Immunogenic effects of a combined recombinant protein formulation based on Brucella Omp19, Omp2b, Omp31, and BP26

Abstract

Brucellosis is a neglected zoonotic disease for which no approved human vaccine is currently available. For Brucella , a typical intracellular pathogen, the success of recombinant protein vaccine development largely depends on whether it can effectively induce immune responses with a strong Th1-associated cellular component. This study aimed to systematically compare the immunological characteristics of single-antigen immunization with rOmp19, rOmp2b, rOmp31, or rBP26 and a four-antigen combined immunization strategy under a uniform CpG adjuvant regimen, and to evaluate the relative advantages of the combined strategy. Female C57BL/6N mice aged 6–8 weeks were randomly assigned to a PBS control group, a CpG control group, four single-antigen immunization groups, and a four-antigen combined immunization group (Combo + CpG). Mice were immunized three times by three-point subcutaneous injection in the abdomen at 2-week intervals. Serum and splenocytes were collected at weeks 1, 2, 4, and 14 after the final immunization. Macrophage polarization, antigen-specific antibody responses, T-cell activation and proliferation, cytokine secretion, and memory T-cell phenotypes were systematically evaluated by ELISA, flow cytometry, and ELISPOT. Functional activity and safety were further assessed through serum complement-dependent bactericidal assays, serum biochemical analysis, and splenic histopathological observation. Under the same CpG adjuvant regimen, all recombinant protein immunization groups induced humoral and cellular immune responses to varying degrees. Compared with single-antigen immunization, the Combo + CpG group showed a more coordinated enhancement across multiple key indicators, including more pronounced changes in macrophage-associated CD11c and CD206 phenotypes during the early and intermediate stages after immunization, higher levels of total IgG and certain IgG subclass responses, more significant activation and proliferation of CD4 + and CD8 + T cells after antigen restimulation, stronger IFN-γ-associated cellular immune responses, and more prominent complement-dependent bactericidal activity in vitro. Meanwhile, the combined immunization group also showed relative advantages in certain CD4 + central memory T-cell (TCM) and CD8 + effector memory T-cell (TEM) subsets. No obvious abnormalities were observed in serum biochemical parameters or splenic histology in any immunization group, suggesting that no detectable systemic toxicity was produced under the conditions of this study. Although the four-antigen combined immunization strategy did not outperform the single-antigen strategies in every individual parameter, it demonstrated a more stable overall advantage in Th1-related cellular immunity, selected humoral immune indicators, in vitro functional activity, and overall immunological coordination. These findings identify the four-antigen combination as an immunogenic formulation warranting further optimization and evaluation in in vivo Brucella challenge models.