RESUMO
BACKGROUND: Understanding immune mechanisms, particularly the role of innate immune markers during latent TB infection remains elusive. The main objective of this study was to evaluate mRNA gene expression patterns of toll-like receptors (TLRs) as correlates of immunity during latent TB infection and further infer their roles as potential diagnostic biomarkers. METHODS: Messenger RNA (mRNA) levels were analysed in a total of 64 samples collected from apparently healthy children and adolescents latently infected with tuberculosis (n = 32) or non-infected (n = 32). Relative expression in peripheral blood of selected genes encoding TLRs (TLR-1, TLR-2, TLR-4, TLR-6 and TLR-9) was determined with a quantitative real-time polymerase chain reaction (qRT-PCR) using specific primers and florescent labelled probes and a comparative threshold cycle method to define fold change. Data were analysed using Graph-Pad Prism 7.01 for Windows and a p-value less than 0.05 was considered statistically significant. RESULTS: An increased mean fold change in the relative expression of TLR-2 and TLR-6 mRNA was observed in LTBI groups relative to non-LTBI groups (p < 0.05), whereas a slight fold decrease was observed for TLR-1 gene. CONCLUSIONS: An increased mRNA expression of TLR-2 and TLR-6 was observed in latently infected individuals relative to those non-infected, possibly indicating the roles these biomarkers play in sustenance of the steady state interaction between the dormant TB bacilli and host immunity.
Assuntos
Tuberculose Latente/imunologia , RNA Mensageiro/metabolismo , Receptores Toll-Like/genética , Adolescente , Biomarcadores/metabolismo , Criança , Diagnóstico Precoce , Feminino , Humanos , Imunidade Inata , Tuberculose Latente/diagnóstico , Masculino , Mycobacterium tuberculosis , RNA Mensageiro/genéticaRESUMO
RATIONALE: Global tuberculosis (TB) control requires effective vaccines in TB-endemic countries, where most adults are infected with Mycobacterium tuberculosis (M.tb). OBJECTIVES: We sought to define optimal dose and schedule of H56:IC31, an experimental TB vaccine comprising Ag85B, ESAT-6, and Rv2660c, for M.tb-infected and M.tb-uninfected adults. METHODS: We enrolled 98 healthy, HIV-uninfected, bacillus Calmette-Guérin-vaccinated, South African adults. M.tb infection was defined by QuantiFERON-TB (QFT) assay. QFT-negative participants received two vaccinations of different concentrations of H56 in 500 nmol of IC31 to enable dose selection for further vaccine development. Subsequently, QFT-positive and QFT-negative participants were randomized to receive two or three vaccinations to compare potential schedules. Participants were followed for safety and immunogenicity for 292 days. MEASUREMENTS AND MAIN RESULTS: H56:IC31 showed acceptable reactogenicity profiles irrespective of dose, number of vaccinations, or M.tb infection. No vaccine-related severe or serious adverse events were observed. The three H56 concentrations tested induced equivalent frequencies and functional profiles of antigen-specific CD4 T cells. ESAT-6 was only immunogenic in QFT-negative participants who received three vaccinations. CONCLUSIONS: Two or three H56:IC31 vaccinations at the lowest dose induced durable antigen-specific CD4 T-cell responses with acceptable safety and tolerability profiles in M.tb-infected and M.tb-uninfected adults. Additional studies should validate applicability of vaccine doses and regimens to both QFT-positive and QFT-negative individuals. Clinical trial registered with www.clinicaltrials.gov (NCT01865487).
