The Unexpected Role of TNFRSF14 Signaling in Promoting Antibody-Dependent Cell-Mediated Cytotoxicity - Vaccine development has been dominated by efforts to elicit neutralizing responses, but this approach has met with limited success for several major pathogens including herpes simplex virus (HSV). We adopted a paradigm-shifting strategy and engineered a single-cycle HSV strain deleted in viral glycoprotein D, designated ∆gD-2. Glycoprotein D, which is required for viral entry and spread, was the primary target of neutralizing antibody responses elicited by the prior failed vaccine efforts. Immunization with this novel ∆gD-2 vaccine strain completely protected mice from lethal challenges with clinical isolates of HSV-1 and HSV-2. Passive transfer studies demonstrated that the protection was mediated by antibodies that had little neutralizing activity but instead mediated antibody-dependent cellular cytotoxicity (ADCC). Monoclonal antibodies from the vaccinated mice were subsequently isolated and characterized. The most potent of these, BMPC-23, had no neutralizing activity but activated mouse FcγRIV, a biomarker of ADCC. A single dose of BMPC-23 administered 24 hours before or after viral challenge provided significant protection when configured as mouse IgG2c and protected mice expressing human FcγRIII when engineered as a human IgG1. Using cryo-electron microscopy, we demonstrated that the epitope recognized by this protective monoclonal antibody resides within domain IV of glycoprotein B. HSV can escape neutralization by spreading across intercellular bridges but cannot escape ADCC. Glycoprotein D binds TNFRSF14, an immunomodulatory protein that functions in signal transduction, and completes with its physiological ligands, resulting in altered signaling. Thus, we hypothesized that HSV utilizes gD- TNFRSF14 engagement as an immune evasion strategy to block ADCC responses. Consistent with this hypothesis, we found that BMPC-23 failed to protect TNFRSF14 knockout mice and similarly, passive transfer of immune serum pooled from ∆gD-2 vaccinated mice (replete with ADCC-mediating activity) protected wild-type but not the knockout mice. The importance of TNFRSF14 in mediating ADCC is generalizable as we obtained similar results using human TNFRSF14 knockout effector cells in ADCC assays with other antigen-antibody complexes such as those formed by rituximab with B cell-presented CD20. The identification of a central role for TNFRSF14 in promoting ADCC provides a unique opportunity to define the signaling pathways and biochemical and structural determinants that mediate ADCC. We will take advantage of human knockout cells, a library of defined TNFRSF14 muteins, structural characterization and monoclonal antibodies that target glycoprotein B but exhibit variable ADCC activity to identify the pathways and the monoclonal antibody characteristics that promote FcγR activation. The results of these studies will provide an enhanced foundation and new strategies for future vaccine and monoclonal antibody design.
