Figure 4.

Evaluation of the expression of synthetic Coxiella burnetii chimeric epitopes and the protective efficacy of the live, recombinant, attenuated Listeria monocytogenes strain expressing Coxiella-specific CD8⁺ T-cell epitopes (Lm-Cb). A, Diagram of the transcription and translation of synthetic C. burnetii epitopes in L. monocytogenes. C. burnetii chimeric epitopes were cloned under a strong cytosolic L. monocytogenes actA promoter and actA N-terminal secretion peptide for efficient expression and secretion of chimeric epitopes after L. monocytogenes escaped into the cytosol of infected cells. B, Lm-Cb was used to infect Raw264.7 cells for 5 hours at a multiplicity of infection of 10. One hour after infection, cells were washed with phosphate-buffered saline (PBS) and supplied with fresh medium containing gentamicin. Five hours after infection, cell lysates were analyzed by Western blot and probed with anti-HA antibody for detection of synthetic C. burnetii epitopes. C, Intracellular localization of C. burnetii CD8⁺ T-cell epitopes in Raw264.7 cells 5 hours after infection with L. monocytogenes. Blue, DAPI-stained nuclear and bacterial DNA; green, HA-Coxiella chimeric epitopes; red, L. monocytogenes. D and E, Spleen weight (D) and C. burnetii load (E) in the spleen of infected mice. Six mice per group were immunized with 5 × 10⁶ colony-forming units of Lm-Cb or L. monocytogenes in 100 µL of PBS twice, with a 2-week interval between immunizations. Mice immunized with PBS were used as a negative control. Four weeks after the boost immunization, each mouse was challenged with 1 × 10⁶ genome equivalents of C. burnetii. Fourteen days after challenge, mice were euthanized, and the spleens were collected. *P < .05 and **P < .01, compared with negative control. Abbreviation: BMDM, bone marrow–derived macrophage.