Identification of Mycobacterium tuberculosis-specific genomic regions encoding antigens inducing protective cellular immune responses

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1 Indian Journal of Experimental Biology Vol. 47, June 2009, pp Identification of Mycobacterium tuberculosis-specific genomic regions encoding antigens inducing protective cellular immune responses Abu Salim Mustafa* & Rajaa Al-Attiyah Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait Received 4 January 2009 Comparative genomic studies have identified 11 regions of difference (RD1, RD4, RD5, RD6, RD7, RD9, RD10, RD11, RD12, RD13 and RD15) in Mycobacterium tuberculosis genome which are absent in all vaccine strains of M. bovis BCG. The proteins encoded by genes predicted in these RDs could be useful as protective vaccines and/or exacerbate the disease process by inducing cellular immune responses involved in protection and pathogenesis of tuberculosis. In our studies, by using pools of overlapping synthetic peptides covering the sequence of putative proteins encoded by genes predicted in each RD, we have determined the cellular immune responses in relation to antigen-induced proliferation and secretion of the protective Th1 cytokine IFN-γ and the pathologic Th2 cytokine IL-10 by peripheral blood mononuclear cells of tuberculosis patients and healthy humans. It has been observed that peptides of RD1 pool induced the highest antigeninduced proliferation and IFN-γ responses, whereas the peptides of RD12 pool and RD13 pool induced the highest IL-10 responses. Furthermore, addition of RD12 pool and RD13 pool to peripheral blood mononuclear cells (PBMCs) cultures inhibited the RD1 pool -induced secretion of IFN-γ by PBMCs of healthy humans. These results suggest the relevance of RD1- encoded proteins in protection and RD12- and RD13-encoded proteins in pathogenesis of tuberculosis. Keywords: IFN-γ, IL-10, Mycobacterium tuberculosis, PBMCs, RD antigens Tuberculosis, the most important bacterial infection, is among the top 10 causes of world-wide mortality. It is estimated that 1/3 rd of the world population is latently infected with the causative agent of tuberculosis, i.e. Mycobacterium tuberculosis. People (8 to 10 million) develop active disease and 1.8 million people die of tuberculosis each year 1. The global problem of tuberculosis is worsening due to co-infection with HIV and the emergence of multidrug-resistant and extensively drug-resistant strains of M. tuberculosis 1. Interestingly, both protection and pathogenesis in tuberculosis are mediated by host s cellular immune responses, which primarily involve the interaction of mononuclear cells, i.e. lymphocytes (mainly T cells) and phagocytes of the monocyte/macrophage lineage 2,3. This interaction is mostly dependent on the interplay of cytokines produced by these cells in response to stimulation by mycobacterial antigens 3. Although, a large number of cytokines may contribute to protective immunity against tuberculosis, T-helper (Th)1 cytokine IFN-γ is *Correspondent author Telephone: ; Fax: abusalim@hsc.edu.kw considered the major player in mediating protective immunity against mycobacterial infections 2-8. On the contrary, Th2 cytokine IL-10 is strongly associated with active disease, reduced resistance, reactivation and chronic progressive tuberculosis In addition, IL-10 deactivates macrophages, inhibits the secretion of the protective Th1 cytokine IFN-γ, and neutralizes the protective effect of IFN-γ 12. Therefore, identification of the antigens of M. tuberculosis, which may preferentially activate secretion of IFN-γ and IL-10 by peripheral blood mononuclear cells (PBMCs), may identify molecules responsible for protection and pathogenesis of tuberculosis. Genomes of complex pathogenic organisms like M. tuberculosis can encode several thousand proteins and most of them are shared with nonpathogenic mycobacteria and the vaccine strains of M. bovis BCG 13. However, genomic comparisons have identified 11 genomic regions in M. tuberculosis, which are deleted in all vaccine strains of M. bovis BCG 14. These regions of difference (RD) encompass >80 kb genomic DNA of M. tuberculosis and are predicted to have 89 open reading frames (ORFs) capable of encoding an equal number of proteins 14,15.

