Vaccination against tuberculosis (TB) should provide long-term protective immunity against Mycobacterium tuberculosis (M.tb). The current TB vaccine, Bacille Calmette-Guerin (BCG), protects against disseminated childhood TB, but protection against lung TB in adolescents and adults is variable and mostly poor. One potential reason for the limited durability of protection may be waning of immunity through gradual attrition of BCG-induced T cells. We determined if a MVA85A viral-vector boost could enhance the durability of mycobacteria-specific T cell responses above those induced by BCG alone.
We describe a long-term follow-up study of persons previously vaccinated with MVA85A. We performed a medical history and clinical examination, a tuberculin skin test and measured vaccine-specific T cell responses in persons previously enrolled as adults, adolescents, children or infants into three different Phase II trials, between 2005 and 2011.
Of 252 potential participants, 183 (72.6%) consented and completed the study visit. Vaccine-induced Ag85A-specific CD4+ T cell responses were remarkably persistent in healthy, HIV-uninfected adults, adolescents, children and infants, up to 6 years after MVA85A vaccination. Specific CD4+ T cells expressed surface markers consistent with either CD45RA−CCR7+ central memory or CD45RA−CCR7− effector memory T cells. Similarly durable Ag85A-specific CD4+ T cell responses were detected in HIV-infected persons who were on successful antiretroviral therapy when MVA85A was administered. By contrast, Ag85A-specific CD4+ T cell frequencies in untreated MVA85A-vaccinated HIV-infected persons were mostly undetectable 3–5 years after vaccination.
MVA85A induces remarkably durable T cell responses in immunocompetent persons. However, results from a recent phase IIb trial of MVA85A, conducted in infants from the same geographic area and study population, showed no vaccine efficacy, suggesting that these durable T cell responses do not enhance BCG-induced protection against TB in infants.
Tuberculosis (TB) is a major public health problem globally. Little is known about TB incidence in adolescents who are a proposed target group for new TB vaccines. We conducted a study to determine the TB incidence rates and risk factors for TB disease in a cohort of school-going adolescents in a high TB burden area in South Africa.
We recruited adolescents aged 12 to 18 years from high schools in Worcester, South Africa. Demographic and clinical information was collected, a tuberculin skin test (TST) performed and blood drawn for a QuantiFERON TB Gold assay at baseline. Screening for TB cases occurred at follow up visits and by surveillance of registers at public sector TB clinics over a period of up to 3.8 years after enrolment.
A total of 6,363 adolescents were enrolled (58% of the school population targeted). During follow up, 67 cases of bacteriologically confirmed TB were detected giving an overall incidence rate of 0.45 per 100 person years (95% confidence interval 0.29–0.72). Black or mixed race, maternal education of primary school or less or unknown, a positive baseline QuantiFERON assay and a positive baseline TST were significant predictors of TB disease on adjusted analysis.
The adolescent TB incidence found in a high burden setting will help TB vaccine developers plan clinical trials in this population. Latent TB infection and low socio-economic status were predictors of TB disease.
New tuberculosis (TB) vaccines are being developed to combat the global epidemic. A phase IIb trial of a candidate vaccine, MVA85A, was conducted in a high burden setting in South Africa to evaluate proof-of-concept efficacy for prevention of TB in infants.
To describe the study design and implementation lessons from an infant TB vaccine efficacy trial.
This was a randomised, controlled, double-blind clinical trial comparing the safety and efficacy of MVA85A to Candin control administered to 4–6-month-old, BCG-vaccinated, HIV-negative infants at a rural site in South Africa. Infants were followed up for 15–39 months for incident TB disease based on pre-specified endpoints.
2797 infants were enrolled over 22 months. Factors adversely affecting recruitment and the solutions that were implemented are discussed. Slow case accrual led to six months extension of trial follow up.
The clinical, regulatory and research environment for modern efficacy trials of new TB vaccines are substantially different to that when BCG vaccine was first evaluated in infants. Future infant TB vaccine trials will need to allocate sufficient resources and optimise operational efficiency. A stringent TB case definition is necessary to maximize specificity, and TB case accrual must be monitored closely.
BCG; Vaccine; Tuberculosis; Lessons learnt; Implementation
Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens. The influence of human TLR6 polymorphisms on susceptibility to infection is only partially understood. Most microbes contain lipopeptides recognized by TLR2/1 or TLR2/6 heterodimers. Our aim was to determine whether single nucleotide polymorphisms (SNPs) in TLR6 are associated with altered immune responses to lipopeptides and whole mycobacteria.
