Abstract
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.
Highlights
Understanding protective human immunity against mycobacteria is critical to developing and evaluating new vaccines against tuberculosis
Colony Forming Units (CFU) were counted using serial dilutions in sterile Phosphate Buffered Saline of 10 μL of the experimental sample plated onto solid culture medium after 21 days
Colony forming units and luminescence of BCG-GFP-LuxFO in liquid culture medium correlated with Spearman rank correlation coefficient r = 0.9714 (Figure 3)
Summary
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, and have the strongest evidence of BCG-inducible protection. Limited amounts of blood can be obtained for research purposes in paediatrics and there is a need for high-yield, low-volume, human immunology assays. Understanding paediatric immune responses is of particular importance as young children are the most susceptible to developing infection, disease, or dying following exposure, and the group for whom there is the strongest evidence of BCG-inducible protection against tuberculosis [11,12,13,14,15]. New methods need to maximise the scientific yield from small volumes of blood from children affected by M. tuberculosis who participate in research studies
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