Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection

Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen‐specific IFN‐γ and IL‐2‐expressing CD4⁺ and CD8⁺ T cells. In exosome‐vaccinated mice, there was a similar T_H1 immune response but a more limited T_H2 response compared to BCG‐vaccinated mice. Using a low‐dose M. tuberculosis mouse aerosol infection model, exosomes from CFP‐treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell‐free vaccine against an M. tuberculosis infection.     

Correlation between culture of Mycobacterium tuberculosis and detection of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis

A retrospective study was done to correlate culture of Mycobacterium tuberculosis and detection of mycobacterial antigen in cerebrospinal fluid (CSF) by an inhibition enzyme-linked immunosorbent assay (ELISA). M. tuberculosis was cultured from CSF of 14 out of 70 patients with a clinical diagnosis of tuberculous meningitis (TBM). Mycobacterial antigens were demonstrated in CSF specimens by inhibition ELISA in all 14 culture-positive patients with antigen concentrations of 14.5-295 ng/ml (mean 158.8 ng/ml). Thus there was positive correlation between the detection of mycobacterial antigen and isolation of M. tuberculosis. Based on this observation, 56 CSF specimens from culture-negative patients with clinically diagnosed TBM were examined for mycobacterial antigen and the data were compared with those from culture positive patients. ELISA gave positive results in 38 specimens, with antigen levels of 12.5-280 ng/ml (mean 152.6 ng/ml). In 70 CSF specimens from patients with non-tuberculous neurological disease (control group), ELISA results were negative. Thus, detection of mycobacterial antigen in CSF specimens by inhibition ELISA had a specificity of 100% and a sensitivity of 67.8% for the diagnosis of TBM and is of potential value in the laboratory diagnosis of TBM.     

Pattern and diversity of cytokine production differentiates between Mycobacterium tuberculosis infection and disease

Tuberculosis (TB) remains a global health problem. The solution involves development of an effective vaccine, but has been limited by incomplete understanding of what constitutes protective immunity during natural infection with Mycobacterium tuberculosis. In this study, M. tuberculosis‐specific responses following an overnight whole‐blood assay were assessed by intracellular cytokine staining and luminex, and compared between TB cases and exposed household contacts. TB cases had significantly higher levels of IFN‐γ⁺TNF‐α⁺IL‐2⁺CD4⁺T cells compared with contacts. TB cases also had a significantly higher proportion of cells single‐positive for TNF‐α, but lower proportion of cells producing IL‐2 alone and these differences were seen for both CD4⁺and CD8⁺ T cells. Cytokine profiles from culture supernatants were significantly biased toward a Th1 phenotype (IFN‐γ and IL‐12(p40)) together with a complete abrogation of IL‐17 secretion in TB cases. Our data indicate that despite a robust response to TB antigens in active TB disease, changes in the pattern of cytokine production between TB infection and disease clearly contribute to disease progression.     

Quantitative T-cell interferon-gamma responses to Mycobacterium tuberculosis-specific antigens in active and latent tuberculosis

The objective was to compare the quantitative T-cell responses measured by the commercial interferon-gamma (IFNγ) release assays (IGRAs) in active and latent tuberculosis (TB) states. T-cell responses of culture-proven TB cases were compared with those of contacts with positive IGRA results and tuberculin skin tests ≥ 15 mm. T-SPOT.TB results in 270 active TB cases and 183 community contacts showed the median spot-forming cells (SFCs) above negative control/2.5 × 10⁵ peripheral blood mononuclear cells to be 27 (−1 to 203) vs 10 (−2 to 174) in response to ESAT-6 (p < 0.001); and 37 (0 to 293) vs 13 (0 to 225) to CFP-10 (p < 0.001). The median IFNγ levels (antigen minus nil control) as measured by QuantiFERON-TB Gold In-tube in 270 cases and 142 contacts in congregate settings was 2.3 IU/ml (−0.58 to 31.44) vs 1.7 IU/ml (0.35 to 26.51, p = 0.98). Quantitative T-cell responses as measured by the T-SPOT.TB may indicate mycobacterial burden and disease activity, but cannot be used to discriminate active from latent TB.     

