# Isolation and characterization of broadly-neutralizing anti-HCMV-gB antibodies from human donors using a prefusion-stabilized HCMV gB variant

## TL;DR

Researchers isolated new antibodies that can neutralize HCMV, a dangerous virus for immunocompromised people and newborns, using a stabilized version of a key viral protein.

## Contribution

A prefusion-stabilized HCMV gB variant was used to isolate twelve new human monoclonal antibodies, including six broadly neutralizing ones.

## Key findings

- Six of the isolated monoclonal antibodies were broadly neutralizing against multiple HCMV strains.
- Most neutralizing antibodies preferentially bound the intermediate form of gB rather than the prefusion form.
- Three distinct neutralization mechanisms were identified based on antibody conformational specificity and epitope location.

## Abstract

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.

Human cytomegalovirus (HCMV) infects the majority of people worldwide but poses the greatest risk to immunocompromised individuals. There is no cure, vaccine, or effective treatment option for HCMV, but human polyclonal antibodies have been tested with limited success. To infect cells, HCMV uses the viral membrane fusogen glycoprotein B (gB), which exists in metastable prefusion, stable postfusion, and highly dynamic intermediate conformations. We engineered a soluble, prefusion-stabilized HCMV gB ectodomain construct and, in the process, also designed the first intermediate HCMV gB construct. We used our prefusion gB construct to isolate twelve new human monoclonal antibodies (mAbs), six of which were broadly neutralizing against multiple HCMV strains. Interestingly, only one mAb preferentially bound the prefusion form while most favored the intermediate form, and this did not correlate with their neutralization potency. We further characterized these mAbs using a variety of biochemical, cell-based, and low-resolution structural approaches and identified their putative neutralization mechanisms. Using our methodology, we also identified neutralization mechanisms for two previously published nAbs. Our constructs introduce new tools into the field of HCMV, and, in the long term, the nAbs isolated in this study could aid in the development of robust HCMV countermeasures.