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

Abstract

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 Å 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.

Prefusion structure of human cytomegalovirus glycoprotein B and structural basis for membrane fusion

“…The last antigenic domain, AD-2, is not resolved in either the prefusion or postfusion gB structure. It is part of the flexible N-terminal 85 residues, which contain the conserved site I (residues 68 to 77) linear epitope, which is the target of mAbs that require bivalency to neutralize (table S2) ( 33 ). The connection of AD-2 to the resolved structure of prefusion gB at T86 is adjacent to the exposed surface of domain IV at the base of the hollow between the tripod legs ( Fig.…”

“…CMV gB is a prominent target of antibodies elicited by infection. Although some neutralize CMV infectivity on both fibroblasts and epithelial cells, inhibiting post-attachment entry events, many antibodies that bind gB are non-neutralizing, and others require complement for their antiviral activity (32)(33)(34). Five gB antigenic domains (AD-1 to AD-5) have been identified.…”

Structural basis for HCMV Pentamer recognition by antibodies and neuropilin 2

“…Previous efforts to characterize the humoral immune response to asymptomatic HCMV infection yielded an extensive panel of neutralizing antibodies directed against gB, the HCMV Trimer, and the HCMV Pentamer 35, 43, 44 . To learn more about the mechanisms of neutralization of high-affinity, Pentamer-directed antibodies, we determined cryo-EM structures of four naturally elicited human antibodies in complex with the Pentamer (Fig.…”

Potent Bispecific Neutralizing Antibody Targeting Glycoprotein B and the gH/gL/pUL128/130/131 Complex of Human Cytomegalovirus

“…We aimed to build a bispecific antibody combining the broad cell-type coverage of the gB-specific MAb 3-25 and the high potency of the pentamer-specific MAb 2-18. We previously demonstrated that bivalency of MAb 3-25 is required for HCMV neutralization ( 33 ). Thus, we chose a tetravalent IgG-scFv as the bispecific antibody format that preserves bivalency of MAb 3-25.…”

“…Among them, one gB-specific antibody, MAb 3-25, and one pentamer-specific antibody, MAb 2-18, demonstrated potential for development as anti-HCMV therapeutics. The MAb 3-25 recognizes a totally conserved epitope on gB AD-2 and neutralizes a group of 14 HCMV strains in both ARPE-19 epithelial cells and MRC-5 fibroblast cells, with a 50% inhibitory concentration (IC 50 ) of 15 to 188.3 ng/ml ( 33 ). MAb 2-18 recognizes a conformational epitope on the pentamer and inhibits HCMV infection of epithelial cells at an IC 50 of 0.9 ng/ml, but it does not inhibit HCMV infection of fibroblast cells ( 32 , 34 ).…”