Delayed BCG Vaccination: Effect on the Infant Immune Response, Unrelated Pathogens and Other EPI Vaccines

Tuberculosis (TB) is one of the leading causes of mortality in developing countries and infants are at particular risk. Currently, the only licensed vaccine is Bacillus Calmette-Guerin (BCG), which has variable efficacy. BCG has been shown to provide heterologous protection against unrelated pathogens and enhance immunity to other Expanded Programme on Immunization (EPI) vaccines when given at birth. However, only one recently published study has shown an enhanced pro-inflammatory effect of BCG among vaccinated neonates and the immunological mechanisms of this effect in infants are not known. Further studies are required if this BCG effect is to be understood. This thesis describes the differences in soluble and cellular responses to vaccine specific and non-specific heterologous antigens and EPI antibody levels between infants vaccinated with BCG at 6 weeks and those delayed until 18 weeks of age using flow cytometry, multiplex cytokine arrays and vaccine antibody assays. We found low plasma cytokine levels and little cytokine production to PPD (the BCG-recall antigen) regardless of vaccination status. However, the BCG vaccinated group had lower production of IL-10 with both Toll like receptor (TLR) agonists and unrelated pathogen stimulation when compared to their unvaccinated counterparts, and this was more prominent in females than males. This contrasts with previous studies showing enhanced pro-inflammatory responses to TLR agonists and killed pathogens following BCG vaccination. The infants received BCG Denmark in these latter studies, whereas BCG Russia was used in our study, therefore strain differences in heterologous effects of BCG may be one explanation for the discrepancy. When cellular responses were analyzed by flow cytometry low cellular responses were observed in both groups. However, we observed a significantly higher proportion of IFNγ+ CD8+ cells 1 week after vaccination compared to the unvaccinated group in response to PPD and Candida albicans stimulation. Despite their different vaccine schedules, protective antibody levels to polio, tetanus and hepatitis B vaccines were obtained by all subjects; although no boosting effect of BCG was seen in contrast to previous studies when BCG was given at birth. A tuberculin skin test (TST) was performed at 18 weeks of age as a functional measure of delayed type hypersensitivity to Mtb. Due to the influence of BCG vaccination on TST induration, the vaccinated infants had significantly higher indurations than the unvaccinated infants and this was more pronounced in males. Our findings are in contrast to a study in South Africa, but support findings from a study in Uganda, where delayed BCG vaccination significantly reduced responses to BCG compared to infants vaccinated at birth, suggesting an impact of environment (including exposure to non-tuberculous mycobacteria) in masking the immune response to BCG, differences in vaccine strains might be a major reason for differences observed in our study and the other South African studies However, two major limitations of our study are the lack of a vaccine group at birth as per the current vaccine schedule, and a postvaccination follow up for the infants vaccinated at 18 weeks to determine differences in immunity to BCG with a further delay in vaccination. In conclusion, BCG vaccination given at 6 weeks of age showed no difference in Th1 responses, but decreased IL-10 responses after TLR agonist and unrelated pathogen stimulation compared to unvaccinated infants. These effects, together with TST induration, were influenced by sex. Importantly, however, delaying vaccination did not have an effect on other EPI vaccine antibody levels, with all infants reaching protective levels by 18 weeks of age. This study provides further insight into BCG effects in infants in a West African setting

A cloud-based bioinformatic analytic infrastructure and Data Management Core for the Expanded Program on Immunization Consortium.

The Expanded Program for Immunization Consortium - Human Immunology Project Consortium study aims to employ systems biology to identify and characterize vaccine-induced biomarkers that predict immunogenicity in newborns. Key to this effort is the establishment of the Data Management Core (DMC) to provide reliable data and bioinformatic infrastructure for centralized curation, storage, and analysis of multiple de-identified "omic" datasets. The DMC established a cloud-based architecture using Amazon Web Services to track, store, and share data according to National Institutes of Health standards. The DMC tracks biological samples during collection, shipping, and processing while capturing sample metadata and associated clinical data. Multi-omic datasets are stored in access-controlled Amazon Simple Storage Service (S3) for data security and file version control. All data undergo quality control processes at the generating site followed by DMC validation for quality assurance. The DMC maintains a controlled computing environment for data analysis and integration. Upon publication, the DMC deposits finalized datasets to public repositories. The DMC architecture provides resources and scientific expertise to accelerate translational discovery. Robust operations allow rapid sharing of results across the project team. Maintenance of data quality standards and public data deposition will further benefit the scientific community

Post-tuberculosis respiratory impairment in Gambian children and adolescents: A cross-sectional analysis.

