Sebastian Gelderbloem

Specific T Cell Frequency and Cytokine Expression Profile Do Not Correlate with Protection against Tuberculosis after Bacillus Calmette-Guérin Vaccination of Newborns

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.

Bacillus Calmette-Guérin Vaccination of Human Newborns Induces T Cells with Complex Cytokine and Phenotypic Profiles

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-γ, IL-2, and TNF-α 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-γ and/or IL-2 and less TNF-α, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-γ. 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-γ+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-γ 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.

Modified vaccinia Ankara-expressing Ag85A, a novel tuberculosis vaccine, is safe in adolescents and children, and induces polyfunctional CD4+ T cells

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‐γ 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‐γ, TNF‐α, IL‐2, IL‐17 and GM‐CSF, were induced. Polyfunctional CD4+ T cells co‐expressing IFN‐γ, TNF‐α 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.

Delaying BCG vaccination from birth to 10 weeks of age may result in an enhanced memory CD4 T cell response

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.

Induction and Regulation of T-Cell Immunity by the Novel Tuberculosis Vaccine M72/AS01 in South African Adults

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).

A Phase IIa Trial of the New Tuberculosis Vaccine, MVA85A, in HIV- and/or Mycobacterium tuberculosis–infected Adults

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.

Dose-Finding Study of the Novel Tuberculosis Vaccine, MVA85A, in Healthy BCG-Vaccinated Infants

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.

Tuberculin skin test and QuantiFERON® assay in young children investigated for tuberculosis in South Africa.

SETTING Although the literature on interferon-gamma release assays on tuberculosis (TB) in children has increased, data pertaining to young children remain relatively limited. OBJECTIVE To compare results from the tuberculin skin test (TST) and the QuantiFERON®-TB Gold In-Tube assay (QFT) in children aged <3 years investigated for TB disease. DESIGN TB suspects were evaluated by medical history and examination, TST, QFT, chest radiography, induced sputum and gastric washings for smear and culture for Mycobacterium tuberculosis. RESULTS A total of 400 children were enrolled. Among 397 children with both test results, 68 (17%) were QFT-positive and 72 (18%) were TST-positive (≥10 mm). Agreement between the tests was excellent (94%, κ = 0.79, 95%CI 0.69-0.89). TB disease was diagnosed in 52/397 (13%) participants: 3 definite, 35 probable and 14 possible TB. QFT sensitivity and specificity for TB disease were respectively 38% and 81%. TST sensitivity and specificity were respectively 35% and 84%. CONCLUSION While TST and QFT had excellent concordance in this population, both tests had much lower sensitivity for TB disease than has been reported for other age groups. Our results suggested equivalent performance of QFT and TST in the diagnosis of TB disease in young children in a high-burden setting.

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry.

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.

Significantly skewed memory CD8+ T cell subsets in HIV-1 infected infants during the first year of life.

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.