RESUMO
We developed a flow cytometry-based assay, termed Differential Leukocyte Counting and Immunophenotyping in Cryopreserved Ex vivo whole blood (DLC-ICE), that allows quantification of absolute counts and frequencies of leukocyte subsets and measures expression of activation, phenotypic and functional markers. We evaluated the performance of the DLC-ICE assay by determining inter-operator variability for processing fresh whole blood (WB) from healthy donors collected at multiple clinical sites. In addition, we assessed inter-operator variability for staining of fixed cells and robustness across different anticoagulants. Accuracy was evaluated by comparing DLC-ICE measurements to real-time cell enumeration using an accredited hematology analyzer. Finally, we developed and tested the performance of a 27-colour immunophenotyping panel on cryopreserved fixed WB and compared results to matched fresh WB. Overall, we observed <20% variability in absolute counts and frequencies of granulocytes, monocytes and lymphocytes (T, B and NK cells) when fresh WB was collected in different anti-coagulant tubes, processed or stained by independent operators. Absolute cell counts measured across operators and anti-coagulants using the DLC-ICE method exhibited excellent correlation with the reference method, complete blood count (CBC) with differential, measured using a hematology analyzer (r2 > 0.9 for majority of measurements). A comparison of leukocyte immunophenotyping on fresh WB versus DLC-ICE processed blood yielded equivalent and linear results over a wide dynamic range (r2 = 0.94 over 10-104 cells/µL). These results demonstrate low variability across trained operators, high robustness, linearity and accuracy, supporting utility of the DLC-ICE assay for large cohort studies involving multiple clinical research sites.
Assuntos
Leucócitos , Monócitos , Humanos , Imunofenotipagem , Contagem de Leucócitos , Células Matadoras Naturais , Citometria de Fluxo/métodosRESUMO
Rationale: Performance of blood transcriptomic tuberculosis (TB) signatures in longitudinal studies and effects of TB-preventive therapy and coinfection with HIV or respiratory organisms on transcriptomic signatures has not been systematically studied. Objectives: We evaluated longitudinal kinetics of an 11-gene blood transcriptomic TB signature, RISK11, and effects of TB-preventive therapy (TPT) and respiratory organisms on RISK11 signature score, in HIV-uninfected and HIV-infected individuals. Methods: RISK11 was measured in a longitudinal study of RISK11-guided TPT in HIV-uninfected adults, a cross-sectional respiratory organisms cohort, or a longitudinal study in people living with HIV (PLHIV). HIV-uninfected RISK11+ participants were randomized to TPT or no TPT; RISK11- participants received no TPT. PLHIV received standard-of-care antiretroviral therapy and TPT. In the cross-sectional respiratory organisms cohort, viruses and bacteria in nasopharyngeal and oropharyngeal swabs were quantified by real-time quantitative PCR. Measurements and Main Results: RISK11+ status was transient in most of the 128 HIV-negative participants with longitudinal samples; more than 70% of RISK11+ participants reverted to RISK11- by 3 months, irrespective of TPT. By comparison, reversion from a RISK11+ state was less common in 645 PLHIV (42.1%). Non-HIV viral and nontuberculous bacterial organisms were detected in 7.2% and 38.9% of the 1,000 respiratory organisms cohort participants, respectively, and among those investigated for TB, 3.8% had prevalent disease. Median RISK11 scores (%) were higher in participants with viral organisms alone (46.7%), viral and bacterial organisms (42.8%), or prevalent TB (85.7%) than those with bacterial organisms other than TB (13.4%) or no organisms (14.2%). RISK11 could not discriminate between prevalent TB and viral organisms. Conclusions: Positive RISK11 signature status is often transient, possibly due to intercurrent viral infection, highlighting potentially important challenges for implementation of these biomarkers as new tools for TB control.