Assuntos
Vacinas contra a Tuberculose/uso terapêutico , Tuberculose/prevenção & controle , Aciltransferases/imunologia , Aciltransferases/uso terapêutico , Adolescente , Adulto , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/uso terapêutico , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/uso terapêutico , Relação Dose-Resposta a Droga , Método Duplo-Cego , Combinação de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/imunologia , Oligodesoxirribonucleotídeos/uso terapêutico , Oligopeptídeos/imunologia , Oligopeptídeos/uso terapêutico , África do Sul , Resultado do Tratamento , Tuberculose/imunologia , Vacinas contra a Tuberculose/imunologia , Adulto JovemRESUMO
C-Tb, a novel Mycobacterium tuberculosis and 6-kDa early secretory antigenic target/10-kDa culture filtrate protein (ESAT-6/CFP-10)-specific skin test, has high specificity in bacille Calmette-Guerin-vaccinated healthy controls. However, the sensitivity of C-Tb has hitherto not been determined. The objective was to determine the sensitivity of C-Tb in patients with active tuberculosis (TB) in comparison with the tuberculin skin test (TST) and QuantiFERON-TB Gold In-Tube (QFT-GIT).C-Tb and TST were randomly administered in a double-blinded fashion to one or the other forearm in 253 patients with active TB with or without HIV co-infection. QFT-GIT testing was performed prior to skin testing.Using a receiver operating characteristic curve-derived cut-point of 5â mm, C-Tb sensitivity was similar to QFT-GIT (73.9 (95% CI 67.8-79.3) versus 75.1 (95% CI 69.3-80.2)), and similar in HIV-infected and HIV-uninfected patients (76.7 (95% CI 69.0-83.3) versus 69.5 (95% CI 59.2-78.5)). However, sensitivity was significantly diminished in HIV-infected patients with CD4 counts <100â cells·mm(-3). C-Tb and QFT-GIT combined had significantly higher sensitivity than C-Tb alone (p<0.0001). C-Tb was safe with no significant adverse events. The 5â mm cut-point corresponded to that found in the previously published specificity study (TESEC-04).C-Tb has similar sensitivity compared with QFT-GIT for the diagnosis of M. tuberculosis infection. Sensitivity was reduced only in HIV-infected patients with severe immunosuppression. Further studies in different settings are required to validate the proposed 5â mm cut-point.
Assuntos
Infecções por HIV/complicações , Teste Tuberculínico/normas , Tuberculose/diagnóstico , Adolescente , Adulto , Coinfecção , Método Duplo-Cego , Feminino , Infecções por HIV/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/isolamento & purificação , Curva ROC , África do Sul , Adulto JovemRESUMO
CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-gamma, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-gamma. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.
Assuntos
Leishmania major/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Células Th1/imunologia , Animais , Humanos , Leishmaniose Cutânea/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: C-Tb, an ESAT-6/CFP-10-based skin test, has similar sensitivity for active TB compared to tuberculin skin test (TST) and QuantiFERON-TB-Gold-In-Tube (QFT). However, data are limited in children and HIV-infected persons. METHODS: Asymptomatic South African contacts <5 years (n = 87; HIV-uninfected), or symptomatic individuals of all ages presenting to clinics with suspected TB (n = 1003; 30% HIV-infected) were recruited from eight South African centres. C-Tb and TST were allocated to either forearm double blinded. Samples for QFT were collected in parallel, and test-positivity rates were compared. RESULTS: In participants with microbiologically confirmed TB (n = 75; 45% HIV-infected) sensitivity of C-Tb, TST and QFT were similar (72% versus 75% versus 73%; p>0.5). All 3 tests had similar positivity rates in HIV-infected participants with active TB, however, positivity rates were reduced when CD4 counts were <100 cells/µL. In participants where active TB was excluded (n = 920), C-Tb (41%), TST (43%), and QFT (44%) also had similar test-positivity rates. Among asymptomatic contacts aged below five, 32% (28/87) tested positive with C-Tb and 32% (28/87) with TST (concordance 89%). Overall, C-Tb and TST showed a similar safety profile. CONCLUSION: C-Tb was safe and showed similar test-positivity rates, compared to TST and QFT, in children and HIV-infected persons with active or latent M. tuberculosis infection. These data inform the utility of C-Tb in clinical practice. TRIAL REGISTRATION: ClinicalTrials.gov NCT01642888. EudraCT 2011-005078-40.
