Fig. 3: Biochemical and antigenicity characterization of JEV and WNV CC_FLE sE mutants.

A Amino acid sequence alignment of the sE N terminus (N-term) and FLE of selected mosquito-born flaviviruses. Residues identical to ZIKV sE are indicated by dots. G5 and G102, mutated to cysteine (C) in the CC_FLE design to form a disulfide linkage, are highlighted in red. Abbreviations: DENV, Dengue Virus; WNV, West Nile Virus; JEV, Japanese Encephalitis Virus; YFV, Yellow Fever Virus. B Size exclusion chromatography profiles of JEV and WNV sE proteins; C Structural characterization of the JEV CC_FLE sE mutant. The overall dimer model is fitted into the Cryo-EM map, with two individual protomers colored in green and gold, respectively. Upper: top view with the interface between the FLE and the N-terminus highlighted in a magenta box. Lower: side view, with N154 glycans indicated; D Close-up view detailing the C5-C102 disulfide linkage (yellow) connecting two protomers (green and gold). E Occlusion of the FLE (green) by the surrounding DI (red) and DIII (blue) domains of the neighboring protomer in the JEV CC_FLE sE dimer, with the C5–C102 disulfide linkage shown in yellow. The superposed FLE loop from the JEV sE dimer (PDB: 5WSN) is shown in light gray. F ELISA binding EC₅₀ value heat map showing attenuated binding to FLE-specific mAbs for JEV CC_FLE sE compared to the WT. G Similar ELISA binding EC₅₀ value heat map for WNV CC_FLE sE compared to the WT. Each sample test was duplicated, and each assay was repeated at least two times. Source data are provided as a Source Data file.