Assuntos
Regras de Decisão Clínica , Perfilação da Expressão Gênica , Transcriptoma , Tuberculose/diagnóstico , Tuberculose/genética , Adolescente , Adulto , Fármacos Anti-HIV/uso terapêutico , Antituberculosos/uso terapêutico , Biomarcadores/sangue , Coinfecção/sangue , Coinfecção/diagnóstico , Coinfecção/genética , Coinfecção/terapia , Estudos Transversais , Feminino , Infecções por HIV/sangue , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Humanos , Modelos Lineares , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Infecções Respiratórias/sangue , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/genética , Infecções Respiratórias/terapia , Medição de Risco , Sensibilidade e Especificidade , Resultado do Tratamento , Tuberculose/sangue , Tuberculose/prevenção & controle , Adulto JovemRESUMO
BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) infection and is a major public health problem. Clinical challenges include the lack of a blood-based test for active disease. Current blood-based tests, such as QuantiFERON (QFT) do not distinguish active TB disease from asymptomatic Mtb infection. METHODS: We hypothesized that TruCulture, an immunomonitoring method for whole-blood stimulation, could discriminate active disease from latent Mtb infection (LTBI). We stimulated whole blood from patients with active TB and compared with LTBI donors. Mtb-specific antigens and live bacillus Calmette-Guérin (BCG) were used as stimuli, with direct comparison to QFT. Protein analyses were performed using conventional and digital enzyme-linked immunosorbent assay (ELISA), as well as Luminex. RESULTS: TruCulture showed discrimination of active TB cases from LTBI (P < .0001, AUC = .81) compared with QFT (P = .45, AUC = .56), based on an interferon γ (IFNγ) readout after Mtb antigen (Ag) stimulation. This result was replicated in an independent cohort (AUC = .89). In exploratory analyses, TB stratification could be further improved by the Mtb antigen to BCG IFNγ ratio (P < .0001, AUC = .91). Finally, the combination of digital ELISA and transcriptional analysis showed that LTBI donors with high IFNγ clustered with patients with TB, suggesting the possibility to identify subclinical disease. CONCLUSIONS: TruCulture offers a next-generation solution for whole-blood stimulation and immunomonitoring with the possibility to discriminate active and latent infection.
Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Ensaio de Imunoadsorção Enzimática , Humanos , Interferon gama , Testes de Liberação de Interferon-gama , Tuberculose Latente/diagnóstico , Tuberculose/diagnósticoRESUMO
BACKGROUND: Recent Mycobacterium tuberculosis infection confers a predisposition to the development of tuberculosis disease, the leading killer among global infectious diseases. H4:IC31, a candidate subunit vaccine, has shown protection against tuberculosis disease in preclinical models, and observational studies have indicated that primary bacille Calmette-Guérin (BCG) vaccination may offer partial protection against infection. METHODS: In this phase 2 trial, we randomly assigned 990 adolescents in a high-risk setting who had undergone neonatal BCG vaccination to receive the H4:IC31 vaccine, BCG revaccination, or placebo. All the participants had negative results on testing for M. tuberculosis infection on the QuantiFERON-TB Gold In-tube assay (QFT) and for the human immunodeficiency virus. The primary outcomes were safety and acquisition of M. tuberculosis infection, as defined by initial conversion on QFT that was performed every 6 months during a 2-year period. Secondary outcomes were immunogenicity and sustained QFT conversion to a positive test without reversion to negative status at 3 months and 6 months after conversion. Estimates of vaccine efficacy are based on hazard ratios from Cox regression models and compare each vaccine with placebo. RESULTS: Both the BCG and H4:IC31 vaccines were immunogenic. QFT conversion occurred in 44 of 308 participants (14.3%) in the H4:IC31 group and in 41 of 312 participants (13.1%) in the BCG group, as compared with 49 of 310 participants (15.8%) in the placebo group; the rate of sustained conversion was 8.1% in the H4:IC31 group and 6.7% in the BCG group, as compared with 11.6% in the placebo group. Neither the H4:IC31 vaccine nor the BCG vaccine prevented initial QFT conversion, with efficacy point estimates of 9.4% (P=0.63) and 20.1% (P=0.29), respectively. However, the BCG vaccine reduced the rate of sustained QFT conversion, with an efficacy of 45.4% (P=0.03); the efficacy of the H4:IC31 vaccine was 30.5% (P=0.16). There were no clinically significant between-group differences in the rates of serious adverse events, although mild-to-moderate injection-site reactions were more common with BCG revaccination. CONCLUSIONS: In this trial, the rate of sustained QFT conversion, which may reflect sustained M. tuberculosis infection, was reduced by vaccination in a high-transmission setting. This finding may inform clinical development of new vaccine candidates. (Funded by Aeras and others; C-040-404 ClinicalTrials.gov number, NCT02075203 .).
