Recognition of a highly conserved glycoprotein B epitope by a bivalent antibody neutralizing HCMV at a post-attachment step

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 angstrom crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody. Author summary HCMV infection is usually asymptomatic in healthy individuals. However, life-threatening diseases frequently accompany HCMV infection in individuals with under-developed or compromised immune systems. Glycoprotein B antigenic domain 2 (AD-2) is a major target for HCMV-neutralizing antibodies that potentially provide immune protection. We report the structure-based study of gB recognition by a potent neutralizing antibody named 3-25 that binds a highly conserved epitope on AD-2. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading. Furthermore, bivalency of 3-25 is required for viral neutralization but not for binding. Our findings advance understanding of gB antibody-mediated HCMV neutralization and facilitate development of gB-targeted vaccines and antibody drugs against HCMV infection.