RESUMO
BACKGROUND: Despite the recent innovations in tuberculosis (TB) and multi-drug resistant TB (MDR-TB) diagnosis, culture remains vital for difficult-to-diagnose patients, baseline and end-point determination for novel vaccines and drug trials. Herein, we share our experience of establishing a BSL-3 culture facility in Uganda as well as 3-years performance indicators and post-TB vaccine trials (pioneer) and funding experience of sustaining such a facility. METHODS: Between September 2008 and April 2009, the laboratory was set-up with financial support from external partners. After an initial procedure validation phase in parallel with the National TB Reference Laboratory (NTRL) and legal approvals, the laboratory registered for external quality assessment (EQA) from the NTRL, WHO, National Health Laboratories Services (NHLS), and the College of American Pathologists (CAP). The laboratory also instituted a functional quality management system (QMS). Pioneer funding ended in 2012 and the laboratory remained in self-sustainability mode. RESULTS: The laboratory achieved internationally acceptable standards in both structural and biosafety requirements. Of the 14 patient samples analyzed in the procedural validation phase, agreement for all tests with NTRL was 90% (P <0.01). It started full operations in October 2009 performing smear microscopy, culture, identification, and drug susceptibility testing (DST). The annual culture workload was 7,636, 10,242, and 2,712 inoculations for the years 2010, 2011, and 2012, respectively. Other performance indicators of TB culture laboratories were also monitored. Scores from EQA panels included smear microscopy >80% in all years from NTRL, CAP, and NHLS, and culture was 100% for CAP panels and above regional average scores for all years with NHLS. Quarterly DST scores from WHO-EQA ranged from 78% to 100% in 2010, 80% to 100% in 2011, and 90 to 100% in 2012. CONCLUSIONS: From our experience, it is feasible to set-up a BSL-3 TB culture laboratory with acceptable quality performance standards in resource-limited countries. With the demonstrated quality of work, the laboratory attracted more research groups and post-pioneer funding, which helped to ensure sustainability. The high skilled experts in this research laboratory also continue to provide an excellent resource for the needed national discussion of the laboratory and quality management systems.
Assuntos
Técnicas de Laboratório Clínico/normas , Laboratórios/economia , Laboratórios/normas , Tuberculose/diagnóstico , Antituberculosos/farmacologia , Técnicas de Laboratório Clínico/economia , Países em Desenvolvimento/economia , Estudos de Viabilidade , Humanos , Laboratórios/organização & administração , Testes de Sensibilidade Microbiana/normas , Garantia da Qualidade dos Cuidados de Saúde , Tuberculose/tratamento farmacológico , Tuberculose/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Uganda/epidemiologiaRESUMO
RATIONALE: Tuberculosis (TB) is a major cause of morbidity and mortality worldwide, thus there is an urgent need for novel TB vaccines. OBJECTIVES: We investigated a novel TB vaccine candidate, M72/AS01, in a phase IIa trial of bacille Calmette-Guérin-vaccinated, HIV-uninfected, and Mycobacterium tuberculosis (Mtb)-infected and -uninfected adults in South Africa. METHODS: Two doses of M72/AS01 were administered to healthy adults, with and without latent Mtb infection. Participants were monitored for 7 months after the first dose; cytokine production profiles, cell cycling, and regulatory phenotypes of vaccine-induced T cells were measured by flow cytometry. MEASUREMENTS AND MAIN RESULTS: The vaccine had a clinically acceptable safety profile, and induced robust, long-lived M72-specific T-cell and antibody responses. M72-specific CD4 T cells produced multiple combinations of Th1 cytokines. Analysis of T-cell Ki67 expression showed that most vaccination-induced T cells did not express Th1 cytokines or IL-17; these cytokine-negative Ki67(+) T cells included subsets of CD4 T cells with regulatory phenotypes. PD-1, a negative regulator of activated T cells, was transiently expressed on M72-specific CD4 T cells after vaccination. Specific T-cell subsets were present at significantly higher frequencies after vaccination of Mtb-infected versus -uninfected participants. CONCLUSIONS: M72/AS01 is clinically well tolerated in Mtb-infected and -uninfected adults, induces high frequencies of multifunctional T cells, and boosts distinct T-cell responses primed by natural Mtb infection. Moreover, these results provide important novel insights into how this immunity may be appropriately regulated after novel TB vaccination of Mtb-infected and -uninfected individuals.Clinical trial registered with www.clinicaltrials.gov (NCT 00600782).