Assuntos
Vacina BCG , Imunização Secundária , Mycobacterium tuberculosis/imunologia , Soroconversão , Vacinas contra a Tuberculose , Tuberculose/prevenção & controle , Adolescente , Anticorpos Antibacterianos/sangue , Vacina BCG/efeitos adversos , Vacina BCG/imunologia , Criança , Feminino , Humanos , Masculino , Modelos de Riscos Proporcionais , Tuberculose/diagnóstico , Tuberculose/transmissão , Vacinas contra a Tuberculose/efeitos adversos , Vacinas contra a Tuberculose/imunologiaRESUMO
BACKGROUND: In human blood, mucosal-associated invariant T (MAIT) cells are abundant T cells that recognize antigens presented on non-polymorphic major histocompatibility complex-related 1 (MR1) molecules. The MAIT cells are activated by mycobacteria, and prior human studies indicate that blood frequencies of MAIT cells, defined by cell surface markers, decline during tuberculosis (TB) disease, consistent with redistribution to the lungs. METHODS: We tested whether frequencies of blood MAIT cells were altered in patients with TB disease relative to healthy Mycobacterium tuberculosis-exposed controls from Peru and South Africa. We quantified their frequencies using MR1 tetramers loaded with 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil. RESULTS: Unlike findings from prior studies, frequencies of blood MAIT cells were similar among patients with TB disease and latent and uninfected controls. In both cohorts, frequencies of MAIT cells defined by MR1-tetramer staining and coexpression of CD161 and the T-cell receptor alpha variable gene TRAV1-2 were strongly correlated. Disease severity captured by body mass index or TB disease transcriptional signatures did not correlate with MAIT cell frequencies in patients with TB. CONCLUSIONS: Major histocompatibility complex (MHC)-related 1-restrictied MAIT cells are detected at similar levels with tetramers or surface markers. Unlike MHC-restricted T cells, blood frequencies of MAIT cells are poor correlates of TB disease but may play a role in pathophysiology.
Assuntos
Células T Invariantes Associadas à Mucosa/imunologia , Mycobacterium tuberculosis/imunologia , Tuberculose/epidemiologia , Tuberculose/imunologia , Adulto , Biomarcadores , Estudos de Casos e Controles , Feminino , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Células T Invariantes Associadas à Mucosa/metabolismo , Prevalência , Vigilância em Saúde Pública , Medição de Risco , Fatores de Risco , Tuberculose/microbiologiaRESUMO
BACKGROUND: Early secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) antigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6-free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6-containing vaccines. We aimed to qualify a recently developed ESAT-6-free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT). METHODS: Participants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6-free IGRA cutoff, test assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen-coated blood collection tubes. RESULTS: ESAT-6-free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. The ESAT-6-free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6-free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74-0.90; McNemar test P = .48). ESAT-6-free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability. CONCLUSIONS: The novel ESAT-6-free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection.
Assuntos
Testes de Liberação de Interferon-gama , Interferon gama/sangue , Kit de Reagentes para Diagnóstico , Tuberculose/diagnóstico , Adolescente , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Mycobacterium tuberculosis/imunologia , Curva ROC , Reprodutibilidade dos Testes , Tuberculose/sangue , Tuberculose/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/imunologiaRESUMO
Diagnostic tests for tuberculosis (TB) usually require collection of sputum, a viscous material derived from human airways. Sputum can be difficult and hazardous to collect and challenging to process in the laboratory. Oral swabs have been proposed as alternative sample types that are noninvasive and easy to collect. This study evaluated the biological feasibility of oral swab analysis (OSA) for the diagnosis of TB. Swabs were tested from South African adult subjects, including sputum GeneXpert MTB/RIF (GeneXpert)-confirmed TB patients (n = 138), sputum GeneXpert-negative but culture-positive TB patients (n = 10), ill non-TB patients (n = 37), and QuantiFERON-negative controls (n = 34). Swabs were analyzed by using a manual, nonnested quantitative PCR (qPCR) targeting IS6110 Two swab brands and three sites within the oral cavity were compared. Tongue swabbing yielded significantly stronger signals than cheek or gum swabbing. A flocked swab performed better than a more expensive paper swab. In a two-phase study, tongue swabs (two per subject) exhibited a combined sensitivity of 92.8% relative to sputum GeneXpert. Relative to all laboratory-diagnosed TB, the diagnostic yields of sputum GeneXpert (1 sample per subject) and OSA (2 samples per subject) were identical at 49/59 (83.1%) each. The specificity of the OSA was 91.5%. An analysis of "air swabs" suggested that most false-positive results were due to contamination of manual PCRs. With the development of appropriate automated methods, oral swabs could facilitate TB diagnosis in clinical settings and patient populations that are limited by the physical or logistical challenges of sputum collection.
