Comparable protective efficacy of Brucella melitensis outer membrane vesicles via intramuscular, subcutaneous, and intranasal immunization routes in mice

Brucella melitensis is a causative agent of chronic infection in both animals and humans. Immunization with outer membrane vesicles (OMVs) released from B. melitensis has been shown to significantly reduce disease burden. This study aimed to investigate the effect of different immunization routes on effect protective immunity conferred by B. melitensis OMVs. OMVs derived from B. melitensis M5 (OMVs-M5) exhibited weak activation of innate immunity and no cytotoxicity in ex vivo assays. Immunization with OMVs-M5 via intramuscular or subcutaneous injection induced severe reactogenicity in mice during the initial days, whereas intranasal administration did not. Mice immunized intramuscularly, intranasally, or subcutaneously with OMVs-M5 showed comparable protective efficacy against challenge with a virulent B. melitensis strain, characterized by significantly reduced bacterial burdens and milder histopathological lesions in the spleen. All three routes of immunization elicited robust humoral immunity and CD4⁺ T cell-mediated immune responses. These findings provide experimental evidence that intramuscular, intranasal or subcutaneous delivery of OMVs-M5 are all effective in protecting mice against B. melitensis infection, with no major differences observed between routes.