T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.     

Evaluation of Rv0220, Rv2958c,Rv2994 and Rv3347c of Mycobacterium tuberculosis for serodiagnosis of tuberculosis

Tuberculosis (TB), the leading cause of death among infectious diseases worldwide, is caused by Mycobacterium tuberculosis (M. tuberculosis). Early accurate diagnosis means earlier prevention, treatment and control of TB. To confirm efficient diagnostic antigens for M. tuberculosis, the serodiagnosis value of four recombinant proteins including Rv0220, Rv2958c, Rv2994 and Rv3347c was evaluated in this study. The specificities and sensitivities of four recombinant proteins were determined based on enzyme‐linked immunosorbent assay (ELISA) by screening sera from smear‐positive pulmonary TB patients (n = 92), uninfected individuals (n = 60) and patients with Mycoplasma pneumoniae (n = 32) that potentially cross‐react with M. tuberculosis. The ELISAs showed that Rv0220, Rv2958c, Rv2994 and Rv3347c exhibited high specificities and sensitivities in detecting immunoglobulin G (IgG) antibody, with 98.3/91.3%, 91.7/85.9%, 93.3/89.1% and 93.3/80.4% respectively. According to the receiver‐operating characteristic (ROC) analysis, the area under the ROC of the target proteins was 0.988, 0.969, 0.929 and 0.945 respectively. Western blot was established to evaluate the immunoreactivities of target proteins to mice and human sera. Results demonstrated that Rv0220, Rv2958c, Rv2994 and Rv3347c could specifically recognize TB‐positive sera and the sera of mice immunized with the corresponding protein. Thus, Rv0220, Rv2958c, Rv2994 and Rv3347c were valuable potential diagnostic antigens for M. tuberculosis.     

结核分枝杆菌Rv3425蛋白的原核表达纯化及其血清学诊断价值

目的：用原核系统表达结核分枝杆菌Rv3425蛋白并纯化,评价该重组蛋白在结核病血清学诊断方面的价值。方法：以结核分枝杆菌H37Rv株基因组为模板,PCR扩增得到Rv3425基因序列,克隆至表达载体pET-28a中,转入大肠杆菌BL21（DE3）进行诱导、表达后纯化,用Western印迹和ELISA法进行抗原性初步评价。结果：在原核系统内经IPTG诱导表达后,Rv3425蛋白主要以包涵体形式存在,经复性和镍柱层析纯化后,纯度达95%以上;Western印迹和ELISA结果证明重组Rv3425具有较强的抗原活性;用纯化的Rv3425蛋白做抗原,临床诊断结核病人血清,阳性率达50%。结论：高纯度的Rv3425蛋白在结核病诊断方面具有很高的应用价值,可作为结核病诊断的备选抗原。     

以蛋白激酶G为靶点的抗结核药物筛选模型的建立和初步应用

结核分枝杆菌可以产生11种丝氨酸/苏氨酸蛋白激酶,其中蛋白激酶G(PknG)对于结核分枝杆菌在巨噬细胞内以"持留"状态长期存活有着重要作用。本研究以结核分枝杆菌基因组DNA为模板,在大肠杆菌中克隆表达了MTBPknG蛋白,并分离纯化得到PknG纯酶。本研究还采用三步级联反应方法测定了PknG酶活性,建立和优化了PknG抑制剂高通量筛选模型。利用此模型共筛选发酵液样品2120个,化合物样品2300个,筛选得到阳性化合物1个,阳性发酵液13个,阳性率0.32%。     

Novel serological biomarker panel using protein microarray can distinguish active TB from latent TB infection