Assuntos
Testes Diagnósticos de Rotina/métodos , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Adulto , Humanos , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , EscarroRESUMO
RATIONALE: Conversion from a negative to positive QuantiFERON-TB test is indicative of Mycobacterium tuberculosis (Mtb) infection, which predisposes individuals to tuberculosis disease. Interpretation of serial tests is confounded by immunological and technical variability. OBJECTIVES: To improve the consistency of serial QuantiFERON-TB testing algorithms and provide a data-driven definition of conversion. METHODS: Sources of QuantiFERON-TB variability were assessed, and optimal procedures were identified. Distributions of IFN-γ response levels were analyzed in healthy adolescents, Mtb-unexposed control subjects, and patients with pulmonary tuberculosis. MEASUREMENTS AND MAIN RESULTS: Individuals with no known Mtb exposure had IFN-γ values less than 0.2 IU/ml. Among individuals with IFN-γ values less than 0.2 IU/ml, 0.2-0.34 IU/ml, 0.35-0.7 IU/ml, and greater than 0.7 IU/ml, tuberculin skin test positivity results were 15%, 53%, 66%, and 91% (P < 0.005), respectively. Together, these findings suggest that values less than 0.2 IU/ml were true negatives. In short-term serial testing, "uncertain" conversions, with at least one value within the uncertainty zone (0.2-0.7 IU/ml), were partly explained by technical assay variability. Individuals who had a change in QuantiFERON-TB IFN-γ values from less than 0.2 to greater than 0.7 IU/ml had 10-fold higher tuberculosis incidence rates than those who maintained values less than 0.2 IU/ml over 2 years (P = 0.0003). By contrast, "uncertain" converters were not at higher risk than nonconverters (P = 0.229). Eighty-seven percent of patients with active tuberculosis had IFN-γ values greater than 0.7 IU/ml, suggesting that these values are consistent with established Mtb infection. CONCLUSIONS: Implementation of optimized procedures and a more rigorous QuantiFERON-TB conversion definition (an increase from IFN-γ <0.2 to >0.7 IU/ml) would allow more definitive detection of recent Mtb infection and potentially improve identification of those more likely to develop disease.
Assuntos
Interferon gama/sangue , Interferon gama/imunologia , Mycobacterium tuberculosis/imunologia , Teste Tuberculínico/métodos , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Teste Tuberculínico/estatística & dados numéricosAssuntos
Biomarcadores/metabolismo , Antígenos HLA-DR/metabolismo , Tuberculose Latente/sangue , Tuberculose Latente/diagnóstico , Linfócitos T/metabolismo , Adulto , Voluntários Saudáveis , Humanos , Tuberculose Latente/imunologia , Leucócitos Mononucleares/metabolismo , Mycobacterium tuberculosis , Fenótipo , Curva ROC , Reprodutibilidade dos Testes , África do Sul , Pesquisa Translacional BiomédicaRESUMO
Tuberculosis (TB) remains a major public health problem and we lack a comprehensive understanding of how Mycobacterium tuberculosis (M. tb) infection impacts host immune responses. We compared the induced immune response to TB antigen, BCG and IL-1ß stimulation between latently M. tb infected individuals (LTBI) and active TB patients. This revealed distinct responses between TB/LTBI at transcriptomic, proteomic and metabolomic levels. At baseline, we identified a novel immune-metabolic association between pregnane steroids, the PPARγ pathway and elevated plasma IL-1ra in TB. We observed dysregulated IL-1 responses after BCG stimulation in TB patients, with elevated IL-1ra responses being explained by upstream TNF differences. Additionally, distinct secretion of IL-1α/IL-1ß in LTBI/TB after BCG stimulation was associated with downstream differences in granzyme mediated cleavage. Finally, IL-1ß driven signalling was dramatically perturbed in TB disease but was completely restored after successful treatment. This study improves our knowledge of how immune responses are altered during TB disease, and may support the design of improved preventive and therapeutic tools, including host-directed strategies.
