Abstract: The ongoing global spread of monkeypox virus (MPXV) poses a persistent public health threat, underscoring the urgent need for novel vaccines that are effective, safe, and highly specific. In this study, we designed and constructed a multivalent subunit vaccine based on the self-assembling mi3 nanoparticle platform, targeting four key MPXV antigens (A35R, B6R, E8L, and M1R) for synergistic immunization. Following codon optimization, the antigen proteins were recombinantly expressed in HEK-293F cells and purified. Using the SpyTag-SpyCatcher covalent conjugation system, these antigens were efficiently and site-specifically displayed on the surface of mi3 nanoparticles, resulting in structurally stable nanovaccines (NP group). A soluble protein mixture group (SU group) was included as a control. Systemic humoral and cellular immune responses were evaluated in BALB/c mouse models. The prepared nanoparticle vaccines showed uniform size distribution and structural integrity. Immunization experiments revealed that the NP group induced significantly higher levels of antigen-specific IgG antibodies compared to the SU group (P<0.01) and elicited a potent Th1/Th2-mixed immune response, as reflected by markedly elevated secretion of multiple cytokines including IL-2, IL-4, IL-5, and IL-6. Transcriptomic analysis further suggested that the nanovaccine may enhance immune responses through modulation of key signaling pathways such as IL-17. In summary, we successfully developed a mi3 nanoparticle-based multivalent subunit vaccine candidate against MPXV, which demonstrated strong immunogenicity in a mouse model, providing an important experimental foundation and a technical platform for the development of next-generation monkeypox vaccines.
Keywords: monkeypox virus; subunit vaccine; nanoparticle; mi3; immunogenicity
（Acta Laser Biology Sinica, 2026, 35(3): 222-232）