Integrated Multi-Omics Strategy for Screening Novel Protective Antigens Against Brucella melitensisand Analysis of Their Immunogenicity

Background: Brucellosis is one of the most severe Class B infectious diseases 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 ani- mals, and the inability to differentiate between natural infection and vaccination (DIVA). Methods: Relying on the ABSL-3 laboratory of the Inner Mongolia Autonomous Region Center for Disease Control and Preven- tion, this study established a chronic infection model in C57BL/6J mice using the virulent strain Brucella melitensisM16. Single- cell RNA sequencing (scRNA-seq) was performed using the 10x Genomics platform to map the heterogeneity of splenic immune cells. Whole-genome next-generation sequencing and pangenomic analysis were conducted on 12 strains with different virulence levels (M16, 544A, M5, 104M, etc.) to screen membrane/secreted proteins unique and conserved in virulent strains as candidate antigens. Candidate antigens were prepared via prokaryotic expression systems, and their humoral and cellular immune levels were detected using indirect ELISA and flow cytometry. Results: A stable chronic infection model was successfully constructed (bacterial load Log10CFU > 4.5). Single-cell sequencing yielded 128,543 high-quality cells, annotated into 12 major immune cell subpopulations, revealing significant expansion of effec- tor CD4+T cells (P< 0.01) and high expression of the Ifnggene. Pangenomic analysis identified three candidate antigens (BMEI0021, BMEI1943, BMEI0367) that are 100% conserved in virulent strains but absent in the M5 vaccine strain. Immuno- genicity assays showed that BMEI1943 induced high levels of IgG2a subtype antibodies (titer Log213.45 ± 0.52) and IFN- γ+CD4+T cell responses (frequency 15.80% ± 2.10%). Conclusion: Through multi-omics integration analysis, this study successfully screened 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.