Assuntos
Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1 , Tuberculose , Humanos , Vacina BCG , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-1/genética , Interleucina-1/imunologia , Redes e Vias Metabólicas , Proteômica , Tuberculose/tratamento farmacológico , Tuberculose/genética , Tuberculose/imunologiaRESUMO
BACKGROUND: A therapeutic vaccine that prevents recurrent tuberculosis would be a major advance in the development of shorter treatment regimens. We aimed to assess the safety and immunogenicity of the ID93â+âGLA-SE vaccine at various doses and injection schedules in patients with previously treated tuberculosis. METHODS: This randomised, double-blind, placebo-controlled, phase 2a trial was conducted at three clinical sites near Cape Town, South Africa. Patients were recruited at local clinics after receiving 4 months of tuberculosis treatment, and screened for eligibility after providing written informed consent. Participants were aged 18-60 years, BCG-vaccinated, HIV-uninfected, and diagnosed with drug-sensitive pulmonary tuberculosis. Eligible patients had completed standard treatment for pulmonary tuberculosis in the past 28 days. Participants were enrolled after completing standard treatment and randomly assigned sequentially to receive vaccine or placebo in three cohorts: 2 µg intramuscular ID93â+â2 µg GLA-SE on days 0 and 56 (cohort 1); 10 µg ID93â+â2 µg GLA-SE on days 0 and 56 (cohort 2); 2 µg ID93â+â5 µg GLA-SE on days 0 and 56 and placebo on day 28 (cohort 3); 2 µg ID93â+â5 µg GLA-SE on days 0, 28, and 56 (cohort 3); or placebo on days 0 and 56 (cohorts 1 and 2), with the placebo group for cohort 3 receiving an additional injection on day 28. Randomisation was in a ratio of 3:1 for ID93â+âGLA-SE and saline placebo in cohorts 1 and 2, and in a ratio of 3:3:1 for (2â×) ID93 + GLA-SE, (3â×) ID93â+âGLA-SE, and placebo in cohort 3. The primary outcomes were safety and immunogenicity (vaccine-specific antibody response and T-cell response). For the safety outcome, participants were observed for 30 min after each injection, injection site reactions and systemic adverse events were monitored until day 84, and serious adverse events and adverse events of special interest were monitored for 6 months after the last injection. Vaccine-specific antibody responses were measured by serum ELISA, and T-cell responses after stimulation with vaccine antigens were measured in cryopreserved peripheral blood mononuclear cells specimens using intracellular cytokine staining followed by flow cytometry. This study is registered with ClinicalTrials.gov, number NCT02465216. FINDINGS: Between June 17, 2015, and May 30, 2016, we assessed 177 patients for inclusion. 61 eligible patients were randomly assigned to receive: saline placebo (n=5) or (2â×) 2 µg ID93â+â2 µg GLA-SE (n=15) on days 0 and 56 (cohort 1); saline placebo (n=2) or (2â×) 10 µg ID93 + 2 µg GLA-SE (n=5) on days 0 and 56 (cohort 2); saline placebo (n=5) on days 0, 28 and 56, or 2 µg ID93 + 5 µg GLA-SE (n=15) on days 0 and 56 and placebo injection on day 28, or (3â×) 2 µg ID93â+â5 µg GLA-SE (n=14) on days 0, 28, and 56 (cohort 3). ID93 + GLA-SE induced robust and durable antibody responses and specific, polyfunctional CD4 T-cell responses to vaccine antigens. Two injections of the 2 µg ID93â+â5 µg GLA-SE dose induced antigen-specific IgG and CD4 T-cell responses that were significantly higher than those with placebo and persisted for the 6-month study duration. Mild to moderate injection site pain was reported after vaccination across all dose combinations, and induration and erythema in patients given 2 µg ID93 + 5 µg GLA-SE in two or three doses. One participant had grade 3 erythema and induration at the injection site. No vaccine-related serious adverse events were observed. INTERPRETATION: Vaccination with ID93â+âGLA-SE was safe and immunogenic for all tested regimens. These data support further evaluation of ID93â+âGLA-SE in therapeutic vaccination strategies to improve tuberculosis treatment outcomes. FUNDING: Wellcome Trust (102028/Z/13/Z).
Assuntos
Imunogenicidade da Vacina , Prevenção Secundária/métodos , Vacinas contra a Tuberculose/efeitos adversos , Tuberculose Resistente a Múltiplos Medicamentos/terapia , Tuberculose Pulmonar/terapia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/efeitos adversos , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Relação Dose-Resposta Imunológica , Método Duplo-Cego , Feminino , Glucosídeos/administração & dosagem , Glucosídeos/efeitos adversos , Glucosídeos/imunologia , Humanos , Lipídeo A/administração & dosagem , Lipídeo A/efeitos adversos , Lipídeo A/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Recidiva , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/sangue , Tuberculose Resistente a Múltiplos Medicamentos/imunologia , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/microbiologia , Adulto JovemRESUMO
We characterize the breadth, function and phenotype of innate and adaptive cellular responses in a prevention of Mycobacterium tuberculosis infection trial. Responses are measured by whole blood intracellular cytokine staining at baseline and 70 days after vaccination with H4:IC31 (subunit vaccine containing Ag85B and TB10.4), Bacille Calmette-Guerin (BCG, a live attenuated vaccine) or placebo (n = ~30 per group). H4:IC31 vaccination induces Ag85B and TB10.4-specific CD4 T cells, and an unexpected NKTlike subset, that expresses IFN-γ, TNF and/or IL-2. BCG revaccination increases frequencies of CD4 T cell subsets that either express Th1 cytokines or IL-22, and modestly increases IFNγ-producing NK cells. In vitro BCG re-stimulation also triggers responses by donor-unrestricted T cells, which may contribute to host responses against mycobacteria. BCG, which demonstrated efficacy against sustained Mycobacterium tuberculosis infection, modulates multiple immune cell subsets, in particular conventional Th1 and Th22 cells, which should be investigated in discovery studies of correlates of protection.
Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle , Adolescente , Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Criança , Citocinas/metabolismo , Humanos , Interferon gama/metabolismo , Mycobacterium tuberculosis/imunologia , Células T Matadoras Naturais/imunologia , Células Th1/imunologia , Tuberculose Pulmonar/imunologiaRESUMO
BACKGROUND: Infants are a key target population for new tuberculosis vaccines. We assessed the safety and immunogenicity of the live-attenuated Mycobacterium tuberculosis vaccine candidate MTBVAC in adults and infants in a region where transmission of tuberculosis is very high. METHODS: We did a randomised, double-blind, BCG-controlled, dose-escalation trial at the South African Tuberculosis Vaccine Initiative site near Cape Town, South Africa. Healthy adult community volunteers who were aged 18-50 years, had received BCG vaccination as infants, were HIV negative, had negative interferon-γ release assay (IGRA) results, and had no personal history of tuberculosis or current household contact with someone with tuberculosis were enrolled in a safety cohort. Infants born to HIV-negative women with no personal history of tuberculosis or current household contact with a person with tuberculosis and who were 96 h old or younger, generally healthy, and had not yet received routine BCG vaccination were enrolled in a separate infant cohort. Eligible adults were randomly assigned (1:1) to receive either BCG Vaccine SSI (5â×â105 colony forming units [CFU] of Danish strain 1331 in 0·1 mL diluent) or MTBVAC (5â×â105 CFU in 0·1 mL) intradermally in the deltoid region of the arm. After favourable review of 28-day reactogenicity and safety data in the adult cohort, infants were randomly assigned (1:3) to receive either BCG Vaccine SSI (2·5â×â105 CFU in 0·05 mL diluent) or MTBVAC in three sequential cohorts of increasing MTBVAC dose (2·5â×â103 CFU, 2·5â×â104 CFU, and 2·5â×â105 CFU in 0·05 mL) intradermally in the deltoid region of the arm. QuantiFERON-TB Gold In-Tube IGRA was done on days 180 and 360. For both randomisations, a pre-prepared block randomisation schedule was used. Participants (and their parents or guardians in the case of infant participants), investigators, and other clinical and laboratory staff were masked to intervention allocation. The primary outcomes, which were all measured in the infant cohort, were solicited and unsolicited local adverse events and serious adverse events until day 360; non-serious systemic adverse events until day 28 and vaccine-specific CD4 and CD8 T-cell responses on days 7, 28, 70, 180, and 360. Secondary outcomes measured in adults were local injection-site and systemic reactions and haematology and biochemistry at study day 7 and 28. Safety analyses and immunogenicity analyses were done in all participants who received a dose of vaccine. This trial is registered with ClinicalTrials.gov, number NCT02729571. FINDINGS: Between Sept 29, 2015, and Nov 16, 2015, 62 adults were screened and 18 were enrolled and randomly assigned, nine each to the BCG and MTBVAC groups. Between Feb 12, 2016, and Sept 21, 2016, 36 infants were randomly assigned-eight to the BCG group, nine to the 2·5â×â103 CFU MTBVAC group, nine to the 2·5â×â104 CFU group, and ten to the 2·5â×â105 CFU group. Mild injection-site reactions occurred only in infants in the BCG and the 2·5â×â105 CFU MTBVAC group, with no evidence of local or regional injection-site complications. Systemic adverse events were evenly distributed across BCG and MTBVAC dose groups, and were mostly mild in severity. Eight serious adverse events were reported in seven vaccine recipients (one adult MTBVAC recipient, one infant BCG recipient, one infant in the 2·5â×â103 CFU MTBVAC group, two in the 2·5â×â104 CFU MTBVAC group, and two in the 2·5â×â105 CFU MTBVAC group), including one infant in the 2·5â×â103 CFU MTBVAC group treated for unconfirmed tuberculosis and one in the 2·5â×â105 CFU MTBVAC group treated for unlikely tuberculosis. One infant died as a result of possible viral pneumonia. Vaccination with all MTBVAC doses induced durable antigen-specific T-helper-1 cytokine-expressing CD4 cell responses in infants that peaked 70 days after vaccination and were detectable 360 days after vaccination. For the highest MTBVAC dose (ie, 2·5â×â105 CFU), these responses exceeded responses induced by an equivalent dose of the BCG vaccine up to 360 days after vaccination. Dose-related IGRA conversion was noted in three (38%) of eight infants in the 2·5â×â103 CFU MTBVAC group, six (75%) of eight in the 2·5â×â104 CFU MTBVAC group, and seven (78%) of nine in the 2·5â×â105 CFU MTBVAC group at day 180, compared with none of seven infants in the BCG group. By day 360, IGRA reversion had occurred in all three infants (100%) in the 2·5â×â103 CFU MTBVAC group, four (67%) of the six in the 2·5â×â104 CFU MTBVAC group, and three (43%) of the seven in the 2·5â×â105 CFU MTBVAC group. INTERPRETATION: MTBVAC had acceptable reactogenicity, and induced a durable CD4 cell response in infants. The evidence of immunogenicity supports progression of MTBVAC into larger safety and efficacy trials, but also confounds interpretation of tests for M tuberculosis infection, highlighting the need for stringent endpoint definition. FUNDING: Norwegian Agency for Development Cooperation, TuBerculosis Vaccine Initiative, UK Department for International Development, and Biofabri.
