Structural and functional characterization of polyclonal antibody responses reveals shared and distinct antigenic features of RSV and hMPV fusion proteins

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), members of the Pneumoviridae family, circulate widely and are leading causes of hospitalization for acute respiratory infections in infants and older adults. The viral fusion (F) protein in its prefusion (preF) conformation is the primary target of neutralizing antibodies, yet how naturally elicited circulating polyclonal antibodies are distributed across its antigenic landscape remains poorly understood. Here, we combined serological profiling, site-specific antibody competition assays and electron microscopy-based polyclonal epitope mapping (EMPEM) to characterize preF-directed immunity to RSV and hMPV in healthy adults. All donors exhibited detectable preF-binding antibodies that positively correlated with virus neutralization and broadly recognized multiple antigenic sites, consistent with immunodominance patterns predicted by a surface-centric computational model. Despite this overall similarity, the distribution of responses across individual antigenic sites differed between RSV and hMPV. EMPEM analyses revealed consistent targeting of the highly neutralization-sensitive preF apex (site O) in both viruses, alongside less abundant responses against equatorial and upper-middle epitopes (sites II, III, IV and V). We additionally observed antibody-bound destabilized preF, supporting a model in which antibody binding promotes transient opening of the preF trimer and may interfere with the conformational rearrangements required for membrane fusion. Together, these findings highlight apex-directed antibodies as a prominent feature of naturally acquired pneumovirus immunity in adults and reveal both shared and distinct patterns of antigenic recognition that may inform next-generation vaccine design and antibody-based prophylactics.