Differential Live Mycobacterium tuberculosis-, M. bovis BCG-, Recombinant ESAT6-, and Culture Filtrate Protein 10-Induced Immunity in Tuberculosis

ABSTRACT The high prevalence of Mycobacterium tuberculosis makes it imperative that immune responses to evaluate could be predictive of infection. We investigated live Mycobacterium- and recombinant antigen-induced cytokine and chemokine responses in patients with active tuberculosis (TB) compared with those of healthy controls from an area where TB is endemic (ECs). M. tuberculosis-, M. bovis BCG-, ESAT6-, and culture filtrate protein 10 (CFP10)-induced responses were determined in peripheral blood mononuclear cells from patients with pulmonary TB (n = 38) and ECs (n = 39). The levels of the cytokines gamma interferon (IFN-γ) and interleukin-10 (IL-10) and the chemokines CCL2, CCL3, and CXCL9 were measured. The levels of M. tuberculosis- and BCG-induced IFN-γ secretion were significantly reduced (P = 0.002 and P < 0.01, respectively), while the amount of IL-10 induced by both virulent (P < 0.01) and avirulent (P = 0.002) mycobacteria was increased in patients with TB. The ESAT6-induced IFN-γ responses were increased in the patients with TB (P = 0.013) compared with those in the EC group. When tuberculin skin test (TST)-negative (TST−; induration, <10 mm) and TST-positive (TST+) donors were studied separately, both TST− and TST+ individuals showed increased IFN-γ responses to M. tuberculosis compared with the responses of the patients with TB (P = 0.037 and P = 0.006, respectively). However, only TST+ ECs showed reduced IFN-γ responses to ESAT6 (P = 0.008) compared with the responses of the patients with TB. The levels of M. tuberculosis-induced CCL2 (P = 0.006) and CXCL9 (P = 0.017) were greater in the patients with TB. The levels of CCL3 secretion in response to Mycobacterium and antigen stimulation were comparable between the two groups. While the levels of ESAT6-induced chemokines did not differ between the patients with TB and the ECs, the levels of CFP10-induced CCL2 (P = 0.01) and CXCL9 (P = 0.001) were increased in the patients. These data indicate differential host IFN-γ, CXCL9, and CCL2 responses to live mycobacteria and mycobacterial antigens and have implications for the identification of potential biomarkers of infection which could be used for the diagnosis of TB.

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Antigenic Equivalence of Human T-Cell Responses toMycobacterium tuberculosis-Specific RD1-Encoded Protein Antigens ESAT-6 and Culture Filtrate Protein 10 and to Mixtures of Synthetic Peptides

Infection and Immunity ◽

10.1128/iai.68.6.3314-3321.2000 ◽

2000 ◽

Vol 68 (6) ◽

pp. 3314-3321 ◽

Cited By ~ 123

Author(s):

Sandra M. Arend ◽

Annemieke Geluk ◽

Krista E. van Meijgaarden ◽

Jaap T. van Dissel ◽

Michael Theisen ◽

...

Keyword(s):

T Cell ◽

Culture Filtrate ◽

Large Scale ◽

Mononuclear Cells ◽

Genomic Region ◽

Protein Antigens ◽

Peripheral Blood Mononuclear ◽

Human T Cell ◽

Peptide Mixtures ◽

Culture Filtrate Protein

ABSTRACT The early secreted antigenic target 6-kDa protein (ESAT-6) and culture filtrate protein 10 (CFP-10) are promising antigens for reliable immunodiagnosis of tuberculosis. Both antigens are encoded by RD1, a genomic region present in all strains of Mycobacterium tuberculosis and M. bovis but lacking in all M. bovis bacillus Calmette-Guérin vaccine strains. Production and purification of recombinant antigens are laborious and costly, precluding rapid and large-scale testing. Aiming to develop alternative diagnostic reagents, we have investigated whether recombinant ESAT-6 (rESAT-6) and recombinant CFP-10 (rCFP-10) can be replaced with corresponding mixtures of overlapping peptides spanning the complete amino acid sequence of each antigen. Proliferation of M. tuberculosis-specific human T-cell lines in response to rESAT-6 and rCFP-10 and that in response to the corresponding peptide mixtures were almost completely correlated (r = 0.96,P < 0.0001 for ESAT-6; r = 0.98,P < 0.0001 for CFP-10). More importantly, the same was found when gamma interferon production by peripheral blood mononuclear cells in response to these stimuli was analyzed (r = 0.89, P < 0.0001 for ESAT-6;r = 0.89, P < 0.0001 for CFP-10). Whole protein antigens and the peptide mixtures resulted in identical sensitivity and specificity for detection of infection with M. tuberculosis. The peptides in each mixture contributing to the overall response varied between individuals with different HLA-DR types. Interestingly, responses to CFP-10 were significantly higher in the presence of HLA-DR15, which is the major subtype of DR2. These results show that mixtures of synthetic overlapping peptides have potency equivalent to that of whole ESAT-6 and CFP-10 for sensitive and specific detection of infection with M. tuberculosis, and peptides have the advantage of faster production at lower cost.