Assuntos
Vacina BCG/uso terapêutico , Vacinas contra a Tuberculose/uso terapêutico , Tuberculose/prevenção & controle , Adolescente , Adulto , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Vias de Administração de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , África do Sul , Tuberculose/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/uso terapêutico , Adulto JovemRESUMO
HIV-infected individuals are at high risk of tuberculosis disease and those with prior tuberculosis episodes are at even higher risk of disease recurrence. A non-sputum biomarker that identifies individuals at highest tuberculosis risk would allow targeted microbiological testing and appropriate treatment and also guide need for prolonged therapy. We determined the utility of a previously developed whole blood transcriptomic correlate of risk (COR) signature for (1) predicting incident recurrent tuberculosis, (2) tuberculosis diagnosis and (3) its potential utility for tuberculosis treatment monitoring in HIV-infected individuals. We retrieved cryopreserved blood specimens from three previously completed clinical studies and measured the COR signature by quantitative microfluidic real-time-PCR. The signature differentiated recurrent tuberculosis progressors from non-progressors within 3 months of diagnosis with an area under the Receiver-operating characteristic (ROC) curve (AUC) of 0.72 (95% confidence interval (CI), 0.58-0.85) amongst HIV-infected individuals on antiretroviral therapy (ART). Twenty-five of 43 progressors (58%) were asymptomatic at microbiological diagnosis and thus had subclinical disease. The signature showed excellent diagnostic discrimination between HIV-uninfected tuberculosis cases and controls (AUC 0.97; 95%CI 0.94-1). Performance was lower in HIV-infected individuals (AUC 0.83; 95%CI 0.81-0.96) and signature scores were directly associated with HIV viral loads. Tuberculosis treatment response in HIV-infected individuals on ART with a new recurrent tuberculosis diagnosis was also assessed. Signature scores decreased significantly during treatment. However, pre-treatment scores could not differentiate between those who became sputum negative before and after 2 months. Direct application of the unmodified blood transcriptomic COR signature detected subclinical and active tuberculosis by blind validation in HIV-infected individuals. However, prognostic performance for recurrent tuberculosis, and performance as diagnostic and as treatment monitoring tool in HIV-infected persons was inferior to published results from HIV-negative cohorts. Our results suggest that performance of transcriptomic signatures comprising interferon stimulated genes are negatively affected in HIV-infected individuals, especially in those with incompletely suppressed viral loads.
RESUMO
Background: Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (TSCM), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (TCM) or effector (TEFF) T cells. Our knowledge of TSCM derives primarily from studies of virus-specific CD8+ TSCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis, generates antigen-specific CD4+ TSCM and to characterize their functional ontology. Methods: We studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT+ adult cohorts; and to bacillus Calmette-Guerin (BCG) vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer+ CD4+ TSCM (CD45RA+ CCR7+ CD27+) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry. Results: M. tb-specific TSCM were not detected in QFT-negative persons. After QFT conversion frequencies of TSCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces TSCM cells. Gene expression (GE) profiling of tetramer+ TSCM showed that these cells were distinct from bulk CD4+ naïve T cells (TN) and shared features of bulk TSCM and M. tb-specific tetramer+ TCM and TEFF cells. These TSCM were predominantly CD95+ and CXCR3+, markers typical of CD8+ TSCM. Tetramer+ TSCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk TN and TSCM cells. M. tb-specific TSCM were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4+ T cell proliferative potential after infant vaccination. Conclusion: Human infection with M. tb induced distinct, antigen-specific CD4+ TSCM cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4+ TSCM should be considered for vaccination approaches that aim to generate long-lived memory T cells against M. tb.
