ABSTRACT
Two candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of colony-forming unit reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study.
Subject(s)
Antigens, Bacterial/immunology , Mycobacterium tuberculosis/immunology , Tuberculosis Vaccines/immunology , Tuberculosis/prevention & control , Vaccines, DNA/immunology , Animals , Antibody Formation/immunology , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Female , Immunophenotyping/methods , Mice , Mice, Inbred C57BL , Mycobacterium tuberculosis/genetics , T-Lymphocytes, Cytotoxic/immunology , Tuberculosis/microbiology , Tuberculosis Vaccines/genetics , Tuberculosis Vaccines/therapeutic use , Vaccines, DNA/genetics , Vaccines, DNA/therapeutic useABSTRACT
Secreted proteins encoded by different regions of difference (RDs) from the genome of Mycobacterium tuberculosis have been considered as attractive candidates for vaccination against tuberculosis owing to their absence in most BCG strains. In this study, the structural gene for the RD2 locus encoding protein CFP21 was PCR amplified and expressed as a fusion protein with hexahistidine residues in Escherichia coli. Expression of CFP21 in E. coli under transcriptional regulation of the T7 promoter yielded a protein located within inclusion bodies. The inclusion bodies were solubilized in the presence of 8M urea and the protein was purified to homogeneity under denaturing conditions at low pH using nitrilotriacetic acid (Ni-NTA) affinity chromatography. The denatured protein was renatured by gradient dialysis against a decreasing concentration of urea. The purified protein was shown to have esterase activity. CFP21 protein was evaluated for immunogenicity in C57BL/6J mice. We observed an elevated T cell proliferative response and production of IFN-gamma and IL-12 (p40). CFP21 also induced an optimum level of cytotoxic T cell activity and induced a strong humoral response as indicated by higher levels of specific IgG1 and IgG2a antibody isotypes. In addition, a moderate level of protection was observed against experimental tuberculosis. This is the first report describing esterase activity of the M. tuberculosis complex-restricted protein CFP21 and its protective potential against experimental tuberculosis.
