Hassan Mahomed

Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial

Summary Background BCG vaccination provides incomplete protection against tuberculosis in infants. A new vaccine, modified Vaccinia Ankara virus expressing antigen 85A (MVA85A), was designed to enhance the protective efficacy of BCG. We aimed to assess safety, immunogenicity, and efficacy of MVA85A against tuberculosis and Mycobacterium tuberculosis infection in infants. Methods In our double-blind, randomised, placebo-controlled phase 2b trial, we enrolled healthy infants (aged 4–6 months) without HIV infection who had previously received BCG vaccination. We randomly allocated infants (1:1), according to an independently generated sequence with block sizes of four, to receive one intradermal dose of MVA85A or an equal volume of Candida skin test antigen as placebo at a clinical facility in a rural region near Cape Town, South Africa. We actively followed up infants every 3 months for up to 37 months. The primary study outcome was safety (incidence of adverse and serious adverse events) in all vaccinated participants, but we also assessed efficacy in a protocol-defined group of participants who received at least one dose of allocated vaccine. The primary efficacy endpoint was incident tuberculosis incorporating microbiological, radiological, and clinical criteria, and the secondary efficacy endpoint was M tuberculosis infection according to QuantiFERON TB Gold In-tube conversion (Cellestis, Australia). This trial was registered with the South African National Clinical Trials Register (DOH-27-0109-2654) and with ClinicalTrials.gov on July 31, 2009, number NCT00953927 Findings Between July 15, 2009, and May 4, 2011, we enrolled 2797 infants (1399 allocated MVA85A and 1398 allocated placebo). Median follow-up in the per-protocol population was 24·6 months (IQR 19·2–28·1), and did not differ between groups. More infants who received MVA85A than controls had at least one local adverse event (1251 [89%] of 1399 MVA85A recipients and 628 [45%] of 1396 controls who received the allocated intervention) but the numbers of infants with systemic adverse events (1120 [80%] and 1059 [76%]) or serious adverse events (257 [18%] and 258 (18%) did not differ between groups. None of the 648 serious adverse events in these 515 infants was related to MVA85A. 32 (2%) of 1399 MVA85A recipients met the primary efficacy endpoint (tuberculosis incidence of 1·15 per 100 person-years [95% CI 0·79 to 1·62]; with conversion in 178 [13%] of 1398 infants [95% CI 11·0 to 14·6]) as did 39 (3%) of 1395 controls (1·39 per 100 person-years [1·00 to 1·91]; with conversion in 171 [12%] of 1394 infants [10·6 to 14·1]). Efficacy against tuberculosis was 17·3% (95% CI −31·9 to 48·2) and against M tuberculosis infection was −3·8% (–28·1 to 15·9). Interpretation MVA85A was well tolerated and induced modest cell-mediated immune responses. Reasons for the absence of MVA85A efficacy against tuberculosis or M tuberculosis infection in infants need exploration. Funding Aeras, Wellcome Trust, and Oxford-Emergent Tuberculosis Consortium (OETC).

International study of temperature, heat and urban mortality: the ‘ISOTHURM’ project

BACKGROUND This study describes heat- and cold-related mortality in 12 urban populations in low- and middle-income countries, thereby extending knowledge of how diverse populations, in non-OECD countries, respond to temperature extremes. METHODS The cities were: Delhi, Monterrey, Mexico City, Chiang Mai, Bangkok, Salvador, São Paulo, Santiago, Cape Town, Ljubljana, Bucharest and Sofia. For each city, daily mortality was examined in relation to ambient temperature using autoregressive Poisson models (2- to 5-year series) adjusted for season, relative humidity, air pollution, day of week and public holidays. RESULTS Most cities showed a U-shaped temperature-mortality relationship, with clear evidence of increasing death rates at colder temperatures in all cities except Ljubljana, Salvador and Delhi and with increasing heat in all cities except Chiang Mai and Cape Town. Estimates of the temperature threshold below which cold-related mortality began to increase ranged from 15 degrees C to 29 degrees C; the threshold for heat-related deaths ranged from 16 degrees C to 31 degrees C. Heat thresholds were generally higher in cities with warmer climates, while cold thresholds were unrelated to climate. CONCLUSIONS Urban populations, in diverse geographic settings, experience increases in mortality due to both high and low temperatures. The effects of heat and cold vary depending on climate and non-climate factors such as the population disease profile and age structure. Although such populations will undergo some adaptation to increasing temperatures, many are likely to have substantial vulnerability to climate change. Additional research is needed to elucidate vulnerability within populations.

Distinct, Specific IL-17- and IL-22-Producing CD4+ T Cell Subsets Contribute to the Human Anti-Mycobacterial Immune Response

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-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.

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.

Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads—that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive 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-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive 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 mo 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 M. tuberculosis-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 M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.

