Hiv blocking anti cd4 antibody

HIV-1 proviral constructs were transduced into Jurkat cells (donor cells) using Amaxa nucleofection as previously described (Amaxa Biosystems). In brief, 5 μg of endotoxin-free HIV-1 proviral plasmids was nucleofected into 6 × 10⁶ Jurkat cells using Cell Line Nucleofector kit V, program S-18. Twenty hours after nucleofection, viable Jurkat cells were purified by centrifugation on a Ficoll-Hypaque density gradient. Twenty-four hours after nucleofection, cells were washed with complete buffer and recovered at 37 °C for co-culture. Unactivated primary CD4⁺ T cells (target cells) were resuspended in serum-free RPMI 1640 and stained with 1 μM Cell-Tracker Orange CMTMR (5-and-6-(((4chloromethyl)benzoyl)amino)tetramethylrhodamine); Molecular Probes) for 45 min at 37 °C, washed, and cultured overnight in complete RPMI medium containing 50 U/ml IL-2. Donor and target cells were mixed at a ratio of approximately 1:1 and cocultured at 37 °C for 3 h before they were treated with trypsin and fixed. Where inhibitor Leu3a, an HIV-blocking anti-CD4 antibody (BD Biosciences) was used, donor and target cells were preincubated separately with equal volumes of inhibitor for 30 min at 37 °C before mixing.

To generate virus-producing donor cells, HIV-1 proviral constructs were transfected into Jurkat cells (donor cells) using Amaxa nucleofection, as previously described (Amaxa Biosystems, Lonza, Basel, Switzerland). In brief, 5 μg of endotoxin-free HIV-1 proviral plasmids was nucleofected into 6 × 10⁶ Jurkat cells using Cell Line Nucleofector kit V, program S-18. Twenty hours after nucleofection, viable Jurkat cells were purified by centrifugation on a Ficoll-Hypaque density gradient, washed with RPMI containing 10% FBS, and recovered at 37 °C for co-culture. Unactivated primary CD4⁺ T cells (target cells) were cultured overnight in complete RPMI medium containing 50 U/mL IL-2. Donor and target cells were mixed at a ratio of approximately 1:1 and cocultured at 37 °C for 3 h before they were treated with trypsin to remove surface-attached virus and fixed. Where inhibitor Leu3a, an HIV-blocking anti-CD4 antibody (BD Biosciences, 1 μg/mL) was used, donor and target cells were preincubated separately with equal volumes of inhibitor for 30 min at 37 °C before mixing.