Efficient Testing of Large Pools of Mycobacterium tuberculosis RD1 Peptides and Identification of Major Antigens and Immunodominant Peptides Recognized by Human Th1 Cells

Abstract

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...

Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice

“…Several reports have suggested that IFN-␥ and IL-17 production by CD4 ϩ T cells is targeted in vaccine-induced immunity to M. tuberculosis infection in mouse models (34,35). The immunogenic IFN-␥-generating T-cell epitopes in PE/PPE family proteins are potential TB vaccines (30) and diagnostic biomarkers (36). In this study, two predominant epitope sites were demonstrated by strong IFN-␥-inducing T cells at amino acids 85 to 102 and 217 to 234.…”

Molecular Cloning, Expression, Purification and Immunological Characterization of Proteins Encoded by Regions of Difference Genes of Mycobacterium tuberculosis

“…The results showed that RD1 was the most important region encoding proteins with Th1-cell reactivity , 2010Mustafa & Al-Attiyah, 2009;Mustafa et al, 2011). The analysis of individual peptide pools of RD1 ORFs identified three major (Rv3873, Rv3874 and Rv3875) and three moderate (Rv3871, Rv3872 and Rv3876) antigens stimulatory for Th1 cells in antigeninduced proliferation and IFN-γassays Mustafa 2005b;Mustafa et al, 2008). However, Rv3873 and Rv3876 proteins were equally good stimulators of Th1 cells from TB patients and healthy donors, whereas Rv3872, Rv3874, Rv3875 proteins were better stimulators in TB patients and Rv3871 protein in healthy donors .…”

“…Interestingly, previous studies using pools of synthetic peptides have shown that all of these proteins (Rv3874, Rv3875 and Rv3619c) are major T cell antigens in humans, and the linear T cell epitopes were scattered throughout the sequence of these proteins Mustafa et al 2000Mustafa et al , 2008. A further analysis of the sequence of these proteins for B cell and T cell epitope prediction using appropriate prediction servers further suggested that B-cell and T-cell epitopes were scattered throughout the sequence of each protein (Hanif et al, 2010a;Mustafa et al, 2008;Mustafa and Shaban, 2006). Thus, both prediction and experimental results confirm the strong immunogenicity of Rv3874, Rv3875 and Rv3619c proteins for inducing antigen-specific immunological reactivity.…”

Proteins and Peptides Encoded by M. tuberculosis-Specific Genomic Regions for Immunological Diagnosis of Tuberculosis

“…Furthermore, testing with peripheral blood mononculaer cells for cellular reactivity in T cell assays identified three proteins of RD1, i.e. PPE68, ESAT6 and CFP10, and two proteins each of RD7 (Rv2346c and Rv2947c) and RD9 (Rv3619 and Rv3620) as the best stimulators of T helper 1 (Th1) cells in antigen-induced proliferation and/or IFN-γ secretion assays [18][19][20][21][22][23].The diagnostic potential of four antigens encoded by M. tuberculosis-specific RD1 region genes (PE35, PPE68, CFP10, ESAT-6) and RD9 region gene Rv3619c was further investigated in cellular assays using DTH responses in guinea pigs [24]. The results showed that mycobacterial sonicates containing species-specific and crossreactive antigens, induced positive DTH responses in M. tuberculosis, M. bovis BCG, M. avium and M. vaccae injected guinea pigs.…”

“…Furthermore, testing with peripheral blood mononculaer cells for cellular reactivity in T cell assays identified three proteins of RD1, i.e. PPE68, ESAT6 and CFP10, and two proteins each of RD7 (Rv2346c and Rv2947c) and RD9 (Rv3619 and Rv3620) as the best stimulators of T helper 1 (Th1) cells in antigen-induced proliferation and/or IFN-γ secretion assays [18][19][20][21][22][23].…”