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Low Induction of Proinflammatory Cytokines Parallels Evolutionary Success of Modern Strains within the Mycobacterium tuberculosis Beijing Genotype

Infection and Immunity ◽

10.1128/iai.00282-13 ◽

2013 ◽

Vol 81 (10) ◽

pp. 3750-3756 ◽

Cited By ~ 39

Author(s):

Arjan van Laarhoven ◽

Jornt J. Mandemakers ◽

Johanneke Kleinnijenhuis ◽

Mimount Enaimi ◽

Ekta Lachmandas ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mononuclear Cells ◽

Ex Vivo ◽

Selective Advantage ◽

Beijing Genotype ◽

Peripheral Blood Mononuclear ◽

Content Type ◽

Tnf Α ◽

Evolutionary Success ◽

Ifn Γ

ABSTRACTOne of the most widespread clades ofMycobacterium tuberculosisworldwide, the Beijing genotype family, consists of ancient (atypical) and modern (typical) strains. Modern Beijing strains outcompete ancient strains in terms of prevalence, while reserving a higher degree of genetic conservation. We hypothesize that their selective advantage lies in eliciting a different host immune response. Bead-disrupted lysates of a collection of differentM. tuberculosisstrains of the modern (n= 7) or ancient (n= 7) Beijing genotype, as well as the Euro-American lineage (n= 6), were used for induction ofex vivocytokine production in peripheral blood mononuclear cells (PBMCs) from 10 healthy individuals. Hierarchical clustering and multivariate regression analyses were used to study possible differences in production of nine cytokines. Modern and ancientM. tuberculosisBeijing genotypes induced different cytokine signatures. Overall induction of interleukin-1β (IL-1β), gamma interferon (IFN-γ), and IL-22 was 38 to 40% lower after stimulation with modern Beijing strains (correctedPvalues of <0.0001, 0.0288, and 0.0002, respectively). Euro-American reactivation strains induced 2-fold more TNF-α production than both types of Beijing strains. The observed differences in cytokine induction point to a reduction in proinflammatory cytokine response as a possible contributing factor to the evolutionary success of modern Beijing strains.

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The Secreted Protein Rv1860 of Mycobacterium tuberculosis Stimulates Human Polyfunctional CD8+T Cells

Clinical and Vaccine Immunology ◽

10.1128/cvi.00554-15 ◽

2016 ◽

Vol 23 (4) ◽

pp. 282-293 ◽

Cited By ~ 2

Author(s):

Vijaya Satchidanandam ◽

Naveen Kumar ◽

Sunetra Biswas ◽

Rajiv S. Jumani ◽

Chandni Jain ◽

...

Keyword(s):

T Cells ◽

Mycobacterium Tuberculosis ◽

T Cell ◽

Mononuclear Cells ◽

Interleukin 10 ◽

T Cell Response ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha ◽

Content Type ◽

Latent Tb Infection

ABSTRACTWe previously reported that Rv1860 protein fromMycobacterium tuberculosisstimulated CD4+and CD8+T cells secreting gamma interferon (IFN-γ) in healthy purified protein derivative (PPD)-positive individuals and protected guinea pigs immunized with a DNA vaccine and a recombinant poxvirus expressing Rv1860 from a challenge with virulentM. tuberculosis. We now show Rv1860-specific polyfunctional T (PFT) cell responses in the blood of healthy latentlyM. tuberculosis-infected individuals dominated by CD8+T cells, using a panel of 32 overlapping peptides spanning the length of Rv1860. Multiple subsets of CD8+PFT cells were significantly more numerous in healthy latently infected volunteers (HV) than in tuberculosis (TB) patients (PAT). The responses of peripheral blood mononuclear cells (PBMC) from PAT to the peptides of Rv1860 were dominated by tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) secretions, the former coming predominantly from non-T cell sources. Notably, the pattern of the T cell response to Rv1860 was distinctly different from those of the widely studiedM. tuberculosisantigens ESAT-6, CFP-10, Ag85A, and Ag85B, which elicited CD4+T cell-dominated responses as previously reported in other cohorts. We further identified a peptide spanning amino acids 21 to 39 of the Rv1860 protein with the potential to distinguish latent TB infection from disease due to its ability to stimulate differential cytokine signatures in HV and PAT. We suggest that a TB vaccine carrying these and other CD8+T-cell-stimulating antigens has the potential to prevent progression of latentM. tuberculosisinfection to TB disease.