Assuntos
Coinfecção/diagnóstico , Infecções por HIV/complicações , Testes Cutâneos/métodos , Tuberculose/complicações , Tuberculose/diagnóstico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Método Duplo-Cego , Feminino , Infecções por HIV/diagnóstico , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: H1/IC31® is a tuberculosis (TB) subunit vaccine candidate consisting of the fusion protein of Ag85B and ESAT-6 (H1) formulated with the IC31® adjuvant. Previous trials have reported on the H1/IC31® vaccine in M. tuberculosis (Mtb)-naïve, BCG-vaccinated and previously Mtb-infected individuals. In this trial, conducted between December 2008 and April 2010, the safety and immunogenicity of H1/IC31® was assessed in participants living in Ethiopia - a highly TB-endemic area. METHODS: Healthy male participants aged 18-25 years were recruited into four groups. Participants in group 1 (N = 12) and group 2 (N = 12) were Tuberculin Skin Test (TST) negative and QuantiFERON-TB Gold in-tube test (QFT) negative (Mtb-naïve groups), participants in group 3 (N = 3) were TST positive and QFT negative (BCG group), and participants in group 4 (N = 12) were both TST and QFT positive (Mtb-infected group). H1 vaccine alone (group 1) or H1 formulated with the adjuvant IC31® (groups 2, 3 and 4) was administered intramuscularly on day 0 and day 56. Safety and immunogenicity parameters were evaluated for up to 32 weeks after day 0. RESULTS: The H1/IC31®vaccine was safe and generally well tolerated. There was little difference among the four groups, with a tendency towards a higher incidence of adverse events in Mtb-infected compared to Mtb-naïve participants. Two serious adverse events were reported in the Mtb-infected group where a relationship to the vaccine could not be excluded. In both cases the participants recovered without sequelae within 72 h. Immunogenicity assays, evaluated in the 29 participants who received both vaccinations, showed a stronger response to TB antigens in the Mtb-naïve group vaccinated with the adjuvant. CONCLUSION: The trial confirmed the need for an adjuvant for the vaccine to be immunogenic and highlighted the importance of early phase testing of a novel TB vaccine candidate in TB-endemic areas. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT01049282. Retrospectively registered on 14 January 2010.
Assuntos
Vacinas contra a Tuberculose/imunologia , Adjuvantes Imunológicos/farmacologia , Adulto , Anticorpos Antibacterianos/sangue , Humanos , Imunoglobulina G/sangue , Masculino , Vacinas contra a Tuberculose/efeitos adversos , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
BACKGROUND: New, simple, and better-performing diagnostic tools are needed for the diagnosis of tuberculosis (TB). Much effort has been invested in developing an antibody-based test for TB, but to date, no such test has performed with sufficient sensitivity and specificity. A key question remaining is the extent to which the disappointing performance of current tests is associated with a high background prevalence of latent TB. METHODS: We compared Mycobacterium tuberculosis-specific ESAT-6 and CFP-10 antibody responses in a total of 565 human serum samples from M. tuberculosis-uninfected donors and donors with latent infection, as well as samples from patients with active TB. Our study included samples from 4 countries, representing environments with low, intermediate, and high TB incidences. RESULTS: We demonstrated significant increases in antibody levels in latently infected contacts, compared with M. tuberculosis-uninfected individuals, and in patients with active TB disease, compared with latently infected contacts. Furthermore, we found a striking increase in the magnitude of the antibody responses in samples obtained from infected Ethiopian individuals (with and without disease), compared with Danish and Brazilian infected individuals; this was presumably the result of higher exposure levels. CONCLUSIONS: Our study confirms the presence of ESAT-6 and CFP-10 antibodies in patients with TB, and we demonstrate that significant antibody responses are not restricted to active TB disease but can reflect latent infection, particularly in areas with high levels of exposure to M. tuberculosis. This finding is important for the understanding of the poor discriminatory power of current serodiagnostic tests in regions of endemicity, and it may have major implications on the future development of serologic tests.
Assuntos
Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Mycobacterium tuberculosis/imunologia , Fragmentos de Peptídeos/imunologia , Tuberculose/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Formação de Anticorpos/imunologia , Brasil , Dinamarca , Etiópia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Sorológicos , Tanzânia , Tuberculose/diagnósticoRESUMO
BACKGROUND: Control of the tuberculosis epidemic requires a novel vaccine that is effective in preventing tuberculosis in adolescents, a key target population for vaccination against TB. METHODS: Healthy adolescents, stratified by M. tuberculosis-infection status, were enrolled into this observer-blinded phase II clinical trial of the protein-subunit vaccine candidate, H1:IC31, comprising a fusion protein (H1) of Ag85B and ESAT-6, formulated with the IC31 adjuvant. Local and systemic adverse events and induced T cell responses were measured after one or two administrations of either 15µg or 50µg of the H1 protein. RESULTS: Two hundred and forty participants were recruited and followed up for 224days. No notable safety events were observed regardless of H1 dose or vaccination schedule. H1:IC31 vaccination induced antigen-specific CD4 T cells, co-expressing IFN-γ, TNF-α and/or IL-2. H1:IC31 vaccination of M.tb-uninfected individuals preferentially drove the emergence of Ag85B and ESAT-6 specific TNF-α+IL-2+CD4 T cells, while H1:IC31 vaccination of M.tb-infected individuals resulted in the expansion of Ag85B-specific but not ESAT-6-specific TNF-α+IL-2+CD4 T cells. CONCLUSIONS: H1:IC31 was safe and immunogenic in uninfected and M.tb-infected adolescents. Two administrations of the 15µg H1:IC31 dose induced the greatest magnitude immune response, and was considered optimal (South African National Clinical Trials Register, DoH-27-0612-3947; Pan African Clinical Trial Registry, PACTR201403000464306).
