Serial T-SPOT.TB responses in Tanzanian adolescents: Transient, persistent and irregular conversions.

BACKGROUND: Prospective studies of interferon-gamma release assays (IGRA) on healthy subjects in tuberculosis-endemic regions have not examined the long-term variability of serial assays. This issue is relevant to the interpretation of tuberculosis (TB) vaccine trials based on prevention of infection. METHODS: T-SPOT.TB assays were performed manually on healthy adolescents during a tuberculosis vaccine trial in Tanzania at 5 intervals over 3 years. Assay results were defined as negative, positive, borderline or invalid. Subsequently, microtiter plates were analyzed by an automated reader to obtain quantitative counts of spot forming cells (SFCs) for the present analysis. RESULTS: 3387 T-SPOT.TB samples were analyzed from 928 adolescents; manual and automated assay results were 97% concordant. Based on the quantitative results 143 (15%) participants were prevalent IGRA-positives at baseline, were ineligible for further study. Among the remaining IGRA-negative participants, the annual rate of IGRA conversion was 2·9%. Among 43 IGRA converters with repeat assays 12 (28%) were persistent converters, 16 (37%) were transient converters, and 15 (35%) comprised a new category defined as irregular converters (≥2 different subsequent results). ESAT-6 and CFP-10 responses were higher in prevalent than incident positives: 53 vs 36 for CFP-10 (p < 0·007); 44 vs 34 for ESAT-6 (p = 0·12). CONCLUSIONS: Definitions of IGRA conversion, reversion, and persistence depend critically on the frequency of testing. Multiple shifts in categories among adolescents in a TB-endemic country may represent multiple infections, variable host responses in subclinical infection, or assay variation. These findings should to be considered in the design and interpretation of TB vaccine trials based on prevention of infection. Household contact studies could determine whether even transient IGRA conversion might represent exposure to an active case of M. tuberculosis disease.

Methylome-wide Analysis Reveals Epigenetic Marks Associated With Resistance to Tuberculosis in Human Immunodeficiency Virus-Infected Individuals From East Africa.

BACKGROUND: Tuberculosis (TB) is the most deadly infectious disease globally and is highly prevalent in the developing world. For individuals infected with both Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV), the risk of active TB is 10% or more annually. Previously, we identified in a genome-wide association study (GWAS) a region on chromosome 5 associated with resistance to TB, which included epigenetic marks that could influence gene regulation. We hypothesized that HIV-infected individuals exposed to Mtb who remain disease free carry epigenetic changes that strongly protect them from active TB. METHODS: We conducted a methylome-wide study in HIV-infected, TB-exposed cohorts from Uganda and Tanzania and integrated data from our GWAS. RESULTS: We identified 3 regions of interest that included markers that were differentially methylated between TB cases and controls with latent TB infection: chromosome 1 (RNF220, P = 4 × 10-5), chromosome 2 (between COPS8 and COL6A3, P = 2.7 × 10-5), and chromosome 5 (CEP72, P = 1.3 × 10-5). These methylation results co-localized with associated single-nucleotide polymorphisms (SNPs), methylation QTLs, and methylation × SNP interaction effects. These markers were in regions with regulatory markers for cells involved in TB immunity and/or lung. CONCLUSIONS: Epigenetic regulation is a potential biologic factor underlying resistance to TB in immunocompromised individuals that can act in conjunction with genetic variants.

DAR-901 vaccine for the prevention of infection with Mycobacterium tuberculosis among BCG-immunized adolescents in Tanzania: A randomized controlled, double-blind phase 2b trial.

BACKGROUND: SRL172 prevented disease due to Mycobacterium tuberculosis in a Phase 3 trial. DAR-901 represents a scalable manufacturing process for SRL172. We sought to determine if DAR-901 would prevent infection with M. tuberculosis among BCG-primed adolescents age 13-15 years in Tanzania. METHODS: Adolescents with a negative T- SPOT.TBR interferon gamma release assay (IGRA) were randomized 1:1 to three intradermal injections of DAR-901 or saline placebo at 0, 2 and 4 months. Repeat IGRAs were performed at 2 months, and at 1, 2, and 3 years. The primary efficacy outcome was time to new TB infection (IGRA conversion to positive); the secondary outcome was time to persistent TB infection (IGRA conversion with repeat positive IGRA). RESULTS: Among 936 participants screened 667 were eligible and randomized to their first dose of vaccine or placebo (safety cohort). At 2 months, 625 participants remained IGRA-negative and were scheduled for the additional two doses (efficacy cohort). DAR-901 was safe and well-tolerated. One DAR-901 recipient developed a vaccine site abscess. Neither the primary nor secondary endpoints differed between the two treatment arms (p = 0.90 and p = 0.20, respectively). DAR-901 IGRA converters had median responses to ESAT-6 of 50.1 spot-forming cells (SFCs) vs. 19.6 SFCs in placebo IGRA converters (p = 0.03). CONCLUSIONS: A three-dose series of 1 mg DAR-901 was safe and well-tolerated but did not prevent initial or persistent IGRA conversion. DAR-901 recipients with IGRA conversion demonstrated enhanced immune responses to ESAT-6. Since protection against disease may require different immunologic responses than protection against infection a trial of DAR-901 to prevent TB disease is warranted. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov as NCT02712424.

