Figure 5.

Evaluation of mutant CHIKV binding and neutralization by mAb 3E7b. (A) Confluent BHK-21 cells were infected with CHIKV-WT, CHIKV-IVT, CHIKV-E24D, CHIKV-N218D or CHIKV-D223G clones at MOI 10. At day 1 p.i., cells were fixed and co-stained with 3E7b IgM and rabbit anti-E2 polyclonal antibody as a positive control. The difference in binding was visualized with goat anti-mouse IgM FITC and anti-rabbit IgG 594 secondary antibodies. Cell nuclei were stained DAPI and images were captured under 10x and 100x magnification. White arrowhead indicates reduced 3E7b binding. Representative images from 3 independent experiments are shown. (B) FITC signal of 3E7b binding is expressed as a percentage over 594 signal that represents the number of infected cells, n, quantitated. Error bars represent ± SD. One-way ANOVA followed by a post-Dunnett test, with statistical significance defined as ***p < 0.001. (C) PRNT of single mutant CHIKV clones was performed where 100 PFU of the respective CHIKV clone was neutralized with serially diluted 3E7b from 3.81 pg/ml to 0.25 μg/ml. Non-linear regression analysis was performed and data was best-fitted to dose-dependent inhibition curves for CHIKV-WT, CHIKV-IVT, CHIKV-E24D and CHIKV-D223G. Due to non-convergence of CHIKV-N218D data points, dose-inhibition curve and IC₅₀ are not applicable. Mean value from at least 3 independent experiments performed in duplicates are presented. Error bars represent ± SEM.CH; CHIKV. a; ambiguous.