Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner, however most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies using viral and recombinant antigens mainly led to the isolation of dominant non-neutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells using a primary ... More
Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner, however most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies using viral and recombinant antigens mainly led to the isolation of dominant non-neutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype cross-neutralizing anti-DENV mAbs, including ultra-potent inhibitory antibodies with neutralizing activity concentrations less than 10 ng/mL. We isolated serotype-specific neutralizing Abs that target diverse regions of the E protein including epitopes present only on the intact fully-assembled viral particle. We also isolated a number of serotype cross-neutralizing mAbs, most of which recognized a region in the E protein domain I/II containing the fusion loop. These data provide insights into targets of the protective Ab-mediated immune response to natural DENV infection, which will prove valuable in the design and testing of new experimental DENV vaccines.
