Developing a safe and effective vaccine is crucial to control the recent worldwide outbreaks of mpox. Here, building upon our previously established lipid nanoparticle (LNP)-encapsulated circRNA vaccine platform, we constructed two bivalent mpox virus (MPXV) circRNA vaccines: cirBA encoding a B6R-A29L tandem antigen and cirAM encoding an A35R-M1R fusion antigen. Both bivalent MPXV circRNA vaccines, whether administered alone or in combination (designated cirMix), could induce robust and durable MPXV antigen-specific humoral and cellular immune responses in mice, conferring complete protection against lethal vaccinia virus Tian Tan strain (VTT) challenge. Moreover, even at low doses (2 µg for cirBA and cirAM, 4 µg for cirMix), all circRNA vaccines could provide 100% protection against lethal VTT challenge. Furthermore, at a higher dose (20 µg), all three vaccine groups induced potent long-term protective immunity lasting for at least 40 weeks, with cirBA achieving 100% protection, a level higher than that observed in the other groups. Collectively, our MPXV multivalent circRNA vaccines exhibit robust immunogenicity and represent promising candidates for further use in humans. The circRNA-based multivalent vaccine platform, capable of co-expressing multiple antigens, can be utilized for prevention of various infectious diseases.