Evaluation of gamma interferon release assays using Mycobacterium tuberculosis antigens for diagnosis of latent and active tuberculosis in Mycobacterium bovis BCG-vaccinated populations

T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) using Mycobacterium tuberculosis-specific antigens have shown higher sensitivity and specificity than the routine tuberculin skin test (TST). However, the effects of Mycobacterium bovis BCG vaccination and anti-tuberculosis (TB) treatment on dynamic T-cell responses to M. tuberculosis-specific antigens in active TB cases have rarely been investigated in regions where TB is endemic. Eighty-nine patients with active pulmonary TB (ATB) and 57 healthy controls (HC) from China were recruited and tested by sputum smear and culture, TSTs, and IGRAs with M. tuberculosis-specific antigens ESAT-6 and CFP-10 (T-SPOT.TB) as well as purified protein derivative (PPD) stimulation. All 146 participants were screened by the T-SPOT.TB assay at recruitment. T-SPOT.TB-positive rates in ATB and HC groups were 87.6% (78/89) and 21.1% (12/57), respectively. Of 38 ATB patients who were both TST and T-SPOT.TB tested, the positive rates were 73.7% (28/38) and 94.7% (36/38), respectively (P = 0.0215), and those in the HC group were 62.3% (33/53) and 18.9% (10/53), respectively (P < 0.0001). The T-SPOT.TB-positive rates declined during TB treatment and were 94.4% (51/54), 86.4% (19/22), and 61.5% (8/13) for ATB patients receiving 0- to 1-month, 1- to 3-month, and 3- to 6-month anti-TB treatment, respectively. The IGRA is a most promising test for both active TB and latent TB infection (LTBI) diagnosis due to the improvement of its specificity and convenience, especially in the Mycobacterium bovis BCG-vaccinated population. Furthermore, the T-SPOT.TB assay using ESAT-6 and CFP-10 in ATB patients during anti-TB treatment could serve as a potential predictor of therapeutic efficacy.     

Lipoprotein antigens of Mycobacterium tuberculosis

Detergent phase separation and metabolic labelling have been used to screen for the presence of lipoproteins amongst the antigens of Mycobacterium tuberculosis. Both techniques indicated that four antigens, with subunit molecular weights of 19, 26, 27 and 38 kilodaltons (kDa), are lipoproteins. This finding is consistent with the presence of conserved cysteine residues characteristic of other bacterial lipoproteins within the amino terminal sequences of the 38 kDa and 19 kDa proteins. It is proposed that lipoproteins are involved in the induction of humoral and cellular immune responses to mycobacteria and have a functional role in the transport of nutrients through the mycobacterial cell wall.     

Recognition of mycobacterial antigens by sera from patients with leprosy.

Mycobacterium leprae sonic extracts prepared from armadillo-derived bacteria were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting (immunoblotting) procedures and probed with serum or plasma samples from 20 patients with lepromatous leprosy and 14 healthy endemic controls. Five proteins of 33, 25, 18, 15, and 12 kilodaltons (kDa) were frequently recognized; the 33- and 15-kDa proteins were, respectively, recognized with high intensity by 16 and 13 of the 20 samples from patients with leprosy, whereas only one healthy donor had antibodies that recognized the 15-kDa protein. By the use of M. leprae-specific murine monoclonal antibodies it was demonstrated that the 33-, 25-, and 15-kDa antigens were different from those bound by the available murine monoclonal antibodies. The 18- and 12-kDa proteins detected had molecular masses similar to those detected by the corresponding murine monoclonal antibodies. The serum and plasma samples from patients with leprosy were also used to probe Western blots of a soluble extract of M. tuberculosis. They recognized, among others, antigens with molecular weights similar to those detected in the M. leprae antigenic preparations, although with less intensity and at a lower frequency.     

Effect of mycobacterial phospholipids on interaction of Mycobacterium tuberculosis with macrophages

This study demonstrates that pretreatment of macrophages with phosphatidylinositol, of either soya bean or mycobacterial origin, results in a down-regulation of the binding and uptake of Mycobacterium tuberculosis by the phagocytes. We also describe the novel observation that cardiolipin induces an increase in the binding and uptake of M. tuberculosis by macrophages. Neither phospholipid interacts with macrophages via the 2F8 epitope of scavenger receptor A, and treatment of macrophages with either phospholipid results in a down-regulation of CR3 function and tumor necrosis factor alpha production by the phagocyte. We have also shown that the ability of macrophages to interact with mycobacteria is greatly affected by an as yet unidentified product from the interaction of chloroform and polypropylene tubes.     

Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by two real-time calibrated PCR assays

Sensitive and specific techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS6110 multicopy element and on the senX3-regX3 intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of Mycobacterium recognition: broader for the IS6110-based assay and restricted to the M. tuberculosis complex for the senX3-regX3-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the M. tuberculosis DNA load measured by the senX3-regX3-based assay. In addition, reduced levels of M. tuberculosis DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens.     

Studies of mycobacterial antigens, with special reference to Mycobacterium leprae.