2 MUSTAFA & AL-ATTIYAH: IDENTIFICATION OF M. TUBERCULOSIS SPECIFIC GENOMIC REGIONS 499 In the recent years, because of specificity to M. tuberculosis, the proteins encoded by various M. tuberculosis RDs have been a major focus for diagnostic applications using both cellular and humoral immune response parameters 16, 17. However, because of their specificity for M. tuberculosis, the antigenic proteins encoded by M. tuberculosis RDs may also be responsible for inducing protective immunity and/or be responsible for disease susceptibility and pathogenesis of M. tuberculosis 17. One of the ways to differentiate between antigens which mediate protective immunity versus those mediating pathogenesis is to study the secretion of protective Th1 cytokine IFN-γ and pathologic Th2 cytokine IL-10 using cell populations containing lymphocytes and monocytes/macrophages, e.g. PBMCs In this study, we have attempted to identify those RDs of M. tuberculosis, which may differentially mediate the protective and pathologic responses in tuberculosis by studying Th1 responses (antigeninduced proliferation and secretion of IFN-γ) and Th2 responses (secretion of IL-10), respectively, by using PBMCs from pulmonary tuberculosis patients and healthy blood donors. In addition, in peptide mixing experiments, we have also determined the effect of IL-10 inducing RDs on secretion of IFN-γ by PBMCs in response to RD that induced primarily Th1 responses. Materials and Methods Overlapping synthetic peptides covering the sequence of putative proteins encoded by genes predicted in M. tuberculosis RDs Overlapping synthetic peptides (25-mers overlapping neighboring peptides by 10 amino acids) covering the sequence of putative proteins encoded by 89 genes predicted in the genomic regions of RD1, RD4, RD5, RD6, RD7, RD9, RD10, RD11, RD12, RD13 and RD15 were designed based on the amino acid sequence deduced from the nucleotide sequence of the respective genes 14, and synthesized by Thermo Hybaid GmbH (Ulm, Germany) using fluonerylmethoxycarbonyl chemistry 18. The stock concentrations (5 mg/ml) of peptides were prepared in normal saline (0 9%) by vigorous pipetting, and the working concentrations were prepared by further dilution in tissue culture medium RPMI-1640, as described earlier 19,20. Peptides corresponding to each RD were pooled and designated as RD1 pool, RD4 pool and RD5 pool etc. (Table 1). Isolation of PBMCs from tuberculosis patients and healthy subjects and in vitro culture for antigeninduced proliferation and cytokine secretion Heparinized venous blood was obtained from 66 pulmonary tuberculosis patients (attending the Chest Diseases Hospital, Kuwait) and 55 healthy subjects (donating blood at the Central Blood Bank, Kuwait). Informed consent was obtained from all the subjects, and the study protocol was approved by the Ethical Committee of the Faculty of Medicine, Kuwait University, Kuwait. Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood by flotation on Lymphoprep gradients using procedures described previously 21,22. The isolated cells were washed three times with 10 ml of tissue culture medium RPMI-1640 and finally suspended in 1 ml complete tissue culture medium [RPMI % human AB serum + penicillin (100 U/mL) + streptomycin (100 μg/ml + gentamycin (40 μg/ml) + fungizone (2 5 μg/ml)]. The cell counts were determined using a Coulter Counter (Coulter Electronics Ltd, Luton, Bedfordshire, UK) and cell viability was determined by trypan blue exclusion technique 18. PBMCs were cultured in 96-well tissue culture plates (Nunc, Denmark) in vitro for peptide-induced proliferation and cytokine secretion in the absence and presence of peptide pools of various RDs, as described earlier 23,24. In brief, PBMCs ( ) suspended in 50 μl of complete tissue culture medium were seeded into the wells of 96-well tissue Table 1 Responders in proliferation with PBMCs obtained from pulmonary tuberculosis patients (n=66) and healthy blood donors (n=45) in response to peptide pools of M. tuberculosis RDs Peptide pool Proliferation responders with PBMCs obtained from Pulmonary tuberculosis Healthy blood donors patients No. positive/ % No. % tested responders positive/tested responders RD1 pool 49/ /45 62 RD4 pool 33/ /45 27 RD5 pool 20/ /45 18 RD6 pool 28/ /45 29 RD7 pool 31/ /45 51 RD9 pool 29/ /45 42 RD10 pool 25/ /45 36 RD11 pool 19/ /45 29 RD12 pool 25/ /45 44 RD13 pool 15/ /45 44 RD15 pool 18/ /45 18

