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-α 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.

Application of a whole blood mycobacterial growth inhibition assay to study immunity against Mycobacterium tuberculosis in a high tuberculosis burden population.

The determinants of immunological protection against Mycobacterium tuberculosis (M.tb) infection in humans are not known. Mycobacterial growth inhibition assays have potential utility as in vitro surrogates of in vivo immunological control of M.tb. We evaluated a whole blood growth inhibition assay in a setting with high burden of TB and aimed to identify immune responses that correlate with control of mycobacterial growth. We hypothesized that individuals with underlying M.tb infection will exhibit greater M.tb growth inhibition than uninfected individuals and that children aged 4 to 12 years, an age during which TB incidence is curiously low, will also exhibit greater M.tb growth inhibition than adolescents or adults. Neither M.tb infection status, age of the study participants, nor M.tb strain was associated with differential control of mycobacterial growth. Abundance and function of innate or T cell responses were also not associated with mycobacterial growth. Our data suggest that this assay does not provide a useful measure of age-associated differential host control of M.tb infection in a high TB burden setting. We propose that universally high levels of mycobacterial sensitization (through environmental non-tuberculous mycobacteria and/or universal BCG vaccination) in persons from high TB burden settings may impart broad inhibition of mycobacterial growth, irrespective of M.tb infection status. This sensitization may mask the augmentative effects of mycobacterial sensitization on M.tb growth inhibition that is typical in low burden settings.

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.

First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected healthy adults.

BACKGROUND: H56:IC31 is a candidate tuberculosis vaccine comprising a fusion protein of Ag85B, ESAT-6 and Rv2660c, formulated in IC31 adjuvant. This first-in-human, open label phase I trial assessed the safety and immunogenicity of H56:IC31 in healthy adults without or with Mycobacterium tuberculosis (M.tb) infection. METHODS: Low dose (15 µg H56 protein in 500 nmol IC31) or high dose (50 µg H56, 500 nmol IC31) vaccine was administered intramuscularly thrice, at 56-day intervals. Antigen-specific T cell responses were measured by intracellular cytokine staining and antibody responses by ELISA. RESULTS: One hundred and twenty-six subjects were screened and 25 enrolled and vaccinated. No serious adverse events were reported. Nine subjects (36%) presented with transient cardiovascular adverse events. The H56:IC31 vaccine induced antigen-specific IgG responses and Th1 cytokine-expressing CD4(+) T cells. M.tb-infected vaccinees had higher frequencies of H56-induced CD4(+) T cells than uninfected vaccinees. Low dose vaccination induced more polyfunctional (IFN-γ(+)TNF-α(+)IL-2(+)) and higher frequencies of H56-specific CD4(+) T cells compared with high dose vaccination. A striking increase in IFN-γ-only-expressing CD4(+) T cells, displaying a CD45RA(-)CCR7(-) effector memory phenotype, emerged after the second high-dose vaccination in M.tb-infected vaccinees. TNF-α(+)IL-2(+) H56-specific memory CD4(+) T cells were detected mostly after low-dose H56 vaccination in M.tb-infected vaccinees, and predominantly expressed a CD45RA(-)CCR7(+) central memory phenotype. Our results support further clinical testing of H56:IC31.

Longitudinal changes in CD4(+) T-cell memory responses induced by BCG vaccination of newborns.

BACKGROUND: Improved vaccination strategies against tuberculosis are needed, such as approaches to boost immunity induced by the current vaccine, BCG. Design of these strategies has been hampered by a lack of knowledge of the kinetics of the human host response induced by neonatal BCG vaccination. Furthermore, the functional and phenotypic attributes of BCG-induced long-lived memory T-cell responses remain unclear. METHODS: We assessed the longitudinal CD4 T-cell response following BCG vaccination of human newborns. The kinetics, function, and phenotype of these cells were measured using flow cytometric whole-blood assays. RESULTS: We showed that the BCG-specific CD4 T-cell response peaked 6-10 weeks after vaccination and gradually waned over the first year of life. Highly activated T-helper 1 cells, predominantly expressing interferon γ, tumor necrosis factor α, and/or interleukin 2, were present at the peak response. Following contraction, BCG-specific CD4 T cells expressed high levels of Bcl-2 and displayed a predominant CD45RACCR7 central memory phenotype. However, cytokine and cytotoxic marker expression by these cells was more characteristic of effector memory cells. CONCLUSIONS: Our findings suggest that boosting of BCG-primed CD4 T cells with heterologous tuberculosis vaccines may be best after 14 weeks of age, once an established memory response has developed.

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.

Optimization of a whole blood intracellular cytokine assay for measuring innate cell responses to mycobacteria.

Innate cells are essential for host defense against invading pathogens, and the induction and direction of adaptive immune responses to infection. We developed and optimized a flow cytometric assay that allows measurement of intracellular cytokine expression by monocytes, dendritic cells (DC) and granulocytes, as well as cellular uptake of green-fluorescent protein (GFP)-expressing mycobacteria, in very small volumes of peripheral blood. We show that innate cell stimulation resulted in increased granularity of monocytes and mDC and decreased granulocyte granularity that precluded flow cytometric discernment of granulocytes from monocytes and myeloid DC by forward and side scatter gating. Anti-CD66a/c/e antibody staining allowed reliable identification and exclusion of granulocytes for subsequent delineation of monocytes and myeloid DC. Intracellular cytokine expression by granulocytes, monocytes and mDC was remarkably sensitive to the dose of mycobacterial inoculum. Moreover, activation of monocytes and mDC with live BCG reduced expression levels of CD14 and CD11c, respectively, necessitating optimization of staining conditions to reliably measure these lineage markers. Finally, we characterized expression of IL-12/23p40, TNF-α, IL-6, and IL-10, by GFP(+) and GFP(-) monocytes and mDC from 25 healthy adults. This assay may be applied to the study of innate cell responses to any GFP-expressing pathogen, and can be performed on blood volumes as low as 200 µL per condition, making the assay particularly suitable for pediatric studies.

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.

HIV-specific gag responses in early infancy correlate with clinical outcome and inversely with viral load.

Many HIV-infected infants progress to AIDS during the first year of life when antiretroviral therapy (ART) is not given. The immune determinants of progression to AIDS are not known. We hypothesized that distinct HIV-specific T cell responses correlate with viral load and survival over the first year of life. Whole blood of infants at 3, 6, 9, and 12 months of age was incubated with HIV antigens Gag and Env. The frequency of specific T cells producing interferon (IFN)-γ was then measured by flow cytometry. Viral load and CD4% in HIV(+) infants were determined at each time point. ART was not available for this population at the time of sample collection. Those infants who survived to 12 months of age (n=12) had lower viral loads and higher Gag-specific CD8(+) T cell responses at 3 months, compared with infants who died (n=8). Furthermore, the frequency of Gag-specific CD4(+) T cells correlated inversely with viral load at 3 and 6 months of age. Together these data indicate that the early presence of quantitatively higher Gag-specific T cell responses in HIV-infected infants is associated with lower viral loads and decreased mortality in the first year of life. Our data support the design of a vaccine that preferentially elicits Gag responses, which may result in lower levels of viremia and possibly improve outcome.

Higher human CD4 T cell response to novel Mycobacterium tuberculosis latency associated antigens Rv2660 and Rv2659 in latent infection compared with tuberculosis disease.

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.

The effect of bacille Calmette-Guérin vaccine strain and route of administration on induced immune responses in vaccinated infants.

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.