Adnan T. Abal

Comparison of Antigen-Specific T-Cell Responses of Tuberculosis Patients using Complex or Single Antigens of Mycobacterium tuberculosis

We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon‐γ (IFN‐γ) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT‐6, nMPT59, nMPT64 and nMPB70) and somatic‐derived (rGroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis. The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole‐cell M. tuberculosis, M. tuberculosis culture filtrate (MT‐CF) and cell wall antigens, as well as the vaccine strain, Mycobacterium bovis bacillus Calmette–Guérin (BCG). In addition, M. tuberculosis and MT‐CF‐induced T‐cell lines were tested in the same assays against the panel of purified and complex antigens. The compiled data from PBMC and T‐cell lines tested for antigen‐induced proliferation and IFN‐γ secretion showed that the most frequently recognized antigen was ESAT‐6, followed by MPT59, GroES, MPB70, MPT64, DnaK, GroEL and PstS. The frequency of ESAT‐6 responders, as measured both by proliferation (18/19) and secretion of IFN‐γ (16/19) was comparable to the results obtained with whole‐cell M. tuberculosis, MT‐CF and M. bovis BCG. We also observed that most of the high responders to complex antigens recognized all of the antigens tested (covariation), demonstrating that the repertoire of human T‐cell specificities induced by natural infection is directed towards several unrelated culture filtrate as well as somatic‐derived protein antigens. In conclusion, the results obtained suggest that the cellular immune response in humans is directed against several important target antigens of M. tuberculosis and that some antigens, such as ESAT‐6, are recognized by a high number of individuals. Such antigens represent candidates to be used for development of specific diagnostic reagents or in subunit vaccines.

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

ABSTRACT Antigen 85B (Ag85B/MPT59) is a major secreted protein fromMycobacterium tuberculosis which is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes from M. tuberculosis Ag85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4+ T-cell lines were established from HLA-typed TB patients and Mycobacterium bovis BCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-γ) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-γ assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the self HLA-DRB1,DRB3, or DRB4 gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from the M. tuberculosisAg85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.

Prevalence of asthma, allergic rhinitis, and eczema in 13- to 14-year-old children in Kuwait : an ISAAC study

OBJECTIVES To determine the prevalence of asthma and allergic diseases in 13 to 14 years old children in Kuwait. DESIGN Supervised self-administered written and video questionnaires of the international study of asthma and allergies in childhood (ISAAC). SUBJECTS Students at third and fourth years from 40 intermediate level schools chosen randomly from across Kuwait. RESULTS 3,110 students were surveyed. The prevalence rates (95% CI) in the written questionnaire for wheeze ever, current wheeze (within the last 12 months), and physician diagnosis of asthma are 25.9% (24.5 to 27.4), 16.1% (15.8 to 17.4), and 16.8% (15.5 to 18.1) respectively. The prevalence rates (95% CI) for symptoms of allergic rhinitis (AR) ever, current symptoms of allergic rhinitis (AR), and diagnosis of AR are 43.9% (42.2 to 45.6), 30.7% (29.1 to 32.4) and 17.1% (14.8 to 18.4) respectively. The prevalence rates (95% CI) for itchy rash ever, current itchy rash, and diagnosis of eczema are 17.5% (16.2 to 18.8), 12.6% (11.4 to 13.8), and 11.3% (10.2 to 12.4) respectively. The prevalence of wheeze ever, wheeze during the last year, and physician diagnosis of asthma are higher in males compared with females, P < .01. In multiple logistic regression: male gender (OR 1.6, 95% CI, 1.3 to 2.0) and diagnosis of AR (OR 1.7, 95% CI, 1.4 to 2.2) were associated with the physician diagnosis of asthma even after controlling for symptoms of asthma. CONCLUSION This is the first study on the prevalence of allergic diseases in Kuwait and it shows that children in Kuwait have a moderate prevalence of asthma, AR, and eczema compared with other countries where the ISAAC study is done. The prevalence of asthma is higher in boys compared with girls.

