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...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2017-06-01
|
| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-02116-1 |
| _version_ | 1797233561574572032 |
|---|---|
| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-24T16:18:08Z |
| format | Article |
| id | doaj.art-e0ea4720731f40618c6dd121172296d2 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-24T16:18:08Z |
| publishDate | 2017-06-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-e0ea4720731f40618c6dd121172296d22024-03-31T11:21:48ZengNature PortfolioScientific Reports2045-23222017-06-017111110.1038/s41598-017-02116-1Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosisChristina Jensen0Line Lindebo Holm1Erik Svensson2Claus Aagaard3Morten Ruhwald4Department of Infectious Disease Immunology, Statens Serum InstitutDepartment of Infectious Disease Immunology, Statens Serum InstitutInternational Reference Laboratory of Mycobacteriology, Statens Serum InstitutDepartment of Infectious Disease Immunology, Statens Serum InstitutDepartment of Infectious Disease Immunology, Statens Serum InstitutAbstract 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.https://doi.org/10.1038/s41598-017-02116-1 |
| spellingShingle | Christina Jensen Line Lindebo Holm Erik Svensson Claus Aagaard Morten Ruhwald Optimisation of a murine splenocyte mycobacterial growth inhibition assay using virulent Mycobacterium tuberculosis Scientific Reports |
| title | 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_short | 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 |
| url | https://doi.org/10.1038/s41598-017-02116-1 |
| work_keys_str_mv | AT christinajensen optimisationofamurinesplenocytemycobacterialgrowthinhibitionassayusingvirulentmycobacteriumtuberculosis AT linelindeboholm optimisationofamurinesplenocytemycobacterialgrowthinhibitionassayusingvirulentmycobacteriumtuberculosis AT eriksvensson optimisationofamurinesplenocytemycobacterialgrowthinhibitionassayusingvirulentmycobacteriumtuberculosis AT clausaagaard optimisationofamurinesplenocytemycobacterialgrowthinhibitionassayusingvirulentmycobacteriumtuberculosis AT mortenruhwald optimisationofamurinesplenocytemycobacterialgrowthinhibitionassayusingvirulentmycobacteriumtuberculosis |