We sequenced the TLR6 coding region in 100 healthy South African adults to assess genetic variation and determined associations between polymorphisms and lipopeptide- and mycobacteria-induced IL-6 production in whole blood. We found 2 polymorphisms, C745T and G1083C that were associated with altered IL-6 secretion. G1083C was associated with altered IL-6 levels in response to lipopeptides, Mycobacterium tuberculosis lysate (Mtb, P = 0.018) and BCG (P = 0.039). The 745T allele was also associated with lower NF-κB signaling in response to di-acylated lipopeptide, PAM2 (P = 0.019) or Mtb (P = 0.026) in a HEK293 cell line reconstitution assay, compared with the 745C allele.
We conclude that TLR6 polymorphisms may be associated with altered lipopeptide-induced cytokine responses and recognition of Mtb. These studies provide new insight into the role of TLR6 variation and the innate immune response to human infection.
Toll-like receptor 6; polymorphism; interleukin 6; tuberculosis; immune response
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 × 107 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.
tuberculosis; HIV-1; vaccine; MVA85A; clinical trial
One third of the world’s population is infected with Mycobacterium tuberculosis (M.tb). A vaccine that would prevent progression to TB disease will have a dramatic impact on the global TB burden. We propose that antigens of M.tb that are preferentially expressed during latent infection will be excellent candidates for post-exposure vaccination. We therefore assessed human T cell recognition of two such antigens, Rv2660 and Rv2659. Expression of these was shown to be associated with non-replicating persistence in vitro. After six days incubation of PBMC from persons with latent tuberculosis infection (LTBI) and tuberculosis (TB) disease, Rv2660 and Rv2659 induced IFN-γ production in a greater proportion of persons with LTBI, compared with TB diseased patients. Persons with LTBI also had increased numbers of viable T cells, and greater specific CD4+ T cell proliferation and cytokine expression capacity. Persons with LTBI preferentially recognize Rv2659 and Rv2660, compared with patients with TB disease. These results suggest promise of these antigens for incorporation into post-exposure TB vaccines.
Mycobacterium tuberculosis; LTBI; TB disease; latency antigens; post-infection vaccine
High antigen load in chronic viral infections has been associated with impairment of antigen-specific T cell responses; however, the relationship between antigen load in chronic Mycobacterium tuberculosis (Mtb) infection and functional capacity of Mtb-specific T cells in humans is not clear. We compared Mtb-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads, i.e., persons with latent Mtb infection (LTBI), with smear − pulmonary tuberculosis (TB), and with smear+ TB. Patients with smear+ TB had decreased polyfunctional IFN-γ+IL-2+TNF-α+ and IL-2-producing specific CD4 T cells and increased TNF-α-single positive cells, when compared with smear − TB and LTBI. TB patients also had increased frequencies of Mtb-specific CD8 T cells, compared with LTBI. Mtb-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear+ TB, and correlated positively with ex vivo IFN-γ+IL-2+TNF-α+ CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 months of anti-TB treatment, specific IFN-γ+IL-2+TNF-α+ CD4 and CD8 T cells increased, whereas TNF-α- and IFN-γ-single positive T cells decreased. These results suggest progressive impairment of Mtb-specific T cell responses with increasing mycobacterial load, and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of Mtb-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. Taken together, these data have potentially significant applications for diagnosis of TB and for identification of T cell correlates of TB disease progression.
Effective provider-parent communication can improve childhood vaccination uptake and strengthen immunisation services in low- and middle-income countries (LMICs). Building capacity to improve communication strategies has been neglected. Rigorous research exists but is not readily found or applicable to LMICs, making it difficult for policy makers to use it to inform vaccination policies and practice.
The aim of this project is to build research knowledge and capacity to use evidence-based strategies for improving communication about childhood vaccinations with parents and communities in LMICs.
Methods and design
This project is a mixed methods study with six sub-studies. In sub-study one, we will develop a systematic map of provider-parent communication interventions for childhood vaccinations by screening and extracting data from relevant literature. This map will inform sub-study two, in which we will develop a taxonomy of interventions to improve provider-parent communication around childhood vaccination. In sub-study three, the taxonomy will be populated with trial citations to create an evidence map, which will also identify how evidence is linked to communication barriers regarding vaccination.
In the project's fourth sub-study, we will present the interventions map, taxonomy, and evidence map to international stakeholders to identify high-priority topics for systematic reviews of interventions to improve parent-provider communication for childhood vaccination. We will produce systematic reviews of the effects of high-priority interventions in the fifth sub-study. In the sixth and final sub-study of the project, evidence from the systematic reviews will be translated into accessible formats and messages for dissemination to LMICs.
