Dengue vaccine trials have revealed deficits in our understanding of the mechanisms of protective immunity, demonstrating a need to measure epitope-specific antibody responses against each DENV serotype. HmAb 5J7 binds to a complex, 3-monomer spanning quaternary epitope in the DENV3 envelope (E) protein, but it is unclear whether all interactions are needed for neutralization. Structure guided design and reverse genetics were used to sequentially transplant larger portions of the DENV3-specific 5J7 mAb epitope into dengue virus serotype 4 (DENV4). We observed complete binding and neutralization only when the entire 3 monomer spanning epitope was transplanted into DENV4, providing empirical proof that cooperative monomer-hmAb 5J7 interactions maximize activity. The rDENV4/3 virus containing the most expanded 5J7 epitope was also significantly more sensitive than WT DENV4 to neutralization by DENV3 primary immune sera. We conclude that the hinge-spanning region of the 5J7 quaternary epitope is a target for serotype-specific neutralizing antibodies after DENV3 infection.
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