A SpyCatcher-assembled mannosylated nanovaccine elicits potent and cross-protective immunity against monkeypox virus

Monkeypox (mpox), caused by the monkeypox virus (MPXV), is a zoonotic disease of global public health concern. Current live-attenuated vaccines suffer from safety concerns and unsatisfactory protection effectiveness. To address these limitations, a protein-based nanovaccine (Ag-M-Spy) was developed to deal with mpox for its high effectiveness and favorable safety profile, incorporating three critical MPXV-derived antigens (A29L, A35R, M1R). Mannose acted as the adjuvant via mannose receptor-mediated endocytosis. Ag-M-Spy was constructed by conjugation of mannose-functionalized antigens with SpyCatcher nanoparticles via the SpyTag-SpyCatcher system. BALB/c mice were immunized intramuscularly on days 0, 14, and 28. Ag-M-Spy evoked a strong antigen-specific antibody response. In addition, Ag-M-Spy induced high levels of splenic Th1- and Th2-type cytokines. Moreover, Ag-M-Spy stimulated robust antigen-specific CD4⁺ and CD8⁺ T cell response, generated durable memory cells, activated T and B cells, enhanced cytotoxic T lymphocyte activity, and promoted dendritic cell maturation. Ag-M-Spy did not render apparent liver, kidney, or heart toxicity to mice. Moreover, it conferred in vivo protective immunity against lethal ectromelia virus challenge in BALB/c mice. In summary, Ag-M-Spy elicited robust, durable humoral and cellular immunity with favorable safety, representing a promising vaccine candidate against life-threatening mpox.