MPXV protein scaffold cum antigen-based macromolecular multi-antigen displaying next-generation vaccine, providing coordinated humoral and cellular response.

The development of next-generation vaccines against emerging orthopoxviruses such as MPXV requires defined macromolecular platforms capable of presenting antigens from both viral forms, intracellular mature virus (IMV) and extracellular enveloped virus (EEV), to elicit coordinated humoral and cellular immune responses. We engineered a MPXV protein scaffold to enable structure-guided antigen display, addressing limitations of conventional multi-antigen co-immunization challenges, with the feasibility to express and accommodate multiple antigens. Utilizing the engineered protein scaffold, we describe the generation of a bivalent protein subunit-based vaccine candidate (BSP; Bivalent Scaffold Polyprotein expressing M1 and A35) with the dual targeting feature of humoral and cellular/cytotoxic T cell responses. The soluble BSP upon immunization in mice generates high-titer anti-MPXV antigen-specific humoral and balanced CD8+ and CD4+ Th1-type cellular responses, where the scaffold protein primarily promotes CTL responses. The protein scaffold, which is otherwise known to be essential for IEV egress and is conserved among orthopoxviruses infecting human, is characterized as an antigen, generating anti-MPXV humoral, cytotoxic T cell, and anti- VACV neutralizing responses. The alum-adjuvanted BSP provides protection against lethal respiratory infections challenged with neurotropic VACV adapted to mice, resulting in complete clearance of the virus from the brain. The modularity demonstrated in this study suggests a flexible, structure-guided platform capable of accommodating rapid antigen substitution for future higher-order multivalent orthopoxvirus vaccine development.