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Therapeutic Efficacy of Antibodies Lacking FcγR against Lethal Dengue Virus Infection Is Due to Neutralizing Potency and Blocking of Enhancing Antibodies

Figure 3

Fusion loop- and A strand-specific modified MAbs can compete for binding with fusion loop-specific MAbs of lesser neutralizing potency.

A. Fusion loop-specific mouse MAb 4G2 was incubated at 1 µg/mL with MAbs E60, 87.1 or E87 at 10, 1 or 0.1 µg/mL human MAb prior to addition to DENV2-virion coated plates (for each MAb concentration, data is represented as mean +/− SEM). Anti-mouse, Fc-specific secondary MAb was then added, followed by PNPP substrate. Optical density (OD) values are shown on the y-axis and were calculated after subtracting the average background (binding of mouse Fcγ-chain specific secondary antibody in the absence of 4G2) from the raw OD. Statistically significant differences in 4G2 binding across the different human MAb concentrations were calculated using a Kruskal-Wallis analysis from triplicate values within each experiment. This data shown is representative of seven independent experiments. B–D. MAb 4G2 was pre-mixed with MAb E60 N297Q, MAb 87.1 LALA or MAb E87 N297Q in ratios of 95% 4G2/5% modified MAb (B), 85% 4G2/15% modified MAb (C), or 75% 4G2/25% modified MAb (D). For each 4G2/modified MAb mixture, a Gaussian distribution was used to fit the enhancement curve (Figure S2). The area under the curve (AUC) was calculated for each curve, and relative infection was expressed by dividing the AUC in the presence of modified MAbs by the AUC measured with 4G2 (no modified MAb) only. The data displayed are the average of three to seven independent experiments +/− SEM. Comparisons between the MAb combinations E87 N297Q/4G2 and E60 N297Q/4G2 or 87.1 LALA/4G2 were performed using a Kruskal-Wallis test. E. AG129 mice (n = 3–6 per group from one or two experiments) were administered an enhancing quantity (20 µg) of 4G2 MAb, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAb. A Kaplan-Meier survival curve is shown, and log-rank analysis was used for statistical comparison.

Figure 3