The mycobacterial secreted antigen 85 complex possesses epitopes that are differentially expressed in human leprosy lesions and Mycobacterium leprae-infected armadillo tissues.

The granulomatous skin lesions in leprosy are thought to be initiated by the immune response to certain antigens of the causative agent, Mycobacterium leprae. The antigen 85 complex is one of the major targets in the immune response to M. leprae infection. In the present study, a panel of previously characterized monoclonal antibodies (MAbs) (3A8, Rb2, A4g4, A2h11, Pe12, and A3c12) reacting with different epitopes of the 85 complex proteins of Mycobacterium tuberculosis and M. leprae was employed in a comparative immunohistological analysis to demonstrate the in situ expression of 85 complex antigenic epitopes in leprosy lesions across the clinical spectrum and in M. leprae-infected armadillo liver tissues. These MAbs showed a heterogeneous staining pattern in a given leprosy lesion. In highly bacilliferous borderline and lepromatous leprosy lesions, MAbs Rb2, A4g4, A2h11, and Pe12 stained clear rod-shaped M. leprae bacilli within macrophages, and the degree of staining correlated with the bacillary index of the lesion. On the other hand, MAbs 3A8 and A3c12 staining was mostly seen as a diffuse staining pattern within interstitial spaces and on the membranes of the infiltrated cells but not the bacilli. In paucibacillary borderline and tuberculoid leprosy lesions, only 3A8, Rb2, and A3c12 showed distinct staining in association with infiltrates in the granuloma. None of these MAbs showed any detectable reaction with control nonleprosy skin lesions, while MAb A3c12 positively stained the granulomas of both leprosy and control specimens. In situ reactivity of these MAbs with M. leprae-infected armadillo liver tissues also showed a heterogeneous staining pattern. Interestingly, a clear difference in expression of these epitopes was observed between armadillo tissues and human leprosy lesions. By immunogold ultracytochemistry, we further showed the differential localization of these MAb-reactive epitopes on the cell surface, in the cytosol, and at the vicinity of M. leprae within Kupffer cells of armadillo liver tissues. Our results indicate that these antigenic epitopes of the antigen 85 complex are differentially expressed in leprosy lesions and infected armadillo tissues and that they could be target determinants in the immunopathological responses during M. leprae infection.     

High antibody levels to the mycobacterial fibronectin-binding antigen of 30-31 kD in tuberculosis and lepromatous leprosy.

Immunoblot assays showed that mycobacterial fibronectin-binding antigens are important targets of the humoral immune response in tuberculosis and leprosy. Using culture filtrate antigens of Mycobacterium tuberculosis, strong reactivity with the fibronectin-binding of 30-31 kD (Fn 30-31) was demonstrated in 55.9% of tuberculosis sera and in 56.5% of lepromatous leprosy sera. Sera from patients with tuberculoid leprosy and control sera gave very weak binding. Reactivity of tuberculosis and lepromatous leprosy sera with the fibronectin-binding antigen of 58-60 kD (Fn 58-60) was less conspicuous. The ability to react with fibronectin of the antigens of 58-60 and 30-31 kD was demonstrated by parallel labelling with a fibronectin-biotin conjugate. Fn 30-31 was purified to homogeneity by a two-step procedure and used for ELISA. Positive titres were found in 63% out of 65 tuberculosis sera and in 60.5% out of 43 lepromatous leprosy sera. Antibody titres in lepromatous leprosy sera were higher than in tuberculosis sera. Our observations indicate indirectly that M. leprae possess a highly immunogenic molecule homologous to M. tuberculosis Fn 30-31, which elicits a high antibody response in lepromatous leprosy but not in tuberculoid leprosy. In this investigation, direct evidence for the presence of this antigen in M. leprae was obtained by immunochemistry of lepromatous leprosy lesions with a monospecific antibody raised against M. tuberculosis Fn 30-31.     