BackgroundRapid laboratory technologies which can effectively distinguish active tuberculosis (ATB) from controls and latent tuberculosis infection (LTBI) are lacked.The objective of this study is to explore MTB biomarkers in serum that can distinguish ATB from LTBI.MethodsWe constructed a tuberculosis protein microarray containing 64 MTB associated antigens. We then used this microarray to screen 180 serum samples, from patients with ATB and LTBI, and healthy volunteer controls. Both SAM (Significance analysis of microarrays) and ROC curve analysis were used to identify the differentially recognized biomarkers between groups. Extra 300 serum samples from patients with ATB and LTBI, and healthy volunteer controls were employed to validate the identified biomarkers using ELISA-based method.ResultsAccording to the results, the best biomarker combinations of 4 proteins (Rv1860, RV3881c, Rv2031c and Rv3803c) were selected. The biomarker panel containing these 4 proteins has reached a sensitivity of 93.3% and specificity of 97.7% for distinguishing ATB from LTBI, and a sensitivity of 86% and specificity of 97.6% for distinguishing ATB from HC.ConclusionThe biomarker combination in this study has high sensitivity and specificity in distinguishing ATB from LTBI, suggesting it is worthy for further validation in more clinical samples.     

Cloning, expression, and purification of the 27 kDa (MPT51, Rv3803c) protein of Mycobacterium tuberculosis

A limited number of proteins of Mycobacterium tuberculosis have been characterized so far for their use as potential candidates for diagnosis and vaccine studies. This study was aimed at cloning, expression, and purification of a 27 kDa protein (otherwise known as the MPT51 or Rv3803c protein) of M. tuberculosis. The Rv3803c gene was PCR amplified using primers that contain specific restriction sites. The amplified product was inserted initially into pTOPO and then sub-cloned into pET15b and pET24d vectors, such that the recombinant protein is predicted to contain an N-terminal or a C-terminal histidine tag, respectively. The recombinant plasmids were introduced into Escherichia coli BL21 (DE3) and the recombinant proteins were purified from the cytosolic fractions of the E. coli sonicates by nickel-NTA chromatography. The purity, molecular mass, and the conformation of the proteins were determined by high performance liquid chromatography (HPLC), matrix assisted laser desorption-ionization-time-of-flight (MALDI-TOF), and circular dichroism (CD) studies, respectively. The purified proteins were found to be immunogenic and useful for immunodiagnostic studies of tuberculosis by enzyme linked immunosorbent assay (ELISA), with a sensitivity of 71% and specificity of 95%.     

Construction of a modified vector for efficient purification of recombinant Mycobacterium tuberculosis proteins expressed in Escherichia coli

A major problem in assessing the vaccine and diagnostic potential of various proteins encoded by Mycobacterium tuberculosis genome is the inability to produce large quantities of these proteins, even when Escherichia coli or other heterologous systems are employed for recombinant protein production. To overcome these barriers, we have constructed a modified expression vector, using pGEX-4T-1 vector as the backbone. In addition to the features offered by the pGEX-4T vectors, the new vector allowed easy purification of recombinant proteins on the highly versatile Ni-NTA-agarose affinity matrix. The utility of the new vector was demonstrated by expressing and purifying, to near homogeneity, two M. tuberculosis proteins, i.e., Rv3872 (a member of the multi-gene PE subfamily) and Rv3873 (a member of the multi-gene PPE subfamily), which are encoded by the RD1 region of M. tuberculosis. The proteins encoded by rv3872 and rv3873 were expressed at high levels as fusion proteins with glutathione-S-transferase in E. coli. The recombinant Rv3872 and Rv3873 proteins were purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose and/or Ni-NTA-agarose affinity matrix and cleavage of the purified fusion proteins by thrombin protease. The recombinant Rv3872 protein was nearly homogeneous (more than 95% pure) while Rv3873 preparation was more than 90% pure. The recombinant Rv3872 and Rv3873 proteins were immunologically active and reacted with antibodies in sera from TB patients. Our results demonstrate the utility of the newly constructed expression vector with two affinity tags for efficient expression and purification of recombinant M. tuberculosis proteins expressed in E. coli, which could be used for further diagnostic and immunological studies.     

Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra

To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.     

A novel method for increasing the expression level of recombinant proteins

Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.     

The Glycosylated Rv1860 Protein of Mycobacterium tuberculosis Inhibits Dendritic Cell Mediated TH1 and TH17 Polarization of T Cells and Abrogates Protective Immunity Conferred by BCG

We previously reported interferon gamma secretion by human CD4⁺ and CD8⁺ T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-α, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-β unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-γ and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-γ and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-α compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.     

