Objective: To construct an autophagy-targeted vaccine harboring the genes encoding Ag85B and microtubule-associated protein light chain-3 (LC3) and to explore its immunoprotection against Mycobacterium tuberculosis (MTB).
Methods: The pCMV-LC3-Ag85B plasmid was constructed and used to transfect RAW264.7 cells. The level of LC3-Ag85B was detected using Western blotting. Then, BALB/c mice were immunized with pCMV, pCMV-Ag85B and pCMV-LC3-Ag85B plasmid, respectively. In vitro, two weeks after the last immunization, the secretion of IL-2, IFN-γ, IL-4 and IL-10 from Ag85B-stimulated lymphocytes was measured by ELISA. Three months after the last immunization, all mice were challenged with MTB H37Rv via the tail vein and the bacterial loads in their spleens and lungs were determined by colony formation assay.
Results: The LC3-Ag85B fusion protein was expressed in RAW264.7 cells that had been transfected with pCMV-LC3-Ag85B and the expression level was in a dose-dependent manner. Compared with the pCMV-Ag85B treatment group, pCMV-LC3-Ag85B-immunized mice showed a significant increase of IL-2 and IFN-γ levels and the lower loads of MTB in the spleens and lungs.
Conclusion: pCMV-LC3-Ag85B can enhance a specific Th1-predominant immunity and a superior immunoprotection against MTB, which may provide a new practical strategy for the development of improved vaccines against MTB.