This project combines evidence mapping, conceptual and taxonomy development, priority setting, systematic reviews, and knowledge transfer. It will build and share concepts, terms, evidence, and resources to aid the development of communication strategies for effective vaccination programmes in LMICs.
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.
mycobacteria immunity; pediatric settings
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-γ, tumor necrosis factor-α, and IL-2 (“polyfunctional” cells). AERAS-402 also induced a potent CD8+ T-cell response, characterized by cells expressing IFN-γ and/or tumor necrosis factor-α, 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).
tuberculosis; vaccine; immunity; CD4; CD8
This study was conducted in a high tuberculosis (TB) burden area in Worcester, South Africa, with a notified all TB incidence rate of 1,400/100,000.
To compare the predictive value of a baseline tuberculin skin test (TST) with that of the QuantiFERON TB Gold (In-tube) assay (QFT) for subsequent microbiologically confirmed TB disease among adolescents.
Adolescents aged 12–18 years were recruited from high schools in the study area. At baseline, blood was drawn for QFT and a TST administered. Participants were followed up for up to 3.8 years for incident TB disease (median 2.4 years).
After exclusions, 5244 (82.4%) of 6,363 adolescents enrolled, were analysed. The TB incidence rate was 0.60 cases per 100 person years (pyrs) (95% CI 0.43–0.82) for baseline TST positive (≥5 mm) participants and 0.64 cases per 100 pyrs (95% CI 0.45–0.87) for baseline QFT positive participants. TB incidence rates were 0.22 per 100 pyrs (0.11–0.39) and 0.22 per 100 pyrs (0.12–0.38) among those with a negative baseline TST and QFT respectively. Sensitivity for incident TB disease was 76.9% for TST and 75.0% for QFT (p = 0.81). Positive predictive value was 1.4% for TST and 1.5% for QFT.
Positive TST and QFT tests were moderately sensitive predictors of progression to microbiologically confirmed TB disease. There was no significant difference in the predictive ability of these tests for TB disease amongst adolescents in this high burden setting. Therefore, these findings do not support use of QFT in preference to TST to predict the risk of TB disease in this study population.
MVA85A is a new tuberculosis vaccine aimed at enhancing immunity induced by BCG. We investigated the safety and immunogenicity of MVA85A in healthy adolescents and children from a tuberculosis 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. Antigen-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 tuberculosis. This includes induction of novel Th1 cell populations which have not been previously described in humans.
MVA85A; tuberculosis; vaccine; polyfunctional; IL-17
The efficacy of BCG may be enhanced by heterologous vaccination strategies that boost the BCG-primed immune response. One leading booster vaccine, MVA85A, has shown promising safety and immunogenicity in UK human trials. We investigated the safety and immunogenicity of MVA85A in mycobacteria-exposed, but Mycobacterium tuberculosis-uninfected, healthy adults from a TB-endemic region of South Africa.
Twenty-four adults were vaccinated with MVA85A. All subjects were followed up for one year for adverse events and for immunological assessment.
MVA85A vaccination was well tolerated and induced potent T cell responses, measured by IFN-γ ELISPOT assay, which exceeded pre-vaccination levels up to 364 days after vaccination. BCG-specific CD4+ T cells boosted by MVA85A comprised of multiple populations expressing combinations of IFN-γ, TNF-α, IL-2 and IL-17, as measured by polychromatic flow cytometry. IFN-γ expressing and polyfunctional IFN-γ+TNF-α+IL-2+ CD4+ T cells were boosted during the peak BCG-specific response 7 days post-vaccination.
The excellent safety profile and quantitative and qualitative immunogenicity data strongly support further trials to assess the efficacy of MVA85A as a boosting vaccine in TB endemic countries.
Vaccination; tuberculosis; T cells; MVA85A; South Africa
Tuberculin skin tests (TSTs) are long-established screening methods for tuberculosis (TB). We aimed to compare agreement between the intradermal Mantoux and multipuncture percutaneous Tine methods and to quantify risk factors for a positive test result.
