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

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 .).

MR1-Independent Activation of Human Mucosal-Associated Invariant T Cells by Mycobacteria.

Tuberculosis (TB) is the leading cause of mortality from a single infectious agent, Mycobacterium tuberculosis Relevant immune targets of the partially efficacious TB vaccine bacille Calmette-Guérin (BCG) remain poorly defined. Mucosal-associated invariant T (MAIT) cells are MHC-related protein 1 (MR1)-restricted T cells, which are reactive against M. tuberculosis, and underexplored as potential TB vaccine targets. We sought to determine whether BCG vaccination activated mycobacteria-specific MAIT cell responses in humans. We analyzed whole blood samples from M. tuberculosis-infected South African adults who were revaccinated with BCG after a six-month course of isoniazid preventative therapy. In vitro BCG stimulation potently induced IFN-γ expression by phenotypic (CD8+CD26+CD161+) MAIT cells, which constituted the majority (75%) of BCG-reactive IFN-γ-producing CD8+ T cells. BCG revaccination transiently expanded peripheral blood frequencies of BCG-reactive IFN-γ+ MAIT cells, which returned to baseline frequencies a year following vaccination. In another cohort of healthy adults who received BCG at birth, 53% of mycobacteria-reactive-activated CD8 T cells expressed CDR3α TCRs, previously reported as MAIT TCRs, expressing the canonical TRAV1-2-TRAJ33 MAIT TCRα rearrangement. CD26 and CD161 coexpression correlated with TRAV1-2+CD161+ phenotype more accurately in CD8+ than CD4-CD8- MAIT cells. Interestingly, BCG-induced IFN-γ expression by MAIT cells in vitro was mediated by the innate cytokines IL-12 and IL-18 more than MR1-induced TCR signaling, suggesting TCR-independent activation. Collectively, the data suggest that activation of blood MAIT cells by innate inflammatory cytokines is a major mechanism of responsiveness to vaccination with whole cell vaccines against TB or in vitro stimulation with mycobacteria (Clinical trial registration: NCT01119521).

Dose Optimization of H56:IC31 Vaccine for Tuberculosis-Endemic Populations. A Double-Blind, Placebo-controlled, Dose-Selection Trial.

RATIONALE: Global tuberculosis (TB) control requires effective vaccines in TB-endemic countries, where most adults are infected with Mycobacterium tuberculosis (M.tb). OBJECTIVES: We sought to define optimal dose and schedule of H56:IC31, an experimental TB vaccine comprising Ag85B, ESAT-6, and Rv2660c, for M.tb-infected and M.tb-uninfected adults. METHODS: We enrolled 98 healthy, HIV-uninfected, bacillus Calmette-Guérin-vaccinated, South African adults. M.tb infection was defined by QuantiFERON-TB (QFT) assay. QFT-negative participants received two vaccinations of different concentrations of H56 in 500 nmol of IC31 to enable dose selection for further vaccine development. Subsequently, QFT-positive and QFT-negative participants were randomized to receive two or three vaccinations to compare potential schedules. Participants were followed for safety and immunogenicity for 292 days. MEASUREMENTS AND MAIN RESULTS: H56:IC31 showed acceptable reactogenicity profiles irrespective of dose, number of vaccinations, or M.tb infection. No vaccine-related severe or serious adverse events were observed. The three H56 concentrations tested induced equivalent frequencies and functional profiles of antigen-specific CD4 T cells. ESAT-6 was only immunogenic in QFT-negative participants who received three vaccinations. CONCLUSIONS: Two or three H56:IC31 vaccinations at the lowest dose induced durable antigen-specific CD4 T-cell responses with acceptable safety and tolerability profiles in M.tb-infected and M.tb-uninfected adults. Additional studies should validate applicability of vaccine doses and regimens to both QFT-positive and QFT-negative individuals. Clinical trial registered with www.clinicaltrials.gov (NCT01865487).

Diagnostic Accuracy of Early Secretory Antigenic Target-6-Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube.

BACKGROUND: Early secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) antigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6-free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6-containing vaccines. We aimed to qualify a recently developed ESAT-6-free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT). METHODS: Participants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6-free IGRA cutoff, test assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen-coated blood collection tubes. RESULTS: ESAT-6-free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. The ESAT-6-free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6-free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74-0.90; McNemar test P = .48). ESAT-6-free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability. CONCLUSIONS: The novel ESAT-6-free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection.