Assuntos
Linfócitos T/imunologia , Vacinas contra a Tuberculose/imunologia , Adulto , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Feminino , Citometria de Fluxo , Humanos , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Interleucina-17/metabolismo , Masculino , África do Sul , Vacinas contra a Tuberculose/administração & dosagem , Adulto JovemRESUMO
RATIONALE: Novel tuberculosis (TB) vaccines should be safe and effective in populations infected with Mycobacterium tuberculosis (M.tb) and/or HIV for effective TB control. OBJECTIVE: To determine the safety and immunogenicity of MVA85A, a novel TB vaccine, among M.tb- and/or HIV-infected persons in a setting where TB and HIV are endemic. METHODS: An open-label, phase IIa trial was conducted in 48 adults with M.tb and/or HIV infection. Safety and immunogenicity were analyzed up to 52 weeks after intradermal vaccination with 5 × 10(7) plaque-forming units of MVA85A. Specific T-cell responses were characterized by IFN-γ enzyme-linked immunospot and whole blood intracellular cytokine staining assays. MEASUREMENTS AND MAIN RESULTS: MVA85A was well tolerated and no vaccine-related serious adverse events were recorded. MVA85A induced robust and durable response of mostly polyfunctional CD4(+) T cells, coexpressing IFN-γ, tumor necrosis factor-α, and IL-2. Magnitudes of pre- and postvaccination T-cell responses were lower in HIV-infected, compared with HIV-uninfected, vaccinees. No significant effect of antiretroviral therapy on immunogenicity of MVA85A was observed. CONCLUSIONS: MVA85A was safe and immunogenic in persons with HIV and/or M.tb infection. These results support further evaluation of safety and efficacy of this vaccine for prevention of TB in these target populations.
Assuntos
Vacinas contra a Tuberculose/uso terapêutico , Tuberculose Pulmonar/terapia , Vacinas Virais/uso terapêutico , Infecções Oportunistas Relacionadas com a AIDS/prevenção & controle , Adolescente , Adulto , Contagem de Linfócito CD4 , Feminino , Infecções por HIV/imunologia , Humanos , Imunidade Celular , Masculino , Pessoa de Meia-Idade , Vacinas contra a Tuberculose/efeitos adversos , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/prevenção & controle , Vacinas de DNA , Carga Viral , Vacinas Virais/efeitos adversos , Vacinas Virais/imunologia , Adulto JovemRESUMO
BACKGROUND: BCG, the only licensed tuberculosis vaccine, affords poor protection against lung tuberculosis in infants and children. A new tuberculosis vaccine, which may enhance the BCG-induced immune response, is urgently needed. We assessed the safety of and characterized the T cell response induced by 3 doses of the candidate vaccine, MVA85A, in BCG-vaccinated infants from a setting where tuberculosis is endemic. METHODS: Infants aged 5-12 months were vaccinated intradermally with either 2.5 × 10(7), 5 × 10(7), or 10 × 10(7) plaque-forming units of MVA85A, or placebo. Adverse events were documented, and T-cell responses were assessed by interferon γ (IFN-γ) enzyme-linked immunospot assay and intracellular cytokine staining. RESULTS: The 3 MVA85A doses were well tolerated, and no vaccine-related serious adverse events were recorded. MVA85A induced potent, durable T-cell responses, which exceeded prevaccination responses up to 168 d after vaccination. No dose-related differences in response magnitude were observed. Multiple CD4 T cell subsets were induced; polyfunctional CD4 T cells co-expressing T-helper cell 1 cytokines with or without granulocyte-macrophage colony-stimulating factor predominated. IFN-γ-expressing CD8 T cells, which peaked later than CD4 T cells, were also detectable. CONCLUSIONS: MVA85A was safe and induced robust, polyfunctional, durable CD4 and CD8 T-cell responses in infants. These data support efficacy evaluation of MVA85A to prevent tuberculosis in infancy. Clinical Trials Registration. NCT00679159.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/prevenção & controle , Aciltransferases/imunologia , Fatores Etários , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Linfócitos T CD8-Positivos/imunologia , Relação Dose-Resposta Imunológica , ELISPOT , Feminino , Humanos , Lactente , Interferon gama/metabolismo , Masculino , Placebos , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/efeitos adversos , Vacinas contra a Tuberculose/normasRESUMO
Modified vaccinia Ankara-expressing Ag85A (MVA85A) is a new tuberculosis (TB) vaccine aimed at enhancing immunity induced by BCG. We investigated the safety and immunogenicity of MVA85A in healthy adolescents and children from a TB endemic region, who received BCG at birth. Twelve adolescents and 24 children were vaccinated and followed up for 12 or 6 months, respectively. Adverse events were documented and vaccine-induced immune responses assessed by IFN-gamma ELISpot and intracellular cytokine staining. The vaccine was well tolerated and there were no vaccine-related serious adverse events. MVA85A induced potent and durable T-cell responses. Multiple CD4+ T-cell subsets, based on expression of IFN-gamma, TNF-alpha, IL-2, IL-17 and GM-CSF, were induced. Polyfunctional CD4+ T cells co-expressing IFN-gamma, TNF-alpha and IL-2 dominated the response in both age groups. A novel CD4+ cell subset co-expressing these three Th1 cytokines and IL-17 was induced in adolescents, while a novel CD4+ T-cell subset co-expressing Th1 cytokines and GM-CSF was induced in children. Ag-specific CD8+ T cells were not detected. We conclude that in adolescents and children MVA85A safely induces the type of immunity thought to be important in protection against TB. This includes induction of novel Th1-cell populations that have not been previously described in humans.
