at 37°C for 1 hr followed by exposure to pseudotyped HIV_JR-FL luciferase reporter virus for 2 hr. DCs were washed and then co-cultured with HeLa-CD4.CCR5 cells for 48 hr before measurement of luciferase activity in HeLa cells. (c) DCs were exposed to pseudotyped HIV_JR-FL luciferase reporter virus for 2 hr at 37°C and then incubated without or with SAMMA at 100 μg/ml for 1 hr at 37°C. DCs were washed four times with PBS and then co-cultured with HeLa-CD4.CCR5 cells in the absence (wash off) or presence (add back) of SAMMA for 48 hr. Luciferase activity in HeLa cells was measured. Data are mean ± SD of triplicated samples and represent three independent experiments.

SAMMA inhibits HIV capture by DCs. MDDC were seeded at 2×10⁵ per well in 96-well flat-bottom plates. HIV-1_BaL (3 ng per well) was pre-incubated in the absence (control) or presence of SAMMA for 1hr at 37°C. DCs were then incubated with virus for 2 hr at 37°C (a) or 4°C (b) in the absence (control) or presence of the inhibitor. Cells were washed four times to remove unbound virus and SAMMA and lysed with 1% Triton X-100. Cell-associated HIV p24 antigens were measured by p24 ELISA (NCI, Frederick). Data are mean ± SD of duplicated samples. In Fig. 4a, results from two donors are shown. In Figs. 4b and 4c, results represent two independent experiments from two DC different donors. (c) DCs were treated without (control) or with SAMMA at 100 μg/ml at 37°C for 1 hr and washed four times with PBS followed HIV exposure for 2 hr. HIV-exposed DCs were washed and lysed. Cell-associated HIV p24 antigen was measured by ELISA. Results represent two independent experiments.