Bacillus Calmette-Guérin (BCG) Revaccination of Adults with Latent Mycobacterium tuberculosis Infection Induces Long-Lived BCG-Reactive NK Cell Responses.

One third of the global population is estimated to be latently infected with Mycobacterium tuberculosis We performed a phase I randomized controlled trial of isoniazid preventive therapy (IPT) before revaccination with bacillus Calmette-Guérin (BCG) in healthy, tuberculin skin test-positive (≥15-mm induration), HIV-negative South African adults. We hypothesized that preclearance of latent bacilli with IPT modulates BCG immunogenicity following revaccination. Frequencies and coexpression of IFN-γ, TNF-α, IL-2, IL-17, and/or IL-22 in CD4 T cells and IFN-γ-expressing CD8 T, γδ T, CD3(+)CD56(+) NKT-like, and NK cells in response to BCG were measured using whole blood intracellular cytokine staining and flow cytometry. We analyzed 72 participants who were revaccinated with BCG after IPT (n = 33) or without prior IPT (n = 39). IPT had little effect on frequencies or cytokine coexpression patterns of M. tuberculosis- or BCG-specific responses. Revaccination transiently boosted BCG-specific Th1 cytokine-expressing CD4, CD8, and γδ T cells. Despite high frequencies of IFN-γ-expressing BCG-reactive CD3(+)CD56(+) NKT-like cells and CD3(-)CD56(dim) and CD3(-)CD56(hi) NK cells at baseline, BCG revaccination boosted these responses, which remained elevated up to 1 y after revaccination. Such BCG-reactive memory NK cells were induced by BCG vaccination in infants, whereas in vitro IFN-γ expression by NK cells upon BCG stimulation was dependent on IL-12 and IL-18. Our data suggest that isoniazid preclearance of M. tuberculosis bacilli has little effect on the magnitude, persistence, or functional attributes of lymphocyte responses boosted by BCG revaccination. Our study highlights the surprising durability of BCG-boosted memory NKT-like and NK cells expressing antimycobacterial effector molecules, which may be novel targets for tuberculosis vaccines.

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-γ 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-γ values less than 0.2 IU/ml. Among individuals with IFN-γ 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-γ 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-γ 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-γ <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.

High-dose rifapentine with moxifloxacin for pulmonary tuberculosis.

BACKGROUND: Tuberculosis regimens that are shorter and simpler than the current 6-month daily regimen are needed. METHODS: We randomly assigned patients with newly diagnosed, smear-positive, drug-sensitive tuberculosis to one of three regimens: a control regimen that included 2 months of ethambutol, isoniazid, rifampicin, and pyrazinamide administered daily followed by 4 months of daily isoniazid and rifampicin; a 4-month regimen in which the isoniazid in the control regimen was replaced by moxifloxacin administered daily for 2 months followed by moxifloxacin and 900 mg of rifapentine administered twice weekly for 2 months; or a 6-month regimen in which isoniazid was replaced by daily moxifloxacin for 2 months followed by one weekly dose of both moxifloxacin and 1200 mg of rifapentine for 4 months. Sputum specimens were examined on microscopy and after culture at regular intervals. The primary end point was a composite treatment failure and relapse, with noninferiority based on a margin of 6 percentage points and 90% confidence intervals. RESULTS: We enrolled a total of 827 patients from South Africa, Zimbabwe, Botswana, and Zambia; 28% of patients were coinfected with the human immunodefiency virus. In the per-protocol analysis, the proportion of patients with an unfavorable response was 4.9% in the control group, 3.2% in the 6-month group (adjusted difference from control, -1.8 percentage points; 90% confidence interval [CI], -6.1 to 2.4), and 18.2% in the 4-month group (adjusted difference from control, 13.6 percentage points; 90% CI, 8.1 to 19.1). In the modified intention-to-treat analysis these proportions were 14.4% in the control group, 13.7% in the 6-month group (adjusted difference from control, 0.4 percentage points; 90% CI, -4.7 to 5.6), and 26.9% in the 4-month group (adjusted difference from control, 13.1 percentage points; 90% CI, 6.8 to 19.4). CONCLUSIONS: The 6-month regimen that included weekly administration of high-dose rifapentine and moxifloxacin was as effective as the control regimen. The 4-month regimen was not noninferior to the control regimen. (Funded by the European and Developing Countries Clinical Trials Partnership and the Wellcome Trust; RIFAQUIN Current Controlled Trials number, ISRCTN44153044.).

