Identification of EmrA and AaeA as Novel Brucella Vaccine Targets and Evaluation of the Immunogenicity of Constructed Multi-Epitope Vaccines

Brucella invades the reproductive systems of various animal species, inducing abortion, infertility, and other reproductive disorders. Although attenuated vaccines are routinely used for livestock prophylaxis, their limitations underscore the urgent need for novel approaches. In this study, the complete protein sequences of 89 Brucella strains of different species, including B. abortus and B. melitensis, were retrieved from the NCBI database for pan-genome analysis. Two highly conserved antigenic proteins, namely p-hydroxybenzoic acid efflux pump subunit AaeA and colistin resistance protein EmrA, were identified from the core genes of Brucella using a subtractive proteomics approach, which could serve as potential vaccine targets. T-cell and B-cell epitopes were subsequently predicted using immunoinformatics tools (IEDB), and a multi-epitope vaccine candidate, Bev353, was constructed by fusing 12 T-cell and 3 B-cell epitopes with suitable adjuvants and using appropriate linkers between epitopes. Animal experiments showed that the recombinant proteins rAaeA, rEmrA, and rBev353 induced robust Th1- and Th2-mediated immune responses in BALB/c mice, as evidenced by significant elevations in cytokine levels (IFN-γ, TNF-α, IL-4, IL-2) and antibody levels (IgG, IgG2a, IgG1). These findings demonstrate that these identified proteins, AaeA, EmrA, and Bev353, can effectively induce both cellular and humoral immune responses. Moreover, no adverse physiological reactions or histopathological damage were observed during immunization, confirming the safety of the vaccine candidate. In summary, this work provides a broad-spectrum protective antigen candidate targeting reproductive disorders caused by Brucella in multiple livestock species, offering a promising strategy for the prevention and control of brucellosis.