Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis

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...

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Autores principales:	Christina Jensen, Line Lindebo Holm, Erik Svensson, Claus Aagaard, Morten Ruhwald
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e0ea4720731f40618c6dd121172296d2
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spelling	oai:doaj.org-article:e0ea4720731f40618c6dd121172296d22021-12-02T16:08:09ZOptimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis10.1038/s41598-017-02116-12045-2322https://doaj.org/article/e0ea4720731f40618c6dd121172296d22017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-02116-1https://doaj.org/toc/2045-2322Abstract 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.Christina JensenLine Lindebo HolmErik SvenssonClaus AagaardMorten RuhwaldNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-11 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Christina Jensen Line Lindebo Holm Erik Svensson Claus Aagaard Morten Ruhwald Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis
description	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.
format	article
author	Christina Jensen Line Lindebo Holm Erik Svensson Claus Aagaard Morten Ruhwald
author_facet	Christina Jensen Line Lindebo Holm Erik Svensson Claus Aagaard Morten Ruhwald
author_sort	Christina Jensen
title	Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis
title_short	Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis
title_full	Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis
title_fullStr	Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis
title_full_unstemmed	Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis
title_sort	optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent mycobacterium tuberculosis
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/e0ea4720731f40618c6dd121172296d2
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Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis

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