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).

Randomized trial of type 1 and type 3 oral monovalent poliovirus vaccines in newborns in Africa.

BACKGROUND: The Global Polio Eradication Initiative aims to eradicate wild poliovirus by the end of 2012. Therefore, more-immunogenic polio vaccines, including monovalent oral poliovirus vaccines (mOPVs), are needed for supplemental immunization activities. This trial assessed the immunogenicity of monovalent types 1 and 3, compared with that of trivalent oral poliovirus vaccine (tOPV), in South Africa. METHODS: We conducted a blinded, randomized, 4-arm controlled trial comparing the immunogenicity of a single dose of mOPV1 (from 2 manufacturers) and mOPV3 (from 1 manufacturer), given at birth, with the immunogenicity of tOPV. RESULTS: Eight hundred newborns were enrolled; 762 (95%) were included in the analysis. At 30 days after vaccine administration, seroconversion to poliovirus type 1 was 73.4% and 76.4% in the 2 mOPV1 arms, compared with 39.1% in the tOPV arm (P < .0000001), and seroconversion to poliovirus type 3 was 58.0% in the mOPV3 arm, compared with 21.2% in the tOPV arm (P < .0000001). The vaccines were well tolerated, and no adverse events were attributed to trial interventions. CONCLUSION: A dose of mOPV1 or mOPV3 at birth was superior to that of tOPV in inducing type-specific seroconversion in this sub-Saharan African population. Our results support continued use of mOPVs in supplemental immunization activities in countries where poliovirus types 1 or 3 circulate. Clinical Trials Registration. ISRCTN18107202.

Moxifloxacin population pharmacokinetics in patients with pulmonary tuberculosis and the effect of intermittent high-dose rifapentine.

We described the population pharmacokinetics of moxifloxacin and the effect of high-dose intermittent rifapentine in patients with pulmonary tuberculosis who were randomized to a continuation-phase regimen of 400 mg moxifloxacin and 900 mg rifapentine twice weekly or 400 mg moxifloxacin and 1,200 mg rifapentine once weekly. A two-compartment model with transit absorption best described moxifloxacin pharmacokinetics. Although rifapentine increased the clearance of moxifloxacin by 8% during antituberculosis treatment compared to that after treatment completion without rifapentine, it did not result in a clinically significant change in moxifloxacin exposure.

Structured approaches for the screening and diagnosis of childhood tuberculosis in a high prevalence region of South Africa.

OBJECTIVE: To measure agreement between nine structured approaches for diagnosing childhood tuberculosis; to quantify differences in the number of tuberculosis cases diagnosed with the different approaches, and to determine the distribution of cases in different categories of diagnostic certainty. METHODS: We investigated 1445 children aged < 2 years during a vaccine trial (2001-2006) in a rural South African community. Clinical, radiological and microbiological data were collected prospectively. Tuberculosis case status was determined using each of the nine diagnostic approaches. We calculated differences in case frequency and categorical agreement for binary (tuberculosis/not tuberculosis) outcomes using McNemar's test (with 95% confidence intervals, CIs) and Cohen's kappa coefficient (Kappa). FINDINGS: Tuberculosis case frequency ranged from 6.9% to 89.2% (median: 41.7). Significant differences in case frequency (P < 0.05) occurred in 34 of the 36 pair-wise comparisons between structured diagnostic approaches (range of absolute differences: 1.5-82.3%). Kappa ranged from 0.02 to 0.71 (median: 0.18). The two systems that yielded the highest case frequencies (89.2% and 70.0%) showed fair agreement (Kappa: 0.33); the two that yielded the lowest case frequencies (6.9% and 10.0%) showed slight agreement (Kappa: 0.18). CONCLUSION: There is only slight agreement between structured approaches for the screening and diagnosis of childhood tuberculosis and high variability between them in terms of case yield. Diagnostic systems that yield similarly low case frequencies may be identifying different subpopulations of children. The study findings do not support the routine clinical use of structured approaches for the definitive diagnosis of childhood tuberculosis, although high-yielding systems may be useful screening tools.

Multidimensional analyses reveal modulation of adaptive and innate immune subsets by tuberculosis vaccines.

