Background: Numerous broadly reactive human monoclonal antibodies (mAbs) against the haemagglutinin (HA) of influenza A viruses have recognised conserved epitopes across HA subtypes or within subtypes. Most heterosubtypic mAbs target the HA stem, the receptor-binding site (RBS), or the trimeric interface. Although at least three H3-specific mAbs recognise epitopes outside these regions, the overall landscape of conserved H3-specific epitopes remains incompletely understood. This study aimed to identify and characterise conserved epitopes on H3-HA to inform the development of vaccines resilient to antigenic change.

Methods: We screened a panel of previously reported H3-HA-reactive human mAbs to identify mAbs recognising conserved epitopes. The candidate clone, 034-10040 4F02 (4F02), was evaluated for neutralising, haemagglutination inhibiting, and HA-mediated fusion-inhibitory, Fc receptor-mediated effector functions in vitro, and for protective efficacy in a lethal mouse challenge model. Cryo-electron microscopy was used to define the structural basis of 4F02 binding. Human sera were screened for antibodies targeting similar epitopes.

Findings: Clone 4F02 recognised the HA of human influenza A (H3N2) viruses that circulated across multiple decades. It neutralised multiple H3N2 viruses, exhibited weak haemagglutination inhibition, blocked HA-mediated fusion activity, activated Fc receptor-mediated signalling, and protected mice against lethal challenge. Cryo-electron microscopy revealed that 4F02 targets the base of the HA head at a head-stem interface spanning antigenic sites C, D, and E. Antibodies targeting similar epitopes were detected, albeit at a low level, in human sera.

Interpretation: Characterisation of the 4F02 epitope reveals a previously underappreciated site of vulnerability at the H3-HA head-stem interface. This finding expands our understanding of conserved epitopes and provides a target for the development of influenza vaccines resilient to antigenic change.