The image processing and 3D reconstruction were performed with a single-particle approach using the EMAN291,92software package. role of prior CBL0137 immunity in JEV and DENV in shaping the breadth of humoral response and provide insights for future vaccination strategies in flavivirus-endemic CBL0137 countries. Subject terms:Viral infection, Molecular medicine, Antibodies An integrated epidemiological, immunological, and structural study suggests that the broadly neutralizing antibodies against Zika virus could CBL0137 be induced from dengue-infected individuals initially primed with Japanese encephalitis virus. == Introduction == Flaviviruses, the largest member of the familyFlaviviridae, present a global threat to public health1. Many of the mosquito-borne flaviviruses are established human pathogens, including the Japanese encephalitis virus (JEV), Yellow fever virus (YFV), West Nile virus (WNV), the four serotypes of dengue virus (DENV), and the re-emerged Zika virus (ZIKV)2. Co-circulation of multiple flaviviruses in the same geographic locations, combined with human global mobility and travel vaccine coverage for YFV and JEV, have increased the likelihood of exposure to multiple flaviviruses within a lifetime3,4. However, how pre-existing immunity will influence the antibody response upon subsequent infections or vaccination with heterologous flaviviruses remains poorly understood. Flaviviruses are enveloped viruses containing a single-strand, positive-sense RNA with an 11-Kb genome and encapsidated by three structural proteins, namely the capsid (C), pre-membrane/membrane (prM/M), and envelope (E) proteins. The immune response to flaviviral infection mainly targets the E proteins and is known to be dominated by transient and highly cross-reactive (CR) antibodies during the acute and early convalescent phase. In the late convalescent phase, the immune response generates type-specific neutralizing antibodies but lacks durable and high cross-neutralizing antibodies against viruses from different serotypes or serocomplexes57. The envelope dimer epitope (EDE) human monoclonal antibodies (huMAbs), which KL-1 broadly neutralize the four dengue virus serotypes by recognizing the quaternary epitopes on the virion surface, have been mostly isolated from individuals exposed to secondary dengue infections8. Recurring huMAbs CR to DENV and ZIKV have also been reported9,10from the regions where both viruses co-circulated. However, the antibody profiles of individuals residing in areas where JEV and DENV co-circulated have not been investigated in detail. Here, we used a unique Taiwan cohort to gain insight into how DENV infection in individuals with pre-existing JEV immunity shapes neutralizing antibody responses against multiple flaviviruses through interdisciplinary and complementary approaches, including epidemiology, immunology, structural biology, and animal studies. The results will guide future vaccination strategies, leading to the generation of broadly neutralizing antibodies against different flavivirus serocomplexes. == Results == == High ZIKV neutralization titers among dengue-infected individuals with pre-existing JEV immunity == Based on the nationwide surveillance system established by the Taiwan Centers for Disease Control, only imported ZIKV cases were detected without local transmission1113. In contrast, JEV is endemic on the island; thus, a nationwide JEV pediatric vaccination program has been implemented since 196814,15. Periodic DENV epidemics have also occurred in Taiwan since 1981, with the largest outbreaks recorded in 20142015 in the southern part of the island16,17. To investigate the antibody profile of DENV-infected patients to different flaviviruses, including ZIKV, plasma of healthy volunteers and DENV-infected febrile (DF) patients were tested for the presence of DENV, JEV, and ZIKV-neutralizing antibodies using 90% foci reduction as the cut-off (FRNT90) (Fig.1a). Expectedly, DF-confirmed samples had significantly elevated DENV FRNT90 titers than the healthy donors. Only four healthy individuals showed DENV FRNT90 titers between 50 and 80, with one donor (1413) showing DENV-1, -2, and -3 neutralizing titers, suggesting previous dengue infection or potential asymptomatic dengue infection given the overlapping residential area of the donors in southern Taiwan. Significantly elevated JEV FRNT90 titers among the DF patients were also observed compared to the healthy controls (Fig.1a), although thirteen healthy donors from the older age group showed varied JEV-neutralizing titers (Supplementary Fig.1a). Surprisingly, the anti-ZIKV-neutralizing titers were substantially higher among DF patients than the healthy individuals (Fig.1a). == Fig. 1. Dengue-immune donors with prior.