Browsing by Author "Nemes, Elisa"

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    Open Access
    Characterisation of Mycobacterium tuberculosis specific T cell immunity with HLA class II tetramers
    (2014) Dintwe, One Bridget; Scriba, Thomas J; Nemes, Elisa
    Tuberculosis (TB) remains a global health burden, with an estimated 1.3 million people dying from the disease in 2012. Protective immunity against TB is thought to depend on specific T cells. However, exactly which T cell characteristics are required for immunological protection is unknown. To gain a better understanding of M. tuberculosis (M.tb)-specific memory T cell immunity, we studied longevity and function of M.tb-specific memory T cells. We reasoned that such knowledge would facilitate rational vaccine design of a TB vaccine. We designed and developed a set of new HLA class II tetramers to perform in-depth studies of M.tb-specific CD4 T cell responses. We studied persons vaccinated with a novel TB vaccine, MVA85A, as well as persons naturally infected with M.tb. Antigen-specific CD4 T cells were detected with HLA class II tetramers and functional and phenotypic attributes of these T lymphocytes characterised by standard flow cytometric techniques. Comprehensive transcriptional analyses of M.tb-specific CD4 T cells, which were also sorted by FACS, were performed by microfluidic quantitative real-time PCR. Early after intradermal vaccination with MVA85A a large proportion of Ag85Aspecific CD4 T cells were highly activated, expressed skin homing markers and displayed an effector T cell phenotype. This effector response waned rapidly and gave way to antigen-specific central memory CD4 T cells with high proliferative potential, which we proposed may be desirable for protection. However, recent results from the first efficacy trial of MVA85A in infants suggested that these cells are not sufficient to enhance protection beyond that induced by BCG vaccination at birth. Further, we characterised surface marker expression and transcriptional signatures of a newly detected and described population of M.tb-specific CD4 T cells, that displayed a CD45RA+CCR7+CD27+ naïve-like T cell phenotype. We hypothesised that these unique M.tb-specific naïve-like CD4 T cells had a transcriptional profile distinct from truly naïve, central memory and effector bulk CD4 T cells, as well as other M.tb-specific memory CD4 T cell subsets. Gene expression of CFP10-specific naïve-like CD4 T cells reflected an mRNA profile that was very distinct from truly naïve bulk CD4 T cells. Rather, naïvelike CD4 T cells clustered with bulk effector CD4 T cells in unsupervised analysis methods such as hierarchical clustering and principle component analyses. Further analyses revealed that naïve-like CFP10-specific CD4 cells expressed mRNAs coding for effector cytokines, cytotoxic molecules and chemokine receptors consistent with effector memory T cells. However, the overall transcriptional profile was more similar to CFP10-specific central memory CD4 T cells than that of the effector CD4 T cells. We concluded that M.tb-specific naïve-like CD4 T cells may possess an ability to traffic to sites of infection or inflammation, where they may contribute to effector function. These hypotheses need confirmation on a protein level. The HLA class II tetramers developed in this thesis are valuabe tools for assessing direct ex vivo M.tb-specific CD4 T cell responses without activation and cell perturbation. Our findings contribute to a more comprehensive understanding of T cell immunity induced by vaccines and/or natural M.tb infection.
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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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    Correlates of risk of TB disease in infants with differential response to BCG vaccination
    (2014) Njikan, Samuel Ayaba; Hanekom, Willem A; Nemes, Elisa; Scriba, Thomas J
    Studying prospective immune correlates of risk of TB disease following BCG vaccination is an important first step towards determining correlates of protection against TB, which can be identified only in a placebo-controlled randomized controlled trial (RCT) of an effective vaccine. To study correlates of risk of TB disease, we collected and stored blood from healthy 10-week old infants vaccinated with BCG at birth. During two years of follow up, infants who developed lung TB were defined as cases, while those who did not develop TB disease were defined as controls. We measured Th1/Th17 cytokine production by BCG-specific T cells, release of pro- and anti-inflammatory mediators, cytotoxic T cell potential and proliferation in response to BCG as potential correlates of risk of TB disease but none of these outcomes were different between cases and controls. However, transcriptional profiling of PBMC revealed two clusters of infants and interestingly, the gene expression profiles from cases and controls in the two clusters were in opposite directions. Based on this, we hypothesised that analysing the two clusters of infants separately will allow discovery of correlates of risk of TB, which were absent when clustering was not taken into account.
