Mzwandile Erasmus

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).

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).

Sequential inflammatory processes define human progression from M. tuberculosis infection to tuberculosis disease

Our understanding of mechanisms underlying progression from Mycobacterium tuberculosis infection to pulmonary tuberculosis disease in humans remains limited. To define such mechanisms, we followed M. tuberculosis-infected adolescents longitudinally. Blood samples from forty-four adolescents who ultimately developed tuberculosis disease (“progressors”) were compared with those from 106 matched controls, who remained healthy during two years of follow up. We performed longitudinal whole blood transcriptomic analyses by RNA sequencing and plasma proteome analyses using multiplexed slow off-rate modified DNA aptamers. Tuberculosis progression was associated with sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis disease diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil gene modules occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells also revealed early suppression of Th17 responses in progressors, relative to M. tuberculosis-infected controls. This was confirmed in an independent adult cohort who received BCG re-vaccination; transcript expression of interferon response genes in blood prior to BCG administration was associated with suppression of IL-17 expression by BCG-specific CD4 T cells 3 weeks post-vaccination. Our findings provide a timeline to the different immunological stages of disease progression which comprise sequential inflammatory dynamics and immune alterations that precede disease manifestations and diagnosis of tuberculosis disease. These findings have important implications for developing diagnostics, vaccination and host-directed therapies for tuberculosis. Trial registration Clincialtrials.gov, NCT01119521

Optimization and Interpretation of Serial QuantiFERON Testing to Measure Acquisition of Mycobacterium tuberculosis Infection

Rationale: Conversion from a negative to positive QuantiFERON‐TB test is indicative of Mycobacterium tuberculosis (Mtb) infection, which predisposes individuals to tuberculosis disease. Interpretation of serial tests is confounded by immunological and technical variability. Objectives: To improve the consistency of serial QuantiFERON‐TB testing algorithms and provide a data‐driven definition of conversion. Methods: Sources of QuantiFERON‐TB variability were assessed, and optimal procedures were identified. Distributions of IFN‐&ggr; response levels were analyzed in healthy adolescents, Mtb‐unexposed control subjects, and patients with pulmonary tuberculosis. Measurements and Main Results: Individuals with no known Mtb exposure had IFN‐&ggr; values less than 0.2 IU/ml. Among individuals with IFN‐&ggr; values less than 0.2 IU/ml, 0.2‐0.34 IU/ml, 0.35‐0.7 IU/ml, and greater than 0.7 IU/ml, tuberculin skin test positivity results were 15%, 53%, 66%, and 91% (P < 0.005), respectively. Together, these findings suggest that values less than 0.2 IU/ml were true negatives. In short‐term serial testing, “uncertain” conversions, with at least one value within the uncertainty zone (0.2‐0.7 IU/ml), were partly explained by technical assay variability. Individuals who had a change in QuantiFERON‐TB IFN‐&ggr; values from less than 0.2 to greater than 0.7 IU/ml had 10‐fold higher tuberculosis incidence rates than those who maintained values less than 0.2 IU/ml over 2 years (P = 0.0003). By contrast, “uncertain” converters were not at higher risk than nonconverters (P = 0.229). Eighty‐seven percent of patients with active tuberculosis had IFN‐&ggr; values greater than 0.7 IU/ml, suggesting that these values are consistent with established Mtb infection. Conclusions: Implementation of optimized procedures and a more rigorous QuantiFERON‐TB conversion definition (an increase from IFN‐&ggr; <0.2 to >0.7 IU/ml) would allow more definitive detection of recent Mtb infection and potentially improve identification of those more likely to develop disease.

H1:IC31 vaccination is safe and induces long-lived TNF-α+IL-2+CD4 T cell responses in M. tuberculosis infected and uninfected adolescents: A randomized trial

