Structural basis for broad orthopoxvirus neutralization by two antibodies targeting distinct L1R epitopes

The 2022 mpox outbreak highlights the persistent threat posed by the mpox virus (MPXV) and related orthopoxviruses (OPXVs). Yet, approved antibody-based therapeutics for OPXVs are lacking. Here, we present two broadly neutralizing monoclonal antibodies (mAbs), G4 and B9, that target the MPXV M1R protein, homologous to L1R in other OPXVs. Both mAbs demonstrate cross-reactive binding and robust neutralization against multiple pathogenic OPXVs. Notably, G4 exhibits exceptional potency, neutralizing mature virions of VACV with an IC₅₀ of 37.93 pM, whereas B9 has an IC₅₀ of 41.13 nM. Cryo-electron microscopy analysis reveals that G4 and B9 target distinct epitopes on M1R. G4 recognizes a conventional conformational epitope but overcomes neutralization escape associated with the Asp35 residue, whereas B9 binds to an epitope on the lateral β-sheets of M1R. Our findings provide two promising candidates with complementary neutralization profiles for the development of broad-spectrum prophylactic and therapeutic interventions against OPXV infections.