Tuberculosis (TB) is among the most important diseases of worldwide distribution with respect to both morbidity and mortality. Annually, TB afflicts about 9 million people with 2 million deaths (18). The rapid spread of TB in developing countries of Africa and Asia is accelerated by the human immunodeficiency virus epidemic and the spread of multi-and extra-drug-resistant TB (46). To protect against TB, vaccination with Mycobacterium bovis BCG has been used for more than 70 years, but its efficacy varies tremendously in different parts of the world (49). In addition, the diagnostic value of the presently used skin test reagent, the purified protein derivative (PPD) of M. tuberculosis, is low due to cross-reactivity with environmental mycobacteria and the vaccine strains of M. bovis BCG (14). Furthermore, vaccination with M. bovis BCG is contraindicated in immunocompromised subjects, including AIDS patients who are usually at a very high risk of developing TB (19). Identification of M. tuberculosis antigens useful for diagnosis and development of new vaccines is therefore an important goal in TB research.The protection against TB requires cellular immune responses mediated by T helper 1 (Th1)-type cells that secrete large quantities of gamma interferon (IFN-␥) (2, 13, 15), and therefore a primary criterion for selecting candidate antigens for vaccine design has been their ability to induce IFN-␥ responses (reviewed in reference 27). M. tuberculosis is rich in antigens that induce IFN-␥ secretion, and the presence of such antigens has been reported in purified cell walls, the cytosolic fraction, and short-term culture filtrates (ST-CF) (reviewed in reference 25). However, several studies have suggested that antigens present in ST-CF are the primary inducers of IFN-␥ secretion and provide protection against TB in mice and guinea pigs (reviewed in reference 26). It has been previously shown that two M. tuberculosis-specific antigens present in ST-CF, i.e., ESAT6 and CFP10, are frequently recognized by human Th1 cells after natural infection and therefore are important in the specific diagnosis of active and latent TB (29,31,47). Interestingly, ESAT6 and CFP10, when tested together in IFN-␥ assays, have been shown to improve the diagnostic efficacy of TB compared to results with each antigen separately (20). In addition, vaccination with ESAT6 and CFP10 has been shown to provide protection against challenge with M. tuberculosis in animal models of TB (50). However, ESAT6 and CFP10 can be used either as vaccines or as diagnostic reagents but not as both. This necessitates the identification of additional M. tuberculosis antigens, some of which could be reserved for diagnosis and others for the development of new vaccines against TB.The analysis of the M. tuberculosis genome sequence has shown that genes encoding ESAT6 and CFP10 are located in region of difference 1 (RD1) that is deleted in all vaccine strains of M. bovis BCG but present in all of the tested strains and isolates of pathogenic M. tuberculosis and M. bov...

RD1 PE35, PPE68, EsxA, EsxB and RD9 EsxV genes are present in Mycobacterium tuberculosis genome but deleted in Mycobacterium bovis BCG. The aim of this study was to clone these genes into DNA vaccine vectors capable of expressing them in eukaryotic cells as fusion proteins, fused with immunostimulatory signal peptides of human interleukin‐2 (hIL‐2) and tissue plasminogen activator (tPA), and evaluate the recombinant DNA vaccine constructs for induction of antigen‐specific cellular immune responses in mice. DNA corresponding to the aforementioned RD1 and RD9 genes was cloned into DNA vaccine plasmid vectors pUMVC6 and pUMVC7 (with hIL‐2 and tPA signal peptides, respectively), and a total of 10 recombinant DNA vaccine constructs were obtained. BALB/c mice were immunized with the parent and recombinant plasmids and their spleen cells were tested for antigen‐induced proliferation with antigens of M. tuberculosis and pure proteins corresponding to the cloned genes. The results showed that antigen‐specific proliferation responses were observed for a given antigen only with spleen cells of mice immunized with the homologous recombinant DNA vaccine construct. The mice immunized with the parent plasmids did not show positive immune responses to any of the antigens of the cloned genes. The ability of the DNA vaccine constructs to elicit cellular immune responses makes them an attractive weapon as a safer vaccine candidate for preventive and therapeutic applications against tuberculosis.

Objective: To identify Th1 cell-stimulating antigens/peptides encoded by the genes predicted in the Mycobacterium tuberculosis-specific genomic region of difference (RD)1, deleted in Mycobacterium bovis Bacille Calmette-Guérin(BCG), by using synthetic peptides and whole blood from tuberculosis (TB) patients. Materials and Methods: Heparinized peripheral blood was obtained from culture-proven pulmonary TB patients (n = 16) attending the Chest Disease Hospital, Kuwait. Whole blood was diluted with tissue culture medium RPMI-1640 and tested for Th1 cell stimulation using antigen-induced proliferation and interferon-γ (IFN-γ) secretion assays. The antigens included a peptide pool of 220 peptides covering the sequence of 12 open reading frames (ORFs) of RD1 (RD1_mix), peptide pools of RD1 ORF5 (ORF5_mix), ORF6 (ORF6_mix) and ORF7 (ORF7_mix), and individual peptides of ORF6 (P6.1–P6.6) and ORF7 (P7.1–P7.6). M. tuberculosis culture filtrate, cell walls and whole-cell M. bovis BCG were used as complex mycobacterial antigens. The results obtained with different antigens and peptides were statistically analyzed for significant differences using Z test. Results: The complex mycobacterial antigens (culture filtrate, cell walls and M.bovis BCG) and RD1_mix induced comparable (p > 0.05) positive antigen-induced proliferation and IFN-γ responses with whole blood from TB patients. However, the positive IFN-γ responses induced by ORF6_mix and ORF7_mix were higher than ORF5_mix. Among the individual peptides, P6.4 and P7.1 of ORF6 and ORF7, respectively, induced the highest IFN-γ responses, suggesting that these peptides represented the immunodominant Th1 cell epitopes of RD1 ORF6 and ORF7 in the patients tested. Conclusion: The whole blood assays with synthetic peptides are useful to identify Th1 cell antigens/peptides encoded by genes located in M. tuberculosis-specific genomic regions.

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The aim of this study was to clone, express and purify three major antigenic proteins, i.e. Rv3874, Rv3875 and Rv3619c, encoded by genes located in regions of difference of Mycobacterium tuberculosis and characterize them for immunogenicity in rabbits. The respective genes were amplified using gene‐specific primers and genomic DNA of M. tuberculosis by polymerase chain reaction. The amplified DNA were cloned into pGEM‐T Easy and subcloned into pGES‐TH‐1 vector for high‐level expression in Escherichia coli and efficient purification. The results showed that the three fusion proteins, i.e. glutathione‐S‐transferase (GST)‐Rv3874, GST‐Rv3875 and GST‐Rv3619c, were expressed at high levels and were purified (free of the GST fusion partner) to homogeneity using glutathione‐Sepharose and Ni‐NTA agarose affinity matrix after cleavage of the column‐bound fusion proteins by thrombin protease. The purified recombinant Rv3874, Rv3875 and Rv3619c proteins were immunogenic and induced antigen‐specific antibodies in rabbits. Testing of the rabbit sera with overlapping synthetic peptides showed that the antibodies were induced to several epitopes that were scattered throughout the sequence of each protein. These results show immunogenicity of all the proteins for inducing antigen‐specific antibodies in rabbits and demonstrate the usefulness of pGES‐TH‐1 vector for obtaining purified recombinant proteins of M. tuberculosis for immunological characterization.