Assuntos
Aciltransferases/imunologia , Adjuvantes Imunológicos/efeitos adversos , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Oligodesoxirribonucleotídeos/efeitos adversos , Oligopeptídeos/efeitos adversos , Vacinas contra a Tuberculose/efeitos adversos , Vacinas contra a Tuberculose/imunologia , Aciltransferases/genética , Adjuvantes Imunológicos/administração & dosagem , Adolescente , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Criança , Combinação de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Voluntários Saudáveis , Humanos , Interleucina-2/metabolismo , Masculino , Oligodesoxirribonucleotídeos/administração & dosagem , Oligopeptídeos/administração & dosagem , Método Simples-Cego , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/genética , Fator de Necrose Tumoral alfa/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: Novel vaccine strategies are required to provide protective immunity in tuberculosis (TB) and prevent development of active disease. We investigated the safety and immunogenicity of a novel TB vaccine candidate, H4:IC31 (AERAS-404) that is composed of a fusion protein of M. tuberculosis antigens Ag85B and TB10.4 combined with an IC31® adjuvant. METHODS: BCG-vaccinated healthy subjects were immunized with various antigen (5, 15, 50, 150µg) and adjuvant (0, 100, 500nmol) doses of the H4:IC31 vaccine (n=106) or placebo (n=18) in two randomized, double-blind, placebo-controlled phase I studies conducted in a low TB endemic setting in Sweden and Finland. The subjects were followed for adverse events and CD4+ T cell responses. RESULTS: H4:IC31 vaccination was well tolerated with a safety profile consisting of mostly mild to moderate self-limited injection site pain, myalgia, arthralgia, fever and post-vaccination inflammatory reaction at the screening tuberculin skin test injection site. The H4:IC31 vaccine elicited antigen-specific CD4+ T cell proliferation and cytokine production that persisted 18weeks after the last vaccination. CD4+ T cell expansion, IFN-γ production and multifunctional CD4+ Th1 responses were most prominent after two doses of H4:IC31 containing 5, 15, or 50µg of H4 in combination with the 500nmol IC31 adjuvant dose. CONCLUSIONS: The novel TB vaccine candidate, H4:IC31, demonstrated an acceptable safety profile and was immunogenic, capable of triggering multifunctional CD4+ T cell responses in previously BCG-vaccinated healthy individuals. These dose-escalation trials provided evidence that the optimal antigen-adjuvant dose combinations are 5, 15, or 50µg of H4 and 500nmol of IC31. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02066428 and NCT02074956.
Assuntos
Vacinas contra a Tuberculose/efeitos adversos , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Aciltransferases/administração & dosagem , Aciltransferases/efeitos adversos , Aciltransferases/imunologia , Adulto , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/efeitos adversos , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/efeitos adversos , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Método Duplo-Cego , Combinação de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Finlândia , Voluntários Saudáveis , Humanos , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/efeitos adversos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/efeitos adversos , Placebos/administração & dosagem , Suécia , Resultado do Tratamento , Vacinas contra a Tuberculose/administração & dosagemRESUMO
There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Testes de Liberação de Interferon-gama/métodos , Vacinas contra a Tuberculose/imunologia , Adulto , Algoritmos , Estudos de Casos e Controles , Estudos de Coortes , Simulação por Computador , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeos/imunologia , Curva ROC , Reprodutibilidade dos Testes , Tuberculose/imunologiaRESUMO
CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection.