Heterogeneity of monoclonal antibody-reactive epitopes on mycobacterial 30-kilodalton-region proteins and the secreted antigen 85 complex and demonstration of antigen 85B on the Mycobacterium leprae cell wall surface.

Proteins of the antigen 85 complex in the 30-kDa region secreted by live mycobacteria are important in the immune response against mycobacterial infections and may play an important biological role in the host-parasite interaction. In the present study, we have characterized epitopes of the 30-kDa-region proteins and the antigen 85 complex by using a panel of 13 monoclonal antibodies (MAbs) reacting with these antigens, 6 of which have not been described before. By using five previously characterized related secreted proteins of Mycobacterium tuberculosis, MPT44 (85A), MPT59 (85B), MPT45 (85C), MPT51 (27 kDa), and MPT64 (26 kDa), we have identified at least 10 different MAb-reactive epitopes on the proteins of the antigen 85 complex. A heterogeneous distribution of epitopes was observed within the components of the antigen 85 complex. Two distinct epitopes specific for antigen 85B and two other epitopes restricted to the 85A and 85B components were recognized. Two of them were shared with a previously unidentified 27-kDa protein present in M. tuberculosis culture fluid from which all MPT proteins were derived. The rest of the MAb-reactive epitopes were found to be present mostly in antigens 85A and 85B and to a lesser extent in antigen 85C. None of these MAbs recognized component 85C alone nor did they bind to the related MPT51 and MPT64 proteins. Interestingly, most of the MAbs reacted with purified native proteins of the antigen 85 complex but not to them in their denatured forms. In contrast, reactivity of the MAbs with the cytosol fraction of M. tuberculosis in immunoblotting revealed that they bound to a closely related cytosolic 30-kDa protein(s) even when they were denatured. Heterogeneity of these MAb-reactive epitopes of the antigen 85 complex was further evident as they were found to be distributed in various patterns among 19 different mycobacterial species. By using fusion proteins of the Mycobacterium leprae 30/31-kDa antigen 85 complex, we have localized at least six different epitopes within amino acid residues 55 to 266 of the M. leprae antigen 85 complex. Finally, by immunohistochemical analysis, we have demonstrated the in situ expression of one of the novel MAb-reactive epitopes specific for antigen 85B on the cell wall surface of M. leprae within macrophages in lepromatous leprosy lesions and thus provide direct evidence for the presence of the B component of the antigen 85 complex on the surface of intact M. leprae.     

Optimization of codon usage enhances the immunogenicity of a DNA vaccine encoding mycobacterial antigen Ag85B

In spite of its many other benefits, DNA vaccine is limited in its application by its insufficient immunogenicity. One promising approach for enhancing its immunogenicity is to maximize its expression in the immunized host. In the current study, we investigated whether codon optimization of the mycobacterial antigen Ag85B gene could enhance the expression and immunogenicity of the Ag85B DNA vaccine. We generated a synthetic humanized Ag85B (hAg85B) gene in which codon usage was optimized for expression in human cells. DNA plasmids with codon-optimized hAg85B increased the level of protein expression in vitro and in vivo. DNA vaccine with hAg85B induced stronger Th1-like and cytotoxic T-cell immune responses in BALB/c mice and generated higher protective immunity in a BALB/c mouse model of Mycobacterium tuberculosis aerosol infection than did the DNA vaccine with wild-type Ag85B. Therefore, our results suggest that codon optimization of mycobacterial antigens (e.g., Ag85B) could improve protein expression and thereby enhance the immunogenicity of DNA vaccines against M. tuberculosis.     

Localization and retention of mycobacterial antigen in lymph nodes of leprosy patients.

