In silico Analysis and Experimental Validation of Mycobacterium tuberculosis-Specific Proteins and Peptides of Mycobacterium tuberculosis for Immunological Diagnosis and Vaccine Development

Comparative analyses of the Mycobacterium tuberculosis genome with the genomes of other mycobacteria have led to the identification of several genomic regions of difference (RDs) between M. tuberculosis and M. bovis BCG. The identification of immunodominant and HLA-promiscuous antigens and peptides encoded by these RDs could be useful for diagnosis and the development of new vaccines against tuberculosis. The analysis of RD proteins and peptides by in silico methods (using computational programs to predict major and HLA-promiscuous antigenic proteins and peptides) and experimental validations (using peripheral blood mononuclear cells and sera from tuberculosis patients and BCG-vaccinated healthy subjects to assess antigen-specific cellular and humoral immune responses in vitro) identified several major antigens and peptides. To evaluate the in vivo potentials, the genes of immunodominant antigens were cloned and expressed in DNA vaccine vectors. Immunizations of experimental animals with the recombinant constructs induced antigen-specific cellular responses. Further experiments showed that each of these proteins had several T and B cell epitopes scattered throughout their sequence, which confirmed their strong immunogenicity. In conclusion, the bioinformatics-based in silico identification of promiscuous antigens and peptides of M. tuberculosis is a useful approach to identify new candidates important for diagnosis and vaccine applications.