Abstract
Tuberculosis (TB) continues to pose a serious global health threat, and the current vaccine, BCG, has variable efficacy. However, the development of a more effective vaccine is severely hampered by the lack of an immune correlate of protection. Candidate vaccines are currently evaluated using preclinical animal models, but experiments are long and costly and it is unclear whether the outcomes are predictive of efficacy in humans. Unlike measurements of single immunological parameters, mycobacterial growth inhibition assays (MGIAs) represent an unbiased functional approach which takes into account a range of immune mechanisms and their complex interactions. Such a controlled system offers the potential to evaluate vaccine efficacy and study mediators of protective immunity against Mycobacterium tuberculosis (M.tb). This review discusses the underlying principles and relative merits and limitations of the different published MGIAs, their demonstrated abilities to measure mycobacterial growth inhibition and vaccine efficacy, and what has been learned about the immune mechanisms involved.
Highlights
Tuberculosis (TB) continues to pose a serious global health threat, and the current vaccine, BCG, has variable efficacy
One alternative to measuring predefined individual parameters is the use of mycobacterial growth inhibition assays (MGIAs), which take into account a range of immune mechanisms and their complex interactions
Monocytes infected with M. microti and pulsed with stimulated lymphocytes for 2 h per day over 3 days Monocytes infected with M.tb in 96 well plate; autologous lymphocytes (PBL) added Monocytes cultured for 6 days overnight infection with BCG; lymphocytes cultured with antigen stimulation and added to monocytes Whole blood infected with BCG lux for 96 h; cells lysed and mycobacteria quantified by relative light units (RLU) Whole blood infected with M.tb for 72 h with 360° mixing; cells lysed and mycobacteria quantified by Bactec Mycobacteria Growth Indicator Tubes (MGIT) TTP Whole blood/peripheral blood mononuclear cells (PBMCs) infected with BCG for 96 h with 360° mixing; cells lysed and mycobacteria quantified by Bactec MGIT TTP Bone marrow macrophages cultured for 7 days; 2 h infection with M.tb addition of splenocytes
Summary
Work by Youmans et al demonstrated that splenocytes from immune mice produce secretory products, or ‘lymphokine’, upon stimulation with mycobacteria. In one of the first examples of an MGIA, addition of this lymphokine to mouse peritoneal macrophages enhanced inhibition of M.tb growth in vitro [15]. Lymphocytes were cultured for 72 h with antigen (trypsin-extracted soluble antigen/tuberculoprotein) to induce lymphokine production. Lymphocytes from immune donors were able to produce lymphokine upon stimulation, and macrophages incubated with this lymphokine showed enhanced inhibition of intracellular bacillary replication. The authors extended their studies to demonstrate the inhibitory effect of vitamin D3 on mycobacterial growth in macrophages [17]; a finding later independently confirmed [18]. A similar assay was used to assess inhibition of mycobacterial growth in murine peritoneal macrophages or human alveolar
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