3 500 INDIAN J EXP BIOL, JUNE 2009 culture plates. Pools of peptides corresponding to each RD were added at optimal concentrations to the wells in triplicates. A set of triplicate wells in each plate lacked the peptides and served as control. The final volume of the culture in each well was adjusted to 200 μl. The plates were incubated at 37 C in a humidified atmosphere containing 5% CO 2 and 95% air. On day 6, culture supernatants were collected from each well and frozen at 20 C until used to determine cytokine concentrations. The remaining cultures were pulsed with radioactive thymidine ( 3 [H]-thymidine), harvested after 4 h of incubation at 37 C and processed to determine the incorporation of radioactive thymidine, as described previously 25,26. Interpretation of peptide-induced proliferation results The radioactivity incorporated by PBMCs in cultures was obtained as counts per minute (cpm). The average cpm was calculated from triplicate cultures stimulated with each peptide pool, as well as from triplicate wells of negative control cultures lacking peptides. The cell proliferation results were calculated as stimulation index (SI), defined as: Sl = cpm in peptide-stimulated cultures/cpm in cultures lacking peptide. A subject was considered responder to a given peptide pool, if PBMCs yielded SI > 3 (Ref. 18). Based on per cent (%) responders (number of subjects responding 100/number of subjects tested) in peptide-induced proliferation assays, the peptide pools were considered strong (>60% responders), median (>40 to 60% responders) or weak (<40% responders) stimulators of PBMCs 24. Immunoassay for quantitation of cytokines in culture supernatants The frozen culture supernatants were thawed and assayed for concentrations of IFN-γ and IL-10 using ELISA and flow cytometry, respectively, using commercial kits, as described previously 18. In response to peptide stimulation, the values with E/C > 3 were considered a positive response 18. E/C is defined as: E/C = Cytokine concentration in peptide-stimulated cultures/cytokine concentration in cultures lacking peptides Based on % responders in peptide-induced cytokine assays, the peptide pools were considered strong (>60% responders), median (>40 to 60% responders) or weak (<40% responders) stimulators of PBMCs 24. Statistical analysis The statistical analysis was performed using Z test to identify significant differences (P< 0.05) with respect to % responders in antigen-induced proliferation, IFN-γ and IL-10 secretion assays in response to the peptide pool of each RD in tuberculosis patients vs. healthy donors. In addition, the same analysis was performed with the data to determine the significant differences (P<0.05) among peptide pools of various RDs in tuberculosis patients and healthy donors. Results In peptide-induced proliferation assays, PBMCs from tuberculosis patients responded strongly to RD1 pool (74%), moderately to RD4 pool (53%), RD6 pool (45%), RD7 pool (51%) and RD9 pool (48%) and weakly (23 to 38%) to all other peptide pools (Table 1). Furthermore, PBMCs from healthy donors responded strongly to RD1 pool (62%), moderately to RD7 pool (51%), RD9 pool (42%), RD12 pool (44%) and RD13 pool (44%) and weakly (18 to 36%) to the remaining peptide pools (Table 1). The statistical analyses of the proliferation data showed that only RD4 pool induced significantly higher responders in tuberculosis patients than in healthy donors (P<0.05), and only RD1 pool, in tuberculosis patients, yielded significantly higher responders than the peptide pools of other RDs (P<0.05). In IFN-γ secretion assays, PBMCs from tuberculosis patients responded moderately to RD1 pool (48%), and