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Differences in Gamma Interferon Production In Vitro Predict the Pace of the In Vivo Response to Leishmania amazonensis in Healthy Volunteers

Infection and Immunity ◽

10.1128/iai.69.12.7453-7460.2001 ◽

2001 ◽

Vol 69 (12) ◽

pp. 7453-7460 ◽

Cited By ~ 37

Author(s):

M. M. L. Pompeu ◽

C. Brodskyn ◽

M. J. Teixeira ◽

J. Clarêncio ◽

J. Van Weyenberg ◽

...

Keyword(s):

Mononuclear Cells ◽

Interleukin 10 ◽

Gamma Interferon ◽

Leishmania Amazonensis ◽

Cell Mediated Immunity ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha ◽

Ifn Γ

ABSTRACT The initial encounter of Leishmania cells and cells from the immune system is fundamentally important in the outcome of infection and determines disease development or resistance. We evaluated the anti-Leishmania amazonensis response of naive volunteers by using an in vitro priming (IVP) system and comparing the responses following in vivo vaccination against the same parasite. In vitro stimulation allowed us to distinguish two groups of individuals, those who produced small amounts of gamma interferon (IFN-γ) (n = 16) (low producers) and those who produced large amounts of this cytokine (n = 16) (high producers). IFN-γ production was proportional to tumor necrosis factor alpha and interleukin 10 (IL-10) levels but did not correlate with IL-5 production. Volunteers who produced small amounts of IFN-γ in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-γ production. However, 6 months after vaccination, all individuals tested produced similarly high levels of IFN-γ upon stimulation of their peripheral blood mononuclear cells with Leishmania promastigotes, indicating that low in vitro producers respond slowly in vivo to vaccination. In high IFN-γ producers there was an increased frequency of activated CD8+ T cells both in vitro and in vivo compared to the frequency in low producers, and such cells were positive for IFN-γ as determined by intracellular staining. Such findings suggest that IVP responses can be used to predict the pace of postvaccination responses of test volunteers. Although all vaccinated individuals eventually have a potent anti-Leishmania cell-mediated immunity (CMI) response, a delay in mounting the CMI response may influence resistance against leishmaniasis.

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Interaction of Mycobacterium tuberculosis-Induced Transforming Growth Factor β1 and Interleukin-10

Infection and Immunity ◽

10.1128/iai.67.11.5730-5735.1999 ◽

1999 ◽

Vol 67 (11) ◽

pp. 5730-5735 ◽

Cited By ~ 59

Author(s):