Assuntos
Antígenos de Diferenciação de Linfócitos T/fisiologia , Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária/imunologia , Tuberculose/imunologia , Aciltransferases/imunologia , Adolescente , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Diferenciação Celular , Citocinas/sangue , Feminino , Humanos , Interferon gama/imunologia , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/patogenicidade , RNA Mensageiro/biossíntese , África do Sul , Tuberculose/microbiologia , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/farmacologia , VacinaçãoRESUMO
BACKGROUND: Targeted screening and treatment of Mycobacterium tuberculosis infection substantially reduces the risk of developing active tuberculosis. C-Tb (Statens Serum Institute, Copenhagen, Denmark) is a novel specific skin test based on ESAT-6 and CFP10 antigens. We investigated the safety and diagnostic potential of C-Tb compared with established tests in the contact-tracing setting. METHODS: Negative controls, close contacts, occasional contacts, and patients with active pulmonary tuberculosis were enrolled at 13 centres in Spain. We compared C-Tb with the QuantiFERON-TB Gold In-Tube ([QFT] Qiagen, Hilden, Germany) interferon γ release assay (IGRA) and the purified protein derivative (PPD) RT 23 tuberculin skin test ([TST] Statens Serum Institute). All participants older than 5 years were tested with QFT. Some participants in the negative control group received C-Tb without the TST to test for potential interactions between C-Tb and PPD RT 23. The rest were randomly assigned in blocks of ten and tested with both C-Tb and TST, with five in each block receiving injection of C-Tb in the right arm and the TST in the left arm and five vice versa. The primary and safety analyses were done in all participants randomly assigned to a group who received any test. This trial is registered with ClinicalTrials.gov, number NCT01631266, and with EudraCT, number 2011-005617-36. FINDINGS: From July 24, 2012, to Oct 2, 2014, 979 participants were enrolled, of whom 263 were negative controls, 299 were occasional contacts, 316 were close contacts, and 101 were patients with tuberculosis. 970 (99%) participants completed the trial. Induration sizes were similar for C-Tb and TST, but TST positivity was affected by BCG vaccination status. We found a strong positive trend towards C-Tb test positivity with increasing risk of infection, from 3% in negative controls to 16% in occasional contacts, to 43% in close contacts. C-Tb and QFT results were concordant in 785 (94%) of 834 participants aged 5 years and older, and results did not differ significantly between exposure groups. The safety profile of C-Tb was similar to that for the TST. INTERPRETATION: C-Tb delivered IGRA-like results in a field-friendly format. Being unaffected by BCG vaccination status, the C-Tb skin test might provide more accurate treatment guidance in settings where the TST is commonly used. FUNDING: Statens Serum Institut.
Assuntos
Testes de Liberação de Interferon-gama/métodos , Teste Tuberculínico/métodos , Tuberculose/diagnóstico , Adolescente , Adulto , Vacina BCG/efeitos adversos , Criança , Pré-Escolar , Método Duplo-Cego , Feminino , Humanos , Masculino , Mycobacterium tuberculosis/imunologia , Espanha , Tuberculose/prevenção & controle , Adulto JovemRESUMO
BACKGROUND: Regulatory T cells (Treg) are an essential arm of adaptive immunity not only in tolerance and autoimmunity but also in infectious diseases. In Tuberculosis (TB), it has been suggested that the frequency of Tregs is higher in the blood of TB patients when compared to healthy controls with subsequent decline after treatment. However, with the discovery that FOXP3, the hallmark marker of Tregs, is not exclusive to Tregs and the lack of specific markers for Tregs, it has been a challenge to fully understand the role of Tregs in TB. METHOD: We isolated PBMC from smear positive TB patients (TB, N = 13) before and after treatment, latent TB infected participants (LTBI, N = 8), and healthy endemic controls (EC, N = 9) and evaluated the frequency of different populations of Tregs and expression of FOXP3 by flowcytometry using six markers. RESULTS: The findings in this study showed that the association of Treg frequency with TB disease depends on the phenotypic markers used. While the frequency of CD4(+)CD25(+/hi) T cells was higher in TB patients compared to LTBI individuals, there was no difference in the frequency of CD4(+)CD25(+)FOXP3(+)CD127(lo) Treg among TB, LTBI, or EC. However, delineation of Tregs into active and naïve subsets revealed a significant increase in FOXP3 expression in active primed Tregs (CD4(+)CD25(+)FOXP3(+)CD127(lo)CD45RO(+)Ki-67(+)) of TB patients compared to LTBI and EC; and a significantly higher frequency of resting primed (CD45RO(+)Ki-67(-)) Treg in QuantiFERON negative EC compared to TB patients. After treatment completion, there was a significant decline in the frequency of active primed Treg, median (IQR) from 12.4% (9.5-21.9) of Tregs to 9.3% (7.0-12.2); P = 0.003 Wilcoxon signed rank test. We conclude that Treg subsets may be differentially regulated and expressed in TB disease, cure, and infection.