Assuntos
Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose/imunologia , Tuberculose/prevenção & controle , Vaccinia virus/genética , Aciltransferases/genética , Adolescente , Antígenos de Bactérias/genética , Criança , Pré-Escolar , Feminino , Humanos , Memória Imunológica , Lactente , Masculino , Vacinas contra a Tuberculose/efeitos adversosRESUMO
RATIONALE: Immunogenicity of new tuberculosis (TB) vaccines is commonly assessed by measuring the frequency and cytokine expression profile of T cells. OBJECTIVES: We tested whether this outcome correlates with protection against childhood TB disease after newborn vaccination with bacillus Calmette-Guérin (BCG). METHODS: Whole blood from 10-week-old infants, routinely vaccinated with BCG at birth, was incubated with BCG for 12 hours, followed by cryopreservation for intracellular cytokine analysis. Infants were followed for 2 years to identify those who developed culture-positive TB-these infants were regarded as not protected against TB. Infants who did not develop TB disease despite exposure to TB in the household, and another group of randomly selected infants who were never evaluated for TB, were also identified-these groups were regarded as protected against TB. Cells from these groups were thawed, and CD4, CD8, and γδ T cell-specific expression of IFN-γ, TNF-α, IL-2, and IL-17 measured by flow cytometry. MEASUREMENTS AND MAIN RESULTS: A total of 5,662 infants were enrolled; 29 unprotected and two groups of 55 protected infants were identified. There was no difference in frequencies of BCG-specific CD4, CD8, and γδ T cells between the three groups of infants. Although BCG induced complex patterns of intracellular cytokine expression, there were no differences between protected and unprotected infants. CONCLUSIONS: The frequency and cytokine profile of mycobacteria-specific T cells did not correlate with protection against TB. Critical components of immunity against Mycobacterium tuberculosis, such as CD4 T cell IFN-γ production, may not necessarily translate into immune correlates of protection against TB disease.
Assuntos
Vacina BCG/imunologia , Tuberculose/prevenção & controle , Contagem de Linfócito CD4 , Estudos de Casos e Controles , Citocinas/sangue , Citocinas/imunologia , Humanos , Imunofenotipagem , Recém-Nascido , Interferon gama/sangue , Estudos Prospectivos , África do Sul , Tuberculose/imunologiaRESUMO
RATIONALE: AERAS-402 is a novel tuberculosis vaccine designed to boost immunity primed by bacillus Calmette-Guérin (BCG), the only licensed vaccine. OBJECTIVES: We investigated the safety and immunogenicity of AERAS-402 in healthy Mycobacterium tuberculosis-uninfected BCG-vaccinated adults from a tuberculosis-endemic region of South Africa. METHODS: Escalating doses of AERAS-402 vaccine were administered intramuscularly to each of three groups of healthy South African BCG-vaccinated adults, and a fourth group received two injections of the maximal dose. Participants were monitored for 6 months, with all adverse effects documented. Vaccine-induced CD4(+) and CD8(+) T-cell immunity was characterized by an intracellular cytokine staining assay of whole blood and peripheral blood mononuclear cells. MEASUREMENTS AND MAIN RESULTS: AERAS-402 was well tolerated, and no vaccine-related serious adverse events were recorded. The vaccine induced a robust CD4(+) T-cell response dominated by cells coexpressing IFN-gamma, tumor necrosis factor-alpha, and IL-2 ("polyfunctional" cells). AERAS-402 also induced a potent CD8(+) T-cell response, characterized by cells expressing IFN-gamma and/or tumor necrosis factor-alpha, which persisted for the duration of the study. CONCLUSIONS: Vaccination with AERAS-402 is safe and immunogenic in healthy adults. The immunity induced by the vaccine appears promising: polyfunctional T cells are thought to be important for protection against intracellular pathogens such as Mycobacterium tuberculosis, and evidence is accumulating that CD8(+) T cells are also important. AERAS-402 induced a robust and durable CD8(+) T-cell response, which appears extremely promising. Clinical trial registered with www.sanctr.gov.za (NHREC no. 1381).
