RESUMO
BACKGROUND: Tuberculosis (TB) remains one of the most deadly infectious diseases. One-third to one-fourth of the human population is estimated to be infected with Mycobacterium tuberculosis (Mtb) without showing clinical symptoms, a condition called latent TB infection (LTBI). Diagnosis of Mtb infection is based on the immune response to a mixture of mycobacterial antigens (PPD) or to Mtb specific ESAT-6/CFP10 antigens (IGRA), highly expressed during the initial phase of infection. However, the immune response to PPD and IGRA antigens has a low power to discriminate between LTBI and PTB. The T-cell response to a group of so-called latency (DosR-regulon-encoded) and Resuscitation Promoting (Rpf) antigens of Mtb has been proved to be significantly higher in LTBI compared to active TB across many populations, suggesting their potential use as biomarkers to differentiate latent from active TB. METHODS: PBMCs from a group LTBI (n = 20) and pulmonary TB patients (PTB, n = 21) from an endemic community for TB of the city of Medellín, Colombia, were in vitro stimulated for 7 days with DosR- (Rv1737c, Rv2029c, and Rv2628), Rpf- (Rv0867c and Rv2389c), the recombinant fusion protein ESAT-6-CFP10 (E6-C10)-, or PPD-antigen. The induced IFNγ levels detectable in the supernatants of the antigen-stimulated cells were then used to calculate specificity and sensitivity in discriminating LTBI from PTB, using different statistical approaches. RESULTS: IFNγ production in response to DosR and Rpf antigens was significantly higher in LTBI compared to PTB. ROC curve analyses of IFNγ production allowed differentiation of LTBI from PTB with areas under the curve higher than 0.70. Furthermore, Multiple Correspondence Analysis (MCA) revealed that LTBI is associated with higher levels of IFNγ in response to the different antigens compared to PTB. Analysis based on decision trees showed that the IFNγ levels produced in response to Rv2029c was the leading variable that best-classified disease status. Finally, logistic regression analysis predicted that IFNγ produced by PBMCs in response to E6-C10, Rv2029c, Rv0867c (RpfA) and Rv2389c (RpfA) antigens correlates best with the probability of being latently infected. CONCLUSIONS: The Mtb antigens E6-C10, Rv2029c (PfkB), Rv0867c (RpfA) and Rv2389c (RpfA), may be potential candidates to discriminate LTBI from PTB.
Assuntos
Proteínas de Bactérias/imunologia , Citocinas/imunologia , Tuberculose Latente/diagnóstico , Mycobacterium tuberculosis/metabolismo , Proteínas Quinases/imunologia , Tuberculose/diagnóstico , Adulto , Área Sob a Curva , Biomarcadores/metabolismo , Colômbia/epidemiologia , Proteínas de Ligação a DNA , Feminino , Humanos , Interferon gama/metabolismo , Tuberculose Latente/epidemiologia , Tuberculose Latente/microbiologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Curva ROC , Sensibilidade e Especificidade , Tuberculose/epidemiologia , Tuberculose/microbiologiaRESUMO
Multifunctional T cells have been shown to be protective in chronic viral infections. In mycobacterial infections, however, evidence for a protective role of multifunctional T cells remains inconclusive. Short-term cultures of peripheral blood mononuclear cells stimulated with the Mycobacterium tuberculosis RD1 antigens 6-kDa early secretory antigenic target (ESAT6) and 10-kDa culture filtrate antigen (CFP10), which are induced in the early infection phase, have been mainly used to assess T cell multifunctionality, although long-term culture assays have been proposed to be more sensitive than short-term assays for assessment of memory T cells, which are essential for long-term immunity. Here we used a long-term culture assay system to study the T cell immune responses to the M. tuberculosis latency-associated DosR antigens and reactivation-associated Rpf antigens, compared to ESAT6 and CFP10, in patients with pulmonary tuberculosis (PTB) and household contacts of PTB patients with long-term latent tuberculosis infection (ltLTBI), in a community in which M. tuberculosis is endemic. Our results showed that the DosR antigens Rv1737c (narK2) and Rv2029c (pfkB) and the Rv2389c (rpfD) antigen of M. tuberculosis induced higher frequencies of CD4+ or CD8+ mono- or bifunctional (but not multifunctional) T cells producing interferon gamma (IFN-γ) and/or tumor necrosis alpha (TNF-α) in ltLTBI, compared to PTB. Moreover, the frequencies of CD4+ and/or CD8+ T cells with a CD45RO+ CD27+ phenotype were higher in ltLTBI than in PTB. Thus, the immune responses to selected DosR and Rpf antigens may be associated with long-term latency, correlating with protection from M. tuberculosis reactivation in ltLTBI. Further study of the functional and memory phenotypes may contribute to further discrimination between the different states of M. tuberculosis infections.
