A neutralizing monoclonal antibody targeting species B adenoviruses largely mimics the DSG2 receptor engagement mechanism

Species B human adenoviruses (B-HAdVs), including HAdV7, HAdV3, and the re-emerging HAdV55, cause severe respiratory disease and engage desmoglein-2 (DSG2) through their fiber knob. Here, we report the identification and structural characterization of two monoclonal antibodies, 5C5 and 6G7, raised against the HAdV7 fiber knob. Both antibodies neutralize HAdV7 and display cross-reactivity toward other B-HAdVs, with nanomolar affinities. Notably, 5C5 recognizes HAdV3 despite limited sequence conservation and exhibits greater neutralization potency than 6G7. Cryo-electron microscopy structures at 1.8 Å and 2.0 Å resolution for 6G7 and 5C5 respectively, reveal distinct binding modes: 6G7 engages the trimeric fiber knob at a 3:3 antibody: knob ratio, whereas 5C5 binds the apex at a 1:3 ratio. The 5C5 epitope overlaps with key DSG2-interacting residues, including a conserved pocket among B-HAdVs, providing a structural basis for its enhanced neutralization through receptor-binding blockade. These findings establish the fiber knob apex as a vulnerable site for antibody-mediated neutralization and identify 5C5 as a candidate targeting pathogenic species B adenoviruses. Vassal-Stermann et al. identified two monoclonal antibodies that neutralize HAdV7 and cross-react with pathogenic species B adenoviruses. Cryo-EM reveals that the potent antibody 5C5 blocks the conserved DSG2 receptor-binding site at the fiber knob apex, defining a vulnerable target for broad neutralization.