weakly (11 to 28%) to all other peptide pools. Whereas, PBMCs from healthy donors responded moderately to RD1 pool (56%), RD7 pool (40%) and RD9 pool (42%) and weakly (2% to 24%) to all other the peptide pools (Table 2). The statistical analysis of IFN-γ data showed that % responders to RD1 pool were significantly higher than all other peptide pools in both groups (P<0.05), except RD7 pool and RD9 pool in healthy donors (P>0.05). In IL-10 secretion assays, PBMCs from tuberculosis patients responded strongly to RD12 pool (83%), moderately to RD9 pool (44%) and RD13 pool (50%) and weakly (22% to 33%) to all other peptide pools (Table 3), whereas, PBMCs from healthy donors responded moderately to both the peptide pools, RD12 pool and RD13 pool (44%) and weakly (6 to 28%). to all other peptide pools. The statistical analysis of IL-10 data showed that, as compared to other peptide pools, RD12 pool induced significantly higher responders in tuberculosis patients (P<0.05). The results presented above showed that RD1 pool was the best stimulator of IFN-γ secretion, whereas RD12 pool and RD13 pool stimulated maximum IL-10

4 MUSTAFA & AL-ATTIYAH: IDENTIFICATION OF M. TUBERCULOSIS SPECIFIC GENOMIC REGIONS 501 Table 2 Responders in IFN-γ secretion assays with PBMCs obtained from pulmonary tuberculosis patients (n=66) and healthy blood donors (n=45) in response to peptide pools of M. tuberculosis RDs Peptide pool IFN -γ responders with PBMCs obtained from Pulmonary tuberculosis Healthy blood donors patients No. % No. % positive/tested responders positive/tested responders RD1 pool 32/ /45 56 RD4 pool 13/ /45 7 RD5 pool 7/ /45 2 RD6 pool 13/ /45 7 RD7 pool 13/ /45 40 RD9 pool 17/ /45 42 RD10 pool 15/ /45 22 RD11 pool 8/ /45 13 RD12 pool 8/ /45 24 RD1 pool 7/ /45 22 RD15 pool 6/66 9 6/45 13 Table 3 Responders in IL-10 secretion assays with PBMCs obtained from pulmonary tuberculosis patients (n=18) and healthy blood donors (n=18) in response to peptide pools of M. tuberculosis RDs Peptide pool IL -10 responders with PBMCs obtained from Pulmonary tuberculosis Healthy blood donors patients No. positive/ % No. positive/ % tested responders tested responders RD1 pool 4/ /18 17 RD4 pool 6/ /18 6 RD5 pool 6/ /18 17 RD6 pool 5/ /18 6 RD7 pool 5/ /18 22 RD9 pool 8/ /18 17 RD10 pool 5/ /18 11 RD11 pool 5/ /18 28 RD12 pool 15/ /18 44 RD13 pool 9/ /18 44 RD15 pool 4/ /18 11 responses with PBMCs of tuberculosis patients and healthy donors. To determine the possible inhibitory effect of IL-10 secreted in response to RD12 pool and RD13 pool on RD1 pool -induced IFN-γ secretion by PBMCs, peptide mixing experiments were performed with PBMCs from 10 additional healthy donors. The results further confirmed that, when tested alone, only RD1 pool induced IFN-γ secretion (IFN-γ concentration = IU/mL), whereas RD12 pool and IL-13 Fig. 1 IFN-γ secretion by PBMCs obtained from healthy donors (n=10) in response to RD1 pool, RD12 pool and RD13 pool, and the effects of adding RD12 pool or RD13 pool on the secretion of IFN-γ by PBMCs in response to RD1 pool. PBMCs obtained from 10 healthy donors were cultured for antigen-induced IFN-γ secretion in the presence of RD1 pool, RD12 pool and RD13 pool alone, and by mixing RD1 pool with RD12 pool or RD13 pool. [Values are mean + SE, represented by bars and vertical lines. IFN-γ concentration is given in IU/mL. The percentage inhibition (IFN-γ concentration in the presence of RD1 pool plus RD12 pool or RD13 pool х 100/IFN-γ concentration in the presence of RD1 pool alone) values are given in numbers on the top of corresponding bars] RD1 pool failed to secrete IFN-γ (IFN-γ concentration = < 1 IU/mL) by the tested PBMCs (Fig. 1). Moreover, addition of RD12 pool and RD13 pool to cultures containing the peptides of RD1 pool inhibited RD1 pool - induced IFN-γ secretion by PBMCs (% inhibition = 53 and 21%, respectively; Fig. 1). Discussion In the present study, PBMCs from pulmonary tuberculosis patients and healthy blood donors were tested for cellular immune responses, i.e. Th1 reactivity (by assessing antigen induced proliferation and IFN-γ secretion) and Th-2 reactivity (by assessing IL-10 secretion) to peptide pools of 89 ORFs corresponding to 11 M. tuberculosis RDs deleted in all vaccine strains of M. bovis BCG 14. The results show that in both donor groups, the highest Th1- responses in terms of % responders were induced by RD1 pool and the highest Th2 responses were induced by RD12 pool and RD13 pool. Because, we included patients with pulmonary tuberculosis, it may be