Assuntos
Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Mycobacterium tuberculosis/imunologia , Proteínas Quinases/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Adulto , Colômbia/epidemiologia , Proteínas de Ligação a DNA , Feminino , Humanos , Memória Imunológica , Interferon gama/imunologia , Tuberculose Latente/imunologia , Tuberculose Latente/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/epidemiologia , Tuberculose/microbiologia , Tuberculose Pulmonar/imunologia , Adulto JovemRESUMO
Immune response to DosR and Rpf antigens from Mycobacterium tuberculosis (Mtb) seems to be important for latency maintenance. Little is known about the dynamics of the immune response to these antigens in an endemic community. Thus, the IFNγ response and cytokine production in response to PPD, Esat6-Cfp10 (E6-C10), DosR and Rpf antigens in healthy HHC of tuberculosis (TB) patients over a 12 (T12) months period (short-term, stLTBI) was investigated. This response was compared with a group of LTBI, who have remained healthy for 5-7 years (long-term, ltLTBI). According to the IFNγ response, two groups of HHCs were identified in stLTBI in response to E6-C10. At T12, E6-C10(+) HHCs displayed a decrease in the IFNγ levels and a generalized decrease in cytokines production. The E6-C10(-) HHC showed an increase in the IFNγ response and cytokine levels. In stLTBI, the responses to E6-C10, DosR, and Rpf may be interpreted as a protective immune response controlling Mtb infection and may be leading to a state of latent infection. Comparing the response of stLTBI and ltLTBI, we observed significant changes in the proportions of CD45RO(+)CD27(+) T cells to specific DosR and Rpf, which may indicate a persistent immune response to Mtb antigens in ltLTBI.
Assuntos
Proteínas de Bactérias/imunologia , Busca de Comunicante , Citocinas/imunologia , Características da Família , Tuberculose Latente/imunologia , Proteínas Quinases/imunologia , Linfócitos T/imunologia , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Idoso , Células Cultivadas , Colômbia , Proteínas de Ligação a DNA , Feminino , Humanos , Tuberculose Latente/diagnóstico , Tuberculose Latente/microbiologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Linfócitos T/microbiologia , Fatores de Tempo , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Adulto JovemRESUMO
Diagnosis of tuberculosis (TB) remains challenging. Serum IgG1 antibodies against Mycobacterium tuberculosis active growth phase antigens (ESAT-6/CFP-10, Rv0717 and Rv3353), DosR regulon-encoded proteins (Rv1733, Rv1737, Rv2628 and Rv2029), and resuscitation-promoting factors (Rv0867 and Rv2389) were evaluated in TB patients using ELISA. Active TB patients showed elevated levels of IgG1 antibodies against ESAT-6/CFP-10, Rv0717, Rv3353, Rv1733, Rv2628, Rv2029 and Rv0867 in comparison to healthy controls (p < 0.001). These levels remained high after the initiation of treatment, while responses to Rv0717 and Rv1733 peaked early during treatment. IgG1 responses to ESAT-6/CFP-10, Rv3353, Rv2628, Rv2029 and Rv0867 declined to control levels after the completion of 6 months chemotherapy. ROC analysis confirmed the good diagnostic performance of Rv0717, Rv1733, Rv3353, Rv2628, Rv2029 and Rv0867antigens. These data suggest that detecting IgG1 antibodies against M. tuberculosis antigens, including DosR and Rpf proteins, may represent an additional tool in the diagnosis of tuberculosis.