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Vacinas contra a Tuberculose/uso terapêutico , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Seguimentos , Humanos , Ativação Linfocitária/imunologia , Masculino , África do Sul , Vacinas contra a Tuberculose/imunologia , Vacinas de DNA , Adulto JovemRESUMO
HIV-1 infection causes a severe T cell compromise; however, little is known about changes in naive, memory, effector and senescent T cell subsets during the first year of life. T cell subsets were studied over the first year of life in blood from 3 infant cohorts: untreated HIV-infected, HIV-exposed but uninfected, and HIV-unexposed. In HIV-infected infants, the frequency of CCR7(+)CD45RA(+) naive CD8(+) T cells was significantly decreased, while the frequency of CCR7(-)CD45RA(-) effector memory CD8(+) T cells was increased, compared with the control cohorts. A larger population of CD8(+) T cells in HIV-infected infants displayed a phenotype consistent with senescence. Differences in CD4(+) T cell subset frequencies were less pronounced, and no significant differences were observed between exposed and unexposed HIV-uninfected infants. We concluded that the proportion of naive, memory, effector and senescent CD8(+) T cells during the first year of life is significantly altered by HIV-1 infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1 , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Contagem de Células , Diferenciação Celular , Feminino , Citometria de Fluxo , Infecções por HIV/transmissão , HIV-1/imunologia , Humanos , Memória Imunológica , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas , Fenótipo , Subpopulações de Linfócitos T/citologiaRESUMO
BACKGROUND: Qualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity. METHODS: Whole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period. RESULTS: The limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4 °C or 20-23 °C) and time (45, 60 or 90 min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells. CONCLUSIONS: The whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.
Assuntos
Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/sangue , Mycobacterium bovis/imunologia , Tuberculose Pulmonar/imunologia , Adulto , Antígenos de Bactérias/imunologia , Criopreservação , Citocinas/imunologia , Citometria de Fluxo , Humanos , Interferon gama/imunologia , Interleucina-17/imunologia , Interleucina-2/imunologia , Limite de Detecção , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Tuberculose Pulmonar/diagnóstico , Fator de Necrose Tumoral alfa/imunologiaRESUMO
We optimized a whole blood intracellular cytokine assay to quantitate the frequency of specific CD4+ and CD8+ T cells in small volumes of whole blood from infants from developing countries. The assay is performed in two steps. First, whole blood is stimulated in the presence of specific antigens for 6-18 h, ending with cryopreservation of fixed white cells. These stimulation steps were specifically adapted to be practical and reliable in a rural, developing country field setting. Later, in a more resourceful setting, interferon-gamma producing CD4+ or CD8+ T cells are detected by flow cytometry. The assay proved sensitive and specific for detecting mycobacteria-specific immunity 10 weeks after Bacillus Calmette-Guerin (BCG) vaccination of newborns from a rural field site.
