Fig 3.

Identifying the epitope mediating non-specific neutralization. (A) Neutralization curves of EBOV-GFP from Huh7 cells in the presence and absence of human complement. (B) Neutralization of Huh7-derived virus vs. HepaRG-derived virus in the presence of complement. (C) Binding of the hit mAbs to glycan epitopes. Glycan array with triplicate antigen spots was probed with the hit antibodies or a negative control antibody. Median fluorescence signals after correction against median signals from the negative control are presented. (D) ELISA against purified ganglioside antigens GM1 and GM2. (E) Influence of ganglioside synthesis inhibition on hit IgM mAb binding to Huh7 cells. Cells were treated with 200 µM miglustat or DMSO as a vehicle control for 4 days. Immunofluorescence analysis was then performed using the hit mAbs or an isotype control as the primary antibodies. (F) Impact of ganglioside synthesis inhibition on virus susceptibility to hit mAb neutralization. After 4 days of pre-treatment with miglustat or vehicle control, EBOV-GFP was passaged on Huh7 cells for 4 days in the presence of the same. Neutralization curves of the resulting virus stocks are presented. (G) Neutralization of virus grown as in F by unheated serum of the B-cell donor. (H) Neutralization of virus grown as in F by the unheated serum panel of Fig. 1A.