Inhalable engineered probiotic outer membrane vesicles co-expressing multiple mpox antigens induce potent specific systemic and mucosal immune responses.

Monkeypox virus (MPXV) is transmitted through respiratory mucosa and close contact, including sexual transmission, especially among men who have sex with men (MSM). Using bioengineering, we constructed an inhaled bacterial outer membrane vesicles (OMVs) vaccine, ABM@OMV, to block MPXV transmission. This nanovaccine consists of OMVs from ΔlpxM EcN strain that co-expressing three MPXV antigens (A9R, B6R and M1R). Our results show that OMVs vector enhances antigen uptake, mediates endosomal escape, and promotes APC maturation. Due to the adjuvant activity of OMVs and the synergistic effects of multi-antigen, ABM@OMV elicits robust systemic and mucosal immunity in both respiratory and vaginal, following intranasal immunization. This is demonstrated by the induction of three high-titer antigen-specific IgG and significant levels of SIgA. Moreover, ABM@OMV with A9R elicited stronger CD4+ and CD8+ T cell responses. The vaccine conferred complete protection against lethal vaccinia virus (VACV, replacing MPXV) challenge, and 100% survival. ABM@OMV is simple to produce, scalable, and easily standardized, offering a novel strategy for needle-free mucosal vaccines against emerging infectious diseases.