Assuntos
Anticorpos Antibacterianos/sangue , Antituberculosos/uso terapêutico , Proteínas de Bactérias/imunologia , Citocinas/imunologia , Imunoglobulina G/sangue , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas Quinases/imunologia , Tuberculose/tratamento farmacológico , Adolescente , Adulto , Idoso , Área Sob a Curva , Biomarcadores/sangue , Estudos Transversais , Proteínas de Ligação a DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Valor Preditivo dos Testes , Curva ROC , Testes Sorológicos , Fatores de Tempo , Resultado do Tratamento , Tuberculose/sangue , Tuberculose/diagnóstico , Tuberculose/imunologia , Tuberculose/microbiologia , Adulto JovemRESUMO
BACKGROUND: Acute inflammatory reactions are a frequently occurring, tissue destructing phenomenon in infectious- as well as autoimmune diseases, providing clinical challenges for early diagnosis. In leprosy, an infectious disease initiated by Mycobacterium leprae (M. leprae), these reactions represent the major cause of permanent neuropathy. However, laboratory tests for early diagnosis of reactional episodes which would significantly contribute to prevention of tissue damage are not yet available. Although classical diagnostics involve a variety of tests, current research utilizes limited approaches for biomarker identification. In this study, we therefore studied leprosy as a model to identify biomarkers specific for inflammatory reactional episodes. METHODS: To identify host biomarker profiles associated with early onset of type 1 leprosy reactions, prospective cohorts including leprosy patients with and without reactions were recruited in Bangladesh, Brazil, Ethiopia and Nepal. The presence of multiple cyto-/chemokines induced by M. leprae antigen stimulation of peripheral blood mononuclear cells as well as the levels of antibodies directed against M. leprae-specific antigens in sera, were measured longitudinally in patients. RESULTS: At all sites, longitudinal analyses showed that IFN-γ-, IP-10-, IL-17- and VEGF-production by M. leprae (antigen)-stimulated PBMC peaked at diagnosis of type 1 reactions, compared to when reactions were absent. In contrast, IL-10 production decreased during type 1 reaction while increasing after treatment. Thus, ratios of these pro-inflammatory cytokines versus IL-10 provide useful tools for early diagnosing type 1 reactions and evaluating treatment. Of further importance for rapid diagnosis, circulating IP-10 in sera were significantly increased during type 1 reactions. On the other hand, humoral immunity, characterized by M. leprae-specific antibody detection, did not identify onset of type 1 reactions, but allowed treatment monitoring instead. CONCLUSIONS: This study identifies immune-profiles as promising host biomarkers for detecting intra-individual changes during acute inflammation in leprosy, also providing an approach for other chronic (infectious) diseases to help early diagnose these episodes and contribute to timely treatment and prevention of tissue damage.
Assuntos
Biomarcadores/análise , Citocinas/imunologia , Hanseníase/imunologia , Mycobacterium leprae/patogenicidade , Bangladesh , Brasil , Citocinas/sangue , Etiópia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Imunidade Humoral/imunologia , Interleucina-10/sangue , Interleucina-17/sangue , Hanseníase/diagnóstico , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/imunologia , Nepal , Estudos ProspectivosRESUMO
Leprosy is not eradicable with currently available diagnostics or interventions, as evidenced by its stable incidence. Early diagnosis of Mycobacterium leprae infection should therefore be emphasized in leprosy research. It remains challenging to develop tests based on immunological biomarkers that distinguish individuals controlling bacterial replication from those developing disease. To identify biomarkers for field-applicable diagnostics, we determined cytokines/chemokines induced by M. leprae proteins in blood of leprosy patients and endemic controls (EC) from high leprosy-prevalence areas (Bangladesh, Brazil, Ethiopia) and from South Korea, where leprosy is not endemic anymore. M. leprae-sonicate-induced IFN-γ was similar for all groups, excluding M. leprae/IFN-γ as a diagnostic readout. By contrast, ML2478 and ML0840 induced high IFN-γ concentrations in Bangladeshi EC, which were completely absent for South Korean controls. Importantly, ML2478/IFN-γ could indicate distinct degrees of M. leprae exposure, and thereby the risk of infection and transmission, in different parts of Brazilian and Ethiopian cities. Notwithstanding these discriminatory responses, M. leprae proteins did not distinguish patients from EC in one leprosy-endemic area based on IFN-γ. Analyses of additional cytokines/chemokines showed that M. leprae and ML2478 induced significantly higher concentrations of MCP-1, MIP-1ß, and IL-1ß in patients compared with EC, whereas IFN-inducible protein-10, like IFN-γ, differed between EC from areas with dissimilar leprosy prevalence. This study identifies M. leprae-unique Ags, particularly ML2478, as biomarker tools to measure M. leprae exposure using IFN-γ or IFN-inducible protein-10, and also shows that MCP-1, MIP-1ß, and IL-1ß can potentially distinguish pathogenic immune responses from those induced during asymptomatic exposure to M. leprae.