Correction to: Successful implementation of isoniazid preventive therapy at a pediatric HIV clinic in Tanzania.

An amendment to this paper has been published and can be accessed via the original article.

Successful implementation of isoniazid preventive therapy at a pediatric HIV clinic in Tanzania.

BACKGROUND: In accordance with international guidance for tuberculosis (TB) prevention, the Tanzanian Ministry of Health recommends isoniazid preventive therapy (IPT) for children aged 12 months and older who are living with HIV. Concerns about tolerability, adherence, and potential mistreatment of undiagnosed TB with monotherapy have limited uptake of IPT globally, especially among children, in whom diagnostic confirmation is challenging. We assessed IPT implementation and adherence at a pediatric HIV clinic in Tanzania. METHODS: In this prospective cohort study, eligible children living with HIV aged 1-15 years receiving care at the DarDar Pediatric Program in Dar es Salaam who screened negative for TB disease were offered a 6-month regimen of daily isoniazid. Patients could choose to receive IPT via facility- or community-based care. Parents/caregivers and children provided informed consent and verbal assent respectively. Isoniazid was dispensed with the child's antiretroviral therapy every 1-3 months. IPT adherence and treatment completion was determined by pill counts, appointment attendance, and self-report. Patients underwent TB symptom screening at every visit. RESULTS: We enrolled 66 children between July and December 2017. No patients/caregivers declined IPT. Most participants were female (n = 43, 65.1%) and the median age was 11 years (interquartile range [IQR] 8, 13). 63 (95.5%) participants chose the facility-based model; due to the small number of participants who chose the community-based model, valid comparisons between the two groups could not be made. Forty-nine participants (74.2%) completed IPT within 10 months. Among the remaining 17, 11 had IPT discontinued by their provider due to adverse drug reactions, 5 lacked documentation of completion, and 1 had unknown outcomes due to missing paperwork. Of those who completed IPT, the average monthly adherence was 98.0%. None of the participants were diagnosed with TB while taking IPT or during a median of 4 months of follow-up. CONCLUSIONS: High adherence and treatment completion rates can be achieved when IPT is integrated into routine, self-selected facility-based pediatric HIV care. Improved record-keeping may yield even higher completion rates. IPT was well tolerated and no cases of TB were detected. IPT for children living with HIV is feasible and should be implemented throughout Tanzania.

The potential of CBC-derived ratios (monocyte-to-lymphocyte, neutrophil-to-lymphocyte, and platelet-to-lymphocyte) to predict or diagnose incident TB infection in Tanzanian adolescents.

BACKGROUND: Ratios of different immune cell populations (i.e., monocyte-to-lymphocyte, neutrophil-to-lymphocyte, and platelet-to-lymphocyte ratios) have been studied as a means of predicting future tuberculosis (TB) disease risk or to assist in the diagnosis of incident TB disease. No studies to-date, however, have evaluated the potential of these ratios to predict or assist in the diagnosis of incident TB infection - the first step in the natural history of TB disease. METHODS: In this prospective study, we evaluated the complete blood count (CBC)-derived metrics of monocyte-to-lymphocyte ratio (MLR), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) as predictors of future TB infection risk or aids in the diagnosis of TB infection among 145 Tanzanian adolescents enrolled in the DAR-901 vaccine trial, using paired CBCs and interferon-gamma release assays (IGRAs) obtained at 0, 60 and 720 days after study enrollment. RESULTS: At baseline, there were no significant differences between study participants who remained persistently IGRA negative throughout the study period and those who subsequently converted to IGRA positive with respect to MLR (0.18 vs 0.17, p = 0.10), NLR (0.88 vs 1.02, p = 0.08), or PLR (115 vs 120, p = 0.28). Similarly, no significant differences were noted with respect to MLR, NLR, and PLR between IGRA converters and time-matched negative controls at the time of IGRA conversion. With respect to other blood cell measures, however, there were modest but significant differences between IGRA negatives and IGRA converters with respect to red blood cell count (4.8 vs 4.6 ×  106 cells/mcL, p = 0.008), hemoglobin (12.6 vs 12.3 g/dL, p = 0.01), and hematocrit (38.8 vs 37.8%, p = 0.005). CONCLUSIONS: In contrast to prior studies that have suggested that the ratios of different immune cell populations are associated with development of TB disease, our present findings do not demonstrate an association between these ratios and the development of TB infection. However, decreased red blood cell measures were associated with the subsequent development of TB infection, suggesting either that dysregulation of iron metabolism may play a role in TB pathogenesis or that following TB infection, iron dysregulation may precede IGRA positivity. TRIAL REGISTRATION: Clinicaltrials.gov NCT02712424 . Date of registration: March 14, 2016.