A blood RNA signature for tuberculosis disease risk: a prospective cohort study

BACKGROUND Identification of blood biomarkers that prospectively predict progression of Mycobacterium tuberculosis infection to tuberculosis disease might lead to interventions that combat the tuberculosis epidemic. We aimed to assess whether global gene expression measured in whole blood of healthy people allowed identification of prospective signatures of risk of active tuberculosis disease. METHODS In this prospective cohort study, we followed up healthy, South African adolescents aged 12-18 years from the adolescent cohort study (ACS) who were infected with M tuberculosis for 2 years. We collected blood samples from study participants every 6 months and monitored the adolescents for progression to tuberculosis disease. A prospective signature of risk was derived from whole blood RNA sequencing data by comparing participants who developed active tuberculosis disease (progressors) with those who remained healthy (matched controls). After adaptation to multiplex quantitative real-time PCR (qRT-PCR), the signature was used to predict tuberculosis disease in untouched adolescent samples and in samples from independent cohorts of South African and Gambian adult progressors and controls. Participants of the independent cohorts were household contacts of adults with active pulmonary tuberculosis disease. FINDINGS Between July 6, 2005, and April 23, 2007, we enrolled 6363 participants from the ACS study and 4466 from independent South African and Gambian cohorts. 46 progressors and 107 matched controls were identified in the ACS cohort. A 16 gene signature of risk was identified. The signature predicted tuberculosis progression with a sensitivity of 66·1% (95% CI 63·2-68·9) and a specificity of 80·6% (79·2-82·0) in the 12 months preceding tuberculosis diagnosis. The risk signature was validated in an untouched group of adolescents (p=0·018 for RNA sequencing and p=0·0095 for qRT-PCR) and in the independent South African and Gambian cohorts (p values <0·0001 by qRT-PCR) with a sensitivity of 53·7% (42·6-64·3) and a specificity of 82·8% (76·7-86) in the 12 months preceding tuberculosis. INTERPRETATION The whole blood tuberculosis risk signature prospectively identified people at risk of developing active tuberculosis, opening the possibility for targeted intervention to prevent the disease. FUNDING Bill & Melinda Gates Foundation, the National Institutes of Health, Aeras, the European Union, and the South African Medical Research Council.Background Identification of blood biomarkers that prospectively predict progression of Mycobacterium tuberculosis infection to tuberculosis disease may lead to interventions that impact the epidemic. Methods Healthy, M. tuberculosis infected South African adolescents were followed for 2 years; blood was collected every 6 months. A prospective signature of risk was derived from whole blood RNA-Sequencing data by comparing participants who ultimately developed active tuberculosis disease (progressors) with those who remained healthy (matched controls). After adaptation to multiplex qRT-PCR, the signature was used to predict tuberculosis disease in untouched adolescent samples and in samples from independent cohorts of South African and Gambian adult progressors and controls. The latter participants were household contacts of adults with active pulmonary tuberculosis disease. Findings Of 6,363 adolescents screened, 46 progressors and 107 matched controls were identified. A 16 gene signature of risk was identified. The signature predicted tuberculosis progression with a sensitivity of 66·1% (95% confidence interval, 63·2–68·9) and a specificity of 80·6% (79·2–82·0) in the 12 months preceding tuberculosis diagnosis. The risk signature was validated in an untouched group of adolescents (p=0·018 for RNA-Seq and p=0·0095 for qRT-PCR) and in the independent South African and Gambian cohorts (p values <0·0001 by qRT-PCR) with a sensitivity of 53·7% (42·6–64·3) and a specificity of 82·8% (76·7–86) in 12 months preceding tuberculosis. Interpretation The whole blood tuberculosis risk signature prospectively identified persons at risk of developing active tuberculosis, opening the possibility for targeted intervention to prevent the disease. Funding Bill and Melinda Gates Foundation, the National Institutes of Health, Aeras, the European Union and the South African Medical Research Council (detail at end of text).

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.

The utility of an interferon gamma release assay for diagnosis of latent tuberculosis infection and disease in children: a systematic review and meta-analysis.

Background: The utility of interferon gamma release assays (IGRAs) has been assessed in adults, but remains unclear in children. We reviewed the literature on the use of a commercial IGRA in immunocompetent children for the diagnosis of both latent tuberculosis infection (LTBI) and TB disease. Methods: We searched PubMed for studies published before January 2010 on the diagnosis of TB in children using an IGRA. We compared the specificity and sensitivity of the tuberculin skin test (TST) and the IGRA for LTBI and conducted a random effects meta-analysis on sensitivity of the IGRA for TB disease. Results: Of 68 studies identified, 20 were included in this review. There was increased specificity of the IGRA for LTBI in children compared with TST, but varying sensitivities. Sensitivity of the IGRA in detecting TB disease in children also varied when compared with TST (mean &kgr; score, 0.57). For all TB cases, the pooled sensitivity was 66% (95% confidence interval [CI], 53%–78%) with heterogeneity (I2 = 74.8%). Stratification by background TB incidence highlighted a significantly reduced IGRA sensitivity of 55% (95% CI, 37%–73%) in high incidence settings when compared with low incidence settings, 70% (95% CI, 53%–84%). Conclusions: There was no clear evidence that IGRAs should replace TST for detecting LTBI in children. Sensitivity of the IGRA for TB disease was no different from TST, and a significantly reduced IGRA sensitivity was found in high-burden TB settings compared with low-burden TB settings. Further studies are needed to determine the value of IGRAs in LTBI and TB disease diagnosis in children.

T-cell activation is an immune correlate of risk in BCG vaccinated infants

Vaccines to protect against tuberculosis (TB) are urgently needed. We performed a case–control analysis to identify immune correlates of TB disease risk in Bacille Calmette–Guerin (BCG) immunized infants from the MVA85A efficacy trial. Among 53 TB case infants and 205 matched controls, the frequency of activated HLA-DR+ CD4+ T cells associates with increased TB disease risk (OR=1.828, 95% CI=1.25–2.68, P=0.002, FDR=0.04, conditional logistic regression). In an independent study of Mycobacterium tuberculosis-infected adolescents, activated HLA-DR+ CD4+ T cells also associate with increased TB disease risk (OR=1.387, 95% CI=1.068–1.801, P=0.014, conditional logistic regression). In infants, BCG-specific T cells secreting IFN-γ associate with reduced risk of TB (OR=0.502, 95% CI=0.29–0.86, P=0.013, FDR=0.14). The causes and impact of T-cell activation on disease risk should be considered when designing and testing TB vaccine candidates for these populations.

Efficacy of percutaneous versus intradermal BCG in the prevention of tuberculosis in South African infants: randomised trial

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.