MOST-110-2313-B-005-041). a lifetime is not uncommon; however, how sequential exposures to different flaviviruses shape the cross-reactive humoral response against an antigen from a different serocomplex has yet to be explored. Here, we statement that dengue-infected individuals in the beginning primed with the Japanese encephalitis computer virus (JEV) showed broad, highly neutralizing potencies against Zika computer virus (ZIKV). We also recognized a rare class of ZIKV-cross-reactive human monoclonal Clarithromycin antibodies with increased somatic hypermutation and broad neutralization Clarithromycin against multiple flaviviruses. One huMAb, K8b, binds quaternary epitopes with heavy and light chains separately interacting with overlapping envelope protein dimer models spanning domains I, II, and III through cryo-electron microscopy and structure-based mutagenesis. JEV virus-like particle immunization in mice further confirmed that such cross-reactive antibodies, mainly IgG3 isotype, can be induced and proliferate through heterologous dengue computer virus (DENV) serotype 2 virus-like particle activation. Our findings spotlight the role of prior immunity in JEV and DENV in shaping the breadth of humoral response and provide insights for future vaccination strategies in flavivirus-endemic countries. Clarithromycin Subject terms: Viral contamination, Molecular medicine, Antibodies An integrated epidemiological, immunological, and structural study suggests that the broadly neutralizing antibodies against Zika computer virus could be induced from dengue-infected individuals in the beginning primed with Japanese encephalitis computer virus. Introduction Flaviviruses, the largest member of the family factor (?2)n.a.?Model composition ?Non-hydrogen atoms ?Protein residues ?Ligands n.a.factors (?2) ?Protein ?Ligand n.a.RMS deviations ?Bond lengths (?) ?Bond angles () n.a.Validation ?MolProbity score ?Clashscore ?Poor rotamers (%) n.a.Ramachandran plot TP15 ?Favored (%) ?Allowed (%) ?Disallowed (%) n.a. Open in a separate windows Refinement was performed by EMAN2 software. The solved cryo-EM structure experienced suboptimal resolution (16.0C19.9??); thus, the requested detailed structural parameters could not be provided with accuracy. answers were not available. The resolution of the K8b-IgG1 immune complex was further enhanced to 12?? using MODELLER to map the potential interacting residues Clarithromycin between the E proteins on an mD2VLP and the heavy or light chain of K8b-IgG1 (Fig.?4e, f). Based on the predicted interacting residues around the E protein of DENV-2, seven residues spanning across domains I, II, and III were mutated into alanine on ZIKV VLP based on the criteria described in Methods (observe SDM and epitope mapping experiments?section), such as R73, T76, and D87 around the bc loop of domain name I, R193 on domain name I and II linker region, T231 around the hi loop of domain name II, G302 on domain name I and III linker region, and T366 on DE loop of domain name III (Fig.?4g and Supplementary Fig.?6a, b). The acknowledgement of K8b-IgG1 to D87A, R193A, T231A, G302A, and T366A mutant antigens were significantly reduced by as much as 50% relative to ZIKV WT VLP (Fig.?4h). In contrast, K8b and K5 binding to residue 101-mutated VLPs were unaffected (Supplementary Fig.?6dCg). The overall binding reactivities of K5-IgG1 to most mutant ZIKV VLPs were much like K8b-IgG1 except for two residues, ZIKV R193A and ZIKV T231A. Mutation of R193 and T231 showed abrogation of K5 binding by 70% (Fig.?4i), indicating substantial functional contribution of both residues to K5 and ZIKV interactions. To validate if antibody geometry could impact the binding and avidity to putative interdomain or inter-dimer epitopes on different E dimers, the full-length bivalent IgG1 of K8b (K8b-IgG1) was designed and expressed as bivalent K8b-F(ab)2 and monovalent K8b-F(ab) (Fig.?5aCd). Parallel binding and neutralization experiments were performed on K8b-IgG1, K8b-F(ab)2, and monovalent K8b-F(ab) against ZIKV, JEV, and the four DENV serotypes. Bivalent K8b-IgG1 and K8b-F(ab)2 have comparable neutralization against DENV-1, DENV-2, DENV-4, ZIKV, and JEV (FRNT50: 7-17?nM), but K8b-F(ab)2 showed a slight improvement in neutralization against DENV-3 (FRNT50: 26-50?nM) (Fig.?5e). Although both VLP and virions form E-dimer on the surface of the icosahedral sphere, the observed VLP binding kinetics for K8b-IgG1 and K8b-F(ab)2 were consistent with neutralization for DENV-2 and ZIKV only but not for JEV, DENV-1, DENV-3, and DENV-4 VLPs probably due to the difference in geometry between VLP.