Assuntos
Tuberculose Latente/imunologia , Linfócitos T Reguladores/imunologia , Tuberculose Pulmonar/imunologia , Adolescente , Adulto , Antituberculosos/uso terapêutico , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Citometria de Fluxo/métodos , Seguimentos , Fatores de Transcrição Forkhead/sangue , Humanos , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/imunologia , Tuberculose Pulmonar/tratamento farmacológico , Adulto JovemRESUMO
BACKGROUND: The role of B cells in human host response to Mycobacterium tuberculosis (Mtb) infection is still controversial, but recent evidence suggest that B cell follicle like structures within the lung may influence host responses through regulation of the local cytokine environment. A candidate for such regulation could be the chemokine CXCL10. CXCL10 is mainly produced by human monocytes, but a few reports have also found CXCL10 production by human B cells. The objective of this study was to investigate CXCL10 production by human B cells in response to in vitro stimulation with Mtb antigens. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed human blood samples from 30 volunteer donors using multiparameter flow cytometry, and identified a subgroup of B cells producing CXCL10 in response to in vitro stimulation with antigens. T cells did not produce CXCL10, but CXCL10 production by B cells appeared to be mediated via IFN-γ and dependent on contact with antigen-specific T cells recognizing the antigen. CONCLUSION: Human B cells are able to produce CXCL10 in an IFN-γ and T cell contact-dependent manner. The present findings suggest a possible mechanism through which B cells in part may influence granuloma formation in human tuberculosis (TB) and participate in infection control.
Assuntos
Subpopulações de Linfócitos B/metabolismo , Quimiocina CXCL10/biossíntese , Subpopulações de Linfócitos T/imunologia , Tuberculose/imunologia , Antígenos de Bactérias/imunologia , Células Cultivadas , Relação Dose-Resposta a Droga , Granuloma/imunologia , Voluntários Saudáveis , Humanos , Imunidade Celular/imunologia , Fatores Imunológicos/farmacologia , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Mycobacterium tuberculosis/imunologiaRESUMO
Tuberculosis (TB) remains a global health problem, with vaccination being a necessary strategy for disease containment and elimination. A TB vaccine should be safe and immunogenic as well as efficacious in all affected populations, including HIV-infected individuals. We investigated the induction and maintenance of vaccine-induced memory CD4(+) T cells following vaccination with the subunit vaccine H1/IC31. H1/IC31 was inoculated twice on study days 0 and 56 among HIV-infected adults with CD4(+) lymphocyte counts of >350 cells/mm(3). Whole venous blood stimulation was conducted with the H1 protein, and memory CD4(+) T cells were analyzed using intracellular cytokine staining and polychromatic flow cytometry. We identified high responders, intermediate responders, and nonresponders based on detection of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) expressing central (TCM) and effector memory CD4(+) T cells (TEM) 182 days after the first immunization. Amplicon-based transcript quantification using next-generation sequencing was performed to identify differentially expressed genes that correlated with vaccine-induced immune responses. Genes implicated in resolution of inflammation discriminated the responders from the nonresponders 3 days after the first inoculation. The volunteers with higher expression levels of genes involved in antiviral innate immunity at baseline showed impaired H1-specific TCM and TEM maintenance 6 months after vaccination. Our study showed that in HIV-infected volunteers, expression levels of genes involved in the antiviral innate immune response affected long-term maintenance of H1/IC31 vaccine-induced cellular immunity. (The clinical trial was registered in the Pan African Clinical Trials Registry [PACTR] with the identifier PACTR201105000289276.).
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Tolerância Imunológica , Imunidade Inata , Memória Imunológica , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Adulto , Citocinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência de DNA , Tuberculose/imunologia , Vacinas contra a Tuberculose/administração & dosagem , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
BACKGROUND: New, more effective vaccines to prevent tuberculosis (TB) disease are needed urgently. H4:IC31 is an investigational vaccine that contains a fusion protein of the immunodominant antigens TB10.4 and Ag85B, formulated in IC31 adjuvant. We assessed the safety and immunogenicity of H4:IC31 in South African adults from a TB endemic setting. METHODS: In this double blind, placebo controlled, phase I trial, Mycobacterium tuberculosis-uninfected, HIV-uninfected, healthy adults with a history of childhood BCG vaccination were randomly allocated to two intramuscular vaccinations with 5, 15, 50 or 150 µg H4 formulated in 500nmol IC31, two months apart. Vaccinees were followed for six months to assess safety; immunogenicity was measured by ELISpot and intracellular cytokine staining assays. RESULTS: Thirty-two participants received H4:IC31 and 8 received placebo. Injection site adverse events were common but mild; mild fatigue was the most common systemic adverse event. Frequencies of adverse events did not differ between dosage groups. Detectable antigen-specific CD4 T cell responses were induced by all doses of H4:IC31, but doses below 50 µg induced the highest frequencies of CD4 T cells, comprised predominantly of IFN-γ(+)TNF-α(+)IL-2(+) or TNF-α(+)IL-2(+) cells. These memory responses persisted up to the end of follow up, on study day 182. CONCLUSIONS: H4:IC31 demonstrated an acceptable safety profile and was immunogenic in South African adults. In this trial, the 15 µg dose appeared to induce the most optimal immune response.
