PLoS One. 2014 Aug 19;9(8):e103679.

Characterization of a cross-reactive, immunodominant and HLA-promiscuous epitope of Mycobacterium tuberculosis-specific major antigenic protein PPE68.


Mustafa AS

Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, Kuwait.



PPE68 (Rv3873), a major antigenic protein encoded by Mycobacterium tuberculosis-specific genomic region of difference (RD)1, is a strong stimulator of peripheral blood mononuclear cells (PBMCs) obtained from tuberculosis patients and Mycobacterium bovis bacillus Calmette Guerin (BCG)-vaccinated healthy subjects in T helper (Th)1 cell assays, i.e. antigen-induced proliferation and interferon-gamma (IFN-γ) secretion. To confirm the antigen-specific recognition of PPE68 by T cells in IFN-γ assays, antigen-induced human T-cell lines were established from PBMCs of M. Bovis BCG-vaccinated and HLA-heterogeneous healthy subjects and tested with peptide pools of RD1 proteins. The results showed that PPE68 was recognized by antigen-specific T-cell lines from HLA-heterogeneous subjects. To further identify the immunodominant and HLA-promiscuous Th1-1 cell epitopes present in PPE68, 24 synthetic peptides covering the sequence of PPE68 were individually analyzed for HLA-DR binding prediction analysis and tested with PBMCs from M. bovis BCG-vaccinated and HLA-heterogeneous healthy subjects in IFN-γ assays. The results identified the peptide P9, i.e. aa 121-VLTATNFFGINTIPIALTEMDYFIR-145, as an immunodominant and HLA-DR promiscuous peptide of PPE68. Furthermore, by using deletion peptides, the immunodominant and HLA-DR promiscuous core sequence was mapped to aa 127-FFGINTIPIA-136. Interestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced strains/species of M. tuberculosis and M. tuberculosis complex, and several other pathogenic mycobacterial species, including M. leprae and M. avium-intracellulare complex. These results suggest that the peptide aa 121-145 may be exploited as a peptide-based vaccine candidate against tuberculosis and other mycobacterial diseases.

PMID: 25136958



Tuberculosis, a disease primarily caused by Mycobacterium tuberculosis, is a major infectious diseases problem of world-wide distribution and ranks among the top 10 causes of death in the world. In spite of global efforts to control tuberculosis, it is estimated that 9.0 million people developed the disease and 1.5 million people died of tuberculosis in 2013 (incidence and deaths up from 7.5 million and 1·3 million, respectively, estimated in 2012) (1). The problem of tuberculosis is complicated due to increase in drug resistance cases and the deadly combination of M. tuberculosis and human immunodeficiency virus (HIV) co-infection [1]. Both of these microorganisms are intracellular pathogens and require effective cellular immunity, primarily mediated by CD4+ T helper (Th)1 cells, for protection of infected humans. However, infection with HIV kills CD4+ Th1 cells, which makes patients highly susceptible to M. tuberculosis infection, and infection with M. tuberculosis results in enhanced intracellular HIV-1 replication. Thus, the dual infection becomes a deadly combination (2).

The impact of current efforts to reduce the global burden of tuberculosis, by means of improved diagnosis and treatment with available drugs, is less than expected (1). Therefore, additional preventive efforts, including effective vaccines are required (3). The only available anti-tuberculosis vaccine for use in humans is the live Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccine. Although, this vaccine has been used since 1921, it has failed to control the global epidemic of tuberculosis. Hence more effective new vaccines are required (3). In order to develop new vaccines against tuberculosis, it is essential to identify appropriate antigens and peptides, capable of inducing protective immunity by activating CD4+ Th1 cells that secrete protective Th1 cytokines (4).

The comparative analyses of M. tuberculosis genome with the genomes of other mycobacteria have shown the presence of several regions of difference (RD) that are specific for M. tuberculosis and absent in many other mycobacteria, including M. bovis BCG (5, 6).

