Extracellular vesicle–guided antigen selection enables a protective multiepitope vaccine against Brucella abortus

Brucellosis remains a major zoonotic disease for which safe and effective subunit vaccines are still unavailable. In this study, we developed a rational multiepitope vaccine against Brucella abortus by exploiting bacterial proteins previously identified in extracellular vesicles released from infected macrophages as a source of biologically relevant vaccine antigens. Proteomic data were used to prioritize immunogenic proteins, from which B-cell, MHC class I, and MHC class II epitopes were predicted and assembled into a recombinant vaccine construct (RecoV) fused to the cholera toxin B subunit.The recombinant protein was expressed, purified, and evaluated in BALB/c mice with or without the Sigma Adjuvant System (SAS). RecoV elicited antigen-specific humoral and cellular immune responses, with the adjuvanted formulation promoting a Th1-biased profile characterized by IgG2a predominance, enhanced IFN-γ secretion by restimulated splenocytes, and increased frequencies of IFN-γ-producing CD4 + and CD8 + T cells. Following challenge with virulent B. abortus 2308, mice immunized with RecoV plus adjuvant exhibited approximately a 1 log 10 reduction in splenic bacterial burden compared with control animals.These findings demonstrate that infection-derived extracellular vesicle proteomes can serve as an effective platform for antigen prioritization and support the use of proteomics-guided reverse vaccinology for the rational development of subunit vaccines against intracellular bacterial pathogens.