Eight individual antigens were detected in soluble antigen preparations from Mycobacterium leprae bacilli by using pools of serum samples from lepromatous leprosy patients as antibody reagents in crossed immunoelectrophoresis. Two of these antigens were analyzed further. Antgent no. 1 gave an elution pattern on Sephadex G-200 corresponding to a molecular weight of 285,000. This antigen was also present in three slow-growing and eight fast-growing mycobacterial species. There was a reaction of complete identity in immunological tests using lepromatous serum pools as well as with rabbit antisera raised against M. leprae and M. smegmatis. Antigen no. 21 of M. leprae showed antigenic heterogeneity when compared with other species. Three types of antigenic determinants were detected; one, called 21A, was shared by all mycobacteria, another, called 21B, was limited to antigen no. 21 of M. leprae; a third, called 21C, was present in all mycobacteria except the leprosy bacillus. This submolecular heterogeneity may indicate a separate taxonomic position of M. leprae among the mycobacteria.     

Recognition of mycobacterial antigens delivered by genetically detoxified Bordetella pertussis adenylate cyclase by T cells from cattle with bovine tuberculosis

The exponential increase in the incidence of tuberculosis in cattle over the last two decades in the British national herd constitutes a significant economic problem. Therefore, research efforts are under way to develop cattle tuberculosis vaccines and specific diagnostic reagents to allow the distinction of vaccinated from infected animals. Mycobacterial antigens like ESAT-6 and CFP10 allow this distinction. This study investigates whether fusion protein of ESAT-6 or CFP10 with genetically detoxified Bordetella pertussis adenylate cyclase (CyaA) are recognized by Mycobacterium bovis-infected cattle more effectively than conventional recombinant proteins are, thus enhancing sensitivity or reducing the amount of antigens required. By measuring the frequencies of gamma interferon (IFN-gamma)-producing cells, we were able to show that the presentation of CFP10 as a CyaA fusion protein enhanced the molecular efficiency of its recognition 20-fold, while the recognition of ESAT-6 was not improved by CyaA delivery. Furthermore, in the whole-blood IFN-gamma test currently used in the field, the delivery of CFP10 and ESAT-6 by fusion to CyaA increased the amount of IFN-gamma produced and hence the proportion of infected animals responding to CFP10. The improved T-cell recognition of CyaA336/CFP10 was found to be dependent upon interaction with CD11b. In addition, presentation of CyaA336/CFP10 to CD4+ T cells was chloroquine sensitive, and CFP10 delivery by CyaA resulted in its accelerated presentation to T cells. In conclusion, the use of CyaA fusion proteins with ESAT-6 and CFP10 has the potential to improve the sensitivity of immunodiagnostic tests detecting bovine tuberculosis in cattle.     

结核分枝杆菌抗原分析及免疫交叉反应研究

目的：筛选和鉴定结核分枝杆菌特异性和保护性抗原，研究结核杆菌的免疫反应特点，以探索结核病诊断和治疗的新途径。方法：采用超声破碎和滤膜抽滤的方法分别得到菌体蛋白和滤液蛋白，通过Western blot试验用结核杆菌的单克隆抗体及结核病人血清来检测蛋白样品，把发生阳性反应的蛋白在BECKMAN LF3200／多肽氨基酸序列测定仪上进行N末端序列分析，并用结核杆菌的单抗对自身抗原组蛋白进行了检测。结果：结核杆菌的31kD和30kD蛋白与结核杆菌的单抗及病人血清反应均呈阳性，但与正常小鼠血清和健康人血清反应呈阴性。31kD和30kD蛋白的N末端序列分别为：Ala Glu Val Asp Trp Leu Val Phe Ala Val和Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr。结核分枝杆菌的单抗与自身抗原组蛋白能发生免疫交叉反应。结论：结核杆菌的31kD和30kD蛋白是免疫保护性抗原，对免疫交叉反应分子基础的进一步研究必将增加对结核免疫机理的了解。     

Nontuberculous mycobacterial infections among patients suspected of pulmonary tuberculosis

The purpose of this study was to present a retrospective analysis of the frequency of nontuberculous mycobacteria (NTM)-related pulmonary infections among the AFB-positive and/or culture-positive patients in the Warsaw region who were suspected of tuberculosis (TB) and hospitalized in the university hospital between 1999 and 2005. All the AFB-positive pulmonary samples were examined with a molecular method using the Amplicor MTB test (Roche) for detection of Mycobacterium tuberculosis complex, and all mycobacterial isolates were speciated by high performance liquid chromatography (HPLC) analysis of mycolic acids. Patients who met clinical, radiological, and bacteriological criteria of mycobacteriosis were classified according to the American Thoracic Society (ATS) guidelines for diagnosis of NTM related disease. Among the 445 smear-positive or/and culture-positive patients, 142 subjects (31.9%) were found to be infected with M. tuberculosis. Among 303 non-TB patients, mycobacteriosis was found in 27 (8.9%) subjects. The frequency of NTM-related lung disease as compared to the bacteriologically-confirmed lung TB was estimated at 1:5. The rapid, precise methods of NTM speciation are necessary for progress in diagnostics of NTM related diseases.     