We characterize the breadth, function and phenotype of innate and adaptive cellular responses in a prevention of Mycobacterium tuberculosis infection trial. Responses are measured by whole blood intracellular cytokine staining at baseline and 70 days after vaccination with H4:IC31 (subunit vaccine containing Ag85B and TB10.4), Bacille Calmette-Guerin (BCG, a live attenuated vaccine) or placebo (n = ~30 per group). H4:IC31 vaccination induces Ag85B and TB10.4-specific CD4 T cells, and an unexpected NKTlike subset, that expresses IFN-γ, TNF and/or IL-2. BCG revaccination increases frequencies of CD4 T cell subsets that either express Th1 cytokines or IL-22, and modestly increases IFNγ-producing NK cells. In vitro BCG re-stimulation also triggers responses by donor-unrestricted T cells, which may contribute to host responses against mycobacteria. BCG, which demonstrated efficacy against sustained Mycobacterium tuberculosis infection, modulates multiple immune cell subsets, in particular conventional Th1 and Th22 cells, which should be investigated in discovery studies of correlates of protection.

Live-attenuated Mycobacterium tuberculosis vaccine MTBVAC versus BCG in adults and neonates: a randomised controlled, double-blind dose-escalation trial.

BACKGROUND: Infants are a key target population for new tuberculosis vaccines. We assessed the safety and immunogenicity of the live-attenuated Mycobacterium tuberculosis vaccine candidate MTBVAC in adults and infants in a region where transmission of tuberculosis is very high. METHODS: We did a randomised, double-blind, BCG-controlled, dose-escalation trial at the South African Tuberculosis Vaccine Initiative site near Cape Town, South Africa. Healthy adult community volunteers who were aged 18-50 years, had received BCG vaccination as infants, were HIV negative, had negative interferon-γ release assay (IGRA) results, and had no personal history of tuberculosis or current household contact with someone with tuberculosis were enrolled in a safety cohort. Infants born to HIV-negative women with no personal history of tuberculosis or current household contact with a person with tuberculosis and who were 96 h old or younger, generally healthy, and had not yet received routine BCG vaccination were enrolled in a separate infant cohort. Eligible adults were randomly assigned (1:1) to receive either BCG Vaccine SSI (5 × 105 colony forming units [CFU] of Danish strain 1331 in 0·1 mL diluent) or MTBVAC (5 × 105 CFU in 0·1 mL) intradermally in the deltoid region of the arm. After favourable review of 28-day reactogenicity and safety data in the adult cohort, infants were randomly assigned (1:3) to receive either BCG Vaccine SSI (2·5 × 105 CFU in 0·05 mL diluent) or MTBVAC in three sequential cohorts of increasing MTBVAC dose (2·5 × 103 CFU, 2·5 × 104 CFU, and 2·5 × 105 CFU in 0·05 mL) intradermally in the deltoid region of the arm. QuantiFERON-TB Gold In-Tube IGRA was done on days 180 and 360. For both randomisations, a pre-prepared block randomisation schedule was used. Participants (and their parents or guardians in the case of infant participants), investigators, and other clinical and laboratory staff were masked to intervention allocation. The primary outcomes, which were all measured in the infant cohort, were solicited and unsolicited local adverse events and serious adverse events until day 360; non-serious systemic adverse events until day 28 and vaccine-specific CD4 and CD8 T-cell responses on days 7, 28, 70, 180, and 360. Secondary outcomes measured in adults were local injection-site and systemic reactions and haematology and biochemistry at study day 7 and 28. Safety analyses and immunogenicity analyses were done in all participants who received a dose of vaccine. This trial is registered with ClinicalTrials.gov, number NCT02729571. FINDINGS: Between Sept 29, 2015, and Nov 16, 2015, 62 adults were screened and 18 were enrolled and randomly assigned, nine each to the BCG and MTBVAC groups. Between Feb 12, 2016, and Sept 21, 2016, 36 infants were randomly assigned-eight to the BCG group, nine to the 2·5 × 103 CFU MTBVAC group, nine to the 2·5 × 104 CFU group, and ten to the 2·5 × 105 CFU group. Mild injection-site reactions occurred only in infants in the BCG and the 2·5 × 105 CFU MTBVAC group, with no evidence of local or regional injection-site complications. Systemic adverse events were evenly distributed across BCG and MTBVAC dose groups, and were mostly mild in severity. Eight serious adverse events were reported in seven vaccine recipients (one adult MTBVAC recipient, one infant BCG recipient, one infant in the 2·5 × 103 CFU MTBVAC group, two in the 2·5 × 104 CFU MTBVAC group, and two in the 2·5 × 105 CFU MTBVAC group), including one infant in the 2·5 × 103 CFU MTBVAC group treated for unconfirmed tuberculosis and one in the 2·5 × 105 CFU MTBVAC group treated for unlikely tuberculosis. One infant died as a result of possible viral pneumonia. Vaccination with all MTBVAC doses induced durable antigen-specific T-helper-1 cytokine-expressing CD4 cell responses in infants that peaked 70 days after vaccination and were detectable 360 days after vaccination. For the highest MTBVAC dose (ie, 2·5 × 105 CFU), these responses exceeded responses induced by an equivalent dose of the BCG vaccine up to 360 days after vaccination. Dose-related IGRA conversion was noted in three (38%) of eight infants in the 2·5 × 103 CFU MTBVAC group, six (75%) of eight in the 2·5 × 104 CFU MTBVAC group, and seven (78%) of nine in the 2·5 × 105 CFU MTBVAC group at day 180, compared with none of seven infants in the BCG group. By day 360, IGRA reversion had occurred in all three infants (100%) in the 2·5 × 103 CFU MTBVAC group, four (67%) of the six in the 2·5 × 104 CFU MTBVAC group, and three (43%) of the seven in the 2·5 × 105 CFU MTBVAC group. INTERPRETATION: MTBVAC had acceptable reactogenicity, and induced a durable CD4 cell response in infants. The evidence of immunogenicity supports progression of MTBVAC into larger safety and efficacy trials, but also confounds interpretation of tests for M tuberculosis infection, highlighting the need for stringent endpoint definition. FUNDING: Norwegian Agency for Development Cooperation, TuBerculosis Vaccine Initiative, UK Department for International Development, and Biofabri.

