Mechanistic blockade of RSV infection by a pre-F targeting single-domain antibody-Fc fusion protein

ABSTRACT Respiratory syncytial virus (RSV) is a primary cause of acute lower respiratory tract infections in children and the elderly. Despite its clinical impact, effective prophylactic options for RSV remain limited. The prefusion conformation of the RSV fusion (F) protein is a critical target for neutralizing antibodies; however, the specific roles of prefusion versus postfusion states in triggering inflammatory responses have remained unclear. We report the development of Hu3F5, a novel prefusion F-specific single-domain antibody-Fc fusion protein. Hu3F5 demonstrates exceptional neutralizing activity in vitro (IC 50 : 0.58–9.15 ng/mL), potent in vivo efficacy, and a favorable safety profile in non-human primates. Crucially, our study reveals that the postfusion conformation, rather than the prefusion state, induces IL-6 elevation. Mechanistic analyses show that Hu3F5 neutralizes RSV by locking the F protein in its prefusion state, independent of Fc-mediated effector functions. This physical stabilization prevents the transition to the postfusion conformation, thereby blocking membrane fusion and subsequent inflammatory signaling, as well as the formation of cytopathic syncytia. By identifying the postfusion state as the driver of RSV-induced inflammation, this study positions Hu3F5 as a promising clinical candidate for prophylaxis. Furthermore, it provides new insights into RSV pathogenesis and the efficacy of “locking” mechanisms in viral neutralization.