Although leprosy, a chronic disease caused by M. leprae, primarily affects skin and peripheral nerves, pathological changes and granulomas have been observed in lymph nodes which are: (a) present in tuberculoid lymph nodes in the absence of acid-fact bacilli and (b) persistent in lepromatous patients even after prolonged treatment. We detected substantial amounts of mycobacterial antigen in 16 leprous lymph nodes using anti-BCG by the peroxidase anti-peroxidase method. The load and distribution of antigen varied along the spectrum and with the duration of treatment. Tuberculoid and long-term treated lepromatous lymph nodes had a similar distribution of antigen in clusters of cells giving a ''speckled'' appearance. The untreated lepromatous had a ''diffuse'' staining of antigen in foamy histiocytes whereas lepromatous lesions with a lower bacillary load had a mixed pattern of ''diffuse'' and ''speckled''. Antigen was also detected in a number of plasma cells along the spectrum but predominantly in lepromatous lymph nodes. Our observations indicate that: (a) antigen exists in lymph nodes despite prolonged chemotherapy which may be responsible for the persistent granuloma and (b) antigen is not confined to any particular anatomical compartment of the lymph node.     

Humoral responses against the 85A and 85B antigens of Mycobacterium bovis BCG in patients with leprosy and tuberculosis.

Immunoglobulin G antibodies against the 85A and 85B components of the Mycobacterium bovis BCG antigen 85 complex separated by isoelectric focusing were investigated in serum samples from 129 patients representing the major forms of leprosy, 111 tuberculous patients, and 153 healthy subjects. For both of the antigens, a higher degree of staining was observed for lepromatous leprosy patients and patients with active tuberculosis than for the other groups. Because sera from some healthy subjects recognized the 85A antigen, we suggest that antigen 85B is the most useful component of the antigen 85 complex for the serodiagnosis of the multibacillary forms of leprosy or of the active forms of tuberculosis.     

Phagosomal processing of Mycobacterium tuberculosis antigen 85B is modulated independently of mycobacterial viability and phagosome maturation

Control of Mycobacterium tuberculosis infection requires CD4 T-cell responses and major histocompatibility complex class II (MHC-II) processing of M. tuberculosis antigens (Ags). We have previously demonstrated that macrophages process heat-killed (HK) M. tuberculosis more efficiently than live M. tuberculosis. These observations suggested that live M. tuberculosis may inhibit Ag processing by inhibiting phagosome maturation or that HK M. tuberculosis may be less resistant to Ag processing. In the present study we examined the correlation between M. tuberculosis viability and phagosome maturation and efficiency of Ag processing. Since heat treatment could render M. tuberculosis Ags more accessible to proteolysis, M. tuberculosis was additionally killed by antibiotic treatment and radiation. Processing of HK, live, radiation-killed (RadK), or rifampin-killed (RifK) M. tuberculosis in activated murine bone marrow macrophages was examined by using an I-A(b)-restricted T-cell hybridoma cell line (BB7) that recognizes an epitope derived from Ag 85B. Macrophages processed HK M. tuberculosis more rapidly and efficiently than they processed live, RadK, or RifK M. tuberculosis. Live, RadK, and RifK M. tuberculosis cells were processed with similar efficiencies for presentation to BB7 T hybridoma cells. Furthermore, phagosomes containing live or RadK M. tuberculosis expressed fewer M. tuberculosis peptide-MHC-II complexes than phagosomes containing HK M. tuberculosis expressed. Since only live M. tuberculosis was able to prevent acidification of the phagosome, our results suggest that regulation of phagosome maturation does not explain the differences in processing of different forms of M. tuberculosis. These findings suggest that the mechanisms used by M. tuberculosis to inhibit phagosomal maturation differ from the mechanisms involved in modulating phagosome Ag processing.     