CD4+ T cell cytokine responses to the DAR-901 booster vaccine in BCG-primed adults: A randomized, placebo-controlled trial.

BACKGROUND: DAR-901 is an inactivated whole cell tuberculosis booster vaccine, prepared using a new scalable, broth-grown method from the master cell bank of SRL172, a vaccine previously shown to prevent tuberculosis. This study examined whether DAR-901 (a) induces CD4+ T cell cytokine profiles previously proposed as correlates of protection and (b) has a specific vaccine-induced immunological signature compared to BCG or placebo. METHODS: We analysed CD4+ T cell cytokine immune responses from 10 DAR-901 recipients, 9 BCG recipients and 9 placebo recipients from the Phase I DAR-901 MDES trial. In that study, HIV-negative, IGRA-negative participants with prior BCG immunization were randomized (double-blind) to receive three intradermal injections of DAR-901 or saline placebo or two injections of saline placebo followed by an intradermal injection of BCG. Antigen-specific functional and phenotypic CD4+ T cell responses along with effector phenotype of responder cells were measured by intracellular cytokine staining. RESULTS: DAR-901 recipients exhibited increased DAR-901 antigen-specific polyfunctional or bifunctional T cell responses compared to baseline. Vaccine specific CD4+ IFNγ, IL2, TNFα and any cytokine responses peaked at 7 days post-dose 3. Th1 responses predominated, with most responder cells exhibiting a polyfunctional effector memory phenotype. BCG induced greater CD4+ T cell responses than placebo while the more modest DAR-901 responses did not differ from placebo. Neither DAR-901 nor BCG induced substantial or sustained Th17 /Th22 cytokine responses. CONCLUSION: DAR-901, a TB booster vaccine grown from the master cell bank of SRL 172 which was shown to prevent TB, induced low magnitude polyfunctional effector memory CD4+ T cell responses. DAR-901 responses were lower than those induced by BCG, a vaccine that has been shown ineffective as a booster to prevent tuberculosis disease. These results suggest that induction of higher levels of CD4+ cytokine stimulation may not be a critical or pre-requisite characteristic for candidate TB vaccine boosters. TRIAL REGISTRATION: ClinicalTrials.gov NCT02063555.

A Breath of Fresh Air: BCG Prevents Adult Pulmonary Tuberculosis.

OBJECTIVES: The efficacy of childhood BCG vaccination in the prevention of adult pulmonary tuberculosis is not universally accepted. METHODS: We reviewed the published literature and summarized studies of BCG vaccination reporting long-term protection against pulmonary TB. RESULTS: We identified 15 papers reporting prospective studies and their long-term follow up, retrospective studies or systematic reviews. CONCLUSIONS: Good quality evidence supports the efficacy of BCG vaccination in the prevention of adult pulmonary tuberculosis.

Sialic acid levels in breast milk from HIV-positive Tanzanian women and impact of maternal diet.