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

Over-producing soluble protein complex and validating protein–protein interaction through a new bacterial co-expression system

Many proteins exert their functions through a protein complex and protein–protein interactions. However, the study of these types of interactions is complicated when dealing with toxic or hydrophobic proteins. It is difficult to use the popular Escherichia coli host for their expression, as these proteins in all likelihood require a critical partner protein to ensure their proper folding and stability. In the present study, we have developed a novel co-expression vector, pHEX, which is compatible with, and thus can be partnered with, many commercially available E. coli vectors, such as pET, pGEX and pMAL. The pHEX contains the p15A origin of replication and a T7 promoter, which can over-produce a His-tagged recombinant protein. The new co-expression system was demonstrated to efficiently co-produce and co-purify heterodimeric protein complexes, for example PE25/PPE41 (Rv2430c/Rv2431c) and ESAT6/CFP10 (Rv3874/Rv3875), from the human pathogen Mycobacterium tuberculosis H37Rv. Furthermore, the system was also effectively used to characterize protein–protein interactions through convenient affinity tags. Using an in vivo pull-down assay, for the first time we have confirmed the presence of three pairs of PE/PPE-related novel protein interactions in this pathogen. In summary, a convenient and efficient co-expression vector system has been successfully developed. The new system should be applicable to any protein complex or any protein–protein interaction of interest in a wide range of biological organisms.     

Hypoxic response of Mycobacterium tuberculosis studied by metabolic labeling and proteome analysis of cellular and extracellular proteins

The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.     

The systematic modeling studies and free energy calculations of the phenazine compounds as anti-tuberculosis agents

Abstract

Phenazine compounds have good activity against Mycobacterium tuberculosis (MTB). Based on the reported activities that were obtained in MTB H37Rv, a three-dimensional quantitative structure–activity relationship (3D-QSAR) model was built to design novel compounds against MTB. A fivefold cross-validation method and external validation were used to analyze the accuracy of forecasting. The model has a cross-validation coefficient q²=0.7 and a non-cross-validation coefficient r² = 0.903, indicating that the model has good predictive possibility. The design of anti-pneumococcus MTB compounds was guided by the obtained 3D-QSAR model, and several compounds with better activity were obtained. To test the activity of these compounds, molecular docking, molecular dynamics simulation, and post-simulation analysis of the already reported drug targets in MTB were carried out. Among the total 15 drug targets, only three targets (Rv2361c, Rv2965c, and Rv3048c) were selected based on the docking results. Initial results reported that these compounds possessed good inhibition activity for Rv2361c. The top nine complexes of Rv2361 ligands were only subjected to MD simulation which resulted in a stable dynamics of the structures and showed a residual fluctuation in inhibitors binding pocket. Free energy reported that overall, the derivatives hold strong energy against the protein target. Energetic contribution results showed that residues, Asp76, Arg80, Asn124, Arg127, Arg244, and Arg250, play a major role in total energy. Systems biology approach validates shortlisted drug effect on the entire system which might be useful to predict potential drug in wet lab as well.

Communicated by Ramaswamy H. Sarma     

结核分枝杆菌Rv3425特异性蛋白片段与CFP10-ESAT6融合蛋白在结核抗体检测中的应用价值

目的:对结核分枝杆菌Rv3425蛋白进行生物信息学分析及抗原表位预测,评价Rv342519-176重组蛋白与CFP10-ESAT6融合蛋白在结核抗体检测中的应用。方法:PCR扩增结核杆菌Rv3425蛋白19～176位的编码基因片段,原核表达并纯化Rv342519-176重组蛋白(rRv342519-176)和CFP10-ESAT6融合蛋白(rCFP10-ESAT6),建立以重组蛋白为抗原的ELISA方法,评价2种蛋白在结核抗体检测中的联合应用价值。结果:具有抗原表位的rRv342519-176与rCFP10-ESAT6在大肠杆菌中高效表达;ELISA结果显示,rRv342519-176的敏感性为39%,特异性为97.5%;rCFP10-ESAT6的敏感性为37%,特异性为95%;2种蛋白联合检测,敏感性提高到57%,特异性为95%。结论:生物信息学预测Rv3425的优势抗原表位,表达并纯化的rRv342519-176和rCFP10-ESAT6在结核抗体检测中具有一定的抗原互补性,在保持特异性的基础上可显著提高检测敏感性。     