Restoration of mycobacterial antigen‐induced proliferation and interferon‐γ responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy

Peripheral blood mononuclear cells (PBMC) were obtained from culture-proven tuberculosis (TB) patients before and after 2 and 6 months of chemotherapy with a multi-drug regimen. PBMC were tested for cellular responses in antigen-induced proliferation and interferon-gamma (IFN-gamma) assays in response to complex mycobacterial antigens (whole cell Mycobacterium bovis BCG and M. tuberculosis, cell walls and short-term culture filtrate [ST-CF] of M. tuberculosis), fractionated ST-CF antigens (fractions F1-F10) and ESAT-6. The responses in TB patients before anti-TB treatment were low (median stimulation index (SI)=1-7, median delta IFN-gamma=0-12 U ml(-1), and percent responders=13-67%) to all the antigenic preparations. Following the administration of anti-TB chemotherapy for 2 months, there were significant (P<0.05) improvements in the cellular responses (median SI=9-76, median delta IFN-gamma=3-70 U ml(-1), and percent responders=33-100%) to most of the antigenic preparations tested. However, concanavalin A-induced proliferation responses of PBMC from the same patients before and after 2 months of chemotherapy were high and comparable (median SI=101 and 114, respectively, P>0.05, 100% responders). A further increase in IFN-gamma responses (median delta IFN-gamma=14-250 U ml(-1) and percent responders=43-100%) to mycobacterial antigens was observed in patients receiving chemotherapy for 6 months. Among the ST-CF fractions, F1 and F2 containing low molecular mass proteins resulted in the highest responses, whereas ESAT-6 showed responses comparable to these fractions only in a minority of the patients. HLA-DR typing of these patients showed heterogeneity in the expression of molecules encoded by HLA-DRB genes. These results show that effective chemotherapy restores cellular responses of TB patients to a large number of M. tuberculosis antigens, which could be useful in monitoring the efficacy of anti-TB treatment.

Human Th1 Cell Lines Recognize the Mycobacterium tuberculosis ESAT-6 Antigen and its Peptides in Association with Frequently Expressed HLA Class II Molecules

We have used a synthetic‐peptide approach to map epitope regions of the Mycobacterium tuberculosis ESAT‐6 antigen recognized by human T cells in relation to major histocompatibility complex (MHC) restriction. ESAT‐6‐specific CD4+ T‐cell lines were established by stimulating peripheral blood mononuclear cells from 25 HLA‐DR‐typed tuberculosis patients with complete antigen in vitro. The established T‐cell lines were then screened for proliferation and interferon‐γ (IFN‐γ) secretion in response to eight overlapping 20‐mer peptides covering the ESAT‐6 sequence. The response of the T‐cell lines to ESAT‐6 and peptides from a human leucocyte antigen (HLA)‐heterogeneous group of donors suggested the presence of multiple epitopes and promiscuous recognition of the antigen. Analysis of antigen and peptide recognition in the presence of anti‐HLA class I and class II antibodies suggested that the T‐cell lines recognized ESAT‐6 in association with HLA‐DR and ‐DQ molecules. Furthermore, testing of selected T‐cell lines with ESAT‐6 and the peptides in the presence of autologous and allogeneic HLA‐DR‐ and ‐DQ‐typed antigen‐presenting cells identified HLA‐DR2, ‐DR52 and ‐DQ2 amongst the HLA molecules involved in the presentation of ESAT‐6 and its peptides to human Th1 cells. In addition, the T‐cell lines were cytotoxic for monocytes and macrophages pulsed with ESAT‐6 and peptides. In conclusion, the recognition of ESAT‐6 by IFN‐γ‐secreting and cytotoxic CD4+ T cells in association with frequently expressed HLA class II molecules supports the application of this antigen to either specific diagnosis or subunit vaccine design.

