Background: Brucellosis, as a global zoonotic disease, has long been constrained in its prevention and control by the inability of traditional vaccines to provide sterilizing immunity and the lack of Differentiating Infected from Vaccinated Animals (DIVA) capabilities. The exquisite intracellular hiding mechanism of Brucellais the core reason for the difficulty in eradicating chronic infections. Methods: This study innovatively combined 10x Visium spatial transcriptomics with single-cell immune profiling (scTCR-seq) to construct a high-dimensional immune atlas of the spleen in Brucella melitensis-infected mice while retaining in situ information. Utilizing CRISPR-Cas9 genome-wide functional screening technology, key host factors regulating the intracellular survival of Brucellawere systematically mined in the RAW264.7 macrophage model. Furthermore, combining AlphaFold2 protein structure prediction with a prokaryotic expression system, novel candidate antigens were designed and prepared, and their immunogenicity was deeply analyzed via high-dimensional flow cytometry (13-color panel). Results: Spatial multi-omics analysis intuitively revealed the central role of the splenic marginal zone (MZ) as a hotspot for bacterial colonization and T cell activation, and discovered that bacteria evade immune surveillance by constructing local “immune-exempt zones” and downregulating MHC class I molecules. CRISPR screening identified for the first time that the actin cytoskeleton regulatory protein Sipa1l1 is the strongest host resistance factor against Brucella(log2FC = -4.85), while Tbk1 plays a dual role in infection homeostasis. The candidate antigen BMEI_RS08405, screened based on AI structure prediction, exhibited excellent epitope exposure (pLDDT > 90%) and strongly polarized Th1-type immunity (IgG2a/IgG1 = 1.85), inducing high-frequency, high-affinity IFN-γ+TNF-α+ double-positive multifunctional T cells (PMI = 0.85). Conclusion: This study breaks the limitation of “black-box” antigen discovery in traditional brucellosis vaccines, revealing the immune escape mechanism of Brucellafrom the dual dimensions of spatial ecology and functional genomics for the first time, and successfully identifies a novel subunit vaccine target with super-strong protective potential, providing a new paradigm for the rational design of other intracellular bacterial vaccines.