5 502 INDIAN J EXP BIOL, JUNE 2009 argued that cells from lung tissue may be more suitable than PBMCs to study cellular and cytokine responses in the patient group. However, it has been shown in mice that detection of antigen-specific IFNγ secretion from blood cultures accurately reflects lung responses, indicating that blood can be an appropriate source of cells to study antigen-specific cellular and cytokine responses in humans as well 8. In the present study, to stimulate PBMCs for Th1 and Th2 responses, we have used overlapping synthetic peptides covering the sequence of ORFs predicted in the RDs studied Other theoretical possibilities include the use of full-length proteins as antigens, which may be purified from cultures of M. tuberculosis 27,28, or produced by recombinant expression and purification However, the isolation of putative full-length proteins, as is the case with most of the RD ORFs, from cultures of M. tuberculosis is almost impossible because of the non-availability of reagents to purify proteins encoded by the predicted genes, many of which are hypothetical. Similarly, the recombinant approach is hampered due to several technological problems, e.g. high G+C content of M. tuberculosis DNA makes it difficult to amplify the large size ORFs prior to cloning them in suitable plasmids, the degradation of foreign proteins by host s proteases and contamination with host proteins etc The approach of synthetic peptides overcomes these problems as peptides could be chemically synthesized and purified using available techniques Furthermore, it has been previously shown that overlapping synthetic peptides can faithfully replace full-length proteins to study cellular immune responses In addition, to minimize missing cellular immune responses to epitopes present in full-length proteins, we have employed 10 aa overlap between the peptides because most mycobacterial epitopes recognized in cellular immune responses are peptides 10 aa in length 40. We have studied peptide-induced proliferation and IFN-γ secretion by PBMCs as indicators of protective Th1-cell reactivity. Although, both of these assays have been used previously as readouts for Th1-cell reactivity 18-28, the importance of IFN-γ as the primary Th1 cytokine that mediates protective immunity against mycobacterial diseases has been clearly demonstrated in various animal models of tuberculosis 2. Furthermore, reduced antigen-specific IFN-γ secretion and low frequencies of IFN-γresponding cells from peripheral blood predict increased risk of tuberculosis disease progression across genetically diverse tuberculosis diseasesusceptible mouse strains, suggesting that similar results may occur in humans 8. This suggestion is actually supported by studies in humans, which show strong IFN-γ responses in PPD-positive healthy subjects and tuberculosis patients with minimal disease, compared to weak IFN-γ responses in tuberculosis patient with advanced disease 5, 13, and the improvement in IFN-γ responses following successful drug therapy of active tuberculosis patients 4. These studies suggest that IFN-γ plays an important role in protection against human tuberculosis and therefore antigens in RD1 pool, which induced highest IFN-γ responses, could be instrumental in inducing protective immunity. The major antigens of RD1 pool, which induce strong Th1 (antigen-induced proliferation and IFN-γ responses) are CFP-10, ESAT-6 and PPE68 (Refs 21-24) and immunizations with preparations containing/expressing CFP-10 and ESAT-6 protect mice against M. tuberculosis