BACKGROUND: Although post-tuberculosis lung disease (PTLD) is a known consequence of pulmonary tuberculosis (pTB), few studies have reported the prevalence and spectrum of PTLD in children and adolescents. METHODS: Children and adolescent (≤19 years) survivors of pTB in the Western Regions of The Gambia underwent a respiratory symptom screening, chest X-ray (CXR) and spirometry at TB treatment completion. Variables associated with lung function impairment were identified through logistic regression models. RESULTS: Between March 2022 and July 2023, 79 participants were recruited. The median age was 15.6 years (IQR: 11.8, 17.9); the majority, 53/79 (67.1%), were treated for bacteriologically confirmed pTB, and 8/79 (10.1%) were children and adolescents living with HIV. At pTB treatment completion, 28/79 (35.4%) reported respiratory symptoms, 37/78 (47.4%) had radiological sequelae, and 45/79 (57.0%) had abnormal spirometry. The most common respiratory sequelae were cough (21/79, 26.6%), fibrosis on CXR (22/78, 28.2%), and restrictive spirometry (41/79, 51.9%). Age at TB diagnosis over ten years, undernutrition and fibrosis on CXR at treatment completion were significantly associated with abnormal spirometry (p = .050, .004, and .038, respectively). CONCLUSION: Chronic respiratory symptoms, abnormal CXR, and impaired lung function are common and under-reported consequences of pTB in children and adolescents. Post-TB evaluation and monitoring may be necessary to improve patient outcomes

Mixed Th1 and Th2 Mycobacterium tuberculosis-specific CD4 T cell responses in patients with active pulmonary tuberculosis from Tanzania.

Mycobacterium tuberculosis (Mtb) and helminth infections elicit antagonistic immune effector functions and are co-endemic in several regions of the world. We therefore hypothesized that helminth infection may influence Mtb-specific T-cell immune responses. We evaluated the cytokine profile of Mtb-specific T cells in 72 individuals with pulmonary TB disease recruited from two Sub-Saharan regions with high and moderate helminth burden i.e. 55 from Tanzania (TZ) and 17 from South Africa (SA), respectively. We showed that Mtb-specific CD4 T-cell functional profile of TB patients from Tanzania are primarily composed of polyfunctional Th1 and Th2 cells, associated with increased expression of Gata-3 and reduced expression of T-bet in memory CD4 T cells. In contrast, the cytokine profile of Mtb-specific CD4 T cells of TB patients from SA was dominated by single IFN-γ and dual IFN-γ/TNF-α and associated with TB-induced systemic inflammation and elevated serum levels of type I IFNs. Of note, the proportion of patients with Mtb-specific CD8 T cells was significantly reduced in Mtb/helminth co-infected patients from TZ. It is likely that the underlying helminth infection and possibly genetic and other unknown environmental factors may have caused the induction of mixed Th1/Th2 Mtb-specific CD4 T cell responses in patients from TZ. Taken together, these results indicate that the generation of Mtb-specific CD4 and CD8 T cell responses may be substantially influenced by environmental factors in vivo. These observations may have major impact in the identification of immune biomarkers of disease status and correlates of protection

Transcriptomic signatures of recurrent tuberculosis disease and treatment response in HIV-infected individuals