Among these regions, the first region, i.e. RD1, appears to be the most important region for vaccine development because it was shown, even before the availability of the comparative genome sequence data in 1999, that RD1 contained genes that encode two small molecular weight (ca 10 kDa) major antigenic proteins of M. tuberculosis, i.e. ESAT-6 and CFP10, capable of activating Th1 cells (7, 8).

The analyses of RD 1 DNA sequence by different groups for probable proteins, using computer-based programs for prediction of open reading frames (ORFs), suggested the existence of variable numbers of proteins/ORFs (Figure 1). Eight ORFs predicted by Mahairas et al (10), nine ORFs by Cole et al (5) and 14 ORFs by Robinson et al (11, 12) (Figure 1). Interestingly, the ORFs predicted by Robinson et al. included all the ORFs predicted by Mahairas et al. and Cole et al. and contained four additional ORFs (ORF4, ORF8, ORF14 and ORF15) (Figure 1). The appropriateness of the of the prediction by Robinson et al. was strengthened when it was shown that ORF14 protein, which was not predicted by Mahairas et al. and Cole et al., was expressed in M. tuberculosis and antibody reactivity to this protein was present in the sera of tuberculosis patients (11, 13). This suggested that the predictions of Robinson et al were more inclusive and immunologically relevant (14). Hence, all of these ORFs were tested for Th1 cell reactivity (15).


ASM Fig1

Fig. 1. M. tuberculosis-specific open reading frames in RD1. The figure shows three predictions (ORF1A to ORF1K by Mahairas et al. (10), Rv3871 to Rv3879c by Cole et al. (5) and ORF2 to ORF14 by Robinson et al. (11, 12) by using various computer-based programs for ORF-predictions.



By using pools of chemically synthesized peptides corresponding to each RD1 protein, it was shown that all of the 14 proteins are immunologically reactive but three of them (ESAT-6, CFP10 and PPE68) are the major antigens (16, 17). However, the antigen PPE68 will have an advantage because it was recognized equally well by tuberculosis patients and M. bovis BCG-vaccinated healthy subjects, suggesting that it will be a better vaccine candidate against tuberculosis (17). Hence, we hypothesized that PPE68 and its immunodominant peptides could be ideal for using as a vaccine against tuberculosis (18).

The immunodominnat epitopes present in PPE68 were identified by testing 24 overlapping synthetic peptides of PPE68 with peripheral blood mononuclear cells from tuberculosis patients and healthy subjects. In both groups, the immunodominant epitope was resrtcited to the peptide aa 121-VLTATNFFGINTIPIALTEMDYFIR-145 of PPE68 (17). The positive cellular responses (antigen-induced proliferation and interferon-γ secretion) from HLA-heterogeneous donors suggested that the immunodominant peptide was HLA-promiscuous (17). Furhtermore, the recognition by Th1 cells was confirmed by establishing human T cells lines from HLA-heterogenuous donors (18). In addition, by using a series of smaller synthetic peptides, the immunodominant and HLA-promiscous core sequence was identified as aa 127-FFGINTIPIA-136 (18). Intrestingly, the core sequence is present in several PPE proteins of M. tuberculosis, and conserved in all sequenced organisms of pathogenic M. tuberculosis and other species of M. tuberculosis complex, including all the vaccine strains of M. Bovis BCG. Furthermore, several other disease-causing mycobacterial organisms, including M. marinum, M. ulcerans and M. leprae and M. avium-intracellulalae complex, also have the core sequence of PPE68 (18).The sequence was predictted to bind multiple HLA calss II and calss II molecules, suggesting that it could be recognized by both CD4+ as well as CD8+ T cells (18). Since the involvement of both CD4+ and CD8+ T cells may be required for optimal protection against mycobacterial disease, the use of crossreactive peptide 121-VLTATNFFGINTIPIALTEMDYFIR-145 of PPE68 may be useful as a peptide-based vaccine against tuberculosis and other mycobacterial diseases.