1512 South African children younger than 5 years of age who were investigated for tuberculosis (TB) during a Bacille Calmette Guerin (BCG) trial were included in this analysis. Children underwent both Mantoux and Tine tests. A positive test was defined as Mantoux ≥15 mm or Tine ≥ Grade 3 for the binary comparison. Agreement was evaluated using kappa (binary) and weighted kappa (hierarchical). Multivariate regression models identified independent risk factors for TST positivity. The Mantoux test was positive in 430 children (28.4%) and the Tine test in 496 children (32.8%, p<0.0001), with observed binary agreement 87.3% (kappa 0.70) and hierarchical agreement 85.0% (weighted kappa 0.66). Among 173 children culture-positive for Mycobacterium tuberculosis, Mantoux was positive in 49.1% and Tine in 54.9%, p<0.0001 (kappa 0.70). Evidence of digit preference was noted for Mantoux readings at 5 mm threshold intervals. After adjustment for confounders, a positive culture, suggestive chest radiograph, and proximity of TB contact were risk factors for a positive test using both TST methods. There were no independent associations between ethnicity, gender, age, or over-crowding, and TST result.
The Tine test demonstrated a higher positive test rate than the Mantoux, with substantial agreement between TST methods among young BCG-vaccinated children. TB disease and exposure factors, but not demographic variables, were independent risk factors for a positive result using either test method. These findings suggest that the Tine might be a useful screening tool for childhood TB in resource-limited countries.
Objective To compare the incidence of tuberculosis over two years in infants vaccinated at birth with intradermal BCG or with percutaneous BCG.
Design Randomised trial.
Setting South Africa.
Participants 11 680 newborn infants.
Interventions Infants were randomised by week of birth to receive Tokyo 172 BCG vaccine through the percutaneous route (n=5775) or intradermal route (n=5905) within 24 hours of birth and followed up for two years.
Main outcome measures The primary outcome measure was documented Mycobacterium tuberculosis infection or radiological and clinical evidence of tuberculosis disease. Secondary outcome measures were rates of adverse events, all cause and tuberculosis specific admissions to hospital, and mortality.
Results The difference in the cumulative incidence of definite, probable, and possible tuberculosis between the intradermal group and the percutaneous group, as defined using study definitions based on microbiological, radiological, and clinical findings was −0.36% (95.5% confidence interval −1.27% to 0.54%). No significant differences were found between the routes in the cumulative incidence of tuberculosis using a range of equivalence of “within 25%.” Additionally, no significant differences were found between the routes in the cumulative incidence of adverse events (risk ratio 0.98, 95% confidence interval 0.91 to 1.06), including deaths (1.19, 0.89 to 1.58).
Conclusion Equivalence was found between intradermal BCG vaccine and percutaneous BCG in the incidence of tuberculosis in South African infants vaccinated at birth and followed up for two years. The World Health Organization should consider revising its policy of preferential intradermal vaccination to allow national immunisation programmes to choose percutaneous vaccination if that is more practical.
Trial registration ClinicalTrials.gov NCT00242047.
We investigated whether the pro-inflammatory T cell cytokines interleukin-17 (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, while bronchoalveolar lavage fluid (BALF) 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-γ 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.
human; T cells; cytokines; bacterial infection
To evaluate the frequency and clinical significance of non-tuberculous mycobacteria (NTM) isolates among children investigated for pulmonary tuberculosis in a rural South African community.
Children were investigated for pulmonary tuberculosis as part of a tuberculosis vaccine surveillance program (2001–2005). The clinical features of children in whom NTM were isolated, from induced sputum or gastric lavage, were compared to those with culture-proven M. tuberculosis.
Mycobacterial culture demonstrated 114 NTM isolates from 109 of the 1,732 children investigated, a crude yield of 6% (95% CI 5–7). The comparative yield of positive NTM cultures from gastric lavage was 40% (95% CI 31–50), compared to 67% (95% CI 58–76) from induced sputum. 95% of children with NTM isolates were symptomatic. Two children were HIV-infected. By contrast, M. tuberculosis was isolated in 187 children, a crude yield of 11% (95% CI 9–12). Compared to those with culture-proven M. tuberculosis, children with NTM isolates were less likely to demonstrate acid-fast bacilli on direct smear microscopy (OR 0.19; 95% 0.0–0.76). Children with NTM were older (p<0.0001), and more likely to demonstrate constitutional symptoms (p = 0.001), including fever (p = 0.003) and loss of weight or failure to gain weight (p = 0.04), but less likely to demonstrate a strongly positive tuberculin skin test (p<0.0001) or radiological features consistent with pulmonary tuberculosis (p = 0.04).
NTM were isolated in 6% of all children investigated for pulmonary tuberculosis and in more than one third of those with a positive mycobacterial culture. NTM may complicate the diagnosis of PTB in regions that lack capacity for mycobacterial species identification. The association of NTM isolates with constitutional symptoms suggestive of host recognition requires further investigation.