Prevalence of latent TB infection and TB disease among adolescents in high TB burden countries in Africa: a systematic review protocol.

INTRODUCTION: Almost a third of the world population has latent tuberculosis (TB) infection (LTBI), ∼10 million of whom develop TB disease annually, despite existence of effective, but lengthy, preventive and curative drug regimens. Although adolescents appear to have a very high force of LTBI, their reported incidence of TB disease is less than that of their corresponding general population. The few available studies on adolescent TB infection and disease prevalence are not sufficient to address the apparent discordance between rates of infection and disease in high TB burden countries in Africa. Therefore, we aim to perform a systematic review to examine the relationship between adolescent LTBI and TB disease, benchmarked against national TB disease burden data. METHODS AND ANALYSIS: A comprehensive literature search will be performed for cross-sectional studies and screening data in cohort studies to determine the prevalence of LTBI and TB disease among adolescents in high TB burden countries in Africa in the following databases: PubMed, Scopus, Cochrane library, Web of Science, Africa Wide, CINAHL and the Africa Index Medicus. This will be supplemented by a search of reference lists of selected articles for potentially relevant articles. We will restrict our search to articles published in the English language between 1990 and 2016 among adolescents in order to obtain estimates reflective of the mature HIV epidemic in most high TB burden countries in Africa that occurred over this critical period. Primary end points are: prevalence of LTBI and TB disease. We will use the random-effects or fixed-effects modelling for our meta-analysis based on heterogeneity estimates. ETHICS AND DISSEMINATION: No ethics approval is required given that this is a systematic review. Findings will be disseminated in a peer-reviewed journal in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA). TRIAL REGISTRATION NUMBER: CRD42015023495.

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).

Antigen Availability Shapes T Cell Differentiation and Function during Tuberculosis.

CD4 T cells are critical for protective immunity against Mycobacterium tuberculosis (Mtb), the cause of tuberculosis (TB). Yet to date, TB vaccine candidates that boost antigen-specific CD4 T cells have conferred little or no protection. Here we examined CD4 T cell responses to two leading TB vaccine antigens, ESAT-6 and Ag85B, in Mtb-infected mice and in vaccinated humans with and without underlying Mtb infection. In both species, Mtb infection drove ESAT-6-specific T cells to be more differentiated than Ag85B-specific T cells. The ability of each T cell population to control Mtb in the lungs of mice was restricted for opposite reasons: Ag85B-specific T cells were limited by reduced antigen expression during persistent infection, whereas ESAT-6-specific T cells became functionally exhausted due to chronic antigenic stimulation. Our findings suggest that different vaccination strategies will be required to optimize protection mediated by T cells recognizing antigens expressed at distinct stages of Mtb infection.

