Figure 4.

Functional characterization of a panel of monoclonal antibodies isolated from ZIKV convalescent individuals. Twenty-four mAbs isolated from ZIKV convalescent individuals were synthesized and expressed as soluble IgGs and used for flavivirus neutralization and antibody-dependent enhancement (ADE) assays. (Left) Heat map of computationally predicted VLP affinities against wild-type ZIKV [ZIKV (wt)], immature ZIKV [ZIKV (pr-mut)], and wild-type YFV [YFV (wt)] virus-like particles. (Center left) Neutralization assays were performed with the indicated mAbs using ZIKV or YFV reporter virus particles (RVPs). Data were analyzed by non-linear regression with a variable slope and constrained to the bottom to estimate the antibody concentration required to inhibit infection by 50% and 90% (EC₅₀ and EC₉₀, respectively). EC₅₀ and EC₉₀ are shown as a heat map and depicts the mean of two independent experiments performed in duplicate technical replicates. The degree of neutralization of ZIKV RVPs observed at the highest concentration of antibody tested is expressed as the percent resistant fraction. (Center right) The sensitivity of neutralization to the maturation state of ZIKV RVPs was analyzed for selected mAbs using standard (Std) and mature (Mat) preparations of RVPs. The EC₅₀ of standard and mature RVPs was estimated for each antibody by non-linear regression analysis with a variable slope not constrained to the bottom. Maturation state sensitivity was expressed as the ratio of the EC₅₀ against standard and mature ZIKV RVPs; mAbs were designated as maturation-state sensitive if the difference in titer was ≥4-fold. Data reflect the mean of two independent experiments performed in duplicate technical replicates per RVP type. Cyan is representative of mAbs that are more sensitive to partially mature virus; blue is representative of mAbs that are more sensitive to mature virus. (Right) Antibody binding was measured using an ADE assay performed with standard ZIKV or YFV RVPs. Antibodies capable of enhancing infection of K562 cells at three times the level of infection observed in the absence of antibody are indicated in green. Data are representative of two independent experiments performed in singlet. (Far right) Antibody heavy and light chain isotype for each mAb analyzed. *mAb-20 was only tested in one experiment for neutralization and ADE studies due to insufficient volume.