IC50 values for the nAbs tested in this study.

Human cytomegalovirus (HCMV) poses a significant risk to immunocompromised individuals and is the leading cause of congenital birth defects worldwide. There is no cure or robust treatment options, although neutralizing antibodies (nAbs) derived from patient sera are being explored as prophylactics with limited success. Glycoprotein B (gB) is a viral membrane fusogen and a major target of the anti-HCMV humoral response in humans. Here, we engineered a soluble, prefusion-stabilized HCMV gB ectodomain variant and used it to isolate twelve new human monoclonal antibodies (mAbs). Seven of these mAbs strongly neutralized at least one strain of HCMV in vitro, whereas six mAbs neutralized both lab-adapted and minimally passaged clinical strains (were broadly neutralizing, bnAbs). All nAbs bound different epitopes within antigenic regions AD-4 or AD-5, and most targeted new sites. Despite being isolated using prefusion-stabilized HCMV gB variant, nAbs varied in their conformational specificity. Only one nAb preferentially bound the prefusion form, and most preferentially bound the intermediate form. The seven nAbs were separated into three classes based on their putative neutralization mechanisms, which were deduced from their conformational specificity, reactivity with gB on the cell surface, and epitope location. Our stabilized prefusion-gB construct provides a tool for isolating potent new nAbs, including prefusion-specific ones, and studying HCMV immunogenicity. Long term, these potent nAbs that arose during natural infections could be developed into potent prophylactics and therapeutics against HCMV diseases.