T-cell recognition of iron-regulated culture filtrate proteins of Mycobacterium tuberculosis in tuberculosis patients and endemic normal controls

Background: Culture filtrate proteins (CFPs) of Mycobacterium tuberculosis are potential vaccine candidates. Objective: The aim was to study the influence of iron levels on CFPs and assess the immuno-protective potential of defined antigenic fractions from high (8 μg Fe/mL) and low iron (0.02 μg Fe / mL) cultures of M. tuberculosis. Materials and Methods: The CFPs of M. tuberculosis from high (CFP-high) and low (CFP-low) iron conditions were first compared to identify iron-regulated proteins and then fractionated to obtain ten antigen pools (CF-Ags H1- H5 and L1-L5) that were used to assess the immune response of TB patients and normal healthy controls. Results: Iron limitation resulted in the up-regulation of two novel iron-regulated low-molecular-weight proteins Irp-1 (in CF-Ag L4) and Irp-2 (in CF-Ag L5) and repression of two ESAT proteins (identified with monoclonal antibody HYB 76.8). The median stimulation indices (SIs) against most of the CF-Ags were high in pulmonary TB patients. The CF-Ags L1 and L2 showed statistically significant SI (P values of 0.0027 and 0.0029 respectively); the % case recognition was high with these antigens as well as with L4 ( P = 0.0275). IFN-γ in response to these CF-Ags was significantly high in the endemic normals; maximal expression was seen with CF-Ag L5 (median value of 233 pg mL -1 ) that was higher than the corresponding H5 (140 pg mL -1 ) and H3 and L3 (205 and 206 pg mL -1 respectively). Conclusions: CF-Ags L5, H3 and L3 showed immuno-protective potential in this geographical location.     

Immunogenicity and safety of Mycobacterium tuberculosis culture filtrate proteins in non-human primates

The development of improved vaccines is considered a high priority in the effort to control tuberculosis (TB) world wide. Results from several studies performed in relevant animal models have demonstrated that Mycobacterium tuberculosis secreted antigens may represent major components of improved TB vaccines. To characterize further the M. tuberculosis secreted antigens as they relate to specific features important for vaccine development, rhesus macaques were immunized with either one of two different preparations containing M. tuberculosis culture filtrate (CF) proteins. These preparations differed in relative protein content and in the presence or absence of lipoarabinomannan. Animals received a total of three monthly intramuscular injections consisting of CF proteins resuspended in RIBI adjuvant and were tested for development of specific antibody and cellular proliferative responses. In addition, all animals were constantly monitored for local and systemic reactions as well as for the development of DTH reactions to intradermal tuberculin injection. Results from this study show that the two CF preparations are relatively safe and immunogenic in non-human primates. These two CF preparations differed in their ability to induce specific antibody responses, but were comparable in their ability to induce specific cellular proliferative responses. Induction of both humoral and cellular responses occurred even in presence of pre-existing antibodies directed against M. tuberculosis antigens. However, these responses appeared to be short-lived. Only one of the four animals produced interferon-gamma (IFN-gamma) in response to immunization with CF proteins. No DTH reaction to intradermal tuberculin injection was observed in any immunized animal. Although it is clear that additional studies are required to design strategies for the improvement of the immunogenicity of CF proteins, our observations support the currently accepted view that secreted protein-based preparations may represent promising vaccine candidates for TB.     

Vaccination of cattle with Mycobacterium bovis culture filtrate proteins and interleukin-2 for protection against bovine tuberculosis

In this study vaccines prepared from culture filtrate proteins (CFP) of Mycobacterium bovis and interleukin-2 (IL-2) were tested in cattle for their capacity to stimulate immune responses and to protect against an intratracheal challenge with virulent M. bovis. Nine groups of cattle were vaccinated with combinations of different doses of CFP and bovine IL-2 mixed with a monophosphoryl lipid A (MPL) adjuvant. An additional group was vaccinated with M. bovis BCG. Immune responses in b1P-IL-2-vaccinated animals differed from those seen in BCG-vaccinated animals by inducing high antigen-specific antibody responses and low levels of gamma interferon and IL-2 released from purified protein derivative-stimulated whole-blood cultures. In a concurrent experiment, additional animals were added to the high-dose CFP-IL-2, MPL control, and BCG groups and these expanded groups of animals were challenged intratracheally with virulent M. bovis. Although the lung lesion scores were significantly lower for both the CFP-IL-2-and BCG-vaccinated groups compared to the MPL control group, the overall level of protection was greatest for the BCG-vaccinated animals. There were more animals with extrathoracic spread of disease in the CFP-IL-2 group than in the other groups. While vaccination of cattle with M. bovis CFP gave an encouraging reduction in tuberculous lesions and did not induce a delayed-type hypersensitivity response to PPD, future CFP vaccines must prevent any extrathoracic spread of disease.     

