Human antibody combinations determined by epitope mapping of orthopoxvirus A33/A35 confer therapeutic protection in mice

Human infections with mpox virus (MPXV) since 2022 have resulted in more than 188,000 cases reported globally as of June 2026, highlighting the urgent need for effective therapeutics. Here, we recruited smallpox vaccinees and donors with incidental vaccinia virus (VACV) exposure and isolated a panel of human anti-A33/A35 monoclonal antibodies (mAbs) from the latter using recombinant MPXV A35 as a cross-reactive bait. These mAbs recognized four distinct nonoverlapping epitopes (epitopes 1 to 4) and one composite epitope (epitope 3 + 4) on MPXV A35 and exhibited cross-reactivity against multiple orthopoxviruses (OPXVs). Cryo–electron microscopy structural analysis revealed that the high-affinity epitope 3 + 4 mAb HD15 engages an epitope at the A35 dimer interface that is conserved across OPXVs. Combinations of mAbs targeting nonoverlapping epitopes mediated synergistic, Fc-dependent neutralization in vitro; conferred therapeutic protection against lethal VACV challenge; and reduced viral burden and disease severity after MPXV challenge in mice. Together, these findings establish a single-antigen, multiepitope strategy for the development of broadly protective therapeutics against OPXVs.