Catherine Othieno ◽

Christina S. Hirsch ◽

Beverly D. Hamilton ◽

Katalin Wilkinson ◽

Jerrold J. Ellner ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

T Cell ◽

Growth Factor ◽

Mononuclear Cells ◽

Transforming Growth Factor ◽

Interleukin 10 ◽

Transforming Growth Factor Β1 ◽

Ifn Γ ◽

Tgf Β1

ABSTRACT Mycobacterium tuberculosis is associated with the activation of cytokine circuits both at sites of active tuberculosis in vivo and in cultures of mononuclear cells stimulated by M. tuberculosis or its components in vitro. Interactive stimulatory and/or inhibitory pathways are established between cytokines, which may result in potentiation or attenuation of the effects of each molecule on T-cell responses. Here we examined the interaction of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) in purified protein derivative (PPD)-stimulated human mononuclear cell cultures in vitro. TGF-β1 induced monocyte IL-10 (but not tumor necrosis factor alpha) production (by 70-fold, P < 0.02) and mRNA expression in the absence but not in the presence of PPD. Both exogenous recombinant (r) IL-10 and rTGF-β1 independently suppressed the production of PPD-induced gamma interferon (IFN-γ) in mononuclear cells from PPD skin test-positive individuals. Synergistic suppression of IFN-γ in cultures containing both rTGF-β1 and rIL-10 was only seen when the responder cell population were peripheral blood mononuclear cells (PBMC) and not monocyte-depleted mononuclear cells and when PBMC were pretreated with rTGF-β1 but not with rIL-10. Suppression of PPD-induced IFN-γ in PBMC containing both rTGF-β1 (1 ng/ml) and rIL-10 (100 pg/ml) was 1.5-fold higher (P< 0.05) than cultures containing TGF-β1 alone and 5.7-fold higher (P < 0.004) than cultures containing IL-10 alone. Also, neutralization of endogenous TGF-β1 and IL-10 together enhanced PPD-induced IFN-γ in PBMC in a synergistic manner. Thus, TGF-β1 and IL-10 together potentiate the downmodulatory effect on M. tuberculosis-induced T-cell production of IFN-γ, and TGF-β1 alone enhances IL-10 production. At sites of active M. tuberculosis infection, these interactions may be conducive to the suppression of mononuclear cell functions.

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Ex Vivo Generation of Donor Antigen-Specific Immunomodulatory Cells

Cell Transplantation ◽

10.1177/0963689718794642 ◽

2018 ◽

Vol 27 (11) ◽

pp. 1692-1704

Author(s):

M. Watanabe ◽

Makiko Kumagai-Braesch ◽

M. Yao ◽

S. Thunberg ◽

D. Berglund ◽

...

Keyword(s):

T Cells ◽

Mononuclear Cells ◽

Ex Vivo ◽

Human Peripheral Blood ◽

Human Leukocyte ◽

Interleukin 10 ◽

Control Group ◽

Peripheral Blood Mononuclear ◽

Foxp3 Mrna ◽

Ifn Γ

Adoptive transfer of alloantigen-specific immunomodulatory cells generated ex vivo with anti-CD80/CD86 mAbs (2D10.4/IT2.2) holds promise for operational tolerance after transplantation. However, good manufacturing practice is required to allow widespread clinical application. Belatacept, a clinically approved cytotoxic T-lymphocyte antigen 4-immunoglobulin that also binds CD80/CD86, could be an alternative agent for 2D10.4/IT2.2. With the goal of generating an optimal cell treatment with clinically approved reagents, we evaluated the donor-specific immunomodulatory effects of belatacept- and 2D10.4/IT2.2-generated immunomodulatory cells. Immunomodulatory cells were generated by coculturing responder human peripheral blood mononuclear cells (PBMCs) (50 × 106 cells) with irradiated donor PBMCs (20 × 106 cells) from eight human leukocyte antigen-mismatched responder–donor pairs in the presence of either 2D10.4/IT2.2 (3 μg/106 cells) or belatacept (40 μg/106 cells). After 14 days of coculture, the frequencies of CD4+ T cells, CD8+ T cells, and natural killer cells as well as interferon gamma (IFN-γ) production in the 2D10.4/IT2.2- and belatacept-treated groups were lower than those in the control group. The percentage of CD19+ B cells was higher in the 2D10.4/IT2.2- and belatacept-treated groups than in the control group. The frequency of CD4+CD25+CD127lowFOXP3+ T cells increased from 4.1±1.0% (preculture) to 7.1±2.6% and 7.3±2.6% (day 14) in the 2D10.4/IT2.2- and belatacept-treated groups, respectively ( p<0.05). Concurrently, delta-2 FOXP3 mRNA expression increased significantly. Compared with cells derived from the no-antibody treated control group, cells generated from both the 2D10.4/IT2.2- and belatacept-treated groups produced lower IFN-γ and higher interleukin-10 levels in response to donor-antigens, as detected by enzyme-linked immunospot. Most importantly, 2D10.4/IT2.2- and belatacept-generated cells effectively impeded the proliferative responses of freshly isolated responder PBMCs against donor-antigens. Our results indicate that belatacept-generated donor-specific immunomodulatory cells possess comparable phenotypes and immunomodulatory efficacies to those generated with 2D10.4/IT2.2. We suggest that belatacept could be used for ex vivo generation of clinical grade alloantigen-specific immunomodulatory cells for tolerance induction after transplantation.

