The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays

Abstract

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays.

Tools for Assessing the Protective Efficacy of TB Vaccines in Humans: in vitro Mycobacterial Growth Inhibition Predicts Outcome of in vivo Mycobacterial Infection

“…Furthermore, we have identified an influence of hemoglobin (Hb) and iron on the direct MGIA (68). While variation in clinical parameters including Hb levels forms part of the overall picture for clinical cohort studies and whole blood is applicable in this context (29,69), it may confound measures of vaccine-induced control of mycobacterial growth and reduce sensitivity to detect a vaccine response (68).…”

The Cross-Species Mycobacterial Growth Inhibition Assay (MGIA) Project, 2010–2014

“…Using samples from the Oxford BCG vaccination study, a positive correlation was observed between mean corpuscular Hb levels and mycobacterial growth in the direct whole-blood MGIA ( 22 ). Experimental addition of Hb or ferric iron to PBMCs from both humans and nonhuman primates (NHPs) resulted in increased mycobacterial growth, an effect reversed by addition of the iron chelator deferoxamine.…”

Mycobacterial growth inhibition is associated with trained innate immunity

“…Development and optimization of the MGIA assay as it is currently used have been recently reviewed at length (10,23). MGIA assays have been used already for cells of human (9, 12, 24-26), nonhuman primate (26), and mouse (11,13,14) origin.…”

“…BCG-vaccinated mice have reduced Mtb loads following infection compared with nonvaccinated animals, and this is paralleled by their splenocytes' capacity to reduce mycobacterial outgrowth in MGIA (11,13), supporting the assay's biological validity. BCG vaccination has mostly been used in such studies, because BCG affords at least partial protection against severe manifestations of TB disease, and efficiently induces IFN-γ-producing T cells (9,11,13,14,25,26). Although many researchers have attempted to identify the cellular subset(s) responsible for mycobacterial growth control, their identity remains inconclusive, and to some extent even elusive.…”

“…Although many researchers have attempted to identify the cellular subset(s) responsible for mycobacterial growth control, their identity remains inconclusive, and to some extent even elusive. Associations have been found with monocyte/lymphocyte ratios (24), multifunctional CD4 + T cells (25), and iron metabolism (26). Increased iron concentrations, in particular in whole blood-based MGIA assays but also in PBMC assays, significantly promoted the in vitro growth capacity of mycobacteria, such that some caution should be applied to interpreting data from whole blood-based MGIA assays (26).…”