An experimentally validated structure-based computational framework for humanisation of anti-orthopoxvirus antibodies

BACKGROUND: The re-emergence of orthopoxviruses, most notably mpox virus (MPXV), poses a growing global public health threat. Well-characterised murine anti-orthopoxvirus antibodies are clinically limited by anti-mouse antibody responses, while traditional sequence-based humanisation often impairs antigen-binding activity. METHODS: We developed an experimentally validated structure-guided computational humanisation framework prioritising 3D architectural congruence over sequence identity, integrating Foldseek-based structural alignment and interface-residue constraints. We applied this framework to humanise two murine anti-orthopoxvirus antibodies (7D11, A27D7), with comprehensive in vitro and in vivo validation. FINDINGS: Structural superimposition confirmed high conformational conservation between the humanised variants (POX1.1 and POX2.1) and their parental mAbs, with root mean square deviation (RMSD) values below 0.6 Å for all variable domains. Both humanised variants retained full epitope specificity with natural humanness profiles. POX1.1 showed enhanced neutralisation potency against vaccinia virus (VACV) and MPXV, compared with the parental 7D11. POX2.1 preserved the broad cross-reactive binding and the extracellular enveloped virion neutralising activity of the parental A27D7. In the lethal VACV mouse model, both monotherapies conferred significant prophylactic and therapeutic protection, reducing pulmonary viral loads and improving survival. The dual-targeting combination of POX1.1 and POX2.1 achieved markedly improved in vivo efficacy compared with individual antibodies, delivering 100% survival even when administered 2 days post-challenge. In the MPXV CAST/EiJ mouse model, the combination significantly reduced splenomegaly and MPXV DNA loads in plasma, spleen and lung tissues, effectively suppressing systemic viral dissemination. INTERPRETATION: These findings establish that the structure-centric workflow enables efficient humanisation of well-characterised murine anti-orthopoxvirus antibodies, providing a validated framework to support the development of countermeasures for orthopoxvirus pandemic. FUNDING: This work was supported by the National Natural Science Foundation of China, the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, the Scientific Research Innovation Capability Support Project for Young Faculty and the National Science and Technology Major Project.