Abstract
In the absence of a validated correlate of protection or robust animal models for human tuberculosis, Mycobacterial growth inhibition assays (MGIAs) aim to assess vaccines ability to inhibit mycobacterial growth in-vitro. We optimised a reproducible murine splenocyte MGIA based on in-vitro infection with virulent Mycobacterium tuberculosis (M.tb) Erdman. We identified splenocyte viability as a problem in state-of-art MGIA protocols, which can be improved by simple changes in culture conditions (viability increase from 21% to 46% at last day of culture). The growth inhibitory potential in mice immunised with either BCG, H56:CAF01 or H56:CAF01 administered side-by-side with BCG was significantly better compared to placebo in all groups (0.3 log10 CFU [±0.2, p = 0.049], 0.5 [±0.2, p = 0.016] and 0.6 [±0.1, p = 0.0007], respectively) corresponding to the levels of in-vivo protection. Unexpectedly the CAF01 adjuvant control group also induced significant growth inhibition of 0.3 log10 CFU (±0.2, p = 0.047). Finally, we explored vaccine-associated T cell effector functions. Despite presence of high levels of vaccine-specific T cells, we found no increase in CD4+ T cell number or cytokine expression profile, nor a difference in cytokine levels in the supernatant after four days culture with or without M.tb. Spontaneous IFN-γ release correlated with growth inhibition levels (p = 0.02), however the cellular source was not found.
Highlights
Over the last two centuries, tuberculosis (TB) is estimated to have killed one billion people, and remains the world’s most lethal infectious disease[1]
Several variations of human and murine Mycobacterial growth inhibition assays (MGIAs) are described in the literature including assays based on human whole blood[7,8,9,10,11] or PBMCs8, 9, 12 and murine assays based on splenocytes[13,14,15] or pre-infected bone marrow derived macrophage target cells in splenocyte co-culture assays (BM/SP-MGIA)[16, 17]
Under the assumptions that Mycobacterium tuberculosis (M.tb) is an intracellular pathogen in vivo and in vitro and that cellular immunity is essential for host control of infection, we focused on the adaptive immune responses
Summary
Over the last two centuries, tuberculosis (TB) is estimated to have killed one billion people, and remains the world’s most lethal infectious disease[1]. Mycobacterial growth inhibition assays (MGIA) have been proposed as simple and unbiased tools to evaluate vaccine efficacy in vitro[5, 6]. Several of the murine MGIAs have demonstrated significant in vitro growth inhibition in a BCG vaccination model corresponding to in vivo protection in parallel challenge studies[14, 15, 17]. A standardised murine MGIA based on direct co-culturing of mouse splenocytes with BCG has been proposed as a robust and simpler version of the BM/SP-MGIA13, 15, 18 This protocol was optimised and qualified with particular emphasis on multiplicity of infection (MOI) for low assay variability and widest window of growth inhibition[15]. Instead of using BCG as the in vitro infectious organism as in the previously described murine splenocyte MGIAs13, 15, the virulent mycobacterial strain M.tb Erdman was used
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