Immunology of mycobacterial infections: with special reference to Mycobacterium avium subspecies paratuberculosis

The interplay between mycobacteria and host determines the outcome of infection. After uptake of mycobacteria by macrophages, several possible scenarios may emerge; mycobacteria may be destroyed immediately or there is establishment of persistent infection. This review is focused around mycobacteria-host interactions with reference to Mycobacterium avium subspecies paratuberculosis (MAP) infection and highlights protective mechanisms involved in order to design vaccines and other control strategies.     

Isolation of Mycobacterium kumamotonense from a patient with pulmonary infection and latent tuberculosis

Mycobacterium kumamotonense is a novel, slow-growing non-chromogenic nontuberculous mycobacterium, which belongs to Mycobacterium terrae complex. We report, for the first time in Greece, the isolation of M. kumamotonense from an immunocompetent patient with pulmonary infection and latent tuberculosis. M. kumamotonense was identified by sequencing analysis of 16S rDNA and 65-kDa heat shock protein genes while by commercial molecular assays it was misidentified as Mycobacterium celatum. Antibiotic susceptibility testing was performed by the reference broth microdilution method. The strain was susceptible to amikacin, clarithromycin, rifampin, ciprofloxacin, moxifloxacin, rifabutin, ethambutol and linezolid.     

Cloning and expression of immunoreactive antigens from Mycobacterium tuberculosis

Four immunoreactive proteins, B.4, B.6, B.10, and B.M, with molecular weights ranging from 16,000 to 58,000, were observed from immunoblots of Mycobacterium tuberculosis total lysates screened with sera from individuals with active tuberculosis. These proteins were identified from microsequence analyses, and genes of proteins with the highest homology were PCR amplified and cloned into the pQE30 vector for expression studies. In addition, a 37.5-kDa protein, designated C17, was identified from a phage expression library of M. tuberculosis genomic DNA. Preliminary immunoblot assays indicated that these five resultant recombinant proteins could detect antibodies in individuals with active pulmonary and extrapulmonary tuberculosis. The overall ranges of sensitivities, specificities, positive predictive values, and negative predictive values for the recombinant antigens were 20 to 58, 88 to 100, 69 to 100, and 56 to 71%, respectively. The B.6 antigen showed preferential reactivity to antibodies in pulmonary compared to nonpulmonary tuberculosis serum specimens. All of these recombinant antigens demonstrated potential for serodiagnosis of tuberculosis.     

Cell-mediated immune responses to mycobacterial antigens in patients with pulmonary tuberculosis and HIV infection

Lymphocyte proliferation and cytokine responses induced by a panel of mycobacterial antigens were compared in Portuguese donors with pulmonary tuberculosis (TB) with or without HIV co-infection, HIV⁺ patients and healthy Mantoux-positive controls. Control donors showed stronger proliferative responses than any of the patient groups, with secreted antigens (Mycobacterium tuberculosis (Mtb) 30 kD and short-term culture filtrate proteins (ST-CFP)), purified protein derivative (PPD) and Mtb H37Rv Sonicate (MtbS) inducing the strongest proliferation. Patients with pulmonary TB showed lower proliferation to PPD or to the 30-kD antigen. Responses to all the antigens (PPD, ST-CFP, MtbS, 70 kD, 65 kD, 38 kD, 30 kD and 10 kD) were higher in TB/HIV patients with CD4 counts 200 CD4⁺ T cells/mm³ compared with HIV alone (CD4 200 T cells/mm³), but were lost in both TB/HIV and HIV patients when CD4 counts fell below 200 T cells/mm³. Measurements of interferon-gamma (IFN-γ) in culture supernatants revealed that PPD, 30 kD, MtbS and ST-CFP induced the strongest Th1 response. Analysis of mRNA for IFN-γ, IL-4 and IL-10 confirmed that IFN-γ production was maintained in patients with pulmonary TB without any concomitant increase in IL-4 or IL-10 mRNA expression, although expression of IL-10 mRNA was increased if HIV infection was present. These results reveal that IFN-γ production is retained in pulmonary TB patients to a broad range of mycobacterial antigens, and that no switch to IL-4 production is seen even with HIV infection. Secreted antigens, and in particular ST-CFP, were the best inducers of IFN-γ secretion, confirming their role in protective responses to Mtb.