Fusion Loop Modified and Mature Dengue Virus Elicits Protective Serum with Minimal Antibody Dependent Enhancement

A balanced and safe vaccine against all four-dengue virus (DENV) serotypes is urgently needed. Although the currently licensed DENV vaccines demonstrate efficacy, they also raise concern about vaccine-associated disease enhancement, particularly in DENV-naive individuals. The conserved, immunodominant fusion loop (FL) epitope is the target of cross-reactive, weakly neutralizing antibodies (Abs) which are associated with antibody-dependent enhancement (ADE). Previously, we developed D2-FLM, a highly mature DENV2 strain containing modification in the FL epitope making it unrecognizable to FL-Abs. Here, we grafted the FLM modification to another serotype and adapted them to replicate in Vero cells for live-attenuated vaccine (LAV) manufacturing while retaining favorable antigenic profiles, generating two new strains: D2-vFLM and D4-vFLM. Deep sequencing revealed mutations at the junction of envelope domains I and II (EDI and EDII) that appeared during adaptation of the engineered FL in mammalian cells. Importantly, both D2-vFLM and D4-vFLM showed no evidence of ADE in the presence of FL-targeting Abs. Sera from D2-vFLM immunized mice displayed strong homotypic and reduced heterotypic neutralization compared to wild-type viruses, with minimal to no ADE potential in vitro. Moreover, D2-vFLM immunization completely protected AG129 mice from lethal challenge with mouse-adapted D220. Collectively, these findings demonstrate that the FLM modification platform is transferable across serotypes and yields strains with favorable immunogenicity and reduced ADE risk. Our FLM approach provides a promising path toward the development of a safer tetravalent DENV LAV.