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. 2019 Nov 26;93(24):e01325-19. doi: 10.1128/JVI.01325-19

FIG 7.

FIG 7

MCG analogs sensitize HIV-1-infected cells to ADCC and viral particles to neutralization by nnAbs. (A and B) Primary CD4 T cells isolated from PBMCs were infected with HIV-1CH58TF for 48 h. For cell surface staining, 5 μg/ml 17b (A) or 1:1,000-diluted HIV+ plasma (n = 15) (B) was used in the presence of the different MCG analogs (50 μM), (+)-BNM-III-170, or an equivalent volume of the vehicle (DMSO). An Alexa Fluor 647-conjugated anti-human IgG secondary Ab was then used for fluorescence labeling. (C) For ADCC, infected cells were used as target cells in a FACS-based ADCC assay that measures the killing of infected (p24+) cells (48). The assay determines susceptibility to ADCC mediated by a 1/1,000 dilution of plasma from 15 HIV-1-infected individuals in the presence of the different MCG analogs (50 μM), (+)-BNM-III-170 (50 μM), or an equivalent volume of the vehicle (DMSO). (D) The correlation between cell surface staining with HIV+ plasma and ADCC was calculated using the Spearman rank correlation. (E) To evaluate the direct virus-neutralizing ability of the analogs, HIV-1CH58TF was incubated with the indicated amounts of different compounds or DMSO for 1 h at 37°C and then added to TZM-bl cells. After incubation for 48 h at 37°C, luciferase activity was measured. Relative infectivity was calculated as the percentage of the value seen in the absence of the compound. (F) To measure the ability of (S)-MCG-IV-210 to sensitize viral particles to neutralization by otherwise nonneutralizing Ab 17b, HIV-1CH58TF was incubated with the indicated amounts of 17b in the presence of 0.5 μM (+)-BNM-III-170, 20 μM (S)-MCG-IV-210, or DMSO for 1 h at 37°C and then added to TZM-bl cells. After incubation for 48 h at 37°C, luciferase activity was measured. Relative infectivity was calculated as the percentage of the value seen in the absence of the compound. Data shown are the means ± SD from at least three independent experiments. Statistical significance was evaluated using a Mann-Whitney unpaired t test (A) or a Wilcoxon paired t test (B and C) (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001).