Abstract:

This study aims to extract Brucella melitensis biotype 3 outer membrane vesicles (OMVs), analyze their protein components, and prepare an OMV subunit vaccine to evaluate its immunogenicity. OMVs were extracted using differential centrifugation, and characterized by SDS-PAGE, transmission electron microscopy, and ZeTA nanoparticle size analysis. The protein components of the OMVs were identified using label-free liquid chromatography-mass spectrometry (LC-MS), followed by Gene Ontology (GO) clustering and KEGG pathway enrichment analysis. After immunizing mice with the OMV subunit vaccine, histopathological analysis of mouse spleens and T cell subpopulation classification were performed. Inflammatory cytokine levels, including IL-2, IFN-γ, TNF-α, IL-4, IL-6, IL-10, and IL-17A, as well as immunoglobulin levels (IgG1, IgG2a, and IgG2b) in mouse serum samples, were measured to assess the pathological changes and cellular immune response post-immunization. Results were as follows: OMVs were successfully extracted with particle sizes ranging from 20 to 200 nm and molecular weights between 10 to 130 ku. Proteomic analysis identified 1 612 proteins in OMVs, including several outer membrane proteins such as Omp2b, Omp25, Omp31, Omp19, and Bp26, as well as lipoproteins and proteins related to ABC transporters. After immunization with the OMV subunit vaccine, no significant lymphocyte disorganization or necrosis was observed in mouse spleens. The vaccine induced high levels of IgG immunoglobulin and elevated the expression of inflammatory cytokines IFN-γ, IL-2, IL-6, IL-17A, and TNF-α, while reducing IL-10 and IL-4 levels. The CD4⁺/CD8⁺T cell ratio also increased. The study demonstrates that OMVs contain several effective immunogenic antigens capable of inducing strong cellular immune responses in mice. This lays the theoretical foundation for identifying the protein components of OMVs and developing Brucella subunit vaccines.

Key words: Brucella, outer membrane vesicles, proteomics, immunogenicity