MTB-PCDB: Mycobacterium tuberculosis proteome comparison database

The Mycobacterium tuberculosis Proteome Comparison Database (MTB-PCDB) is an online database providing integrated access to proteome sequence comparison data for five strains of Mycobacterium tuberculosis (H37Rv, H37Ra, CDC 1551, F11 and KZN 1435) sequenced completely so far. MTB-PCDB currently hosts 40252 protein sequence comparison data obtained through inter-strain proteome comparison of five different strains of MTB. 2373 proteins were found to be identical in all 5 strains using MTB H(37)Rv as reference strain. To enable wide use of this data, MTB-PCDB provides a set of tools for searching, browsing, analyzing and downloading the data. By bringing together, M. tuberculosis proteome comparison among virulent & avirulent strains and also drug susceptible & drug resistance strains MTB-PCDB provides a unique discovery platform for comparative proteomics among these strains which may give insights into the discovery & development of TB drugs, vaccines and biomarkers. AVAILABILITY: The database is available for free at http://www.bicjbtdrc-mgims.in/MTB-PCDB/     

结核分枝杆菌感染人源树突状细胞的蛋白质表达谱

在结核分枝杆菌(Mycobacteriumtuberculosis,MTB)的感染中,树突状细胞(Dendriticcells ,DCs)的应答反应是机体起始免疫应答的关键。因此,利用双向电泳技术(Two_dimensionalelectrophoresis,2_DE)对人源树突状细胞受结核分枝杆菌H37RvATCC 2 72 94菌株感染前后的全细胞蛋白表达图谱进行差异比较和分析,发现其中产生差异的有4 5个蛋白质斑点,利用基质辅助激光解析电离串联飞行时间质谱技术对其中4个表达明显上调的蛋白质斑点进行分析鉴定,获得4个明确的肽指纹图谱,通过在数据库中检索分析,确定这4个蛋白质分别为人亚砷酸诱导ATP酶I(HumanArsenite_stimulatedATPase ,hASNA_I) ,膜联蛋白IV(AnnexinIV) ,γ_肌动蛋白(γ_actin) ,热休克蛋白2 7(Heatshockprotein2 7,HSP2 7)。上述发现有助于了解结核分枝杆菌入侵早期导致的树突状细胞蛋白质组表达变化,为深入研究结核分枝杆菌 宿主相互作用提供了探索方向     

Evidence for a rhesus monkey model of asymptomatic tuberculosis

Rhesus monkeys (RM) were inoculated intrabronchially with graded doses of Mycobacterium tuberculosis (MTB) strains Erdman and H37Rv in an effort to produce a model of asymptomatic tuberculosis infection. Erdman strain produced active disease within 7-11 weeks regardless of dose. Low doses of H37Rv resulted in asymptomatic infections; high doses produced active disease within 11 weeks. Over a 4-month period of post-inoculation study, MTB culture-filtrate protein (CFP)-stimulated bronchoalveolar lavage cells (BALC) and blood mononuclear cells (PBMC) from monkeys with active disease (30 cfu Erdman-inoculated) or asymptomatic infection (200 cfu H37Rv-inoculated) produced similar significant quantities of mRNA encoding for IFN-gamma or TNF-alpha, but insignificant quantities of IL-4 mRNA. Differences were observed in antigen-induced in vitro blastogenic responses and serum anti-lipoarabinomannan (LAM) antibody responses in animals with active compared with asymptomatic MTB infections. The results indicate that RM are a good model for the study of asymptomatic tuberculosis infections using low doses of H37Rv.     

A Broad Profile of Co-Dominant Epitopes Shapes the Peripheral Mycobacterium tuberculosis Specific CD8+ T-Cell Immune Response in South African Patients with Active Tuberculosis

We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.