LAUSR

The outbreak of clade II monkeypox virus (MPXV) and the additional outbreak in Central Africa of clade I virus from 2023 have attracted worldwide attention. The development of a scalable and effective vaccine against the ongoing epidemic of mpox is urgently needed. We previously constructed two bivalent MPXV mRNA vaccines, LBA (B6R‐A29L) and LAM (A35R‐M1R), and a quadrivalent mRNA vaccine, LBAAM (B6R‐A35R‐A29L‐M1R). These vaccines at a 20 µg dose could induce potential MPXV antigen‐specific immune responses and provide protection against lethal VACV challenge. Compared with the individual bivalent mRNA vaccines, the two quadrivalent vaccines LBAAM and LBA& LAM displayed superior protective effects. To characterize these vaccines further, we monitored long‐term immunity and protection as long as 28 weeks after initial immunization and optimized the immunization dosages to decrease the cost of production for future clinical use. Our results demonstrated that both the bivalent MPXV mRNA vaccine LAM (A35R‐M1R) and the two tetravalent vaccines LBAAM and LBA& LAM could elicit long‐lasting antigen‐specific IgG antibodies as well as neutralizing antibodies against VACV and MPXV. They all provided complete protection against VACV challenge until 28 weeks post prime immunization. Moreover, the immunogenicity and protective efficacy of the two tetravalent vaccines (LBAAM and LBA& LAM) are dose dependent, and even the low‐dose (1 µg) vaccine could provide sufficient protection against lethal VACV challenge. These results provide valuable clues for the further production of MPXV mRNA vaccines for use in humans.