Abstract
Respiratory syncytial virus (RSV) is the leading cause of respiratory infection-related hospitalizations in children younger than 5 years. Neutralizing nanobody-based interventions represent a promising strategy against RSV. Here, we identify a novel nanobody (4-H1) targeting the RSV prefusion F (pre-F) protein, which demonstrates potent neutralization against both RSV A and B subtypes. Epitope characterization via binning assays, molecular docking, and mutational analyses revealed that 4-H1 interacts with a unique region within antigenic site Ø by engaging critical residues L207, K209, and the K65-N67-C69 cluster. To improve the in vivo efficacy and stability of the 4-H1 nanobody, we engineered a heterotrimeric bispecific nanobody (4-H1-anti-HSA-4-H1). This single-chain molecule contains two anti-RSV F nanobody domains and one anti-human serum albumin (HSA) domain, resulting in a trivalent molecule with dual specificity. This construct demonstrated sub-nanogram per milliliter (sub-ng/mL) neutralization potency against both RSV A and B subtypes, with prolonged in vivo half-life. Notably, intranasal administration of this construct before exposure conferred robust protection against RSV challenge in BALB/c mice. These results underscore the potential of 4-H1-anti-HSA-4-H1 as a respiratory-delivered prophylactic against RSV.IMPORTANCERSV is the leading cause of infant respiratory hospitalizations, highlighting the urgent need for effective prophylaxis. Here, we engineered a potent bispecific nanobody (4-H1-anti-HSA-4-H1) that exhibits exceptional neutralization against both RSV A and B subtypes with prolonged serum persistence. Prophylactic intranasal delivery of this construct conferred robust protection against RSV challenge in mice, indicating its potential as a respiratory-delivered prophylactic candidate against RSV.