Assuntos
Antígenos de Bactérias/imunologia , Memória Imunológica , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Células Th1/imunologia , Células Th17/imunologia , Tuberculose/imunologia , Adolescente , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Proliferação de Células , Estudos Transversais , Citocinas/imunologia , Feminino , Humanos , Masculino , Receptores de Quimiocinas/imunologia , Células Th1/patologia , Células Th17/patologia , Tuberculose/patologiaRESUMO
BACKGROUND: A vaccine that prevents pulmonary tuberculosis in adults is needed to halt transmission in endemic regions. This trial aimed to assess the safety and immunogenicity of three administrations at varying doses of antigen and adjuvant of an investigational vaccine (ID93â+âGLA-SE) compared with placebo in previously BCG-vaccinated healthy adults in a tuberculosis endemic country. METHODS: In this randomised, double-blind, placebo-controlled phase 1 trial, we enrolled HIV-negative, previously BCG-vaccinated adults (aged 18-50 years), with no evidence of previous or current tuberculosis disease, from among community volunteers in the Worcester region of Western Cape, South Africa. Participants were randomly assigned to receive varying doses of ID93â+âGLA-SE or saline placebo at day 0, day 28, and day 112. Enrolment into each cohort was sequential. Cohort 1 participants were Mycobacterium tuberculosis uninfected (as defined by negative QuantiFERON [QFT] status), and received 10 µg ID93 plus 2 µg GLA-SE, or placebo; in cohorts 2-4, QFT-negative or positive participants received escalating doses of vaccine or placebo. Cohort 2 received 2 µg ID93 plus 2 µg GLA-SE; cohort 3 received 10 µg ID93 plus 2 µg GLA-SE; and cohort 4 received 10 µg ID93 plus 5 µg GLA-SE. Dose cohort allocation was sequential; randomisation within a cohort was according to a randomly-generated sequence (3 to 1 in cohort 1, 5 to 1 in cohorts 2-4). The primary endpoint was safety of ID93â+âGLA-SE as defined by solicited and unsolicited adverse events up to 28 days after each study injection and serious adverse events for the duration of the study. Specific immune responses were measured by intracellular cytokine staining, flow cytometry, and ELISA. All analyses were done according to intention to treat, with additional per-protocol analyses for immunogenicity outcomes. This trial is registered with ClinicalTrials.gov, number NCT01927159. FINDINGS: Between Aug 30, 2013, and Sept 4, 2014, 227 individuals consented to participate; 213 were screened (three participants were not included as study number was already met and 11 withdrew consent before screening occurred, mostly due to relocation or demands of employment). 66 healthy, HIV-negative adults were randomly allocated to receive the vaccine (n=54) or placebo (n=12). All study participants received day 0 and day 28 study injections; five participants did not receive an injection on day 112. ID93â+âGLA-SE was well tolerated; no severe or serious vaccine-related adverse events were recorded. Vaccine dose did not affect frequency or severity of adverse events, but mild injection site adverse events and flu-like symptoms were common in M tuberculosis-infected participants compared with uninfected participants. Vaccination induced durable antigen-specific IgG and Th1 cellular responses, which peaked after two administrations. Vaccine dose did not affect magnitude, kinetics, or profile of antibody and cellular responses. Earlier boosting and greater T-cell differentiation and effector-like profiles were seen in M tuberculosis-infected than in uninfected vaccinees. INTERPRETATION: Escalating doses of ID93â+âGLA-SE induced similar antigen-specific CD4-positive T cell and humoral responses, with an acceptable safety profile in BCG-immunised, M tuberculosis-infected individuals. The T-cell differentiation profiles in M tuberculosis-infected vaccinees suggest priming through natural infection. While cohort sample sizes in this phase 1 trial were small and results should be interpreted in context, these data support efficacy testing of two administrations of the lowest (2 µg) ID93 vaccine dose in tuberculosis endemic populations. FUNDING: Aeras and the Paul G Allen Family Foundation.