Importance of the study: The presence of immunodominant and HLA-promisculus peptide (aa 121-145) of PPE68 in M. tuberculosis and several other pathogenic mycobacterial species suggest that this peptide may be useful as a peptide-based vaccine candidate to control the world-wide problem of tuberculosis and many other diseases caused by mycobacterial organisms.



  1. Zumla A, George A, Sharma V, Herbert RH; Baroness Masham of Ilton, Oxley A, Oliver M (2015) The WHO 2014 Global tuberculosis report—further to go. Lancet Glob Health 3:e10-2.
  2. Espert L, Beaumelle B, Vergne I (2015) Autophagy in Mycobacterium tuberculosis and HIV infections. Front Cell Infect Microbiol 5:49.
  3. Mustafa AS (2012) What’s new in the development of tuberculosis vaccines. Med Princ Pract 21:195-196.
  4. Mustafa AS (2005) Mycobacterial gene cloning and expression, comparative genomics, bioinformatics and proteomics in relation to the development of new vaccines and diagnostic reagents. Medical Principles and Practice 14 (Suppl. 1), 27-34.
  5. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence Nature 393:537-44,
  6. Behr MA, Wilson MA, Gill WP, Salamon H, Schoolnik GK, Rane S, Small PM (1999)
  7. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284:1520-1523.
  8. Mustafa AS, Amoudy HA, Wiker HG, Abal AT, Ravn P, Oftung F, Andersen P. (1998) Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis. Scand J Immunol 48:535-543.
  9. Berthet FX, Rasmussen PB, Rosenkrands I, Andersen P, Gicquel B (1998)
  10. A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate protein (CFP-10) Microbiology 144:3195-3203.
  11. Amoudy HA, Al-Turab MB, Mustafa AS (2006) Identification of transcriptionally active open reading frames within the RD1 genomic segment of Mycobacterium tuberculosis. Med Princ Pract 15:137-144.
  12. Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover K (1996) Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 178: 1274–1282.
  13. Ahmad S, Amoudy HA, Thole JE, Young DB, Mustafa AS (1999) Identification of a novel protein antigen encoded by a Mycobacterium tuberculosis-specific RD1 region gene. Scand J Immunol 49:515-522.
  14. Mustafa AS (2001) Biotechnology in the development of new vaccines and diagnostic reagents against tuberculosis. Curr Pharm Biotechnol 2: 157–173.
  15. Amoudy HA, Ahmad S, Thole JE, Mustafa AS (2007) Demonstration of in vivo expression of a hypothetical open reading frame (ORF-14) encoded by the RD1 region of Mycobacterium tuberculosis. Scand J Immunol 66:422-425.
  16. Amoudy HA, Mustafa AS (2008) Amplification of six putative RD1 genes of Mycobacterium tuberculosis for cloning and expression in Escherichia coli and purification of expressed proteins. Med Princ Pract 17:378-384.
  17. Mustafa AS (2002) Development of new vaccines and diagnostic reagents against tuberculosis. Mol Immunol 39:113–119.
  18. Hanif SN, El-Shammy AM, Al-Attiyah R, Mustafa AS (2008) Whole blood assays to identify Th1 cell antigens and peptides encoded by Mycobacterium tuberculosis-specific RD1 genes. Med Princ Pract 17:244-249.
  19. Mustafa AS, Hanif SNM, Shaban FA (2008) Efficient testing of large pools of Mycobacterium tuberculosis RD1 peptides and identification of major antigens and immunodominant peptides recognized by human Th1 cells. Clin Vaccine Immunol 15;916-924.
  20. Mustafa AS (2014) Characterization of a cross-reactive, immunodominant and HLA-promiscuous epitope of Mycobacterium tuberculosis-specific major antigenic protein PPE68. PLoS One 9:e103679.


Acknowledgments: The study was supported by Kuwait University Research Sector grants MI01/10 and SRUL02/13.



Abu Salim Mustafa, Ph.D., FRCPath

Professor and Consultant

Department of Microbiology, Faculty of Medicine, Kuwait University, PO Box 24913, Safat 13110, Kuwait



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