二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗加强BCG免疫中的效应研究

目的 探讨二甲基三十六烷基铵(dimo-thylidioctyl ammonium bromide,DDA)和卡介苗多糖核酸(BCC-PSN)佐剂在结核融合蛋白疫苗加强卡介苗(BCG)免疫中的不同免疫辅助效应.方法 选择DDA、BCG-PSN作为融合蛋白AMM(Ag85B-MPT64190-198-Mtb8.4)的佐剂,在BCG初免小鼠后,AMM疫苗加强免疫两次,其中一组联合使用两种佐剂(DDA/BCG-PSN),另一组单独以DDA作为佐剂,同时设立BCG或磷酸缓冲液(PBS)免疫组为对照.应用ELISA及ELISPOT检测免疫小鼠的体液与细胞免疫反应,最后一次加强免疫后第12周,以H37Rv尾静脉攻毒并检测小鼠肺脾组织细菌载量和病理改变,评价不同佐剂疫苗的保护效果.结果 在BCG初免基础上,联合佐剂组(AMM/DDA/BCG-PSN)和单独佐剂组(AMM/DDA)加强免疫两次后,脾脏淋巴细胞经抗原Ag85B和PPD(purified protein derivative)刺激后,皆可产生分泌较BCG组高的IFN-γ.毒力株攻击后菌落形成单位(colony-forming unit,CFU)计数显示,联合佐剂组(AMM/DDA/BCG-PSN)脾脏荷菌量少于PBS组和BCG组(P<0.05);而单独佐剂组(AMM/DDA)肺部荷菌量少于PBS组和BCG组(P<0.05).组织病理分析结果 表明AMM/DDA/BCG-PSN组肺组织病理损伤较轻,而AMM/DDA组病理损伤个体差异较大.结论 DDA是较为理想的结核亚单位疫苗佐剂,能诱导较强的细胞免疫和免疫保护作用;BCG-PSN可能具有免疫调节作用,可以减轻免疫病理损伤.     

Evasion and subversion of antigen presentation by Mycobacterium tuberculosis

Mycobacterium tuberculosis is one of the most successful of human pathogens and has acquired the ability to establish latent or progressive infection and persist even in the presence of a fully functioning immune system. The ability of M. tuberculosis to avoid immune-mediated clearance is likely to reflect a highly evolved and coordinated program of immune evasion strategies, including some that interfere with antigen presentation to prevent or alter the quality of T-cell responses. Here, we review an extensive array of published studies supporting the view that antigen presentation pathways are targeted at many points by pathogenic mycobacteria. These studies show the multiple potential mechanisms by which M. tuberculosis may actively inhibit, subvert or otherwise modulate antigen presentation by major histocompatibility complex class I, class II and CD1 molecules. Unraveling the mechanisms by which M. tuberculosis evades or modulates antigen presentation is of critical importance for the development of more effective new vaccines based on live attenuated mycobacterial strains.     

Circulating, Mycobacterium tuberculosis-specific lymphocytes from PPD skin test-negative patients with tuberculosis do not secrete interferon-gamma (IFN-gamma) and lack the cutaneous lymphocyte antigen skin-selective homing receptor

Individuals with a negative intradermal reaction to tuberculin PPD have long been described in the Mycobacterium tuberculosis exposed, immune-competent population. Here, we studied PPD-specific blood T lymphocytes from these subjects for phenotypic markers relevant to skin migration, including the expression of the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen (CLA). Out of 82 patients with active tuberculosis we identified four subjects who were repeatedly PPD skin test-negative. CD4 T lymphocytes specific to mycobacterial antigens were derived from these individuals, which (i) proliferated in vitro to M. tuberculosis antigens comparably to those from PPD+ patients; (ii) secreted comparable amounts of IL-2 but lower amounts of IFN-gamma; (iii) were confined within the CLA-negative T cell subset. We conclude that the negative tuberculin reaction in a small subset of patients exposed to mycobacteria is associated with impaired production of IFN-gamma by circulating PPD-specific T cells that are lacking CLA expression. On this basis in vitro proliferation to PPD can discriminate bona fide non-responders from infected patients with a deficit in the margination of M. tuberculosis-specific T lymphocytes.     