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    Human myeloid cell and innate lymphocyte responses to mycobacterial vaccination or infection
    (2022) Murphy, Melissa; Nemes, Elisa; Scriba, Thomas J
    We investigated immune responses beyond conventional T cells in the context of BCG vaccination and tuberculosis disease.
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    Human newborn bacille Calmette–Guérin vaccination and risk of tuberculosis disease: a case-control study
    (2016) Fletcher, Helen A; Filali-Mouhim, Ali; Nemes, Elisa; Hawkridge, Anthony; Keyser, Alana; Njikan, Samuel; Hatherill, Mark; Scriba, Thomas J; Abel, Brian; Kagina, Benjamin M; Veldsman, Ashley; Agudelo, Nancy Marín; Kaplan, Gilla; Hussey, Gregory D; Sekaly, Rafick-Pierre; Hanekom, Willem A
    : An incomplete understanding of the immunological mechanisms underlying protection against tuberculosis (TB) hampers the development of new vaccines against TB. We aimed to define host correlates of prospective risk of TB disease following bacille Calmette-Guérin (BCG) vaccination. : In this study, 5,726 infants vaccinated with BCG at birth were enrolled. Host responses in blood collected at 10 weeks of age were compared between infants who developed pulmonary TB disease during 2 years of follow-up (cases) and those who remained healthy (controls). : Comprehensive gene expression and cellular and soluble marker analysis failed to identify a correlate of risk. We showed that distinct host responses after BCG vaccination may be the reason: two major clusters of gene expression, with different myeloid and lymphoid activation and inflammatory patterns, were evident when all infants were examined together. Cases from each cluster demonstrated distinct patterns of gene expression, which were confirmed by cellular assays. : Distinct patterns of host responses to Mycobacterium bovis BCG suggest that novel TB vaccines may also elicit distinct patterns of host responses. This diversity should be considered in future TB vaccine development.
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    Multivariate analysis of the immune response upon recent acquisition of Mycobacterium tuberculosis infection
    (2021) Lloyd, Tessa; Little, Francesca; Nemes, Elisa; Steigler, Pia
    Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis (M.tb), is the leading cause of mortality due to an infectious agent worldwide. Based on data from an adolescent cohort study carried out from May 2005 to February 2009, we studied and compared the immune responses of individuals from four cohorts that were defined based on their longitudinal QFT results: the recent QFT converters, the QFT reverters, the persistent QFT positives and negatives. Analysis was based on the integration of different arms of the immune response, including adaptive and “innaptive” responses, measured on the cohorts. COMPASS was used to filter the adaptive dataset and identify bioligically meaningful subsets, while, for the innaptive dataset, we came up with a novel filtering method. Once the datasets were integrated, they were standardized using variance stabilizing (vast) standardization and missing values were imputed using a multiple factor analysis (MFA)-based approach. We first set out to define a set of immune features that changed during recent M.tb infection. This was achieved by employing the kmlShape clustering algorithm to the recent QFT converters. We identified 55 cell subsets to either increase or decrease post-infection. When we assessed how the associations between these changed pre- and post-infection using correlation networks, we found no notable differences. By comparing the recent QFT converters and the persistent QFT positives, a blood-based biomarker to distinguish between recent and established infection, namely ESAT6/CFP10-specific expression of HLA-DR on total Th1 cells, was identified using elastic net (EN) models (average AUROC = 0.87). The discriminatory ability of this variable was confirmed using two tree-based models. Lastly, to assess whether the QFT reverters are a biologically distinct group of individuals, we compared them to the persistent QFT positive and QFT negative individuals using a Projection to Latent Space Discriminant Analysis (PLS-DA) model. The results indicated that reverters appeared more similar to QFT negative individuals rather than QFT positive. Hence, QFT reversion may be associated with clearance of M.tb infection. Immune signatures associated with recent infection could be used to refine end-points of clinical trials testing vaccine efficacy against acquisition of M.tb infection, while immune signatures associated with QFT reversion could be tested as correlates of protection from M.tb infection.