Evaluation of Xpert® MTB/RIF Assay in Induced Sputum and Gastric Lavage Samples from Young Children with Suspected Tuberculosis from the MVA85A TB Vaccine Trial.

OBJECTIVE: Diagnosis of childhood tuberculosis is limited by the paucibacillary respiratory samples obtained from young children with pulmonary disease. We aimed to compare accuracy of the Xpert® MTB/RIF assay, an automated nucleic acid amplification test, between induced sputum and gastric lavage samples from young children in a tuberculosis endemic setting. METHODS: We analyzed standardized diagnostic data from HIV negative children younger than four years of age who were investigated for tuberculosis disease near Cape Town, South Africa [2009-2012]. Two paired, consecutive induced sputa and early morning gastric lavage samples were obtained from children with suspected tuberculosis. Samples underwent Mycobacterial Growth Indicator Tube [MGIT] culture and Xpert MTB/RIF assay. We compared diagnostic yield across samples using the two-sample test of proportions and McNemar's χ2 test; and Wilson's score method to calculate sensitivity and specificity. RESULTS: 1,020 children were evaluated for tuberculosis during 1,214 admission episodes. Not all children had 4 samples collected. 57 of 4,463[1.3%] and 26 of 4,606[0.6%] samples tested positive for Mycobacterium tuberculosis on MGIT culture and Xpert MTB/RIF assay respectively. 27 of 2,198[1.2%] and 40 of 2,183[1.8%] samples tested positive [on either Xpert MTB/RIF assay or MGIT culture] on induced sputum and gastric lavage samples, respectively. 19/1,028[1.8%] and 33/1,017[3.2%] admission episodes yielded a positive MGIT culture or Xpert MTB/RIF assay from induced sputum and gastric lavage, respectively. Sensitivity of Xpert MTB/RIF assay was 8/30[26.7%; 95% CI: 14.2-44.4] for two induced sputum samples and 7/31[22.6%; 11.4-39.8] [p = 0.711] for two gastric lavage samples. Corresponding specificity was 893/893[100%;99.6-100] and 885/890[99.4%;98.7-99.8] respectively [p = 0.025]. CONCLUSION: Sensitivity of Xpert MTB/RIF assay was low, compared to MGIT culture, but diagnostic performance of Xpert MTB/RIF did not differ sufficiently between induced sputum and gastric lavage to justify selection of one sampling method over the other, in young children with suspected pulmonary TB. TRIAL REGISTRATION: ClinicalTrials.gov NCT00953927.

The Role of Clinical Symptoms in the Diagnosis of Intrathoracic Tuberculosis in Young Children.

BACKGROUND: Childhood tuberculosis (TB) is usually Mycobacterium tuberculosis (MTB) culture negative. Furthermore, clinical presentation may be altered by active case finding, isoniazid prophylaxis and early treatment. We aimed to establish the value of presenting symptoms for intrathoracic TB case diagnosis among young children. METHODS: Healthy, HIV-uninfected, South African infants in an efficacy trial of a novel TB vaccine (MVA85A) were followed for 2 years for suspected TB. When suspected, investigation followed a standardized algorithm comprising symptom history, QuantiFERON Gold-in-Tube, chest radiography (CXR), MTB culture and Xpert MTB/RIF from paired gastric lavage and induced sputa. Adjusted odds ratios and 95% confidence intervals describe the associations between symptoms and positive MTB culture or Xpert MTB/RIF, and CXR compatible with intrathoracic TB. RESULTS: Persistent cough was present in 172/1017 (16.9%) of the children investigated for TB. MTB culture/Xpert MTB/RIF was positive in 38/1017 children (3.7%); and CXR was positive, that is, compatible with intrathoracic TB, in 131/1017 children (12.9%). Children with persistent cough had more than triple the odds of a positive MTB culture/Xpert MTB/RIF (adjusted odds ratios: 3.3, 95% confidence interval: 1.5-7.0) and positive CXR (adjusted odds ratios: 3.5, 95% confidence interval: 2.2-5.5). Persistent cough was the only symptom that differentiated children with severe (56.5%) from nonsevere intrathoracic TB disease (28.2%; P = 0.001). CONCLUSION: Persistent cough was the cardinal diagnostic symptom associated with microbiologic and radiologic evidence, and disease severity, of intrathoracic TB. Symptom-based definitions of TB disease for diagnostic, preventive and therapeutic studies should prioritize persistent cough above other symptoms compatible with childhood TB.