challenge Moreover, vaccinations of mice and guinea-pigs with RD1-constructs of BCG and M. microti induce ESAT-6 and CFP-10-specific immune responses and provide better protection against M. tuberculosis challenge than protection afforded by vaccination with BCG and M. microti alone 44,45. However, CFP-10 and ESAT-6 have also been implicated in the virulence of M. tuberculosis 46, and an RD1-construct of BCG grows more rapidly in severe combined immunodeficient mice, an extreme model of immunodeficiency, than does its parental BCG strain 47, but in immunocompetent mice and guinea-pigs only a slight increase in persistence and no increase in pathology has been observed 30. The property of RD1 to make recombinant BCG to persist longer than the parent BCG may be beneficial rather than harmful, because a longer persistent may be induce long-lasting immunity to RD1 antigens encoded by recombinant BCG 3. With respect to Th2 reactivity, stimulation of PBMCs with RD12 pool and RD13 pool resulted into highest IL-10 responders in both tuberculosis patients and healthy donors (Table 3). In addition, peptide mixing experiments performed to determine the effect of IL-10 secretion by PBMCs in response to RD12 pool and RD13 pool on RD1 pool induced IFN-γ secretion showed that RD12 pool and RD13 pool inhibited secretion of IFN-γ by PBMCs of healthy donors in response to

6 MUSTAFA & AL-ATTIYAH: IDENTIFICATION OF M. TUBERCULOSIS SPECIFIC GENOMIC REGIONS 503 RD1 pool (Fig. 1). Interestingly, the extent of this inhibition correlated with their ability to induce secretion of IL-10, i.e. the inhibition was stronger with RD12 pool than the inhibition observed with RD13 pool (Fig. 1), and quantitatively, RD12 pool induced secretion of more IL-10 than RD13 pool 18. Because IL-10 helps maintain mycobacterial infections by acting primarily at the level of macrophages and overrides anti-mycobacterial signals delivered by IFN-γ 12. Therefore it is imperative that IL-10 inducing antigens present in RD12 pool and RD13 pool should be avoided in any future vaccine formulations against tuberculosis. In conclusion, our study suggests that RDs of M. tuberculosis can be divided into two major groups. The first group, represented by RD1, activates PBMCs to preferentially secrete the protective cytokine IFN-γ, and the second group, represented by RD12 and RD13, activates preferential secretion of the pathologic cytokine IL-10 and suppresses the secretion of IFN-γ in response to peptides of the first group. Therefore, the first group may have roles in protection and the second group in pathogenesis of tuberculosis. Acknowledgement This work was supported by the Kuwait Foundation for the Advancement of Sciences (KFAS) grant no We acknowledge with thanks the support from Drs. F. Al Saidi and B. Jayakrishnan from the Chest Diseases Hospital, Ministry of Health, Kuwait for providing blood from tuberculosis patients, and the Director of Central Blood Bank, Kuwait for providing blood from healthy donors. References 1 Lönnroth K & Raviglione M, Global epidemiology of tuberculosis: Prospects for control, Semin Respir Crit Care Med, 29 (2008) Flynn J, Immunology of tuberculosis and implications in vaccine development, Tuberculosis, 4 (2004) Mustafa A S, Progress towards the development of new antituberculosis vaccines, in Focus on Tuberculosis Research, edited by Lucy T Smithe (Nova Science Publishers Inc, NY, USA) 2005, Al-Attiyah R, Mustafa A S, Abal A T, Madi N M & Andersen P, Restoration of mycobacterial antigen-induced proliferation and interferon-gamma responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy, FEMS Immunol Med Microbiol, 38 (2003) Al-Attiyah R, Madi N, El-Shamy A S, Herald W, Andersen P & Mustafa A S, Cytokine profiles in tuberculosis patients and healthy subjects in response to complex and single antigens of Mycobacterium tuberculosis, FEMS Immunol Med Microbiol, 47 (2006) Al-Attiyah R, El-Shazly A & Mustafa A S, Assessment of in vitro immunity to Mycobacterium tuberculosis in a human peripheral blood