Sterilized protective immunity induced by DAM and DAM+ in mouse models for both VACV and MPXV.

The increasing prevalence of mpox calls for the development of safer and more accessible next-generation vaccines. Based on a structure-guided "two-in-one" immunogen design strategy, we have previously developed an innovative protein-based monkeypox virus (MPXV) vaccine, DAM (Double A35 and M1), which addresses the issues of imbalanced bioavailability associated with cocktail vaccines and elicits superior antiviral immunity with a safety profile. In this study, we iteratively designed two "four-in-one" chimeric immunogens, DAM+s, using four MPXV antigens, M1, A29, A35, and B6. Although DAM+s elicited broader immune responses against four antigens, no additional benefit in either in vitro neutralization or in vivo protection against poxviruses was detected compared to DAM. Notably, vaccination-related tissue damage was observed in the live virus vaccine group, whereas all protein-based vaccines showed better safety and protection against lethal vaccinia virus (VACV) challenge. Together, these further demonstrate that DAM, with a minimal protein subunit of two components, is a promising immunogen to be further clinically developed.