Browsing by Author "Scriba, Thomas"

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    BCG-specific T cell proliferation and cytotoxic capacity in infants as risk of tuberculosis disease, following newborn BCG vaccination
    (2014) Keyser, Alana; Hanekom, Willem A; Scriba, Thomas
    BCG is the only vaccine against tuberculosis and has been used for over 90 years. BCG efficacy is variable, especially in countries with high TB prevalence, where over a million deaths due to tuberculosis, are still reported annually. New TB vaccines are under development to either replace or boost the BCG vaccine. However, our understanding of the immune response required for protection against TB disease, remains inadequate. Identification of a protective immune response is only possible in a clinical trial of an efficacious vaccine, allowing comparison of vaccine-induced immune responses in protected and unprotected individuals. In the absence of such a vaccine, as is the case with TB, we can only explore biomarkers of risk of disease. The most commonly measured outcomes of anti-mycobacterial immunity in clinical trials, specific Th1 cells, are typically thought to be protective in TB. However, to date, human mycobacteria-specific Th1 responses have not correlated with risk of TB disease. New approaches are urgently required to identify other factors at play in conferring protection against TB. In this thesis, we explored BCG-specific cytotoxic T cells as candidate correlates of risk of TB disease in BCG-vaccinated infants. We hypothesized that reduced production of cytotoxic molecules by T cells in response to BCG are associated with risk of developing TB disease. We designed a case/control study nested within a large trial of newborn BCG-vaccination.Blood was collected at 10 weeks and infants, were followed up for two years.We compared outcomes in infants ultimately diagnosed with TB (at risk of TB disease) and two groups of healthy infants (not at risk of TB disease), the first group had household contact with TB cases, the second group were randomly selected from the community, which is endemic for TB. Amongst these groups, we designated a training and a test cohort to allow validation of candidate correlates of risk of TB.
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    Characterisation of T cell specificity, functional, activation and memory profiles associated with QuantiFERON TB Gold conversion and reversion
    (2021) Mpande, Cheleka Anne-Marie; Nemes, Elisa; Scriba, Thomas; Rozot, Virginie
    Recent acquisition of Mycobacterium tuberculosis (M.tb) infection is associated with a higher risk of tuberculosis disease, compared with remote, asymptomatic infection. M.tb infection, defined by a positive tuberculin skin test (TST) and/or IFN--release assay [IGRA e.g. QuantiFERON TB GOLD (QFT)], is commonly thought to be a chronic state. However, longitudinal studies have demonstrated the dynamic nature of M.tb infection, whereby TSTs and IGRAs revert from a positive to a negative test in some individuals, possibly an indication of bacterial clearance. Despite the first observation of discordant serial TST results over 80 years ago and the wide use of TSTs/IGRAs, there is still a limited understanding of immunological features associated with different stages of M.tb infection and discordant serial TST/IGRA results. Most studies of M.tb-specific immune responses in humans are based on cross-sectional comparisons between M.tb infection and active disease, with very few large cohort studies enabling a longitudinal assessment of different phases of infection. Thus, the main objective of this thesis was to gain a better understanding of changes in M.tbspecific CD4 T cell functional, memory and activation profiles associated with QFT conversion (acquisition of M.tb infection) and reversion (potential M.tb clearance). Our first aim was to characterise the homing, cytotoxic and functional capacity of M.tbspecific memory CD4+ T cells during recent and remote M.tb infection, with a special focus on stem cell memory T (TSCM) cells. TSCM cells play a critical role in maintaining long-lasting immunity, demonstrated by their superior longevity, proliferation and differentiation capacity compared to central memory (TCM) and effector (TE) cells. Before this study, our knowledge of TSCM cells was primarily based on virus-specific CD8+ TSCM cells. We demonstrate that M.tb-specific CD4+ TSCM cells are induced upon recent M.tb infection and maintained at steady-state during established infection. Despite being the least differentiated M.tb-specific memory subset and representing 2 years) M.tb infection, we also aimed to define an M.tb- specific T cell biomarker that can distinguish between the two infection states as current diagnostics fail to do so. Our second major finding demonstrated that recently infected individuals have lower proportions of highly differentiated IFN-+TNF+KLRG-1+ CD4+ TE cells and higher proportions of early differentiated IFN--TNF+IL2+KLRG-1- CD4+ T cells than remotely infected individuals in response to M.tb lysate but not CFP-10/ESAT-6 stimulation. Akin to their recent M.tb exposure, recently infected individuals had higher levels of T cell activation, regardless of M.tb antigen specificity, than remotely infected individuals. The degree of M.tb-specific CD4 T cell activation was identified as the best candidate biomarker for recent infection. The very same biomarker could also distinguish between progressors and non-progressors and identify individuals with active tuberculosis disease among healthy individuals with remote M.tb infection. We propose that, upon large-scale clinical validation, the T cell activation biomarker could be used as a screening test in conjunction with current tuberculosis diagnostics to guide the provision of either preventive or full tuberculosis therapy. These results have very important implications for targeting provision of preventive treatment to M.tb infected individuals at high-risk of tuberculosis, which is one of the top 10 strategies required to achieve tuberculosis elimination targets. Based on data from observational studies conducted during the pre-antibiotic era and guinea pig tuberculosis models, TST/IGRA reversion in humans is hypothesised to be associated with spontaneous (natural) clearance