Optimisation, harmonisation and standardisation of the direct mycobacterial growth inhibition assay using cryopreserved human peripheral blood mononuclear cells

Rachel Tanner, Steven G Smith, Krista E van Meijgaarden, Federico Giannoni, Morven Wilkie, Lucia Gabriele, Carla Palma, Hazel M Dockrell, Tom H M Ottenhoff, Helen McShane

Research output: Contribution to journalArticlepeer-review

Abstract

A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2 ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-γ) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be <5% coefficient of variation (CV), intermediate precision to be <20% CV, and inter-site reproducibility to be <30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development.

Original languageEnglish
Pages (from-to)1-10
Number of pages10
JournalJournal of Immunological Methods
Volume469
DOIs
Publication statusPublished - Jun 2019

Keywords

  • Bacteriological Techniques/standards
  • Cell Culture Techniques/standards
  • Cells, Cultured
  • Cryopreservation/standards
  • Drug Development/standards
  • Host-Pathogen Interactions
  • Humans
  • Interferon-gamma/metabolism
  • Interferon-gamma Release Tests/statistics & numerical data
  • Leukocytes, Mononuclear/immunology
  • Mycobacterium bovis/drug effects
  • Reproducibility of Results
  • Tuberculosis Vaccines/pharmacology

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