Brucellosis is an infectious disease caused by different Brucella species. The outer membrane proteins 25 and 31 play a significant role in stimulation of immunity against Brucella. Herein, the humoral and cellular immune responses of selected recombinant proteins emulsified in chitosan nanoparticles as individual (univalent) and simultaneous (divalent) injections were assessed. The humoral and cellular immune responses were measured by enzyme-linked immunosorbent assay in 12 groups (individual and simultaneous injections of rOMP25 and rOMP31 in different protein concentrations) and lymphocyte proliferation was measured using 3–4,5-dimethylthiazol-2-yl2,5-diphenyltetrazolium bromide (MTT) assay based on average Optical density (OD) of stimulated cells/average OD of unstimulated cells. Finally, data were analyzed using one-way analysis of variance. rOMP25 + rOMP31 group stimulated higher titer of INF-γ than other groups, whilst there were no statistically significant differences between all uni/divalent immunized groups. Tumor necrosis factor alpha titer showed no significant difference between divalent immunized groups (except rOMP31 + rOMP25(1) and rOMP31 + rOMP25(3)) and positive control group. Interleukin-4 analysis results demonstrated that there were no significant differences between positive control and uni/divalent vaccinated groups. In addition, analysis of antibody responses revealed rOMP25 + rOMP31, rOMP25 + rOMP31(2), and rOMP25 + rOMP31(3) groups induced higher level of total antibody compared to other immunized groups, although both univalent and divalent immunized mice induced higher IgG2a titer than IgG1 with the mean of IgG2a/IgG1 ratio ~1.01 indicating strong bent of Th1 immune response. The cell proliferation assay demonstrated the vaccination with rOMP25 + rOMP3, rOMP25 + rOMP31(2), and rOMP25 + rOMP31(3) elicited vigorous antigen-specific cell proliferative. rOMP25 + rOMP3, rOMP25 + rOMP31(2), and rOMP25 + rOMP31(3) treatments could be used as potential candidates for developing new subunit vaccines.

Keywords