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Cell Envelope Protein PPE68 Contributes to Mycobacterium tuberculosis RD1 Immunogenicity Independently of a 10-Kilodalton Culture Filtrate Protein and ESAT-6

Infection and Immunity ◽

10.1128/iai.72.4.2170-2176.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 2170-2176 ◽

Cited By ~ 62

Author(s):

Caroline Demangel ◽

Priscille Brodin ◽

Paul J. Cockle ◽

Roland Brosch ◽

Laleh Majlessi ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Culture Filtrate ◽

Envelope Protein ◽

Protective Efficacy ◽

Cell Envelope ◽

Genomic Region ◽

Low Molecular Weight ◽

Ifn Γ ◽

Culture Filtrate Protein ◽

Low Molecular Weight Proteins

ABSTRACT The protective efficacy of Mycobacterium bovis BCG can be markedly augmented by stable integration of Mycobacterium tuberculosis genomic region RD1. BCG complemented with RD1 (BCG::RD1) encodes nine additional proteins. Among them, 10-kDa culture filtrate protein (CFP-10) and ESAT-6 (6-kDa early secreted antigenic target) are low-molecular-weight proteins that induce potent Th1 responses. Using pools of synthetic peptides, we have examined the potential immunogenicity of four other RD1 products (PE35, PPE68, Rv3878, and Rv3879c). PPE68, the protein encoded by rv3873, was the only one to elicit gamma interferon (IFN-γ)-producing cells in C57BL/6 mice infected with M. tuberculosis. Anti-PPE68 T cells were predominantly raised against an epitope mapped in the N-terminal end of the protein. Importantly, inactivation of rv3873 in BCG::RD1 did not modify CFP-10 and ESAT-6 secretion. Moreover, the generation of IFN-γ responses to these antigens following immunization with BCG::RD1 was independent of PPE68 expression. Taken together, these results show that PPE68 is an immunogenic product of the RD1 region, which does not interfere with the secretion and immunogenicity of CFP-10 and ESAT-6.

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Regulation of Interferon-γ receptor (IFN-γR) expression in macrophages during Mycobacterium tuberculosis infection

BioMolecular Concepts ◽

10.1515/bmc-2020-0006 ◽

2020 ◽

Vol 11 (1) ◽

pp. 76-85

Author(s):

Gunjan Kak ◽

Brijendra K Tiwari ◽

Yogendra Singh ◽

Krishnamurthy Natarajan

Keyword(s):

Mycobacterium Tuberculosis ◽

Mononuclear Cells ◽

Second Messengers ◽

Surface Expression ◽

Interferon Γ ◽

Fine Tuning ◽

Mycobacterium Tuberculosis Infection ◽

Peripheral Blood Mononuclear ◽

Negative Role ◽

Ifn Γ

AbstractInterferon-gamma (IFN-γ) is a key cytokine that mediates immunity to tuberculosis (TB). Mycobacterium tuberculosis (M. tb) is known to downregulate the surface expression of IFN-γ receptor (IFN-γR) on macrophages and peripheral blood mononuclear cells (PBMCs) of patients with active TB disease. Many M. tb antigens also downmodulate IFN-γR levels in macrophages when compared with healthy controls. In the current study, we aimed at deciphering key factors involved in M. tb mediated downregulation of IFN-γR levels on macrophage surface. Our data showed that both M. tb H37Rv and M. bovis BCG infections mediate downmodulation of IFN-γR on human macrophages. This downmodulation is regulated at the level of TLR signaling pathway, second messengers such as calcium and cellular kinases i.e. PKC and ERK-MAPK, indicating that fine tuning of calcium response is critical to maintaining IFN-γR levels on macrophage surface. In addition, genes in the calcium and cysteine protease pathways which were previously identified by us to play a negative role during M. tb infection, also regulated IFN-γR expression. Thus, modulations in IFN-γR levels by utilizing host machinery may be a key immune suppressive strategy adopted by the TB pathogen to ensure its persistence and thwart host defense.