Assuntos
Citocinas/sangue , Hanseníase/epidemiologia , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Adulto , Idoso , Antígenos de Bactérias/imunologia , Bangladesh/epidemiologia , Biomarcadores/sangue , Brasil/epidemiologia , Citocinas/biossíntese , Citocinas/genética , Etiópia/epidemiologia , Feminino , Humanos , Interferon gama/biossíntese , Interferon gama/sangue , Interferon gama/genética , Hanseníase/diagnóstico , Masculino , Pessoa de Meia-Idade , República da Coreia/epidemiologia , Células Th1/imunologia , Células Th1/microbiologia , Células Th2/imunologia , Células Th2/microbiologia , Adulto JovemRESUMO
Mycobacterium tuberculosis DosR regulon-encoded proteins elicit strong immune T-cell responses in individuals with latent tuberculosis (LTBI). Also, resuscitation (Rpf) proteins can induce such responses. However, variations in the immunogenicity of the DosR and Rpf proteins have been observed in European and African populations, and no data are published from other geographic areas. In Colombian LTBI and patients with recently diagnosed PTB, we therefore studied the immune response to DosR, Rpf, stress, and nominal antigens from Mtb, in 7-day stimulated cultures. Three DosR (Rv1737c, Rv2029c, Rv2628c) and 2 Rpf (Rv0867 and Rv2389c) antigens were recognized most prominently on the basis of the net IFNγ production (DosR) or the percentage of responding individuals (Rpf). Results show that the selected DosR antigens induced a higher proportion of CD4-T cells producing IFNγ from LTBI, compared to pulmonary TB patients (PTB), while there were no differences in the proportion of CD8-T cells. An increased frequency of CD4, but not CD8 T-cells with a CD45RO(+)CD27(+) phenotype was observed in LTBI in response to Rv2029c, Rv0867c, and Rv2389c, compared to PTB. The levels of cytokines and chemokines in the supernatants of stimulated cells, showed that the DosR and Rpf antigens induced higher levels of IFNγ in cultures from LTBI compared to PTB, although the induced pattern of cytokines and chemokines was also antigen dependent. In summary, our results are consistent with the significant immunogenicity of Mtb DosR and Rpf antigens in LTBI individuals, and confirm and extend previously reported data from other TB affected human populations.
Assuntos
Proteínas de Bactérias/imunologia , Citocinas/imunologia , Tuberculose Latente/imunologia , Proteínas Quinases/imunologia , Subpopulações de Linfócitos T/imunologia , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Idoso , Antígenos de Bactérias/imunologia , Relação CD4-CD8 , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Quimiocinas/biossíntese , Colômbia , Citocinas/biossíntese , Proteínas de Ligação a DNA , Feminino , Humanos , Memória Imunológica , Imunofenotipagem , Interferon gama/biossíntese , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Adulto JovemRESUMO
Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.
