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. 2023 Jan 10;19(1):e1010814. doi: 10.1371/journal.ppat.1010814

Fig 3. An engineered N67 glycan on ZIKV E protein blocks G9E binding and neutralization.

Fig 3

(A) Structural overlay of ZIKV E/G9E complex on to DENV2 E-protein. Introducing a ‘DENV-like’ N-67 glycan on ZIKV E protein was predicted to block G9E binding. DENV E (cyan, PDB 1OAN), ZIKV E (purple), G9E heavy-chain (green), and G9E light-chain (gold) are shown in cartoon representation. DENV N-67 glycan is shown as spheres. (B) G9E does not bind to a recombinant ZIKV with N-linked glycan on EDII. Comparison of ZIKV mAbs binding to ZIKV H/PF/2013 and ZIKV H/PF/2013 with engineered glycan at position 67 (rZIKV-D67N). ZIKV type-specific mAbs ZV67 and ZKA-190 bind to EDIII. ZIKV type-specific mAbs G9E and Z20 target quaternary structure epitopes on EDII. MAbs EDE-C8 and EDE-C10 target quaternary structure E dimer dependent epitopes that are conserved between ZIKV and DENVs. mAb 1M7 binds to a highly conserved epitope near the fusion loop. DV4-141 is a DENV4 specific mAb used as a negative control. Antibody binding was evaluated on a ZIKV capture ELISA. Each mAb was tested in 3–4 independent experiments and the graph depicts representative data from one experiment. Within each experiment, the antibodies were tested in duplicate, and the binding signal was normalized to ZIKV EDIII targeting mAb ZKA-190. The graph displays the normalized mean OD and standard deviation (error bars). (C) Comparison of virus neutralization against ZIKV and rZIKV-D67N by a panel of ZIKV mAbs. While the integrity of the epitopes targeted by ZIKV mAbs is maintained, G9E failed to neutralize r-ZIKV-D67N. Each graph depicts representative results from 1 of two independent experiments, except for MAb 1M7 and EDE-C8, which were tested once. Within each experiment, the antibodies were tested at different concentrations in duplicate, and the graphs display mean % neutralization and standard deviation (error bars). (D) Convalescent sera from Zika patients show reduced neutralization against rZIKV-D67N. Convalescent sera from 10 individuals who experienced primary ZIKV were tested for neutralization WT ZIKV and rZIKV-D67N. Subjects 1–4 were tested in 2–3 independent experiments. Because of the small volumes of sera available, subjects 5–10 were tested just once. Paired neutralization titers against WT and D67N ZIKVs were compared using the Wilcoxon matched pairs test.