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    Outcome selection in longitudinal analysis of immunological data
    (2025) Holcroft, Shannon; Little, Francesca; Nemes, Elisa
    Immunological research often compares subgroups defined by exposure variables known (or hypothesised) to influence continuous immune responses. Many immune outcomes are measured over time, often in a small number of patients. Effective outcome selection ensures that research focuses on immune outcomes with the strongest signals for subgroup differences. This dissertation explores an outcome selection technique for longitudinal immunological data, addressing current methodological limitations and proposing improvements. The approach integrates statistical modelling with dimension reduction to identify immune outcomes with the most evidence for subgroup differences. By focusing on these subsets, fewer statistical hypotheses are tested simultaneously, preserving power when stricter significance thresholds are applied to reduce type-I error inflation. The dissertation examines the suitability of different longitudinal modelling frameworks. Generalised linear mixed-effects models are better suited to the characteristics of immunological data and research than linear mixed-effects models. Two dimension reduction techniques are compared: principal component analysis (PCA) and hierarchical cluster analysis (HCA) followed by PCA. PCA identifies the largest sources of variance across all outcomes, while HCA followed by PCA identifies variance within groups of similar outcomes. These techniques influence the definition of families of tests for false discovery rate (FDR) corrections. When outcomes are selected via PCA-only dimension reduction, more tests are performed simultaneously and require correction. It was hypothesised that HCA followed by PCA would yield more significant discoveries after FDR control. However, fewer simultaneous comparisons did not reliably correspond with more statistically significant discoveries. The methodology was applied to a dataset from the South African Tuberculosis Vaccine Initiative (SATVI), focusing on 33 immune outcomes and three exposures: MVA85A priming, maternal Mycobacterium tuberculosis sensitisation (measured by a positive QuantiFERONTB Gold test), and combinations of feeding practices and cotrimoxazole treatment. The analysis shows that different dimension reduction techniques lead to different outcome selections and families of tests, emphasising the need to align analysis objectives with outcome selection techniques. This dissertation contributes to outcome selection methodology in high-dimensional, longitudinal settings, with broader applications in biomedical research.
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    Statistical modelling to determine the influence of vaccine dose and prior Mycobacterium tuberculosis exposure on antigen-specific T cell responses
    (2024) Williams, Kelly; Little, Francesca; Nemes, Elisa; Gela , Anele
    This dissertation investigates the effects of two subunit vaccines H1:IC31 and H56:IC31 as well as prior M.tb sensitization on the immune responses of three cohorts of South African adolescents and adults. The primary outcomes are frequencies of antigen-specific CD4 T cells expressing different combinations of immunological markers over three time points. Two M.tb antigens are investigated: Ag85B and ESAT-6. The dissertation compares the results produced by the standard procedures that would typically be employed in the immunology research community to investigate these aims with the results produced by employing a mixed effect modelling approach. Not only is it of interest to investigate whether the results agree, but also to investigate the difference in inference that one can make and whether the mixed effect modelling approach is able to provide greater insight into the data. Methods typically employed by the immunology community that are used in this thesis are non-parametric pair-wise tests and the data analysis pipelines mixture models for single-cell assays (MIMOSA) and combinatorial polyfunctionality analysis of single cells (COMPASS). For the mixed effect modelling approach, generalized linear mixed effect models with various hierarchical structures as well as latent variable models are employed. Results suggest that 5 μg of the vaccine induces the strongest immune response. The mixed effect modelling approach showed good potential in terms of depth of analysis and ease of interpretation, however many model assumptions were violated making inference difficult. The standard approaches where much more cumbersome to implement and interpret and resulted in significant multiple testing concerns.