OBJECTIVE: To quantify total sialic acid in milk from HIV-positive Tanzanian mothers and to determine the impact of maternal diet on milk sialic acid levels. DESIGN: Milk samples were analyzed from 74 HIV-positive, Tanzanian women enrolled in a randomized, controlled clinical study of a dietary macronutrient supplement. Women were provided with a daily protein-calorie supplement and a micronutrient supplement or micronutrient supplement only during the last trimester of pregnancy and up to the first 6 months of breastfeeding. METHODS: Milk samples were collected at approximately 2 weeks and at least 3 months postpartum and assayed for total sialic acid. Milk sialic acid was assessed relative to maternal macronutrient intake, age, BMI, CD4+ cell count and infant birth weight. RESULTS: The mean concentration of milk sialic acid was highest in the first 2 weeks postpartum (6.89 ±â€Š2.79 mmol/l) and declined rapidly by 3 months (2.49 ±â€Š0.60 mmol/l). Sialic acid content in milk was similar between both treatment arms of the study, and did not correlate with maternal macronutrient intake. No correlation was found between maternal age, BMI, CD4+ cell count or infant birth weight and total milk sialic acid concentration. CONCLUSION: Milk sialic acid levels in HIV-positive, Tanzanian women without malnutrition are comparable with reported values for women of European descent and show a similar temporal decline during early lactation. These findings suggest that total milk sialic acid is maintained despite macronutrient deficiencies in maternal diet and support a conserved role for milk sialic acid in neonatal development.

Pre- and post-natal macronutrient supplementation for HIV-positive women in Tanzania: Effects on infant birth weight and HIV transmission.

OBJECTIVE: To determine if a protein-calorie supplement (PCS) plus a micronutrient supplement (MNS) improves outcomes for HIV-infected lactating women and their infants. DESIGN: Randomized, controlled trial. SETTING: Dar es Salaam, Tanzania. SUBJECTS, PARTICIPANTS: Pregnant HIV-infected women enrolled in PMTCT programs who intended to breastfeed for 6 months. INTERVENTION: Randomization 1:1 to administration of a PCS plus MNS versus MNS alone among 96 eligible women beginning in the third trimester and continuing for 6 months of breast-feeding. MAIN OUTCOME MEASURE(S): Primary: infant weight at 3 months. Secondary: maternal BMI at 6 months. RESULTS: PCS resulted in significant increases in daily energy intake compared to MNS at all time points (range of differences: +388-719 Kcal); and increases in daily protein intake (range of differences: +22-33 gm). Infant birth weight (excluding twins) was higher in the PCS than MNS groups: 3.30 kg vs 3.04 kg (p = 0.04). Infant weight at 3 months did not differ between PCS and MNS groups: 5.63 kg vs 5.99 kg (p = 0.07). Maternal BMI at 6 months did not differ between PCS and MNS groups: 24.3 vs 23.8 kg/m2 (p = 0.68). HIV transmission occurred in 0 infants in the PCS group vs 4 in the MNS group (p = 0.03). CONCLUSIONS: In comparison to MNS the PCS + MNS intervention was well tolerated, increased maternal energy and protein intake, and increased infant birth weight, but not weight at 3 months or maternal BMI at 6 months. Reduced infant HIV transmission in the PCS + MNS group was observed. TRIAL REGISTRATION: Clinical Trials.Gov NCT01461863.

Fine epitope signature of antibody neutralization breadth at the HIV-1 envelope CD4-binding site.

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs-specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades.

A chromosome 5q31.1 locus associates with tuberculin skin test reactivity in HIV-positive individuals from tuberculosis hyper-endemic regions in east Africa.

One in three people has been infected with Mycobacterium tuberculosis (MTB), and the risk for MTB infection in HIV-infected individuals is even higher. We hypothesized that HIV-positive individuals living in tuberculosis-endemic regions who do not get infected by Mycobacterium tuberculosis are genetically resistant. Using an "experiment of nature" design that proved successful in our previous work, we performed a genome-wide association study of tuberculin skin test positivity using 469 HIV-positive patients from prospective study cohorts of tuberculosis from Tanzania and Uganda to identify genetic loci associated with MTB infection in the context of HIV-infection. Among these individuals, 244 tested were tuberculin skin test (TST) positive either at enrollment or during the >8 year follow up, while 225 were not. We identified a genome-wide significant association between a dominant model of rs877356 and binary TST status in the combined cohort (Odds ratio = 0.2671, p = 1.22x10-8). Association was replicated with similar significance when examining TST induration as a continuous trait. The variant lies in the 5q31.1 region, 57kb downstream from IL9. Two-locus analyses of association of variants near rs877356 showed a haplotype comprised of rs877356 and an IL9 missense variant, rs2069885, had the most significant association (p = 1.59x10-12). We also replicated previously linked loci on chromosomes 2, 5, and 11. IL9 is a cytokine produced by mast cells and TH2 cells during inflammatory responses, providing a possible link between airway inflammation and protection from MTB infection. Our results indicate that studying uninfected, HIV-positive participants with extensive exposure increases the power to detect associations in complex infectious disease.

