Figure 4. Characterization of the epitope recognized by 3BC176 and 3BC315. (A) MFIs of the antibodies 3BC176, 3BC315, and controls measured at 20 µg/ml on GFP-293TBaL wt and the indicated mutants (N160K, D368R, and I420R). (B) ELISA for binding to SF162 wt (left) and to SF162K160N mutant (right) by PG9, PG16, 3BC176, and 3BC315. Graphs show OD405 nm (y axis) and antibody concentration in µg/ml (x axis). (C) Pseudovirus neutralization measured in TZM-bl assay. Graphs show percent neutralization (y axis) by increasing concentrations of 3BC176 or 3BC315 (x axis) of wt SF162 and mutants lacking the V1 or the V2 loop (SF162 DV1 and SF162 DV2, respectively). (D) IC50 of 3BC176 and 3BC315 neutralization of SF162 wt and 11 SF162 pseudoviruses carrying single mutations at different glycosylation sites or the K160N mutatio.n Position of mutated glycosylation site (x axis) according to HXBc2. For both antibodies, the fold increase or decrease of the IC50 values are visualized. (E) Microarray analyses of 3BC176, 3BC315, and 2G12 binding using a set of 50 oligosaccharide probes as neoglycolipids. Table S4 gives designations of the oligosaccharide probes. The binding signals (fluorescence intensities) shown are the mean values of duplicate spots, printed at 2 and 5 fmol per spot (the error bars represent half of the difference between the two values). (F) Graph shows enhancement of Alexa Fluor 647–labeled antibody binding to GFP-293TBaL in the presence of sCD4. Staining intensity is measured by MFI (y axis), and the starting value normalized to MFI 1,000. 3–67 is a CD4-induced site (CD4i) antibody and was used as control (Scheid et al., 2011). (G) Inhibition of 3BC176 and 3BC315 binding to gp160 ΔcBaL-transfected 293T cells in the presence of sCD4 (10 µg/ml). Graphs show inhibition (in percentage) of Alexa Fluor 647–labeled 3BC176 or 3BC315 (y axis) in the presence of increasing concentrations of the indicated antibodies (x axis). All experiments were at least performed in duplicate, and results of representative experiments (D and G) are shown. Standard errors are shown for A–C and F.
Figure 4.

Characterization of the epitope recognized by 3BC176 and 3BC315. (A) MFIs of the antibodies 3BC176, 3BC315, and controls measured at 20 µg/ml on GFP-293TBaL wt and the indicated mutants (N160K, D368R, and I420R). (B) ELISA for binding to SF162 wt (left) and to SF162K160N mutant (right) by PG9, PG16, 3BC176, and 3BC315. Graphs show OD405 nm (y axis) and antibody concentration in µg/ml (x axis). (C) Pseudovirus neutralization measured in TZM-bl assay. Graphs show percent neutralization (y axis) by increasing concentrations of 3BC176 or 3BC315 (x axis) of wt SF162 and mutants lacking the V1 or the V2 loop (SF162 DV1 and SF162 DV2, respectively). (D) IC50 of 3BC176 and 3BC315 neutralization of SF162 wt and 11 SF162 pseudoviruses carrying single mutations at different glycosylation sites or the K160N mutatio.n Position of mutated glycosylation site (x axis) according to HXBc2. For both antibodies, the fold increase or decrease of the IC50 values are visualized. (E) Microarray analyses of 3BC176, 3BC315, and 2G12 binding using a set of 50 oligosaccharide probes as neoglycolipids. Table S4 gives designations of the oligosaccharide probes. The binding signals (fluorescence intensities) shown are the mean values of duplicate spots, printed at 2 and 5 fmol per spot (the error bars represent half of the difference between the two values). (F) Graph shows enhancement of Alexa Fluor 647–labeled antibody binding to GFP-293TBaL in the presence of sCD4. Staining intensity is measured by MFI (y axis), and the starting value normalized to MFI 1,000. 3–67 is a CD4-induced site (CD4i) antibody and was used as control (Scheid et al., 2011). (G) Inhibition of 3BC176 and 3BC315 binding to gp160 ΔcBaL-transfected 293T cells in the presence of sCD4 (10 µg/ml). Graphs show inhibition (in percentage) of Alexa Fluor 647–labeled 3BC176 or 3BC315 (y axis) in the presence of increasing concentrations of the indicated antibodies (x axis). All experiments were at least performed in duplicate, and results of representative experiments (D and G) are shown. Standard errors are shown for A–C and F.

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