HIV-infected persons are at particularly high risk of tuberculosis (TB) disease, especially in TB endemic countries where M. tuberculosis transmission is common. Although antiretroviral therapy (ART) reduces risk of TB, it does not return to that of HIV-uninfected persons. In addition, a previous history of TB disease significantly increases the risk of recurrent TB disease. Identification of HIV-infected individuals at greatest risk of recurrent TB for highly targeted therapy before disease manifestation would be a major advance in the fight against TB. This would also allow the provision of TB treatment in persons with subclinical TB. Diagnosis of TB in HIV-infected persons is markedly undermined by the paucibacillary nature of HIV-associated disease. A non-sputum based diagnostic test that is highly sensitive and specific, such as a blood-based RNA signature, would be an important new tool. Such a test may also facilitate monitoring of TB treatment, opening the possibility for customizing the duration of TB treatment to that necessary for cure. We previously discovered and validated a 16-gene transcriptomic signature with promising prognostic and diagnostic utility for TB in HIV-uninfected persons. The transcriptomic signature could predict progression to active TB disease up to a year before diagnosis and was shown to be a useful tool for TB treatment response monitoring in a treatment cohort of HIV-uninfected persons. The signature was reduced to an 11-gene signature to improve throughput with equivalent prognostic and diagnostic performance. In addition, we developed a smaller, 6-gene signature in preparation for translation to point-of-care testing. In this thesis, we aimed to determine (1) the diagnostic performance of these two transcriptomic signatures in HIV-infected persons, (2) whether they could predict recurrent TB disease in HIV infected persons, and (3) their utility to monitor TB treatment response in HIV infected persons. To assess aim 1, we designed a cross-sectional study of HIV-infected (n=40) and uninfected persons (n=60), each comprising equal numbers of active TB cases and QuantiFERON-positive controls. To assess aims 2 and 3, we designed retrospective substudies among participants enrolled into two clinical studies previously completed by our collaborators at CAPRISA, namely the TRuTH and IMPRESS studies. In the TRuTH cohort participants who developed recurrent TB diagnosis, diagnosed by microbiological testing of induced sputum, were assigned as progressors (n=43), while those who remained asymptomatic were assigned as non-progressors (n=86). In the IMPRESS cohort, participants with a new diagnosis of recurrent TB who initiated TB treatment were stratified into early (n=44) and late (n=19) converters based on time to sputum culture conversion from diagnosis. RNA was isolated from cryopreserved PBMC or PAXgene whole blood and gene expression measured by microfluidic qRT-PCR. Signature scores were generated using in-house customised scripts in R and performance of the signatures was measured using receiver operating characteristic area under the curve (ROC AUC), calculated using the pROC and verification packages in R. The 11-gene and 6-gene signatures could diagnose active TB disease in HIV infected persons with good accuracy (AUC = 0.83 and 0.92, respectively), although performances were lower than those observed in HIV-uninfected persons (AUC = 0.97 and 0.96). Signature performance was decreased in HIV-infected persons due to higher signature scores, reflecting high expression of IFN-stimulated genes, especially in HIV-infected controls. In the TRuTH cohort, these signatures could identify those with recurrent TB within 3 months of diagnosis (AUC = 0.77, p = 0.003), suggesting detection of subclinical disease. Scores of both signatures decreased during TB treatment in the IMPRESS cohort, in participants with early or late sputum conversion. Importantly, two months after initiating TB treatment, the ACS 11-gene signature could differentiate early from late converters. Detectable plasma viral load was associated with higher signature scores in both cohorts, leading to a decrease in signature specificities. We show that the 11-gene and 6-gene signatures performed well as blood-based diagnostic tests for active TB disease in HIV-infected persons. The signatures could detect recurrent TB disease during the subclinical phase of disease progression and demonstrated promise as treatment response markers in HIV-infected persons. The signatures performed best in persons with effectively suppressed HIV load, highlighting the importance of ART adherence and integration of HIV and TB care for effective clinical management of TB

Plasma Type I IFN Protein Concentrations in Human Tuberculosis

International audienceTuberculosis (TB) remains one of the leading causes of mortality worldwide, and a lack of understanding of basic disease pathogenesis is hampering development of new vaccines and treatments. Multiple studies have previously established a role for type I interferon (IFN) in TB disease. Type I IFNs are critical immune mediators for host responses to viral infection, yet their specific influence in bacterial infection remains unclear. As IFN-stimulated genes (ISGs) can have both stimulatory and inhibitory effects on immune function, clarifying the role of type I interferon in TB remains an important question. The quantification of interferon proteins in the circulation of patients has been restricted until the recent development of digital ELISA. To test the hypothesis that patients with active TB disease have elevated circulating type I IFN we quantified plasma IFNα and β proteins with Simoa digital ELISA in patients with active disease and asymptomatic M. tuberculosis infection. Strikingly no differences were observed between these two groups, while plasma from acute influenza infection revealed significantly higher plasma levels of both IFNα and IFNβ proteins. These results suggest a discordance between ISG mRNA expression by blood leukocytes and circulating type I IFN in TB

A phase I randomized clinical trial of candidate human immunodeficiency virus type 1 vaccine MVA. HIVA administered to Gambian infants

Background A vaccine to decrease transmission of human immunodeficiency virus type 1 (HIV-1) during breast-feeding would complement efforts to eliminate infant HIV-1 infection by antiretroviral therapy. Relative to adults, infants have distinct immune development, potentially high-risk of transmission when exposed to HIV-1 and rapid progression to AIDS when infected. To date, there have been only three published HIV-1 vaccine trials in infants. Trial Design We conducted a randomized phase I clinical trial PedVacc 001 assessing the feasibility, safety and immunogenicity of a single dose of candidate vaccine MVA.HIVA administered intramuscularly to 20-week-old infants born to HIV-1-negative mothers in The Gambia. Methods Infants were followed to 9 months of age with assessment of safety, immunogenicity and interference with Expanded Program on Immunization (EPI) vaccines. The trial is the first stage of developing more complex prime-boost vaccination strategies against breast milk transmission of HIV-1. Results From March to October 2010, 48 infants (24 vaccine and 24 no-treatment) were enrolled with 100% retention. The MVA.HIVA vaccine was safe with no difference in adverse events between vaccinees and untreated infants. Two vaccine recipients (9%) and no controls had positive ex vivo interferon-γ ELISPOT assay responses. Antibody levels elicited to the EPI vaccines, which included diphtheria, tetanus, whole-cell pertussis, hepatitis B virus, Haemophilus influenzae type b and oral poliovirus, reached protective levels for the vast majority and were similar between the two arms. Conclusions A single low-dose of MVA.HIVA administered to 20-week-old infants in The Gambia was found to be safe and without interference with the induction of protective antibody levels by EPI vaccines, but did not alone induce sufficient HIV-1-specific responses. These data support the use of MVA carrying other transgenes as a boosting vector within more complex prime-boost vaccine strategies against transmission of HIV-1 and/or other infections in this age group. Trial Registration ClinicalTrials.gov NCT00982579 The Pan African Clinical Trials Registry PACTR200812000090411