Risk of Disease After Isoniazid Preventive Therapy for Mycobacterium tuberculosis Exposure in Young HIV-uninfected Children.

BACKGROUND: The risk of developing tuberculosis (TB) disease in HIV-uninfected children after isoniazid preventive therapy (IPT) for a positive QuantiFERON-TB Gold In-Tube test (QFT-GIT) is unknown. The aim of this study was to evaluate risk of TB disease after IPT in young HIV-uninfected children with a positive QFT-GIT result, or household TB contact. METHODS: HIV-uninfected South African infants aged 4-6 months were screened for enrolment in a TB vaccine trial. Baseline household TB contact and positive QFT-GIT result were exclusion criteria, and these infants were referred for IPT. Outcome data are reported for 36 months after IPT referral. RESULTS: Four thousand seven hundred forty-nine infants were screened. Household TB contact was reported in 131 (2.8%) infants; 279 (6.0%) were QFT-GIT positive, and 138 of these 410 infants (34.0%) started IPT. Forty-four cases of TB disease (11.0%) were recorded within 991 child years of observation. TB disease incidence was 4.8 versus 3.6 per 100 child years in household exposed versus QFT-GIT-positive children [incidence rate ratio: 1.35; 95% confidence interval (CI): 0.67-2.88] and 2.4 versus 5.5 per 100 child years in children who received versus did not receive IPT, respectively (incidence rate ratio: 0.44; 95% CI: 0.17-0.96). Adjusted hazard ratio (Cox regression) for TB disease was 0.48 (95% CI: 0.21-1.05) for those who received IPT. CONCLUSION: In young HIV-uninfected children, the effect of IPT on risk of TB disease is similar, whether TB exposure was defined by household contact history or by positive QFT-GIT result. International IPT guidelines for HIV-uninfected children with a positive QFT-GIT result should be updated.

A randomized clinical trial in adults and newborns in South Africa to compare the safety and immunogenicity of bacille Calmette-Guérin (BCG) vaccine administration via a disposable-syringe jet injector to conventional technique with needle and syringe.

INTRODUCTION: Intradermal bacille Calmette-Guérin (BCG) vaccination by needle-free, disposable-syringe jet injectors (DSJI) is an alternative to the Mantoux method using needle and syringe (NS). We compared the safety and immunogenicity of BCG administration via the DSJI and NS techniques in adults and newborn infants at the South African Tuberculosis Vaccine Initiative (SATVI) research site in South Africa. METHOD: Thirty adults and 66 newborn infants were randomized 1:1 to receive intradermal BCG vaccine (0.1 mL in adults; 0.05 mL in infants) via DSJI or NS. Wheal diameter (mm) and skin fluid deposition at the site of injection (SOI) were measured immediately post-vaccination. Adverse events and SOI reactogenicity data were collected 30 min and 1, 2, 4, and 12 weeks after vaccination for adults and at 30 min and 4, 10, and 14 weeks for infants. Blood was collected in infants at 10 and 14 weeks to assess BCG-specific T-cell immune responses. RESULTS: More infant BCG vaccinations by DSJI deposited >5 µL fluid on the skin surface, compared to NS (49% versus 9%, p=0.001). However, all 12 infant vaccinations that did not produce any SOI wheal occurred in the NS group (36%, p<0.001). Median wheal diameter, in participants for which an SOI wheal formed, did not differ significantly between groups in infants (combined 3.0mm IQR 2.0 to 4.0, p=0.59) or in adults (combined 9.0mm IQR 7.0 to 10.0, p=0.13). Adverse events were similar between study arms. Proportion of participants with BCG scars after three months did not differ in adults (combined 97%, p=0.67) or infants (combined 62%, p=0.13). Frequencies of BCG-specific clusters of differentiation 4 (CD4) and clusters of differentiation 8 (CD8) T-cells co-expressing IFN-γ, TNF-α, IL-2, and/or IL-17 were not different in the DSJI and NS groups. CONCLUSION: BCG vaccination of newborn infants via DSJI was more likely to deliver an appropriate intradermal wheal at the SOI as compared to NS, despite leaving more fluid on the surface of the skin. Safety, reactogenicity, and antigen-specific T-cell immune responses did not differ between DSJI and NS techniques.