Subtype-specific differences in susceptibility to monoclonal antibodies and vaccines among contemporary RSV-A and RSV-B isolates

ABSTRACT Respiratory syncytial virus (RSV) continues to circulate at high levels despite the introduction of new monoclonal antibodies (mAbs) and vaccines targeting the pre-fusion F (pre-F) protein. We analyzed the viral genome sequences of 133 RSV clinical samples collected during the 2022–2023 and 2023–2024 seasons and selected representative isolates for phenotypic testing. We selected four RSV-A and four RSV-B replication-competent, sequence-verified stocks and then assessed them for replication kinetics in vitro and neutralization sensitivity with a panel of F-targeting mAbs and polyclonal sera using a rabbit vaccination model. Isolates retained sensitivity to all mAbs tested. We detected no mutations in mAb binding sites in the isolates tested. RSV-A isolates were slightly less susceptible to multiple mAbs than RSV-B isolates. Antigenic cartography revealed a separation of antibody responses by subtype: RSV-A isolates clustered together and aligned with lower neutralization by antibodies such as MPE8 and 101F, whereas RSV-B isolates formed a distinct cluster associated with higher mAb susceptibility. In a rabbit vaccination model, RSV-A-only sera efficiently neutralized all RSV-A isolates and the RSV-B1 reference strain but showed diminished activity against contemporary RSV-B isolates. RSV-B-only sera displayed balanced neutralization across both subtypes. Combined RSV-A + RSV-B immunization produced uniformly strong responses to all isolates, suggesting that multivalent exposure may overcome subtype-specific antibody polarization. Collectively, our results demonstrate consistent antigenic divergence of RSV subtypes and underscore the importance of using contemporary isolates, rather than legacy reference strains, to guide F-directed immunoprophylaxis and vaccine design. IMPORTANCE Respiratory syncytial virus (RSV) remains a major cause of lower respiratory tract infections. Although new monoclonal antibodies (mAbs) and vaccines have been approved in recent years, the impact of circulating genetic diversity on these therapeutics is incompletely understood. In this study, we demonstrate how recent RSV isolates respond to a panel of mAbs and whether sera from rabbits vaccinated with mRNA vaccines effectively neutralize these isolates. We observe that RSV-A isolates are slightly more resistant to neutralization by mAbs than RSV-B isolates and that a bivalent vaccine elicits broader neutralizing responses than the monovalent vaccine. This study provides insights into how viral diversity may influence antibody-mediated protection and suggests that recent RSV-B isolates may respond differently from the laboratory-adapted strains commonly used in research, an important consideration for the design of next-generation vaccines and antibody therapeutics.