In vitro cellular immune responses to complex and newly defined recombinant antigens of Mycobacterium tuberculosis

The immunological diagnosis and development of new antituberculosis vaccines require the characterization of Mycobacterium tuberculosis antigens inducing cell‐mediated immune responses. In this study, we have tested peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients (n = 43) and Bacille Calmette–Guérin (BCG)‐vaccinated healthy subjects (n = 24) for in vitro cellular immune responses, as indicated by antigen‐induced proliferation and interferon (IFN)‐γ secretion, in response to a panel of complex (culture filtrate and cell wall preparations) and single recombinant antigens (Mtb8·4, Mtb9·8, Mtb9·9, Mtb32A, Mtb39A, Mtb40, Mtb41 and Ag85B) of M. tuberculosis. The results of cellular responses showed that the majority (ranging from 70 to 98%) of TB patients and healthy donors responded to the complex antigens in antigen‐induced proliferation and IFN‐γ secretion assays. However, when PBMC from the same groups of patients and healthy donors were tested with the recombinant antigens, TB patients showed strong recognition (>50% responders) of Mtb9·8 and Mtb39A in proliferation assays (median SI = 6·2 and 6·4, respectively) and of Mtb9·8, Mtb39A, Mtb40 and Ag85B in IFN‐γ assays (median delta IFN‐γ= 15·5, 10·8, 7·8 and 8·1 U/ml, respectively). BCG‐vaccinated healthy donors showed weak (<30% responders) to moderate (31–50% responders) responses to all of the recombinant antigens in both assays. When PBMC of a subset of TB patients (n = 11) were tested for secretion of protective Th1 cytokines [IFN‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐12] and the immunosuppressive cytokine IL‐10, the complex CF and CW antigens as well as the recombinant Mtb9·8, Mtb9·9, Mtb40 and Ag85B induced the secretion of both types of cytokines. On the other hand, Mtb41 induced only IL‐10, while Mtb8·4, Mtb32Aand Mtb39A induced the secretion of one or more of Th1 cytokines, but not IL‐10. In conclusion, the recombinant antigens inducing the secretion of Th1 cytokines could be useful as subunit vaccine candidates against TB.

Variations in the Occurrence of the S315T Mutation Within the katG Gene in Isoniazid-Resistant Clinical Mycobacterium tuberculosis Isolates from Kuwait

The worldwide threat of drug-resistant tuberculosis (TB) to human health has led to the development of molecular methods for rapidly determining the resistance of clinical Mycobacterium tuberculosis isolates to the two front-line antituberculous drugs, isoniazid and rifampin. The prevalence of the S315T mutation within the katG gene, which confers clinically significant resistance to isoniazid, was determined in isoniazid-resistant clinical M. tuberculosis isolates recovered from TB patients in Kuwait. A total of 67 isoniazid-resistant and 18 susceptible clinical M. tuberculosis isolates were tested. The mutation S315T was found in 46 (69%) of the 67 resistant strains, whereas none of the susceptible strains contained this mutation. The prevalence of this mutation was highest (32 of 40, 80%) in isolates recovered from patients of South Asian origin and lowest in isolates from patients of Middle Eastern origin (8 of 18, 44%). The genotyping performed on isolates carrying the S315T mutation showed that the isolates belong to several different types as they exhibited unique DNA banding patterns. The results point to a varying prevalence of the S315T mutation within the katG gene in clinical M. tuberculosis isolates recovered from patients of different ethnic groupings within the same country. The results also suggest that detection of the S315T mutation in the katG gene may be used as a rapid screening method for identifying isoniazid-resistant clinical M. tuberculosis isolates recovered from majority of patients in some ethnic groupings.