infection model using a luciferase reporter construct of M. tuberculosis H37Rv, Clin Exp Immunol, 145 (2006) Bai, X, Wilson S E, Chmura K, Feldman N E & Chan E D, Morphometric analysis of Th(1) and Th(2) cytokine expression in human pulmonary tuberculosis, Tuberculosis, 84 (2004) Beamer G L, Flaherty D K, Vesosky B &Turner J, Peripheral blood gamma interferon release assay predict lung responses and Mycobacterium tuberculosis disease outcome in mice, Clin Vaccine Immunol, 15 (2008) Turner J, Gonzalez-Juarrero M, Ellis D L, Basarba R J, A. Kipnis A, Orme I M & Cooper A M, In vivo IL-10 production reactivates chronic pulmonary tuberculosis in C57BL/6 mice, J Immunol, 169 (2002) Beamer G L, Flaherty D K, Assogba B D, Stromberg P, Gonzalez-Juarrero M, de Waal Malefyt R, Vesosky B &Turner J, Interleukin-10 promotes Mycobacterium tuberculosis disease progression in CBA/J mice, J Immunol, 181 (2008) Sai Priya V H, Anuradha B, Latha Gaddam S, Hasnain S E, Murthy K J & Valluri V L, In vitro levels of interleukins IL-10 and IL-12 in response to Mycobacterium bovis BCG r32kda antigen after treatment for tuberculosis, Clin Vaccine Immunol, 16 (2009) Murray P J & Young R A, Increased antimycobacterial immunity in interleukin-10 deficient mice, Infect Immun, 67 (1999) Mustafa A S, Biotechnology in the development of new vaccines and diagnostic reagents against tuberculosis, Curr Pharm Biotechnol, 2 (2001) Behr M A, Wilson M A, Gill W P, Salamon H, Schoolnik G K, Rane S & Small P M, Comparative genomics of BCG vaccines by whole-genome DNA microarray, Science, 284 (1999) Mustafa A S, Development of new vaccines and diagnostic reagents against tuberculosis, Mol Immunol, 39 (2002) Mustafa A S, Recombinant and synthetic peptides to identify Mycobacterium tuberculosis antigens and epitopes of diagnostic and vaccine relevance, Tuberculosis (Edinb), 85 (2005) Mustafa A S, Mycobacterial gene cloning and expression, comparative genomics, bioinformatics and proteomics in relation to the development of new vaccines and diagnostic reagents, Med Princ Pract, 14 Suppl 1 (2005) Al-Attiyah R & Mustafa A S, Characterization of human cellular immune responses to novel Mycobacterium tuberculosis antigens encoded by genomic regions absent in Mycobacterium bovis BCG, Infect Immun, 76 (2008) Mustafa A S, Abal A T, Shaban F, El-Shamy A M & Amoudy H A, HLA-DR binding prediction and experimental evaluation of mycolyltransferase (Ag85B), a major secreted antigen of Mycobacterium tuberculosis, Med Princ Pract, 14 (2005) 140.

7 504 INDIAN J EXP BIOL, JUNE Mustafa A S & Shaban F A, Propred analysis and experimental evaluation of promiscuous Th1 cell epitopes of three major secreted antigens of Mycobacterium tuberculosis, Tuberculosis (Edinb), 86 (2006) Mustafa A S, El-Shamy A M, Madi N M, Amoudy H A & Al-Attiyah R, Cell-mediated immune responses to complex and single mycobacterial antigens in tuberculosis patients with diabetes, Med Princ Pract, 17 (2008) Mustafa A S, Th1-cell reactivity and HLA-DR binding prediction for promiscuous recognition of MPT63 (Rv1926c), a major secreted protein of Mycobacterium tuberculosis, Scand J Immunol, 69 (2009) Hanif S N M, El-Shamy A M, Al-Attiyah R & Mustafa A S, Whole blood assays to identify Th1 cell antigens and peptides encoded by Mycobacterium tuberculosis-specific RD1 genes, Med Princ Pract, 17 (2008) Mustafa A S, Al-Attiyah R, Hanif S N M & Shaban F A, 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 (2008) Mustafa A S, Shaban F A, Abal A T, Al-Attiyah R, Wiker HG, Lundin K E A, Oftung F & Huygen K, Identification and HLA restriction of naturally derived Th1-cell epitopes from the secreted Mycobacterium tuberculosis antigen 85B recognized by antigen-specific human CD4+ T cell lines, Infect Immun, 68 (2000) Al-Attiyah R, Mustafa A S, Abal A T, El-Shamy A M, Dalemans W & Skeiky Y A W, In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis, Clin Exp Immunol, 138 (2004) Mustafa A S, Amoudy H A, Wiker H G, Abal A T, Ravn P, Oftung F & Andersen P, Comparison of antigen-specific T- cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis, Scand J Immunol, 48 (1998) Al-Attiyah R, Shaban F A, Wiker H G, Oftung F & Mustafa A S, Synthetic peptides identify promiscuous human Th1 cell epitopes of the secreted mycobacterial antigen MPB70, Infect Immun, 71 (2003) Mustafa A S, Oftung F, Amoudy H A, Madi N M, Abal A T, Shaban F, Rosen Krands I & Andersen P, Multiple epitopes from the Mycobacterium tuberculosis ESAT-6 antigen are recognized by antigen-specific human T cell lines, Clin Infect Dis, 30 Suppl 3 (2000) S Mustafa A S, Cockle P J, Shaban F, Hewinson R G & Vordermeier H M, Immunogenicity of Mycobacterium tuberculosis RD1 region gene products in infected cattle, Clin Exp Immunol, 130 (2002) Mustafa A S, Shaban F A, Al-Attiyah R, Abal A T, El- Shamy A M, Andersen P & Oftung F, Human Th1 cell lines recognize the Mycobacterium tuberculosis ESAT-6 antigen and its peptides in association with frequently expressed HLA class II molecules, Scand J Immunol, 57 (2003) Mustafa A S, Skeiky Y A, Al-Attiyah R, Alderson M R, Hewinson R G & Vordermeier H M, Immunogenicity of Mycobacterium tuberculosis antigens in Mycobacterium bovis BCG-vaccinated and M. bovis-infected cattle, Infect Immun, 74 (2006) Ahmad S, Amoudy H A, Thole J E, Young D B & Mustafa A S, Identification of a novel protein antigen encoded by a Mycobacterium tuberculosis-specific RD1 region gene, Scand J Immunol, 49 (1999) Ahmad S, Akbar P K, Wiker H G, Harboe M & Mustafa A S, Cloning, expression and immunological reactivity of two mammalian cell entry proteins encoded by the mce1 operon of Mycobacterium tuberculosis, Scand J Immunol, 50 (1999) Ahmad S, Ali M M & Mustafa A S, Construction of a modified vector for efficient purification of recombinant Mycobacterium tuberculosis proteins expressed in Escherichia coli, Protein Exp Purif, 29 (2003) Ahmad S, El-Shazly S, Mustafa A S & Al-Attiyah R, Mammalian cell-entry proteins encoded by the mce3 operon of Mycobacterium tuberculosis are expressed during natural infection in humans, Scand J Immunol, 60 (2004) Ahmad S, El-Shazly S, Mustafa A S & Al-Attiyah R, The six mammalian cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during in vitro growth of Mycobacterium tuberculosis, Scand J Immunol, 62 (2005) Amoudy H A, Al-Turab M B & Mustafa A S, Identification of transcriptionally active open reading frames within the RD1 genomic segment of Mycobacterium tuberculosis, Med Princ Pract, 15 (2006) Amoudy H A & Mustafa A S, 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 (2008) Mustafa A S, HLA-restricted immune response to mycobacterial antigens: Relevance to vaccine design, Hum Immunol, 61 (2000) Fan X, Gao Q & Fu R, DNA vaccine encoding ESAT-6 enhances the protective efficacy of BCG against Mycobacterium tuberculosis infection in mice, Scand J Immunol, 66 (2007) Kalra M, Grover A, Mehta N, Singh J, Kaur J, Sable S B, Behera D, Sharma P, Verma I & Khuller G K, Supplementation with RD antigens enhances the protective efficacy of BCG in tuberculous mice, Clin Immunol, 125 (2007) Wu Y, Woodworth J S, Shin D S, Morris S & Behar S M, Vaccine-elicited 10-kilodalton culture filtrate protein-specific CD8+ T cells are sufficient to mediate protection against Mycobacterium tuberculosis infection, Infect Immun, 76 (2008) Pym A S, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths K E, Marchal G, Leclerc C & Cole S T, Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis, Nat Med, 9 (2003) Brodin P, Majlessi L, Brosch R, Smith D, Bancroft G, Clark S, Williams A, Leclerc C & Cole S T, Enhanced protection against tuberculosis by vaccination with recombinant Mycobacterium microti vaccine that induces T cell immunity against region of difference 1 antigens, J Infect Dis, 190 (2004) Ganguly N, Siddiqui I & Sharma P, Role of M. tuberculosis RD-1 region encoded secretory proteins in protective response and virulence, Tuberculosis (Edinb), 88 (2008) Pym A S, Brodin P, Brosch R, Huerre M & Cole S T, Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti, Mol Microbiol, 46 (2002) 709.