Assuntos
Citocinas/sangue , Espaço Intracelular/química , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Vacina BCG/imunologia , Coleta de Amostras Sanguíneas , Criopreservação , Países em Desenvolvimento , Citometria de Fluxo , Infecções por HIV/sangue , Infecções por HIV/imunologia , Humanos , Memória Imunológica/imunologia , Lactente , Interferon gama/análise , Interferon gama/metabolismo , Mycobacterium tuberculosis/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Subpopulações de Linfócitos T/imunologia , Fatores de TempoRESUMO
BACKGROUND: In most tuberculosis (TB) endemic countries, bacillus Calmette-Guérin (BCG) is usually given around birth to prevent severe TB in infants. The neonatal immune system is immature. Our hypothesis was that delaying BCG vaccination from birth to 10 weeks of age would enhance the vaccine-induced immune response. METHODS: In a randomized clinical trial, BCG was administered intradermally either at birth (n=25) or at 10 weeks of age (n=21). Ten weeks after vaccination, and at 1 year of age, vaccine-specific CD4 and CD8 T cell responses were measured with a whole blood intracellular cytokine assay. RESULTS: Infants who received delayed BCG vaccination demonstrated higher frequencies of BCG-specific CD4 T cells, particularly polyfunctional T cells co-expressing IFN-gamma, TNF-alpha and IL-2, and most strikingly at 1 year of age. CONCLUSIONS: Delaying BCG vaccination from birth to 10 weeks of age enhances the quantitative and qualitative BCG-specific T cell response, when measured at 1 year of age.
Assuntos
Vacina BCG/imunologia , Linfócitos T CD4-Positivos/imunologia , Esquemas de Imunização , Memória Imunológica/imunologia , Tuberculose/prevenção & controle , Humanos , Lactente , Recém-Nascido , Interferon gama/imunologia , Interleucina-2/imunologia , Tuberculose/imunologia , Fator de Necrose Tumoral alfa/imunologia , VacinaçãoRESUMO
BACKGROUND: Worldwide, most infants born to mothers infected with human immunodeficiency virus (HIV) receive bacille Calmette-Guérin (BCG) vaccine. Tuberculosis is a major cause of death among infants infected with HIV in sub-Saharan Africa, and it should be prevented. However, BCG may itself cause disease (known as "BCGosis") in these infants. Information regarding the immunogenicity of BCG is imperative for the risk/benefit assessment of BCG vaccination in HIV-infected infants; however, no such data exist. METHODS: We compared BCG-induced CD4 and CD8 T cell responses, as assessed by flow cytometry, in HIV-infected (n=20), HIV-exposed but uninfected (n=25), and HIV-unexposed (n=23) infants, during their first year of life. RESULTS: BCG vaccination of the 2 HIV-uninfected groups induced a robust response, which was characterized by CD4 T cells expressing interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and/or interleukin (IL)-2. In contrast, HIV-infected infants demonstrated a markedly lower response throughout the first year of life. These infants also had significantly reduced numbers of polyfunctional CD4 T cells coexpressing IFN-gamma, TNF-alpha, and IL-2, a finding that is thought to indicate T cell quality. CONCLUSIONS: Infection with HIV severely impairs the BCG-specific T cell response during the first year of life. BCG may therefore provide little, if any, vaccine-induced benefit in HIV-infected infants. Considering the significant risk of BCGosis, these data strongly support not giving BCG to HIV-infected infants.
Assuntos
Vacina BCG/imunologia , Infecções por HIV/imunologia , HIV-1 , Adulto , Vacina BCG/efeitos adversos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Infecções por HIV/transmissão , Humanos , Lactente , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas , GravidezRESUMO
The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-gamma, IL-2, and TNF-alpha in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-gamma and/or IL-2 and less TNF-alpha, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-gamma. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA-CCR7-CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-gamma+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-gamma production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.
Assuntos
Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Diferenciação Celular/imunologia , Citocinas/biossíntese , Imunofenotipagem , Recém-Nascido/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Pré-Escolar , Citocinas/classificação , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/sangue , Humanos , Lactente , Interferon gama/biossíntese , Interferon gama/sangue , Interleucina-10/biossíntese , Interleucina-2/biossíntese , Interleucina-2/sangue , Subpopulações de Linfócitos T/citologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND: The efficacy of bacille Calmette-Guérin (BCG) may be enhanced by heterologous vaccination strategies that boost the BCG-primed immune response. One leading booster vaccine, MVA85A (where "MVA" denotes "modified vaccinia virus Ankara"), has shown promising safety and immunogenicity in human trials performed in the United Kingdom. We investigated the safety and immunogenicity of MVA85A in mycobacteria-exposed--but Mycobacterium tuberculosis-uninfected--healthy adults from a region of South Africa where TB is endemic. METHODS: Twenty-four adults were vaccinated with MVA85A. All subjects were monitored for 1 year for adverse events and for immunological assessment. RESULTS: MVA85A vaccination was well tolerated and induced potent T cell responses, as measured by interferon (IFN)-gamma enzyme-linked immunospot assay, which exceeded prevaccination responses up to 364 days after vaccination. BCG-specific CD4+ T cells boosted by MVA85A were comprised of multiple populations expressing combinations of IFN-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, and IL-17, as measured by polychromatic flow cytometry. IFN-gamma-expressing and polyfunctional IFN-gamma+TNF-gamma+IL-2+ CD4+ T cells were boosted during the peak BCG-specific response, which occurred 7 days after vaccination. CONCLUSION: The excellent safety profile and quantitative and qualitative immunogenicity data strongly support further trials assessing the efficacy of MVA85A as a boosting vaccine in countries where TB is endemic. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00460590.
