Biological nanoparticles from Brucella abortus_{ΔeipB∆perΔwadC} elicit protective immunity against brucellosis

Brucellosis, caused by bacteria of the genus Brucella, is a globally prevalent zoonotic disease that poses serious risks to both animal and human health. Brucella abortus releases the biological nanoparticles (outer-membrane-vesicles, OMVs), which contribute to chronic infection by promoting host invasion and immune evasion. In this study, we performed a detailed characterization of OMVs derived from the wild-type B. abortus strain. These OMVs enhanced bacterial invasion and intracellular survival in macrophages ex vivo and increased pathogenicity in mice by modulating macrophage polarization in vivo. However, native OMVs showed low yield and limited immunogenicity. To overcome these limitations, we engineered a recombinant B. abortus strain Bru-M3, by deleting the eipB, per, and wadC genes (Brucella abortus_{∆eipB∆per∆wadC}), which remodels noncanonical lipopolysaccharide (LPS). The Bru-M3 strain produced OMVs (OMV_Bru-M3) at significantly higher yields and with enhanced immunogenicity. Immunization of mice via subcutaneous or intranasal routes with OMV_Bru-M3 conferred better effective protection against both low and high-dose intraperitoneal challenges with wild-type B. abortus. Vaccination with OMV_Bru-M3 also elicited stronger humoral responses and IFN-γ-producing Th1 CD4⁺ T cells, resulting in improved bacterial clearance, reduced tissue damage, and elevated inflammatory cytokine production compared to OMVs from wild-type B. abortus. These findings support the potential of OMV_Bru-M3 as a promising candidate for brucellosis vaccine development.