Figure 2. Label-free quantification of dengue fusion inhibition by a monoclonal antibody.

(A) Schematic of the mode of action for the DENV neutralizing monoclonal antibody 4G2, which binds to the fusion peptide, FP. After acidification, molecular rearrangement of the E protein exposes the hydrophobic FP (green), which associates with the host cell membrane. 4G2 binding to the exposed FP prevents membrane insertion and therefore blocks membrane fusion. (B) Measurement of the anti-fusion effects of 4G2 using cellular impedance. Cells were infected at an MOI of 2 or mock infected. Twenty-four hour post infection the culture media was replaced with fresh media at pH 6 and the CI recorded. The cell impedance measurement at 1.5 h post media change is shown. The presence of 4G2 (500 μg/ml) within the low pH media was sufficient to completely abrogate the fusion signal. An isotype matched antibody control, 9C4 (specific for the RSV F protein) had no significant effect. (C) Parallel fusion assay setup to (B) was observed using live cell bright field microscopy. Similar anti-fusion effects of 4G2 were observed, with fusion extent inversely proportional to cell number. (D) A dose response analysis of the inhibitory effect of 4G2 using both cell impedance and live cell microscopy revealed near identical activity profiles. The CEI measurement is indicated on the left axis and BFM measurements are plotted on an inverted right axis to allow direct comparison of the dose response curves observed. (E) 4G2 inhibition of DENV cell infection analyzed using PRNT analysis. Control antibody 9C4 was observed to have no antiviral activity in this assay format.