Fig 8. L403F and L438V modulate bNAb resistance by altering binding to SR-B1.

(A) Binding of serial dilutions of strain 1a154 (H77) soluble E2 (sE2) to CHO cells expressing human CD81 (blue peaks) or to wild type CHO cells that do not express HCV receptors (pink peaks), measured by flow cytometry. (B) Binding of serial dilutions of 1a154, 1a154_L403F, or 1a154_L438V sE2 to SR-B1-CHO cells or CD81-CHO cells. Each point was calculated from 10e4 events. Background binding to wild type CHO cells was subtracted from mean fluorescence intensity (MFI) values. sE2 supernatants were normalized for relative sE2 concentration (shown in S3 Fig) prior to dilution. One experiment that is representative of two independent experiments is shown. The second experiment is shown in S4 Fig. (C) Percent inhibition of sE2 binding to SR-B1 or CD81 by HC33.4. MFI of binding of a fixed, equivalent concentration of 1a154 or 1a154_L403F sE2 to SR-B1-CHO or CD81-CHO cells in the presence of serial dilutions of mAb HC33.4 or nonspecific human IgG was used to calculate percent inhibition of binding. Each point was calculated from 10e4 events. Background binding to wild type CHO cells was subtracted from mean fluorescence intensity (MFI) values. IC₅₀ was calculated by nonlinear regression. (D) Binding of equivalent concentrations of sE2 to SR-B1-CHO cells in the presence of serial dilutions of HC33.4. Each point was calculated from 10e4 events. Background binding to wild type CHO cells was subtracted from mean fluorescence intensity (MFI) values. Binding after incubation of each sE2 with 100 μg/mL of nonspecific IgG is shown for reference. (E) Binding of serial dilutions of 1a154, 1a154_L403F, or 1a154_L438V sE2 to SR-B1-CHO cells after preincubation of sE2 with 40 μg/mL of HC33.4. Each point was calculated from 10e4 events. Background binding to wild type CHO cells was subtracted from mean fluorescence intensity (MFI) values. sE2 supernatants were normalized for relative sE2 concentration prior to dilution.