Safety and immunogenicity of an inactivated whole cell tuberculosis vaccine booster in adults primed with BCG: A randomized, controlled trial of DAR-901.

BACKGROUND: Development of a tuberculosis vaccine to boost BCG is a major international health priority. SRL172, an inactivated whole cell booster derived from a non-tuberculous mycobacterium, is the only new vaccine against tuberculosis to have demonstrated efficacy in a Phase 3 trial. In the present study we sought to determine if a three-dose series of DAR-901 manufactured from the SRL172 master cell bank by a new, scalable method was safe and immunogenic. METHODS: We performed a single site, randomized, double-blind, controlled, Phase 1 dose escalation trial of DAR-901 at Dartmouth-Hitchcock Medical Center in the United States. Healthy adult subjects age 18-65 with prior BCG immunization and a negative interferon-gamma release assay (IGRA) were enrolled in cohorts of 16 subjects and randomized to three injections of DAR-901 (n = 10 per cohort), or saline placebo (n = 3 per cohort), or two injections of saline followed by an injection of BCG (n = 3 per cohort; 1-8 x 106 CFU). Three successive cohorts were enrolled representing DAR-901 at 0.1, 0.3, and 1 mg per dose. Randomization was performed centrally and treatments were masked from staff and volunteers. Subsequent open label cohorts of HIV-negative/IGRA-positive subjects (n = 5) and HIV-positive subjects (n = 6) received three doses of 1 mg DAR-901. All subjects received three immunizations at 0, 2 and 4 months administered as 0.1 mL injections over the deltoid muscle alternating between right and left arms. The primary outcomes were safety and immunogenicity. Subjects were followed for 6 months after dose 3 for safety and had phlebotomy performed for safety studies and immune assays before and after each injection. Immune assays using peripheral blood mononuclear cells included cell-mediated IFN-γ responses to DAR-901 lysate and to Mycobacterium tuberculosis (MTB) lysate; serum antibody to M. tuberculosis lipoarabinomannan was assayed by ELISA. RESULTS: DAR-901 had an acceptable safety profile and was well-tolerated at all dose levels in all treated subjects. No serious adverse events were reported. Median (range) 7-day erythema and induration at the injection site for 1 mg DAR-901 were 10 (4-20) mm and 10 (4-16) mm, respectively, and for BCG, 30 (10-107) mm and 38 (15-55) mm, respectively. Three mild AEs, all headaches, were considered possibly related to DAR-901. No laboratory or vital signs abnormalities were related to immunization. Compared to pre-vaccination responses, three 1 mg doses of DAR-901 induced statistically significant increases in IFN-γ response to DAR-901 lysate and MTB lysate, and in antibody responses to M. tuberculosis lipoarabinomannan. Ten subjects who received 1 mg DAR-901 remained IFN-γ release assay (IGRA) negative after three doses of vaccine. CONCLUSIONS: A three-injection series of DAR-901 was well-tolerated, had an acceptable safety profile, and induced cellular and humoral immune responses to mycobacterial antigens. DAR-901 is advancing to efficacy trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT02063555.

The volatile molecule signature of four mycobacteria species.

Mycobacteria are the leading cause of death from infectious disease worldwide and limitations in current diagnostics are hampering control efforts. In recent years, the use of small volatile molecules as diagnostic biomarkers for mycobacteria has shown promise for use in the rapid analysis of in vitro cultures as well as ex vivo diagnosis using breath or sputum. In this study, 18 strains from four mycobacteria species (Mycobacterium avium, M. bovis BCG, M. intracellulare and M. xenopi) were analyzed for the first time using two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS). This study represents the first time volatile molecules associated with M. intracellulare and M. xenopi have ever been reported. A total of 217 chromatographic features were identified and 58 features were selected that discriminate between these four species. Putative identifications are provided for 17 of the 58 discriminatory features, three of which have been reported previously in mycobacteria. The identification of mycobacteria-associated volatile biomarker suites could reduce the time-to-diagnosis for mycobacterial infections, either from in vitro cultures prior to the visualization of colonies or directly from ex vivo specimens, thereby shortening the empiric treatment window and potentially improving outcomes.

Novel approaches to tuberculosis vaccine development.

Tuberculosis (TB) remains the deadliest infectious disease. The widely used bacille Calmette-Guérin (BCG) vaccine offers only limited protection against TB. New vaccine candidates for TB include subunit vaccines and inactivated whole-cell vaccines, as well as live mycobacterial vaccines. Current developments in TB vaccines are summarized in this review.