Mapping of murine Th1 helper T-Cell epitopes of mycolyl transferases Ag85A,Ag85B,and Ag85C from Mycobacterium tuberculosis

BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice were infected intravenously with Mycobacterium tuberculosis H37Rv or vaccinated intramuscularly with plasmid DNA encoding each of the three mycolyl transferases Ag85A, Ag85B, and Ag85C from M. tuberculosis. Th1-type spleen cell cytokine secretion of interleukin-2 (IL-2) and gamma interferon (IFN-gamma) was analyzed in response to purified Ag85 components and synthetic overlapping peptides covering the three mature sequences. Tuberculosis-infected C57BL/6 mice reacted strongly to some peptides from Ag85A and Ag85B but not from Ag85C, whereas tuberculosis-infected BALB/c mice reacted only to peptides from Ag85A. In contrast, spleen cells from both mouse strains produced elevated levels of IL-2 and IFN-gamma following vaccination with Ag85A, Ag85B, and Ag85C DNA in response to peptides of the three Ag85 proteins, and the epitope repertoire was broader than in infected mice. Despite pronounced sequence homology, a number of immunodominant regions contained component specific epitopes. Thus, BALB/c mice vaccinated with all three Ag85 genes reacted against the same amino acid region, 101 to 120, that was also immunodominant for Ag85A in M. bovis BCG-vaccinated and tuberculosis-infected H-2(d) haplotype mice, but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241 to 260 and 261 to 280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA. Our results underline the need for comparative analysis of all three Ag85 components in future vaccination studies.     

Immunocytochemical detection of mycobacterial antigen in extrapulmonary tuberculosis

The aim of the study is to determine whether immunostaining for mycobacterial antigen can contribute to the cytological diagnosis of extrapulmonary tuberculosis (EPTB). The study was carried out on aspirated material of lymph nodes, and other accessible sites, from 65 patients with clinical diagnosis of tuberculosis (TB). Twenty patients, diagnosed by fine‐needle aspiration, with non‐tuberculous granulomas served as controls. The diagnosis of TB was based on the demonstration of acid‐fast bacilli (AFB), culture positivity for Mycobacterium tuberculosis (M. tuberculosis), or response to treatment with standard anti‐tubercular therapy. Immunostaining was done using polyclonal antibody to mycobacteria. AFB positivity by Ziehl Neelsen (ZN) staining was 21%, 65.38%, and 68% respectively in Pattern 1 (granulomas alone), in Pattern 2 (granulomas with necrosis), and in Pattern 3 (necrosis alone). Overall AFB positivity was 56.92%. Twenty‐eight of 65 cases were negative for AFB on direct smear. Culture was positive in 46% (13/28). Sensitivity and specificity of immunostaining were 96.92% (63/65) and 95%, respectively. Immunoreactivity was seen in 26 (92.8%) of 28 cases which were negative by ZN staining. Except in the case of leprosy, in which cross reactivity was seen, there was no immunoreactivity in the control group. Immunocytochemistry (ICC) had high sensitivity (96.2%) and specificity (95%) in the diagnosis of EPTB. ICC may be a useful adjunct to evaluation of cytomorphology and ZN staining. Diagn. Cytopathol 2014;42:391–395. © 2013 Wiley Periodicals, Inc.     

Localization and retention of mycobacterial antigen in lymph nodes of leprosy patients

Although leprosy, a chronic disease caused by M. leprae, primarily affects skin and peripheral nerves, pathological changes and granulomas have been observed in lymph nodes which are: (a) present in tuberculoid lymph nodes in the absence of acid-fact bacilli and (b) persistent in lepromatous patients even after prolonged treatment. We detected substantial amounts of mycobacterial antigen in 16 leprous lymph nodes using anti-BCG by the peroxidase anti-peroxidase method. The load and distribution of antigen varied along the spectrum and with the duration of treatment. Tuberculoid and long-term treated lepromatous lymph nodes had a similar distribution of antigen in clusters of cells giving a 'speckled' appearance. The untreated lepromatous had a 'diffuse' staining of antigen in foamy histiocytes whereas lepromatous lesions with a lower bacillary load had a mixed pattern of 'diffuse' and 'speckled'. Antigen was also detected in a number of plasma cells along the spectrum but predominantly in lepromatous lymph nodes. Our observations indicate that: (a) antigen exists in lymph nodes despite prolonged chemotherapy which may be responsible for the persistent granuloma and (b) antigen is not confined to any particular anatomical compartment of the lymph node.     

