| Brucella is an important gram negative and faculative intracellular pathogen capable of causing brucellosis in many animal species and humans. Several research groups have made many efforts to develop vaccine against brucellosis. Bacterial ghosts are nonliving bacterial cell envelopes while maintaining their native surface antigenic structures. Therefore, bacterial ghosts might be used as excellent vaccine candidates against Brucella infection.In this study, lysis gene E and its regulatory systemλpL/pR-cI857 were amplified by PCR from pBV220::E and inserted into pBBR1MCS-2 vector forming the temperature-sensitive bacteriolysis plasmid pBBR1MCS-E. Plasmid pBBR1MCS-E was electrical transformed into Brucella suis strain S2 and grown at 28℃until mid log-phase, followed by incubation at 42℃to induce the expression of gene E. Then the Brucella ghost was generated. The OD600 value of cultural medium reached the peak in 6h and began to gentlely decline. After 2 days later, the OD600 value reached a low point (about 1.6) at which point the bacterial ghosts were harvested. In contrast, the growth curve incubated at 28°C was normal. The viable bacteria rapidly decreased after expression of lysis gene E. Induction efficiency of bacterial ghosts, as measured by viable colony counts, were 99.99%. Electron microscopy of B. suis ghosts revealed that they retained the basic cell surface structures. However, as bacterial cytoplasmic material escaped through lysis tunnel, the bacterial ghosts surface significantly shrank and the cell morphology have changed.To study the safety of Brucella ghost, mice inoculated intraperitoneally with B. suis strain S2 exhibited some clinical symptoms, such as apathetic, towering hair, shaking and death. Mice inoculated with B. suis ghosts did not exhibited clinical symptoms, no mice died and gained weight normally. Mice inoculated with S2 manifested spleen enlargement and the mean spleen weight showed significant difference with that of mice inoculated with saline, bacterial ghosts and FKB (formalin killed Brucella).The specific IgG antibodies were examined by ELISA. Specific anti-B.suis IgG antibodies were detected in the serum of mice immunized with either B. suis S2, BSG or FKB. No antibody reactivity could be detected in the serum collected from control mice. Antibody titer of animals immunized with BSG (Brucella suis ghosts) slightly higher than that of animals immunized with S2, but no significant difference between them. Mice immunized with S2 and BSG showed significantly higher IgG than mice immunized with FKB. Animals immunized with BSG showed the highest IgG titers (about 6400) in serum samples collected on 6-7 weeks post-immunization.To evaluate the natural capacity of the bacterial ghosts to induce effective T cell mediated immunity against B. suis, the percentage of CD3+, CD4+ and CD8+ cells in the spleen T lymphocyte of mice were determined by flow cytometry. Compared to that of mice treated with FKB and saline, the percentage of CD3+ and CD4+ cells increased significantly in spleen T lymphocyte of mice immunized with S2 and BSG. The percentage of CD3+ and CD4+ cells showed no significant difference between mice immunized with S2 and BSG. To CD8+ T cells, there was no significant difference among four groups. The values of CD4+/CD8+ of mice immunized with S2 (2.301) and BSG (2.175) are significant higher than that of mice treated with FKB (1.316) and saline (1.503).Following challenge with lethal B. suis, 5/20, 13/20 and 2/20 mice immunized with S2, BSG and FKB survived, respectively. In contrast, none of the control group mice (injected with saline) survived after challenge. BSG-immunized mice showed the highest survival rates (13/20, 65% survival).Altogether, these results suggest that Brucella ghost possess favorable safety and immunogenicity, and might be used as a vaccine candidate to against Brucella infection. |
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