Structural and Functional Characterization of a Cross‐Reactive Dengue Virus Neutralizing Antibody That Recognizes a Cryptic Epitope

Understanding the molecular basis of the neutralizing antibody response to dengue virus (DENV) is an essential component in the design and development of effective vaccines and immunotherapeutics. We previously identified a number of cross‐reactive neutralizing antibodies isolated from mice immunized with domain III (DIII) of the dengue envelope (E) protein. Here we present the structure of one such antibody, 3E31, in complex with DIII derived from DENV‐4. Co‐crystallization revealed 3E31 recognises a conserved, cryptic epitope on DIII that is not available in any of the known conformations of E on the dengue virion. This finding was further validated by functional assays, which revealed that virion binding and neutralization is temperature‐sensitive. We observed that 3E31 inhibits E‐mediated membrane fusion, suggesting that the antibody is able to neutralize virus through binding an as yet uncharacterized intermediate conformation of DENV E and sterically block trimer formation and the fusion process. Finally, we assessed the capacity of 3E31 for antibody‐dependent enhancement (ADE) of infection, and showed that, unlike cross‐reactive fusion peptide‐specific antibodies, 3E31 does not promote infectious virus entry at sub‐neutralizing concentrations. Our results highlight the 3E31 epitope on the E protein DIII as a promising target for immunotherapeutics or vaccine design.