结核分枝杆菌DNA疫苗pVS85B的构建及免疫保护实验研究

评价构建的结核DNA疫苗pVS85B免疫小鼠后脾细胞体外产生细胞因子和抵抗结核分枝杆菌H37Rv攻击的能力。利用基因操作技术将结核菌ag85b插入pVAX1载体,构建表达结核菌Ag85B分泌型蛋白的DNA疫苗pVS85B。将雌性C57BL/6小鼠分成5组,每组20只,分别用pVS85B、pVAX1、pIL2S和PBS分别免疫3次,均间隔2周,以相同剂量加强免疫2次。另一组用BCG(105CFU)免疫1次。每组10只鼠在最后一次加强免疫后,无菌取脾培养,检测上清细胞因子。另10只鼠用结核菌H37Rv经静脉攻击,2周后取脾、肝和肺培养结核菌并进行菌落计数。结果是pVS85B免疫组鼠脾淋巴细胞培养上清的mIL-2和mIFN-γ平均含量分别为(311.3±46.2)和(273.6±55.4)pg/ml,显著高于3个阴性对照组(P<0.001),与BCG免疫组无显著性差异(P>0.05)。5个组的平均mIL-6和mIL-10无显著性差异。pVS85B免疫组小鼠的脾、肝和肺的平均结核菌载量分别为(47 716.6±5 689.0)、(50 113.4±6 532.2)和(51 095.3±2 788.9)CFU/g,分别低于pVAX1、pIL2S和PBS三个阴性对照组的相应器官的结核菌载量(P<0.001)。pVS85B免疫组脾、肝和肺的结核菌载量显著高于BCG免疫对照组。提示,表达结核菌分泌型Ag85B的DNA疫苗pVS85B能够刺激机体产生抗结核菌所需的Th1型免疫应答,免疫鼠获得抵抗H37Rv攻击的能力。     

Modulation of expression of delayed hypersensitivity by mycobacterial antigen 85 fibronectin-binding proteins.

Although demonstration of delayed hypersensitivity to purified protein derivative of tuberculin (PPD) is an important element in the diagnosis of infection with Mycobacterium tuberculosis, many patients with tuberculosis are anergic. Several possible mechanisms for this specific lack of response have been described. We have now uncovered an additional one. T-cell fibronectin (FN), a lymphokine secreted by activated T cells, is closely associated with the initiation of delayed hypersensitivity reactions. Mycobacterial antigen 85 (Ag85) proteins have been shown to bind to plasma FN. The ability of Ag85 to bind to T-cell FN and modulate expression of delayed hypersensitivity was therefore studied. Purified Ag85 proteins from M. tuberculosis, Mycobacterium bovis BCG, or Mycobacterium kansasii bound to T-cell FN, fibroblast FN, and plasma FN in vitro. Purified 65-kDa heat shock protein (hsp65) from M. bovis BCG did not bind to any FN. Ag85, but not hsp65, inhibited the ability of T-cell FN to agglutinate monocytes in vitro in a dose-dependent manner. In vivo, mixtures of PPD or dinitrophenyl-ovalbumin and purified M. tuberculosis or M. bovis BCG Ag85 proteins elicited significantly smaller delayed hypersensitivity inflammatory reactions in sensitized guinea pigs than did PPD or dinitrophenyl-ovalbumin alone. Purified hsp65 did not inhibit expression of delayed hypersensitivity to PPD or dinitrophenyl-ovalbumin. We suggest that Ag85 proteins could inhibit in vivo expression of delayed hypersensitivity during mycobacterial infections because of their interaction with T-cell FN.     

Evidence for three separate genes encoding the proteins of the mycobacterial antigen 85 complex.

The secreted Mycobacterium bovis BCG antigen 85 complex, which is known to bind to human fibronectin, consists of three closely related cross-reacting antigens. Amino-terminal sequence analysis of the purified proteins showed distinct differences. Data are presented to show that the three components are produced by individual cells, which indicates that three separate genes are involved.     