of infection. Similarly, individuals recently exposed to patients with tuberculosis who did not convert TST/IGRA (termed resistors) nor develop disease had M.tb-specific T cell responses that did not include IFN- production. However, clearance of M.tb infection is virtually impossible to demonstrate in healthy individuals. We clearly illustrated that acquisition infection is associated with induction of CD4+ Th1 functional and memory T cell subsets associated with increased antigen burden. We thus hypothesised that if reversion represents natural M.tb infection clearance then immune responses post-QFT reversion, if detectable, would be predominantly TSCM/TCM cells that have an IFN- independent cytokine expression profile and low T cell activation levels. Interestingly, QFT reversion was not associated with a decrease in CFP-10/ESAT-6-specific IFN-+ CD4 T cell responses detected by flow cytometry. Overall, CD4 T cell responses to CFP-10/ESAT-6 in reverters were of intermediate magnitude between non-converters and remotely infected individuals. These responses were very low in most reverters (regardless of QFT status), which may explain fluctuations around the QFT assay cut-off resulting in reversion of the test. In the reverters who had low but robustly detectable responses, CFP-10/ESAT-6-specific CD4 T cells showed low levels of M.tb-specific T cell activation, maintenance of both IFN- dependent and independent Th1 cytokine co-expressing profiles and a predominantly TTM/TE phenotype. Memory and functional profiles detected in reverters in response to M.tb lysate shared more characteristics with non-converters than persistently infected (QFT+) individuals. Based on these results, we conclude that QFT reverters represent a heterogenous population in the tuberculosis spectrum who may experience very low or no in vivo antigen exposure. Altogether, these results indicate that not everyone with a QFT+ test likely experiences ongoing in vivo M.tb exposure, as suggested by much lower T cell activation observed during remote M.tb infection and QFT reversion compared to recent M.tb infection. Whether ongoing in vivo antigen exposure is required to maintain memory responses against M.tb remains to be determined. It is possible that key features of T cell responses against M.tb, including magnitude and differentiation, are shaped by the antigen load experienced during primary infection, regardless of whether infection is subsequently cleared. Answering these questions is critical to inform the interpretation of the current immunodiagnostic assays and to determine who could be spared from preventive tuberculosis therapy. On the other hand, here we defined a biomarker of recent infection and tuberculosis disease, which could enable the provision of targeted treatment to those who would benefit the most.
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    Investigations of mycobacteria-specific memoryy/effector T cell responses in HIV infected children receiving antiretroviral therapy
    (2011) Tena-Coki, Nontobeko Gwendoline; Hanekom, Willem; Scriba, Thomas; Kampmann, Beate
    Human immunodeficiency virus (HIV) infected children are at greater risk of developing tuberculosis disease, and might benefit from vaccination with novel TB vaccines. However, little is known about the effect of HIV-infection on function and phenotype of T cell responses to mycobacterial antigens in children. This study compares both CD4 and CD8 T cell cytokine expression and memory phenotype in children, following in vitro stimulation with mycobacterial antigens, also contained in novel anti-TB vaccines that are currently undergoing clinical trials.
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    Optimisation of a flow cytometry antibody panel to detect BCG-induced innate responses in infants
    (2023) Mwambene, Temwa Dango; Scriba, Thomas
    The outcome of exposure to Mycobacterium tuberculosis is highly variable ranging from clearance, latency to a wide spectrum of subclinical and clinical tuberculosis (TB) disease. The underlying basis of the variable disease outcomes to mycobacterial exposure is largely unknown and is thought to involve a complex interplay between genetic variation in both host and pathogen. This is further convoluted by factors such as age, geography, coinfections including human immunodeficiency virus (HIV) and previous Bacillus Calmette–Guérin (BCG) vaccination. As it stands, other than inborn errors in key mycobacterial susceptibility genes both animal and human studies have not found many genetic polymorphisms that are strongly and reproducibly associated with increased mycobacterial disease susceptibility and/or BCG vaccine efficacy. Given the complexity of the interplay between the variability in disease susceptibility, BCG vaccine efficacy and human exposure to genetically diverse pathogens there is a need to study both host and pathogen genetic factors that contribute to increased TB disease susceptibility. This will facilitate rational design of more broadly efficacious interventions. This MSc project is nested in a project which aims to uncover the genetic factors that result in variable responses to BCG vaccination and in so doing, expand the basis of learning for novel TB vaccine candidates. The overall aim of this MSc was to optimise a flow cytometry panel to measure the BCG-induced innate immune responses in infants. The innate response to BCG was characterised by whole blood intracellular cytokine staining of banked, previously stimulated immune cells from 25, 10-week-old, HIV-uninfected, infants. The cohort is a subset of participants of a randomised control trial conducted in Worcester, South Africa. A 13-colour flow cytometry antibody panel was successfully optimised for enumerating and measuring cytokine and cell maturation marker expression by neutrophils, monocytes, pDCs, mDCs and T cells in response to BCG stimulation. Expression of cytokines (IFNg, IL-6, TNF) and maturation markers (CD40, PD-L1) by these cell subsets to BCG stimulation were quantified, allowing identification of single nucleotide polymorphisms that associate with variability in innate responses to BCG. Results from this MSc thus inform the bigger project which aims to determine genetic determinants of innate responses to BCG and TB disease risk.