Inoculum size and traits of the infecting clinical strain define the protection level against Mycobacterium tuberculosis infection in a rabbit model

Host protective immunity against pathogenic Mycobacterium tuberculosis (Mtb) infection is variable and poorly understood. Both prior Mtb infection and BCG vaccination have been reported to confer some protection against subsequent infection and/or disease. However, the immune correlates of host protection with or without BCG vaccination remain poorly understood. Similarly, the host response to concomitant infection with mixed Mtb strains is unclear. In this study, we used the rabbit model to examine the host response to various infectious doses of virulent Mtb HN878 with and without prior BCG vaccination, as well as simultaneous infection with a mixture of two Mtb clinical isolates HN878 and CDC1551. We demonstrate that both the ability of host immunity to control pulmonary Mtb infection and the protective efficacy of BCG vaccination against the progression of Mtb infection to disease is dependent on the infectious inoculum. The host response to infection with mixed Mtb strains mirrors the differential responses seen during infection with each of the strains alone. The protective response mounted against a hyperimmunogenic Mtb strain has a limited impact on the control of disease caused by a hypervirulent strain. This preclinical study will aid in predicting the success of any vaccination strategy and in optimizing TB vaccines.     

T cell reactivity against antigen 85 but not against the 18- and 65-kD heat shock proteins in the early stages of acquired immunity against Mycobacterium leprae.

T cell proliferation and interferon-gamma (IFN-gamma) production of peripheral blood mononuclear cells (PBMC) from 20 household contacts were tested against the 18- and 65-kD heat shock proteins from Mycobacterium leprae (ML18 and ML65 respectively) and antigen 85 from Myco. bovis bacille Calmette-Guérin (BCG) (Ag 85) during a 12-months follow-up study. Among the eight contacts that became positive, eight showed positive reactivity against Ag 85, 5/8 against ML65 and 4/8 against ML18 at the end of the study. Of the 16 contacts who were lepromin-positive either at first or second testing, all responded to Ag 85, 11 to ML 65, but only eight reacted to ML18 antigen. Contacts who were lepromin-positive at first testing developed responses to ML18 only at second testing. In contrast, among the four contacts that remained lepromin-negative during the follow up, three proliferated to Ag 85 either at first or second testing, but only one produced IFN-gamma against Ag 85 at the end of the study. These results demonstrated that T cell reactivity and particularly IFN-gamma secretion against Ag 85, but not against ML18 and ML65, might be a predominant mechanism in the early stages of acquired protective immunity against Myco. leprae.     

Circulating levels of IL-11 and leukaemia inhibitory factor (LIF) do not significantly participate in the production of acute-phase proteins by the liver

To investigate the contribution of IL-11 and LIF to acute-phase protein (APP) production, we first analysed the effects of IL-11 and LIF on production of C-reactive protein (CRP), fibrinogen, and haptoglobin by human primary hepatocytes. We also measured the serum levels of IL-11, LIF, and CRP in serum from patients with inflammatory rheumatic diseases to assess the role of these cytokines in the APP response in vivo. We included patients with conditions associated with a high APP response such as rheumatoid arthritis (RA) or spondylarthropathy (SpA), and others usually associated with a weak APP response such as systemic lupus erythematosus (SLE), in order to investigate whether these cytokines could account for the differences in APP responses. Our results showed that IL-11 and LIF induced only minimal stimulation on production of APP by human primary hepatocytes compared with IL-6, known as the major inducer. Serum levels of CRP were elevated in RA and SpA, and significantly higher than in SLE patients. Despite the presence of a high APP response in some of our patients and despite the fact that we used sensitive assays to measure IL-11 and LIF, serum levels of both cytokines were not detected in any of the tested sera. In conclusion, our results show that circulating levels of IL-11 or LIF do not contribute significantly to the production of APP in vivo, and that they do not account for the difference in APP response between SLE and other inflammatory rheumatic diseases.     

IFN‐γ against the 38‐kDa antigen of Mycobacterium tuberculosis discriminates pulmonary tuberculosis from infection and infection from exposure: evidence from a study of human population in a high endemic setting

Mycobacterium tuberculosis (Mtb) 38‐kDa antigen is an immunogenic lipoprotein that induces strong T‐cell responses in experimental animals. However, there is limited information on the role of this antigen in human population. In this article, we present the dynamics of pro‐inflammatory (IFN‐γ and TNF‐α) and anti‐inflammatory cytokine (IL‐10) against the 38 kDa in cohorts of pulmonary TB (PTB) patients, household contacts (HHCs), and community controls (CCs) in a high endemic setting. Whole blood assay was used to determine the levels of cytokines in 149 patients, 149 HHCs, and 68 CCs at baseline, 6 months, and 12 months. At baseline, the level of IFN‐γ was significantly (p < 0.0001) higher in CCs and HHCs than in untreated patients. CCs had significantly (p < 0.05) higher level of IFN‐γ than HHCs. There was no significant difference between treated and untreated patients, and there was no significant change in HHCs over 12 months. At baseline, the levels of IL‐10 and TNF‐α were significantly (p < 0.0001) higher in patients than in HHCs and CCs. No significant change was observed between treated patients and untreated patients and HHCs over time. The study shows that IFN‐γ against the 38 kDa discriminates clinical TB from infection and infection from exposure, suggesting its potential for immune protection and diagnosis.