Abstract
Background Brucellosis is one of the most severe Class B infectious diseases prevalent in the agricultural and pastoral areas of northern China. Current attenuated live vaccines (e.g., M5, S19) have defects such as residual virulence, causing abortion in pregnant animals, and the inability to differentiate between natural infection and vaccination (DIVA). Methods Relying on the ABSL-3 laboratory of the Inner Mongolia Center for Disease Control and Prevention, this study established a chronic infection model in C57BL/6J mice using the virulent strain Brucella melitensis M16. Single-cell transcriptome sequencing (10x Genomics) was employed to map the heterogeneity of splenic immune cells. Whole-genome scanning and pangenomic analysis were performed on four strains with different virulence levels (M16, 544 A, M5, 104 M) using second-generation sequencing. Membrane/secreted proteins unique and conserved in virulent strains were screened as candidate antigens, prepared via prokaryotic expression systems, and their humoral and cellular immune levels were detected by indirect ELISA and flow cytometry. Results A stable chronic infection model was successfully constructed (bacterial load Log10 CFU > 4.5). Single-cell sequencing yielded 45,231 cells, annotated into 12 immune cell subpopulations, revealing significant expansion of Effector CD4 + T cells ( P Ifng gene. Pangenomic analysis identified three candidate antigens (BMEI0021, BMEI1943, BMEI0367) that are 100% conserved in virulent strains but absent in the vaccine strain M5. Immunogenicity assays showed that BMEI1943 induced high levels of IgG2a subtype antibodies (titer Log2 13.45 ± 0.52) and IFN-γ + CD4 + T cell responses (frequency 15.80% ± 2.10%). Conclusion Through multi-omics integration analysis, this study successfully identified a novel candidate antigen, BMEI1943, with strong Th1-type immunogenicity, providing an experimental basis for the development of safe and efficient subunit vaccines against brucellosis.