Assuntos
Citocinas/imunologia , Epitopos de Linfócito T/imunologia , Mycobacterium leprae/patogenicidade , Virulência/imunologia , Proteínas de Bactérias/imunologia , Brasil , Biologia Computacional , Mapeamento de Epitopos , Etiópia , Humanos , Mycobacterium leprae/imunologia , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/virologia , Nepal , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes/imunologiaRESUMO
Detection of specific antibodies may represent an additional tool in diagnosis of tuberculosis (TB). Herein, levels of serum IgG antibodies against early secreted antigenic target (ESAT-6), culture filtrate antigen-10 (CFP-10) and 16 kDa Mycobacterium tuberculosis antigens were measured in 33 active pulmonary TB patients (0M-TB), in 47 patients after 1-3 months of treatment (3M-TB) and in 22 patients who had completed 6 months of chemotherapy (6M-TB). The control group consisted of 38 BCG-vaccinated healthy controls (HC). In addition, IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-6, IL-2, IL-4 and IL-10 production in PBMC cultures from 20 patients were measured following stimulation with the M. tuberculosis-specific fusion protein ESAT-6/CFP-10. Elevated levels of IgG against ESAT-6, CFP-10 and 16 kDa antigens were detected in 0M-TB and 3M-TB patients in comparison to the HC and 6M-TB groups. Receiver operating characteristic analysis indicated sensitivity of 85, 94 and 61% and specificity of 89, 87 and 89% for serum IgG against ESAT-6, CFP-10 and 16 kDa, respectively. A predominant IgG1 response to ESAT-6 and CFP-10 was observed in 0M-TB patients, together with ESAT-6/CFP-10-specific IFN-gamma, TNF-alpha and IL-6 that were produced at lower levels in the 6M-TB group. These data indicate that a T(h)1 phenotype against early phase Mtb antigens appears to be dominant in the peripheral blood of patients with active pulmonary TB that is reduced after chemotherapy. Taken together, ESAT-6/CFP-10 cytokine tests together with detecting IgG antibodies specific to ESAT-6 and CFP-10 may be the useful TB disease biomarkers in monitoring treatment success.
Assuntos
Antígenos de Bactérias/imunologia , Antituberculosos/uso terapêutico , Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose Pulmonar/tratamento farmacológico , Adolescente , Adulto , Idoso , Células Cultivadas , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Imunoglobulina G/genética , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/patogenicidade , Resultado do Tratamento , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
The detection of hundreds of thousands of new cases of leprosy every year suggests that transmission of Mycobacterium leprae infection still continues. Unfortunately, tools for identification of asymptomatic disease and/or early-stage M. leprae infection (likely sources of transmission) are lacking. The recent identification of M. leprae-unique genes has allowed the analysis of human T-cell responses to novel M. leprae antigens. Antigens with the most-promising diagnostic potential were tested for their ability to induce cytokine secretion by using peripheral blood mononuclear cells from leprosy patients and controls in five different areas where leprosy is endemic; 246 individuals from Brazil, Nepal, Bangladesh, Pakistan, and Ethiopia were analyzed for gamma interferon responses to five recombinant proteins (ML1989, ML1990, ML2283, ML2346, and ML2567) and 22 synthetic peptides. Of these, the M. leprae-unique protein ML1989 was the most frequently recognized and ML2283 the most specific for M. leprae infection/exposure, as only a limited number of tuberculosis patients responded to this antigen. However, all proteins were recognized by a significant number of controls in areas of endemicity. T-cell responses correlated with in vitro response to M. leprae, suggesting that healthy controls in areas where leprosy is endemic are exposed to M. leprae. Importantly, 50% of the healthy household contacts and 59% of the controls in areas of endemicity had no detectable immunoglobulin M antibodies to M. leprae-specific PGL-I but responded in T-cell assays to >or=1 M. leprae protein. T-cell responses specific for leprosy patients and healthy household contacts were observed for ML2283- and ML0126-derived peptides, indicating that M. leprae peptides hold potential as diagnostic tools. Future work should concentrate on the development of a sensitive and field-friendly assay and identification of additional peptides and proteins that can induce M. leprae-specific T-cell responses.