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beta-Glucan Increases IFN-gamma and IL-12 Production of Peripheral Blood Mononuclear Cells with/without Induction of Mycobacterium tuberculosis Wild-type/Mutant DNA

The Indonesian Biomedical Journal ◽

10.18585/inabj.v11i2.688 ◽

2019 ◽

Vol 11 (2) ◽

pp. 200-4

Author(s):

Meira Erawati ◽

Nyoman Suci Widyastiti ◽

Tri Indah Winarni ◽

Edi Dharmana

Keyword(s):

Mycobacterium Tuberculosis ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Wild Type ◽

Peripheral Blood Mononuclear ◽

Significant Difference ◽

Blood Mononuclear Cells ◽

Type Mutant ◽

Ifn Γ

BACKGROUND: In tuberculosis infections, the immune system is weakened and cannot produce enough cytokines to against the infection. b-glucan is a potent immunomodulator that induces cytokine production in various bacterial infections. This study aimed to determine the effects of b-glucan on the production of interferon (IFN)-γ and interleukin (IL)-12 in peripheral blood mononuclear cells (PBMCs) induced by Mycobacterium tuberculosis DNA.METHODS: PBMCs were isolated from 11 healthy subjects. PBMCs were treated with/without 5 μg/mL b-glucan and M. tuberculosis rpoB wild-type or mutant DNA. The production of IFN-γ and IL-12 in the supernatant was performed with enzyme-linked immune-sorbent assay (ELISA).RESULTS: b-glucan increased significantly (p<0.05) IFN-γ of M. tuberculosis mutant DNA-induced PBMCs, M. tuberculosis wild-type DNA-induced PBMCs, and non-induced PBMCs. b-glucan also increased significantly (p<0.05) IL-12 of M. tuberculosis mutant DNA-induced PBMCs, M. tuberculosis wild-type DNA-induced PBMCs, and non-induced PBMCs. There were not any significant difference between male and female groups for IL-12 and IFN-γ in all treatment groups (p>0.05, ANOVA test).CONCLUSION: This in vitro study indicates that b-glucan increases the performance of PBMCs to produce IFN-γ and IL-12, with/without induction of M. tuberculosis wild-type/ mutant DNA.KEYWORDS: b-glucan, IFN-γ, IL-12, M. tuberculosis, rpoB

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Serodiagnosis Efficacy and Immunogenicity of the Fusion Protein of Mycobacterium tuberculosis Composed of the 10-Kilodalton Culture Filtrate Protein, ESAT-6, and the Extracellular Domain Fragment of PPE68

Clinical and Vaccine Immunology ◽

10.1128/cvi.05708-11 ◽

2012 ◽

Vol 19 (4) ◽

pp. 536-544 ◽

Cited By ~ 14

Author(s):

Jia-Nan Xu ◽

Jian-Ping Chen ◽

Da-Li Chen

Keyword(s):

Mycobacterium Tuberculosis ◽

Fusion Protein ◽

Culture Filtrate ◽

Immunological Response ◽

Extracellular Domain ◽

Enzyme Linked Immunosorbent Assay ◽

Content Type ◽

Prokaryotic Expression Vector ◽

Ifn Γ ◽

Culture Filtrate Protein

ABSTRACTIn order to identify immunodominant antigens ofMycobacterium tuberculosisthat may be used in the serodiagnosis of active tuberculosis (TB), we designed anM. tuberculosisfusion protein consisting of CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secreted antigenic target), and the extracellular domain fragment of PPE68 (PPE68′). Then, the coding sequences of the three proteins were inserted into a prokaryotic expression vector, pET-32a(+). To enhance the immunological response, the proteins were linked together. The fusion proteins with a 6×His tag were successfully overexpressed inEscherichia coliBL21 and purified. The purified proteins were applied for detection of the total IgG titer by using an enzyme-linked immunosorbent assay (ELISA) with human sera from well-characterized TB cases and the control cases, and results were compared to those with purified protein derivative tuberculin (PPD). The ELISA results showed that among 140 cases of confirmed active TB and 70 control cases, CFP-10–ESAT-6–PPE68′ had a sensitivity of 73.3% and specificity of 94.3%, compared to a sensitivity of 66.7% and specificity of 74.3% for PPD and a sensitivity of 65% and specificity of 91.4% for CFP-10–ESAT-6. In addition, the fusion protein CFP-10–ESAT-6–PPE68′ stimulated a higher level of antigen-specific gamma interferon (IFN-γ) release for active-TB patients than PPD and CFP-10–ESAT-6. After immunization of C57BL/6 mice, the findings indicated that the total IgG titers and the concentrations of IFN-γ in mice immunized by CFP-10–ESAT-6–PPE68′ were high and induced strong, long-term humoral immunity compared to results with PPD and CFP-10–ESAT-6. Thus, our study indicates that the fusion protein CFP-10–ESAT-6–PPE68′ may be useful as an immunodominant antigen for the serodiagnosis of active TB.

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