BACKGROUND Control of the tuberculosis epidemic requires a novel vaccine that is effective in preventing tuberculosis in adolescents, a key target population for vaccination against TB. METHODS Healthy adolescents, stratified by M. tuberculosis-infection status, were enrolled into this observer-blinded phase II clinical trial of the protein-subunit vaccine candidate, H1:IC31, comprising a fusion protein (H1) of Ag85B and ESAT-6, formulated with the IC31 adjuvant. Local and systemic adverse events and induced T cell responses were measured after one or two administrations of either 15μg or 50μg of the H1 protein. RESULTS Two hundred and forty participants were recruited and followed up for 224days. No notable safety events were observed regardless of H1 dose or vaccination schedule. H1:IC31 vaccination induced antigen-specific CD4 T cells, co-expressing IFN-γ, TNF-α and/or IL-2. H1:IC31 vaccination of M.tb-uninfected individuals preferentially drove the emergence of Ag85B and ESAT-6 specific TNF-α+IL-2+CD4 T cells, while H1:IC31 vaccination of M.tb-infected individuals resulted in the expansion of Ag85B-specific but not ESAT-6-specific TNF-α+IL-2+CD4 T cells. CONCLUSIONS H1:IC31 was safe and immunogenic in uninfected and M.tb-infected adolescents. Two administrations of the 15μg H1:IC31 dose induced the greatest magnitude immune response, and was considered optimal (South African National Clinical Trials Register, DoH-27-0612-3947; Pan African Clinical Trial Registry, PACTR201403000464306).

Four-Gene Pan-African Blood Signature Predicts Progression to Tuberculosis

Rationale: Contacts of patients with tuberculosis (TB) constitute an important target population for preventive measures because they are at high risk of infection with Mycobacterium tuberculosis and progression to disease. Objectives: We investigated biosignatures with predictive ability for incident TB. Methods: In a case‐control study nested within the Grand Challenges 6‐74 longitudinal HIV‐negative African cohort of exposed household contacts, we employed RNA sequencing, PCR, and the pair ratio algorithm in a training/test set approach. Overall, 79 progressors who developed TB between 3 and 24 months after diagnosis of index case and 328 matched nonprogressors who remained healthy during 24 months of follow‐up were investigated. Measurements and Main Results: A four‐transcript signature derived from samples in a South African and Gambian training set predicted progression up to two years before onset of disease in blinded test set samples from South Africa, the Gambia, and Ethiopia with little population‐associated variability, and it was also validated in an external cohort of South African adolescents with latent M. tuberculosis infection. By contrast, published diagnostic or prognostic TB signatures were predicted in samples from some but not all three countries, indicating site‐specific variability. Post hoc meta‐analysis identified a single gene pair, C1QC/TRAV27 (complement C1q C‐chain / T‐cell receptor‐&agr; variable gene 27) that would consistently predict TB progression in household contacts from multiple African sites but not in infected adolescents without known recent exposure events. Conclusions: Collectively, we developed a simple whole blood‐based PCR test to predict TB in recently exposed household contacts from diverse African populations. This test has potential for implementation in national TB contact investigation programs.

Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCG Revaccination

Background Recent Mycobacterium tuberculosis (M.tb) infection predisposes to tuberculosis disease, the leading global infectious disease killer. We tested safety andefficacy of H4:IC31® vaccination or Bacille Calmette-Guerin (BCG) revaccination for prevention of M.tb infection. Methods QuantiFERON-TB Gold In-tube (QFT) negative, HIV-uninfected, remotely BCG-vaccinated adolescents were randomized 1:1:1 to placebo, H4:IC31® or BCG revaccination (NCT02075203). Primary outcomes were safety and acquisition of M.tb infection, defined by initial QFT conversion tested 6-monthly over two years. Secondary outcomes were immunogenicity and sustained M.tb infection, defined by sustained QFT conversion without reversion three and six months post-conversion. Statistical significance for efficacy proof-of-concept was set at 1-sided p<0.10. Results 990 participants were enrolled. Both vaccines had acceptable safety profiles and were immunogenic. QFT conversion occurred in 134 and sustained conversion in 82 participants. Neither H4:IC31® nor BCG prevented initial QFT conversion, with efficacy point estimates of 9.4% (95% confidence interval: -36.2, 39.7; one-sided p=0.32) and 20.1% (-21.0, 47.2; one-sided p=0.14), respectively. However, BCG did prevent sustained QFT conversion with an efficacy of 45.4% (6.4, 68.1; one-sided p=0.013); H4:IC31® efficacy was 30.5% (-15.8, 58.3; one-sided p=0.08). QFT reversion rate from positive to negative was 46% in BCG, 40% in H4:IC31 and 25% in placebo recipients. Conclusions This first proof-of-concept, prevention of M.tb infection trial showed that sustained infection can be prevented by vaccination in a high-transmission setting and confirmed feasibility of this strategy to inform clinical development of new vaccine candidates. Evaluation of BCG revaccination to prevent tuberculosis disease in M.tb- uninfected populations is warranted.BACKGROUND Recent Mycobacterium tuberculosis infection confers a predisposition to the development of tuberculosis disease, the leading killer among global infectious diseases. H4:IC31, a candidate subunit vaccine, has shown protection against tuberculosis disease in preclinical models, and observational studies have indicated that primary bacille Calmette–Guérin (BCG) vaccination may offer partial protection against infection. METHODS In this phase 2 trial, we randomly assigned 990 adolescents in a high‐risk setting who had undergone neonatal BCG vaccination to receive the H4:IC31 vaccine, BCG revaccination, or placebo. All the participants had negative results on testing for M. tuberculosis infection on the QuantiFERON‐TB Gold In‐tube assay (QFT) and for the human immunodeficiency virus. The primary outcomes were safety and acquisition of M. tuberculosis infection, as defined by initial conversion on QFT that was performed every 6 months during a 2‐year period. Secondary outcomes were immunogenicity and sustained QFT conversion to a positive test without reversion to negative status at 3 months and 6 months after conversion. Estimates of vaccine efficacy are based on hazard ratios from Cox regression models and compare each vaccine with placebo. RESULTS Both the BCG and H4:IC31 vaccines were immunogenic. QFT conversion occurred in 44 of 308 participants (14.3%) in the H4:IC31 group and in 41 of 312 participants (13.1%) in the BCG group, as compared with 49 of 310 participants (15.8%) in the placebo group; the rate of sustained conversion was 8.1% in the H4:IC31 group and 6.7% in the BCG group, as compared with 11.6% in the placebo group. Neither the H4:IC31 vaccine nor the BCG vaccine prevented initial QFT conversion, with efficacy point estimates of 9.4% (P=0.63) and 20.1% (P=0.29), respectively. However, the BCG vaccine reduced the rate of sustained QFT conversion, with an efficacy of 45.4% (P=0.03); the efficacy of the H4:IC31 vaccine was 30.5% (P=0.16). There were no clinically significant between‐group differences in the rates of serious adverse events, although mild‐to‐moderate injection‐site reactions were more common with BCG revaccination. CONCLUSIONS In this trial, the rate of sustained QFT conversion, which may reflect sustained M. tuberculosis infection, was reduced by vaccination in a high‐transmission setting. This finding may inform clinical development of new vaccine candidates. (Funded by Aeras and others; C‐040‐404 ClinicalTrials.gov number, NCT02075203.)

The cross-species Mycobacterial Growth Inhibition Assay (MGIA) project, 2010–2014

ABSTRACT The development of a functional biomarker assay in the tuberculosis (TB) field would be widely recognized as a major advance in efforts to develop and to test novel TB vaccine candidates efficiently. We present preliminary studies using mycobacterial growth inhibition assays (MGIAs) to detect Mycobacterium bovis BCG vaccine responses across species, and we extend this work to determine whether a standardized MGIA can be applied in characterizing new TB vaccines. The comparative MGIA studies reviewed here aimed to evaluate robustness, reproducibility, and ability to reflect in vivo responses. In doing so, they have laid the foundation for the development of a MGIA that can be standardized and potentially qualified. A major challenge ahead lies in better understanding the relationships between in vivo protection, in vitro growth inhibition, and the immune mechanisms involved. The final outcome would be a MGIA that could be used with confidence in TB vaccine trials. We summarize data arising from this project, present a strategy to meet the goals of developing a functional assay for TB vaccine testing, and describe some of the challenges encountered in performing and transferring such assays.

Safety and Immunogenicity of Newborn MVA85A Vaccination and Selective, Delayed Bacille Calmette-Guerin for Infants of Human Immunodeficiency Virus-Infected Mothers: A Phase 2 Randomized, Controlled Trial

Newborn MVA85A prime vaccination was safe and induced an early immune response that did not interfere with immunogenicity of subsequent bacille Calmette-Guérin vaccination. New tuberculosis vaccine candidates should be tested using this strategy, which appears safe regardless of infant human immunodeficiency virus exposure.