Epitope focus, clonal composition and Th1 phenotype of the human CD4 response to the secretory mycobacterial antigen Ag85

Lymphoproliferation of healthy donors was tested against mycobacterial antigens (PPD, Ag85, Ag85 peptides). All PPD responders recognized the secretory antigen Ag85 and the peptide specificity for Ag85B was defined. Peptide 91-108 was recognized by 85% of donors. In addition, all CD4 T cell lines generated from 12 donors against PPD or Ag85 responded to 91-108. When this peptide was used to generate T cell lines, the cells responded also to tuberculins from atypical mycobacterial species. Thus the cross-reactive peptide behaved as quasi-universal. The analysis of TCR-BV gene usage by cell lines showed that most Ag85-specific T cells correspond to 91-108-specific clonotypes. Intracytoplasmic staining of cell lines after phorbol myristate acetate stimulation resulted in dominance of interferon-gamma (IFN-gamma)-IL-4 double-positive cells, whereas antigen stimulation resulted in production of IFN-gamma only. The data show that peptide 91-108 is the major focus of the CD4 response to mycobacterial antigens in peripheral blood mononuclear cells and in T cell lines from PPD responders.     

Delayed hypersensitivity reactions by the mycobacterial antigen A60 and cutaneous testing in tuberculosis

Antigen A60 has been purified from the cytoplasm of Mycobacterium bovis BCG, and its composition has been determined: it has proved to be able to elicit immune reactions of both humoral and cellular type. Inoculation of A60 into the footpad of mice previously sensitized with the same antigen, or with whole mycobacterial cells produced a footpad swelling showing a peak at 24 h. Similar delayed hypersensitivity reactions were induced in sensitized guinea-pigs by subcutaneous injection of an A60 dose of 0.01 g (minimal revealing dose). A quantity thousandfold higher (15 g A60) was unable to induce in unsensitized guinea pigs the mounting of a cellular immunisation against A60, as shown by negative cutaneous testings 1 month later. Our results show that A60 preparations satisfied the requirements of the European Pharmacopoeia Commission and met the WHO recommandations for new tuberculins. Handicaps of old tuberculin and PPD (heterogeneous mixtures titrated biologically and unstable in solution) can be overcome by A60 preparations (a single antigen spectrophoretically measurable and stable).Abbreviations BCG Mycobacterium bovis, strain Calmette Guérin - A60 Antigen 60 of BCG (classification of Closs et al. [4] - CMN Group of Gram-positive bacteria including genera Corynebacterium, Mycobacterium and Nocardia - TMA (thermostable macromolecular antigens): a group of mycobacterial components including A60 of BCG and A7 of M. leprae - Elisa (enzyme-linked immunosorbent assay) immunoenzymometric method - Ph.Eur.U. European Pharmacopoeia Units of tuberculins     

腺病毒为载体的新型结核病疫苗Ad5Ag85A

结核病是一个在全球范围内严重威胁人类健康的呼吸道传染性疾病.据世界卫生组织(WHO)估计,世界上约有1/3的人口受到结核分枝杆菌(Mycobacterium tuberculosis)的感染,其中1/10的人可能会最终患上结核病.全球每年的新病例超过900万,约200万人死于结核病[1].结核病负担最重的地区主要是东南亚(占总报告病例的35％)、非洲(24％)和西太平洋地区(22％).中国的结核病年发病患者数约为130万,占全球发病的14.3％,是世界第二大结核病高发国家(仅次于印度)[2].     

