A bivalent Mpox nanoparticle vaccine induces robust immune response and provides long-lasting protection against vaccinia virus challenge.

The 2022 and 2024 monkeypox (mpox) outbreak highlighted the urgent need for effective, durable, and safe vaccines. In addition to the traditional smallpox vaccines that could bring cross-protection against mpox, mRNA and protein-subunit mpox vaccines were extensively studied after the outbreak of mpox. In this study, we engineered monkeypox virus (MPXV) nanoparticle vaccines by conjugating the M1R and A35R, two well-characterized protective antigens to the mi3 nanoparticle using the SpyTag-SpyCatcher system, generating mi3-M1R and mi3-A35R constructs. An equimolar mixture of mi3-M1R and mi3-A35R formed a bivalent MPXV vaccine candidate, termed mi3-AM. When administered intraperitoneally with the Mn adjuvant, the mi3-AM vaccine induced robust humoral and antigen-specific cellular immune responses. Notably, the mi3-AM vaccine provided long-lasting protection against a lethal challenge with vaccinia virus Western Reserve strain (VACV-WR) in mice. With ongoing mpox outbreaks and the limitations of current vaccines, our candidate represents a promising, deployable solution with potential to bridge existing gaps in global orthopoxvirus prevention.