Establishment and evaluation of a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex

OBJECTIVE To establish a multiplex polymerase chain reaction for detection of mycobacteria and specific identification of Mycobacterium tuberculosis complex and to evaluate the test in the diagnosis of tuberculosis. DESIGN Three sets of primers were used to amplify 383 bp, 240 bp and 131 bp DNA fragments from the genes encoding the 65 kDa, MPB64 and the 19 kDa proteins of M. tuberculosis in a single reaction tube. Reaction conditions were optimized with respect to the requirement of DMSO, concentration of MgCl2, annealing and denaturation temperatures and number of amplification cycles. Inhibitory activity in clinical samples was identified by amplifying a 500 bp DNA fragment of the phage lambda along with the mycobacterial targets within the same reaction tube. The multiplex PCR was evaluated in differentiating M. tuberculosis complex from other mycobacteria and in the diagnosis of tuberculosis by testing clinical specimens. RESULTS Amplification of the 383 bp DNA fragments was specific to the genus Mycobacterium. The 240 bp DNA fragment was amplified from M. tuberculosis complex and M. fortuitum and the 131 bp DNA fragment was amplified from the mycobacteria of M. tuberculosis complex and M. scrofulaceum. All the three bands were amplified only from M. tuberculosis complex. Applicability of the multiplex PCR is demonstrated in differentiating M. tuberculosis complex from other mycobacteria by using standard strains and clinical isolates. The multiplex PCR was also useful in the detection of inhibitory activity and in the identification of M. tuberculosis complex directly in clinical samples. CONCLUSION The multiplex PCR established in this study could differentiate M. tuberculosis complex from other mycobacteria. This test may also be helpful in the early and specific diagnosis of tuberculosis.

HLA-DR binding prediction and experimental evaluation of T-cell epitopes of mycolyl transferase 85B (Ag85B), a major secreted antigen of Mycobacterium tuberculosis.

Objective: To identify T-cell epitopes of Ag85B by analysis of its sequence for prediction to bind HLA-DR alleles and evaluate the predicted peptides for recognition by T cells in antigen-induced proliferation assays. Materials/Subjects and Methods: The complete sequence of Ag85B was analyzed for HLA-DR binding prediction to 51 HLA-DR alleles by using a virtual matrix-based prediction program (ProPred). Synthetic peptides covering the sequence of mature Ag85B were also analyzed for binding to HLA-DR alleles, and evaluated for recognition in antigen-induced proliferation assays with Ag85B-specific T-cell lines established from the peripheral blood mononuclear cells of 10 HLA-DR-heterogeneous tuberculosis patients. Results: The ProPred analysis of the full-length Ag85B (325 aa), signal peptide (40 aa) and the mature protein (285 aa) predicted their binding to 100, 76 and 98% of the 51 HLA-DR alleles, respectively. The analysis of 31 synthetic peptides for binding to HLA-DR alleles showed that 4 of them could bind >50% HLA-DR alleles, and were considered promiscuous. Testing of Ag85B-specific T-cell lines with synthetic peptides showed that all of the T-cell lines responded to one or more peptides of Ag85B, and 9 of the 10 cell lines responded to one or more of the four peptides considered promiscuous for binding to HLA-DR alleles. Conclusion: The ProPred program was useful in predicting the HLA-DR alleles binding regions of Ag85B and identifying the promiscuous peptides recognized by T cells.

Molecular fingerprinting of isoniazid-resistant Mycobacterium tuberculosis isolates from chest diseases hospital in Kuwait.

Touchdown double‐repetitive‐element‐PCR (DRE‐PCR) was carried out for typing 38 consecutive isoniazid‐resistant Mycobacterium tuberculosis strains isolated at Chest Diseases Hospital, Kuwait, during 1998–2000. The polymorphism at codon 463 in the katG gene was also determined and correlated with genotypic relationships among the isolates. The isolates exhibited 21 distinct patterns in DRE‐PCR. Nearly half of the isolates (18 of 38) exhibited unique patterns. Majority of isolates (16 of 20) yielding multiple DNA fragments in DRE‐PCR were unique strains while most of the isolates (16 of 18) yielding a single DNA fragment in DRE‐PCR clustered together. The prevalence of L463 in the katG gene was much higher in isolates from Middle Eastern (mostly Kuwaiti) patients than is reported for this ethnic group. The data indicate the possibility of some strains of South Asian/Southeast Asian origin spreading among local populations.