A19Δbpe275 emerges as a safer live attenuated vaccine candidate

Brucellosis, a significant zoonotic disease caused by Brucella spp., remains a major global challenge to both animal production and public health. Current live attenuated vaccines, such as Brucella abortus A19, are limited in their application due to residual virulence and interference with serodiagnosis. The intracellular survival and immune evasion of Brucella critically depend on effector proteins delivered by the Type IV Secretion System (T4SS), yet the functions of many of these effectors remain poorly defined. In this study, we constructed a markerless deletion mutant, A19Δ bpe275 , in the B. abortus A19 background and comprehensively evaluated its phenotype, virulence, and vaccine potential. The A19Δ bpe275 mutant retained wild-type morphology, smooth lipopolysaccharide (LPS) structure, in vitro growth kinetics, and genetic stability, but exhibited significantly impaired long-term intracellular survival in macrophages. In murine infection models, A19Δ bpe275 displayed markedly attenuated virulence, characterized by consistently lower splenic bacterial loads, milder histopathological lesions, and accelerated clearance compared to the parental A19 strain. Immunologically, infection with A19Δ bpe275 elicited a robust and sustained Th1-biased immune response. Notably, A19Δ bpe275 conferred comparable protective efficacy with improved safety against challenge with the virulent B. abortus 2308 and demonstrated cross-protection against B. melitensis 16M . Uniquely, the bpe275 deletion provides a molecular signature for a DIVA-compatible real-time PCR assay, enabling differentiation between vaccinated and infected animals. Collectively, by achieving an optimal balance between enhanced safety and preserved immunogenicity, A19Δ bpe275 emerges as a promising candidate for next-generation live attenuated brucellosis vaccines.