Assuntos
Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Mycobacterium tuberculosis/imunologia , Oligodesoxirribonucleotídeos/administração & dosagem , Oligopeptídeos/administração & dosagem , Vacinas contra a Tuberculose/imunologia , Adolescente , Adulto , Antígenos de Bactérias/administração & dosagem , Citocinas/análise , Método Duplo-Cego , Combinação de Medicamentos , ELISPOT , Feminino , Humanos , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/efeitos adversos , Oligopeptídeos/efeitos adversos , Placebos/administração & dosagem , África do Sul , Coloração e Rotulagem , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/efeitos adversos , Adulto JovemRESUMO
BACKGROUND: H56:IC31 is a candidate tuberculosis vaccine comprising a fusion protein of Ag85B, ESAT-6 and Rv2660c, formulated in IC31 adjuvant. This first-in-human, open label phase I trial assessed the safety and immunogenicity of H56:IC31 in healthy adults without or with Mycobacterium tuberculosis (M.tb) infection. METHODS: Low dose (15 µg H56 protein in 500 nmol IC31) or high dose (50 µg H56, 500 nmol IC31) vaccine was administered intramuscularly thrice, at 56-day intervals. Antigen-specific T cell responses were measured by intracellular cytokine staining and antibody responses by ELISA. RESULTS: One hundred and twenty-six subjects were screened and 25 enrolled and vaccinated. No serious adverse events were reported. Nine subjects (36%) presented with transient cardiovascular adverse events. The H56:IC31 vaccine induced antigen-specific IgG responses and Th1 cytokine-expressing CD4(+) T cells. M.tb-infected vaccinees had higher frequencies of H56-induced CD4(+) T cells than uninfected vaccinees. Low dose vaccination induced more polyfunctional (IFN-γ(+)TNF-α(+)IL-2(+)) and higher frequencies of H56-specific CD4(+) T cells compared with high dose vaccination. A striking increase in IFN-γ-only-expressing CD4(+) T cells, displaying a CD45RA(-)CCR7(-) effector memory phenotype, emerged after the second high-dose vaccination in M.tb-infected vaccinees. TNF-α(+)IL-2(+) H56-specific memory CD4(+) T cells were detected mostly after low-dose H56 vaccination in M.tb-infected vaccinees, and predominantly expressed a CD45RA(-)CCR7(+) central memory phenotype. Our results support further clinical testing of H56:IC31.
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Anticorpos Antibacterianos/sangue , Linfócitos T CD4-Positivos/imunologia , Mycobacterium tuberculosis/imunologia , Profilaxia Pós-Exposição/métodos , Subpopulações de Linfócitos T/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Aciltransferases/administração & dosagem , Aciltransferases/imunologia , Adolescente , Adulto , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Citocinas/biossíntese , Combinação de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Voluntários Saudáveis , Humanos , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/administração & dosagem , Oligopeptídeos/administração & dosagem , Resultado do Tratamento , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/efeitos adversos , Adulto JovemRESUMO
BACKGROUND: Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1) formulated with the adjuvant IC31, was evaluated in HIV-infected adults. METHODS: HIV-infected adults with CD4+ T cell counts >350/mm3 and without evidence of active tuberculosis were enrolled and followed until day 182. H1/IC31 vaccine or placebo was randomly allocated in a 5:1 ratio. The vaccine was administered intramuscularly at day 0 and 56. Safety assessment was based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was determined by a short-term whole blood intracellular cytokine staining assay. RESULTS: 47 of the 48 randomised participants completed both vaccinations. In total, 459 mild or moderate and 2 severe adverse events were reported. There were three serious adverse events in two vaccinees classified as not related to the investigational product. Local injection site reactions were more common in H1/IC31 versus placebo recipients (65.0% vs. 12.5%, p = 0.015). Solicited systemic and unsolicited adverse events were similar by study arm. The baseline CD4+ T cell count and HIV viral load were similar by study arm and remained constant over time. The H1/IC31 vaccine induced a persistent Th1-immune response with predominately TNF-α and IL-2 co-expressing CD4+ T cells, as well as polyfunctional IFN-γ, TNF-α and IL-2 expressing CD4+ T cells. CONCLUSION: H1/IC31 was well tolerated and safe in HIV-infected adults with a CD4+ Lymphocyte count greater than 350 cells/mm3. The vaccine did not have an effect on CD4+ T cell count or HIV-1 viral load. H1/IC31 induced a specific and durable Th1 immune response. TRIAL REGISTRATION: Pan African Clinical Trials Registry (PACTR) PACTR201105000289276.