Minimal Sex-Differential Modulation of Reactivity to Pathogens and Toll-Like Receptor Ligands following Infant Bacillus Calmette–Guérin Russia Vaccination

Bacillus Calmette–Guérin (BCG), the only licensed vaccine against tuberculosis, has been shown to provide heterologous protection against unrelated pathogens and enhance antibody responses to several routine expanded program on immunization (EPI) vaccines. Understanding these heterologous effects is important for the development of optimal vaccination strategies. We set out to assess the effect of vaccination with BCG Russia of 6-week-old infants on in vitro reactivity to a panel of toll-like receptor (TLR) agonists (TLR2, 4, and 7/8) and heat-killed pathogens [Streptococcus pneumoniae, Candida albicans (CA), and Escherichia coli], and antibody responses to other EPI vaccines compared to BCG naïve infants. We observed no effect of BCG vaccination on innate (TNF-α) or Th2 (IL-4) cytokine responses, but found enhanced CA-specific CD8+IFN-γ+ responses in BCG vaccinated males and females 1 week after vaccination and decreased IFN-γ:IL4 ratio to SP in females. By 12 weeks (but not 1 week) of post-vaccination, there was significant downmodulation of Th1 cytokine responses in BCG vaccinated infants; and TLR-stimulated IL-10 and IL-17 responses declined in BCG vaccinated females but not males. Significant changes also occurred in the BCG naïve group, mainly at 18 weeks, including decreased Th1 and increased IL-10 responses. The effects at 18 weeks were most likely a result of immune modulation by the intervening EPI vaccines given at 8, 12, and 16 weeks of age. There was no effect of BCG vaccination on EPI antibody levels at either time point. Taken together, our results support minimal early heterologous immune modulation by BCG Russia vaccination that did not persist 12 weeks after vaccination

A multi-cohort study of the immune factors associated with M. tuberculosis infection outcomes

Most infections with Mycobacterium tuberculosis (Mtb) manifest as a clinically asymptomatic, contained state, known as latent tuberculosis infection, that affects approximately one-quarter of the global population. Although fewer than one in ten individuals eventually progress to active disease, tuberculosis is a leading cause of death from infectious disease worldwide. Despite intense efforts, immune factors that influence the infection outcomes remain poorly defined. Here we used integrated analyses of multiple cohorts to identify stage-specific host responses to Mtb infection. First, using high-dimensional mass cytometry analyses and functional assays of a cohort of South African adolescents, we show that latent tuberculosis is associated with enhanced cytotoxic responses, which are mostly mediated by CD16 (also known as FcγRIIIa) and natural killer cells, and continuous inflammation coupled with immune deviations in both T and B cell compartments.\ua0Next, using cell-type deconvolution of transcriptomic data from several cohorts of different ages, genetic backgrounds, geographical locations and infection stages, we show that although deviations in peripheral B and T cell compartments generally start at latency, they are heterogeneous across cohorts. However, an increase in the abundance of circulating natural killer cells in tuberculosis latency, with a corresponding decrease during active disease and a return to baseline levels upon clinical cure are features that are common to all cohorts. Furthermore, by analysing three longitudinal cohorts, we find that changes in\ua0peripheral levels of natural killer cells can inform disease progression and treatment responses, and inversely correlate with the inflammatory state of the lungs of patients with active tuberculosis. Together, our findings offer crucial insights into the underlying pathophysiology of tuberculosis latency, and identify factors that may influence infection outcomes

Erratum to: A multi-cohort study of the immune factors associated with M. tuberculosis infection outcomes (Nature, (2018), 560, 7720, (644-648), 10.1038/s41586-018-0439-x)

The spelling of author Qianting Yang was corrected; the affiliation of author Stephanus T. Malherbe was corrected; and graphs in Fig. 4b and c were corrected owing to reanalysis of the data into the correct timed intervals