结核分枝杆菌Ag85A蛋白的原核表达、纯化和免疫反应性

目的通过原核表达获得结核分枝杆菌Ag85A蛋白。方法用PCR从结核分枝杆菌H37Rv菌株中扩增出编码Ag85A的fbpA基因,克隆入原核表达载体pProEXHTb,产生重组质粒pPro85A后,转化至大肠杆菌感受态细胞BL21并诱导大量表达。用镍纯化系统纯化重组Ag85A蛋白,用不同分枝杆菌感染的小鼠血清通过ELISA确定其免疫反应性。利用PCR技术鉴定fbpA基因在不同分枝杆菌的分布。结果32 ku的Ag85A蛋白获得高效表达和纯化。表达Ag85A蛋白的fbpA基因在结核分枝杆菌H37Rv、H37Ra、BCG、草分枝杆菌、土地分枝杆菌、耻垢分枝杆菌和次要分枝杆菌中均有表达,但在牝牛分枝杆菌中未表达。结核病患者和结核分枝杆菌毒株H37Rv感染小鼠血清所产生的抗Ag85A抗体滴度最高。结论重组Ag85A蛋白已成功表达纯化,并保留了免疫反应性。     

结核分支杆菌Ag85A DNA疫苗免疫治疗作用的研究

目的:研究结核分支杆菌Ag85A DNA疫苗的免疫治疗作用。方法:用结核分支杆菌H37Rv腹腔内注射BALB/c小鼠 2个月后,将小鼠随机分为 5组,分别用生理盐水(A)、载体质粒(B)、卡介苗(C)、维卡菌苗(D)和 Ag85A DNA疫苗(E)免疫小鼠;用ELISA法检测血清抗体;免疫治疗2月和5月时,分别取肺、肝和脾脏观察病理改变、称取重量、作菌落计数、肺组织涂片及脾淋巴细胞转化试验。结果:DNA疫苗组抗原特异性抗体不同程度升高。免疫治疗2月时,各组鼠脾淋巴细胞转化率无显著差异;各治疗组肺组织涂片细菌数和肺菌落计数均比对照组不同程度地减少;C和B组的肺病变程度较轻。免疫治疗5月时,各治疗组鼠脾淋巴细胞转化率均显著增高;各组肺、脾细菌数无显著差别比和D组的肺未见明显病变。结论:DNA疫苗似乎具有一定的免疫治疗效果。     

Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis.

The T-cell response of human donors to secreted antigen fractions of Mycobacterium tuberculosis was investigated. The donors were divided into five groups: active pulmonary tuberculosis (TB) patients with minimal and with advanced disease, Mycobacterium bovis BCG-vaccinated donors with and without contact with TB patients, and nonvaccinated individuals. We found that patients with active minimal TB responded powerfully to secreted antigens contained in a short-term culture filtrate. The response to secreted antigens was mediated by CD4+ Th-1-like lymphocytes, and the gamma interferon release by these cells was markedly higher in patients with active minimal TB than in healthy BCG-vaccinated donors. Patients with active advanced disease exhibited depressed responses to all preparations tested. The specificity of the response to secreted antigens was investigated by stimulating lymphocytes with narrow-molecular-mass fractions of short-term culture filtrate obtained by the multielution technique. Considerable heterogeneity was found within the donor groups. Patients with active minimal TB recognized multiple secreted targets, but interestingly, six of eight patients demonstrated a predominant recognition of a low-mass (< 10-kDa) protein fraction which induced high levels of gamma interferon release in vitro. Only a few of 12 previously characterized secreted antigens were recognized by T cells isolated from TB patients, suggesting the existence of a number of as yet undefined antigenic targets among secreted antigens.     

Characterization of the secreted MPT53 antigen of Mycobacterium tuberculosis

MPT53 is a secreted protein of Mycobacterium tuberculosis. Southern transfer and hybridization showed mpt53 to be conserved in the M. tuberculosis complex and to have homology with DNA from Mycobacterium avium and other nontuberculous mycobacteria. However, anti-MPT53 polyclonal antibodies detected no antigen in the culture filtrates of M. avium and other nontuberculous mycobacteria. MPT53 of M. tuberculosis induced strong, tuberculosis-specific antibody responses in guinea pigs but induced no delayed-type hypersensitivity. Involvement in immune responses during human tuberculosis was very modest.