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Infecções por HIV/complicações , Vacinas contra a Tuberculose/uso terapêutico , Tuberculose/prevenção & controle , Aciltransferases/imunologia , Adjuvantes Imunológicos/uso terapêutico , Adulto , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Contagem de Linfócito CD4 , Método Duplo-Cego , Feminino , Infecções por HIV/imunologia , Humanos , Masculino , Proteínas Recombinantes de Fusão/imunologia , Tuberculose/complicações , Vacinas contra a Tuberculose/efeitos adversos , Vacinas contra a Tuberculose/imunologia , Carga Viral/efeitos dos fármacosRESUMO
Here, we report on a first-in-man trial where the tuberculosis (TB) vaccine Ag85B-ESAT-6 (H1) was adjuvanted with escalating doses of a novel liposome adjuvant CAF01. On their own, protein antigens cannot sufficiently induce immune responses in humans, and require the addition of an adjuvant system to ensure appropriate delivery and concomitant immune activation. To date no approved adjuvants are available for induction of cellular immunity, which seems essential for a number of vaccines, including vaccines against TB. We vaccinated four groups of human volunteers: a non-adjuvanted H1 group, followed by three groups with escalating doses of CAF01-adjuvanted H1 vaccine. All subjects were vaccinated at 0 and 8 weeks and followed up for 150 weeks. Vaccination did not cause local or systemic adverse effects besides transient soreness at the injection site. Two vaccinations elicited strong antigen-specific T-cell responses which persisted after 150 weeks follow-up, indicating the induction of a long-lasting memory response in the vaccine recipients. These results show that CAF01 is a safe and tolerable, Th1-inducing adjuvant for human TB vaccination trials and for vaccination studies in general where cellular immunity is required.
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Adjuvantes Imunológicos/administração & dosagem , Memória Imunológica , Lipossomos/administração & dosagem , Mycobacterium tuberculosis/imunologia , Linfócitos T/imunologia , Vacinas contra a Tuberculose/imunologia , Adjuvantes Imunológicos/efeitos adversos , Adolescente , Adulto , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/toxicidade , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/efeitos adversos , Adulto JovemRESUMO
BACKGROUND: Tuberculin skin testing is simple and relatively inexpensive, but the specificity of PPD is affected by BCG vaccination. OBJECTIVE: Determine optimal dose and specificity of recombinant ESAT-6 and CFP-10 (C-Tb) produced in Lactococcus lactis for diagnosis of M. tuberculosis infection. METHODS: In a dose finding phase I trial 0.01 or 0.1 µg preserved and unpreserved C-Tb was injected by Mantoux technique in 38 patients with active tuberculosis and induration responses measured. In a phase II specificity trial in 151 uninfected, BCG vaccinated participants 0.1 µg C-Tb was compared to 2 TU PPD. RESULTS: 0.1 µg C-Tb gave a median induration of 15 mm after 2 days. Phenol preservation did not affect the response. The specificity of C-Tb was 99.3% (95% CI 96-100%) regarding indurations ≥5 mm as a positive outcome. This was higher than the specificity of PPD (63% using a cut-off of 5 mm or 92% using a cut-off of 15 mm to adjust for non-specific BCG responses). Local adverse reactions following C-Tb injection included transient itching and discomfort as expected components of the immune response. CONCLUSION: C-Tb offers a simple and convenient skin test to diagnose M. tuberculosis infection using a single, universal cut-off unaffected by BCG vaccination. TRIAL REGISTRATION: ClinicalTrials.gov NCT01033929 and NCT01241188.