Assuntos
Antígenos de Bactérias/imunologia , Vacina BCG/efeitos adversos , Vacina BCG/imunologia , Imunidade Celular/efeitos dos fármacos , Vaccinia virus/metabolismo , Adulto , Antígenos Virais/química , Vacina BCG/administração & dosagem , Vacina BCG/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunidade Celular/imunologia , Interferon gama/análise , Masculino , Mycobacterium tuberculosis/imunologia , Segurança , África do Sul , Tuberculose/prevenção & controle , Vacinas Sintéticas/imunologiaRESUMO
We investigated whether the proinflammatory T cell cytokines IL-17 and IL-22 are induced by human mycobacterial infection. Remarkably, >20% of specific cytokine-producing CD4(+) T cells in peripheral blood of healthy, mycobacteria-exposed adults expressed IL-17 or IL-22. Specific IL-17- and IL-22-producing CD4(+) T cells were distinct from each other and from Th1 cytokine-producing cells. These cells had phenotypic characteristics of long-lived central memory cells. In patients with tuberculosis disease, peripheral blood frequencies of these cells were reduced, whereas bronchoalveolar lavage fluid contained higher levels of IL-22 protein compared with healthy controls. IL-17 was not detected in this fluid, which may be due to suppression by Th1 cytokines, as PBMC IL-17 production was inhibited by IFN-gamma in vitro. However, Th1 cytokines had no effect on IL-22 production in vitro. Our results imply that the magnitude and complexity of the anti-mycobacterial immune response have historically been underestimated. IL-17- and IL-22-producing CD4(+) T cells may play important roles in the human immune response to mycobacteria.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interleucina-17/metabolismo , Interleucinas/metabolismo , Mycobacterium tuberculosis/imunologia , Subpopulações de Linfócitos T/imunologia , Tuberculose Pulmonar/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Humanos , Memória Imunológica , Interleucina-17/análise , Interleucinas/análise , Masculino , Células Th1/imunologia , Interleucina 22RESUMO
In 10-week-old infants vaccinated at birth with Japanese Mycobacterium bovis BCG, the number of dermal needle penetrations correlated positively with frequency of proliferating CD4(+) T cells in whole blood following BCG stimulation for 6 days but did not correlate with secreted cytokine levels after 7 h or interferon CD4(+) T-cell frequency after 12 h of BCG stimulation.
Assuntos
Vacina BCG/imunologia , Recém-Nascido/imunologia , Tuberculose/prevenção & controle , Relação Dose-Resposta Imunológica , Humanos , Lactente , Tuberculose/imunologiaRESUMO
Vaccination with Mycobacterium bovis bacille Calmette-Guerin (BCG) has variable efficacy in preventing tuberculosis. Both BCG strain and route of administration have been implicated in determining efficacy; however, these variables are not considered in current clinical recommendations for vaccine choice. We evaluated antigen-specific immunity after percutaneous or intradermal administration of Japanese BCG or intradermal administration of Danish BCG. Ten weeks after vaccination of neonates, percutaneous Japanese BCG had induced significantly higher frequencies of BCG-specific interferon- gamma -producing CD4(+) and CD8(+) T cells in BCG-stimulated whole blood than did intradermal Danish BCG. Similarly, percutaneous vaccination with Japanese BCG resulted in significantly greater secretion of the T helper 1-type cytokines interferon- gamma, tumor necrosis factor- alpha , and interleukin-2; significantly lower secretion of the T helper 2-type cytokine interleukin-4; and greater CD4(+) and CD8(+) T cell proliferation. Thus, BCG strain and route of neonatal vaccination confer different levels of immune activation, which may affect the efficacy of the vaccine.