Engineered attenuated probiotic Escherichia coli oral vaccine provides effective protection against monkeypox virus by eliciting specific systemic and mucosal immunity.

Currently available mpox vaccines (actually smallpox vaccines) face numerous challenges including supply shortages, limited specificity, and adverse reactions. Given that monkeypox virus (MPXV) transmits through respiratory (Clade I) and intimate contact (Clade IIb, primarily sexual behavior), developing vaccines capable of eliciting mucosal immunity is crucial for effective mpox prevention. This study developed an oral mpox vaccine, ABMΔEcN, based on attenuated Escherichia coli Nissle 1917 (ΔEcN). This engineered probiotic stably expresses three MPXV antigens (A9R/B6R/M1R) derived from extracellular enveloped and intracellular mature viruses, while tolerating stomach acid and intestinal fluids. The combination of MPXV antigens with ΔEcN's natural adjuvant properties synergistically enhances both innate and adaptive immune responses following oral administration in mice. This regimen elicits robust humoral and cellular immunity at systemic and mucosal sites with immune memory effect. In VACV and MPXV challenge models, ABMΔEcN conferred potent protection across mucosal sites by improving survival, reducing viral loads and mitigating tissue inflammatory damage. Moreover, ABMΔEcN was found to improve gut microbiota dysbiosis in virus-infected mice, indicating an interaction between mucosal immunity and microbiota homeostasis. This study proposes a novel oral vaccine strategy that enhances immune accessibility through a needle-free administration while achieving comprehensive prevention against mpox.