An Auto-luminescent Fluorescent BCG Whole Blood Assay to Enable Evaluation of Paediatric Mycobacterial Responses Using Minimal Blood Volumes
Introduction: Understanding protective human immunity against mycobacteria is critical to developing and evaluating new vaccines against tuberculosis. Children are the most susceptible population to infection, disease, and death from tuberculosis, but also have the strongest evidence of BCG-inducibl...
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doaj-a723b0a8fefe47b4940111b4489223152020-11-25T02:50:07ZengFrontiers Media S.A.Frontiers in Pediatrics2296-23602019-04-01710.3389/fped.2019.00151431822An Auto-luminescent Fluorescent BCG Whole Blood Assay to Enable Evaluation of Paediatric Mycobacterial Responses Using Minimal Blood VolumesRobindra Basu Roy0Robindra Basu Roy1Basil Sambou2Iria Uhía3Sophie Roetynck4Brian D. Robertson5Beate Kampmann6Beate Kampmann7Beate Kampmann8Department of Paediatrics, Centre for International Child Health, Imperial College London, London, United KingdomVaccines & Immunity Theme, MRC Unit The Gambia at the London School of Hygiene and Tropical Medicine (LSHTM), Banjul, The GambiaVaccines & Immunity Theme, MRC Unit The Gambia at the London School of Hygiene and Tropical Medicine (LSHTM), Banjul, The GambiaMRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United KingdomVaccines & Immunity Theme, MRC Unit The Gambia at the London School of Hygiene and Tropical Medicine (LSHTM), Banjul, The GambiaMRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United KingdomDepartment of Paediatrics, Centre for International Child Health, Imperial College London, London, United KingdomVaccines & Immunity Theme, MRC Unit The Gambia at the London School of Hygiene and Tropical Medicine (LSHTM), Banjul, The GambiaFaculty of Infectious and Tropical Diseases, The Vaccine Centre, London School of Hygiene and Tropical Medicine, London, United KingdomIntroduction: Understanding protective human immunity against mycobacteria is critical to developing and evaluating new vaccines against tuberculosis. Children are the most susceptible population to infection, disease, and death from tuberculosis, but also have the strongest evidence of BCG-inducible protection. Limited amounts of blood can be obtained for research purposes in paediatrics and therefore there is a need for high-yield, low-volume, human immunology assays.Methods: We transformed BCG Danish with plasmids encoding luciferase full operon derived from Photorhabdus luminescens together with Green Fluorescent Protein and antibiotic selection markers. We characterised the luminescent and fluorescent properties of this recombinant BCG strain (BCG-GFP-LuxFO) using a luminometer and flow cytometry and developed a paediatric whole blood in vitro infection model.Results: Luminescence of BCG-GFP-LuxFO correlated with optical density (Spearman Rank Correlation coefficient r = 0.985, p < 0.0001) and colony forming units (CFUs) in liquid culture medium (r = 0.971, p < 0.0001). Fluorescence of BCG-GFP-LuxFO in paediatric whole blood was confirmed by flow cytometry in granulocytes and monocytes 1 h following infection. Luminescence of BCG-GFP-LuxFO in whole blood corresponded with CFUs (r = 0.7123, p < 0.0001).Conclusion: The BCG-GFP-LuxFO assay requires 225 μL whole blood per sample, from which serial luminescence measurements can be obtained, together with biochemical analysis of supernatants and cellular assay applications using its fluorescent properties. This offers the opportunity to study human-mycobacterial interactions using multiple experimental modalities with only minimal blood volumes. It is therefore a valuable method for investigating paediatric immunity to tuberculosis.https://www.frontiersin.org/article/10.3389/fped.2019.00151/fulltuberculosispaediatricBCGimmunologymycobacterial growth inhibition assay |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Robindra Basu Roy Robindra Basu Roy Basil Sambou Iria Uhía Sophie Roetynck Brian D. Robertson Beate Kampmann Beate Kampmann Beate Kampmann |
spellingShingle |
Robindra Basu Roy Robindra Basu Roy Basil Sambou Iria Uhía Sophie Roetynck Brian D. Robertson Beate Kampmann Beate Kampmann Beate Kampmann An Auto-luminescent Fluorescent BCG Whole Blood Assay to Enable Evaluation of Paediatric Mycobacterial Responses Using Minimal Blood Volumes Frontiers in Pediatrics tuberculosis paediatric BCG immunology mycobacterial growth inhibition assay |
author_facet |
Robindra Basu Roy Robindra Basu Roy Basil Sambou Iria Uhía Sophie Roetynck Brian D. Robertson Beate Kampmann Beate Kampmann Beate Kampmann |
author_sort |
Robindra Basu Roy |
title |
An Auto-luminescent Fluorescent BCG Whole Blood Assay to Enable Evaluation of Paediatric Mycobacterial Responses Using Minimal Blood Volumes |
title_short |
An Auto-luminescent Fluorescent BCG Whole Blood Assay to Enable Evaluation of Paediatric Mycobacterial Responses Using Minimal Blood Volumes |
title_full |
An Auto-luminescent Fluorescent BCG Whole Blood Assay to Enable Evaluation of Paediatric Mycobacterial Responses Using Minimal Blood Volumes |
title_fullStr |
An Auto-luminescent Fluorescent BCG Whole Blood Assay to Enable Evaluation of Paediatric Mycobacterial Responses Using Minimal Blood Volumes |
title_full_unstemmed |
An Auto-luminescent Fluorescent BCG Whole Blood Assay to Enable Evaluation of Paediatric Mycobacterial Responses Using Minimal Blood Volumes |
title_sort |
auto-luminescent fluorescent bcg whole blood assay to enable evaluation of paediatric mycobacterial responses using minimal blood volumes |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Pediatrics |
issn |
2296-2360 |
publishDate |
2019-04-01 |
description |
Introduction: Understanding protective human immunity against mycobacteria is critical to developing and evaluating new vaccines against tuberculosis. Children are the most susceptible population to infection, disease, and death from tuberculosis, but also have the strongest evidence of BCG-inducible protection. Limited amounts of blood can be obtained for research purposes in paediatrics and therefore there is a need for high-yield, low-volume, human immunology assays.Methods: We transformed BCG Danish with plasmids encoding luciferase full operon derived from Photorhabdus luminescens together with Green Fluorescent Protein and antibiotic selection markers. We characterised the luminescent and fluorescent properties of this recombinant BCG strain (BCG-GFP-LuxFO) using a luminometer and flow cytometry and developed a paediatric whole blood in vitro infection model.Results: Luminescence of BCG-GFP-LuxFO correlated with optical density (Spearman Rank Correlation coefficient r = 0.985, p < 0.0001) and colony forming units (CFUs) in liquid culture medium (r = 0.971, p < 0.0001). Fluorescence of BCG-GFP-LuxFO in paediatric whole blood was confirmed by flow cytometry in granulocytes and monocytes 1 h following infection. Luminescence of BCG-GFP-LuxFO in whole blood corresponded with CFUs (r = 0.7123, p < 0.0001).Conclusion: The BCG-GFP-LuxFO assay requires 225 μL whole blood per sample, from which serial luminescence measurements can be obtained, together with biochemical analysis of supernatants and cellular assay applications using its fluorescent properties. This offers the opportunity to study human-mycobacterial interactions using multiple experimental modalities with only minimal blood volumes. It is therefore a valuable method for investigating paediatric immunity to tuberculosis. |
topic |
tuberculosis paediatric BCG immunology mycobacterial growth inhibition assay |
url |
https://www.frontiersin.org/article/10.3389/fped.2019.00151/full |
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