Determination of HIV-1 drug susceptibility

The sensitivity of infectious clones to IND02 and the IND02-trimer was determined in the multinuclear activation of the galactosidase indicator (MAGI) assay [32] with some modifications using viral preparations titrated as previously described [33]. Briefly, target cells (HeLa CD4/CCR5-LTR/β-gal; 10⁴/well) were plated in 96-well flat microtiter culture plates. On the following day, the medium was removed, and the cells were inoculated with HIV-1 NL4-3 clone or with the reference HIV-1-LAI strain (70 MAGI units/well, which gave 70 blue cells after 48 h of incubation) and cultured in the presence of various concentrations of the drug in fresh medium. The test drugs were added initially and viruses were immediately inoculated onto the cells. Forty-eight hours after viral exposure, all blue cells in each well were counted. The cytotoxicity of the compound was determined by the MTT method as previously described [34]. All experiments were performed in triplicate and AZT was used as reference anti-HIV-1 molecule. The 50% effective concentration (EC₅₀ in μM) required to inhibit 50% of HIV-1 replication was calculated.

In order to confirm antiretroviral activity of the compounds, using a cell model closer to the pathophysiology, Peripheral Blood Mononuclear Cells (PBMCs) were separated from a buffy-coat from healthy HIV-, HCV-, HBV-seronegative blood donors by Ficoll-Hypaque density gradient centrifugation. The PBMCs were activated by incubation with 1 μg/ml phytohemagglutinin-P (PHA-P; Difco Laboratories) for three days in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS, +56 ◦C for 45 min), 2mM l-glutamine and a 1% antibiotic cocktail (penicillin, streptomycin, neomycin). After this mitogen activation, PBMCs were cultivated in the same culture medium supplemented with recombinant human interleukin-2 (rHuIL-2; 20 IU/mL) and treated and/or infected. PBMCs were infected either with the reference lymphotropic (-X4) HIV-1 LAI strain [35], the reference macrophage-tropic (-R5) HIV-1 BaL strain [36] or with 4 different clinical isolates (subtype A, B or C). All these viruses were amplified in vitro with PHA-P-activated blood mononuclear cells and 50% tissue culture infectious doses (TCID₅₀) were calculated using Kärber’s formula [37], after limited-dilution titration. Viruses (125 TCID₅₀) were incubated with a range of concentrations (0–20 μM) of the IND02-trimer molecule and added to PBMCs (m.o.i. ~ 0.001). Cell supernatants were collected at day 7 post-infection and stored at -20°C. Viral replication was measured by quantifying reverse transcriptase (RT) activity in the cell culture supernatants using the Lenti RT Activity Kit (Cavidi) and AZT was used as reference anti-HIV-1 molecule. EC₅₀ were then calculated and expressed in μM.