Clinically relevant RSV anti-F antibodies exhibit comparable inhibition of syncytium formation despite marked differences in neutralization potency

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants, immunocompromised individuals, and older adults. A hallmark of RSV infection is the formation of multinucleated syncytia through fusion between infected and adjacent uninfected cells. This process is mediated by the RSV fusion protein (RSV F) expressed on infected cells, which interacts with receptors on neighboring cells to drive membrane fusion. Syncytium formation may facilitate cell-to-cell spread while reducing virion exposure to extracellular neutralizing antibodies and immune cells. Clinically used monoclonal antibodies (mAbs) target RSV F and are routinely evaluated using conventional virus neutralization assays. However, their ability to inhibit syncytium formation remains poorly characterized because these assays primarily measure virus-to-cell entry rather than cell-to-cell fusion. Here, we present a quantitative flow cytometry-based assay to measure RSV-induced syncytium formation during infection. Using this assay, we evaluated clinically relevant anti-F mAbs and compared their potency in syncytium inhibition and conventional virus neutralization assays in HEp-2 and A549 cells. Palivizumab, nirsevimab, and clesrovimab inhibited syncytium formation in a dose-dependent manner, with only modest differences in potency. In contrast, nirsevimab and clesrovimab exhibited substantially greater virus neutralization potency than palivizumab. These findings indicate that inhibition of RSV-induced cell-to-cell fusion represents a distinct functional property of anti-F antibodies that is not fully captured by conventional neutralization assays. Our assay provides a quantitative approach to assess syncytium inhibition and highlights the value of complementary functional readouts for evaluating antibodies targeting RSV and other syncytium-forming viruses.IMPORTANCERespiratory syncytial virus (RSV) is a leading cause of respiratory disease in infants, older adults, and immunocompromised individuals. A hallmark of RSV infection is syncytium formation, although its contribution to viral spread and disease pathogenesis remains incompletely understood. Increased fusogenicity has been associated with disease severity, and syncytium formation may promote cell-to-cell spread while limiting virion exposure to extracellular neutralizing antibodies. Although RSV fusion (F)-specific monoclonal antibodies (mAbs) are increasingly used for disease prevention, their ability to inhibit syncytium formation remains unexplored, as conventional neutralization assays primarily measure virus-to-cell entry. Here, we describe a quantitative flow cytometry-based assay to measure RSV-induced syncytium formation and evaluate inhibition by clinically relevant anti-F mAbs. Our results indicate that syncytium inhibition represents a distinct functional property of anti-F antibodies that is not fully captured by conventional neutralization assays, highlighting the value of complementary functional readouts for evaluating antibodies targeting RSV and other syncytium-forming viruses.