Background: In the absence of a validated animal model and/or an immune correlate which predict vaccinemediated\nprotection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis\ncandidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed.\nMethods: We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine\nMycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit\nmycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-GuÃ?©rin\n(BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with\nMycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen.\nResults: Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3ââ?¬â??0.8 log10 after co-culture with\nmurine splenocytes from BCG vaccinated versus naÃ?¯ve C57BL/6 mice. BCG vaccination in our hands led to a reduction\nin bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1\nlog10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition.\nAn increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used,\ncompared to wild type controls, indicating that immune mechanisms may also be investigated using this assay.\nConclusions: We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess\nvaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of\nunderlying immune mechanisms.
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