Transcriptomic blood responses to mycobacteria in a BCG challenge model
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ABSTRACT: Cattle were vaccinated at week 0 with the live attenuated M. bovis BCG SSI vaccine strain; some animals remain as non-vaccinated controls. After eight weeks animals were challenged intranodally with 108 BCG Tokyo cfu. Lymph nodes were harvested at week 11 and bacterial load in lymph nodes evaluated. Some BCG vaccinates had bacterial loads similar to those found in non-vaccinated animals (not-protected) and some animals had lower bacterial loads (protected) than non-vaccinated animals. Immune responses to mycobacteria were monitored in vitro by stimulation of whole blood with medium, purified protein derivative from M. bovis (PPD-B) or live BCG Tokyo longitudinally. Blood samples from 6 BCG-protected, 6 BCG-not-protected and 6 not-vaccinated. Challenged cattle stimulated with mycobacterial antigens or not were used to prepare RNA for RNAseq analysis at weeks 0 (vaccination), 8 (challenge), 9, 10 and 11. The outcome of this project would help not only in the selection of vaccine candidates but would also inform on the formulation of novel vaccines/vaccination strategies.
Project description:Micro RNA profiling was performed on in vitro PPDB and nil stimulated bovine PBMCs isolated from BCG vaccinated and unvaccinated cattle before and after challenge with M. bovis.
Project description:In the present study, we applied microarray technology to define a biosignature from the whole genome expression in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M. bovis. Further, Our aim was to define these markers to be detectable without in vitro antigenic challenge. After BCG vaccination, we defined a specific pulmonary gene expression signature related to the connective tissue development and function network that predicted vaccine success before M. bovis challenge. In addition, a Th17-related cytokine profile was found that correlated with vaccine-induced protective immunity following infection with virulent M. bovis in the lung as well as additional genes that were up-regulated in the spleens of vaccinated animals post-infection that was related to neutrophil biology and inflammation. This study has therefore prioritized both biomarkers predicting vaccination success before challenge and bio-signatures that are associated with protective immune responses that will be useful to evaluate future vaccine candidates. Two groups of 20 mice each were immunised by a single intradermal injection of 2 x 10 to 5 CFU of M. bovis BCG (Vaccinated), or Hanks buffered salt solution (HBSS) (Unvaccinated). Six weeks later 5 mice from each group were euthanized for immunological analyses and the remaining mice from each group were challenged with approx 600 CFU M. bovis via the intranasal route. At days 3 and 14 post challenge five mice per group were euthanized and spleens and lungs harvested.
Project description:The aim of this experiment was to investigate differential gene expression in splenocytes stimulated with BCG from naïve and BCG vaccinated mice. The differences between naïve and BCG vaccinated mice might indicate the mechanisms by which BCG vaccination confers an enhanced ability of splenocytes from BCG vaccinated mice to inhibit growth of BCG in splenocyte cultures as compared with splenocytes from naive animals.
Project description:The aim of this experiment was to investigate differential gene expression in splenocytes stimulated with BCG from naïve and BCG vaccinated mice. The differences between naïve and BCG vaccinated mice might indicate the mechanisms by which BCG vaccination confers an enhanced ability of splenocytes from BCG vaccinated mice to inhibit growth of BCG in splenocyte cultures as compared with splenocytes from naive animals. Splenocytes from 4 naïve and 4 BCG vaccinated female C57bl/6 mice were cultured for 12 hours with BCG (MOI 1:1) or without (unstimulated control). Each animal had a stimulated and an unstimulated sample hybridised to a beadchip.
Project description:In the present study, we applied microarray technology to define a biosignature from the whole genome expression in lung and spleen samples after BCG vaccination and M. bovis infection of BALB/c mice. The aims were two-fold, namely to define biosignatures that could predict vaccine success before challenge, and biomarker patterns that correlated with anamnestic protective responses following exposure to virulent M. bovis. Further, Our aim was to define these markers to be detectable without in vitro antigenic challenge. After BCG vaccination, we defined a specific pulmonary gene expression signature related to the connective tissue development and function network that predicted vaccine success before M. bovis challenge. In addition, a Th17-related cytokine profile was found that correlated with vaccine-induced protective immunity following infection with virulent M. bovis in the lung as well as additional genes that were up-regulated in the spleens of vaccinated animals post-infection that was related to neutrophil biology and inflammation. This study has therefore prioritized both biomarkers predicting vaccination success before challenge and bio-signatures that are associated with protective immune responses that will be useful to evaluate future vaccine candidates.
Project description:Gene expression profiles in PPD-stimulated and unstimulated peripherhal blood mononuclear cells isolated from chinese cynomolgus macaques before and after BCG vaccination and before and after M. tuberculosis challenge were compared in animals able to control TB infection and those that developed TB disease in order to identify potential correlates of protection and/or biomarkers of disease. 6 macaques received BCG vaccination prior to challenge and were able to control TB infection (vaccinated controllers). 3 unvaccinated macaques were also able to control TB infection after challenge (unvaccinated controllers) and 3 other unvaccinated macaques developed TB disease and reached humane endpoint criteria (unvaccinated progressors). PBMCs isolated at 8 and 18 weeks post BCG-vaccination from the vaccinated controllers and at 6 weeks post M.tb challenge and at 2 time-points when the animals were naive in all animals were stimulated with PPD. RNA was extracted from the cells and hybridised to and Agilent rhesus macaque GE microarray in a one-colour hybridisation. Gene expression post-vaccination and/or post-challenge was compared with expression before vaccination/challenge when the animals were naive.
Project description:Transcriptional profiling of splenic lymphocytes derived from vaccinated mice, and ex-vivo exposed to M.tb.-infected macrophages in culture. Splenocytes from mice innoculated with Mycobacterium bovis BCG Pasteur (PAS), or M. bovis BCG Copenhagen (SSI), or heat killed BCG SSI (HK), or uninfected control, were ex vivo co-cultured with mouse bone marrow macrophages previously infected with M. tb.
Project description:Despite wide scale vaccination with Mycobacterium bovis BCG, the prevalence of tuberculosis remains high, reflecting the global variable efficacy of this vaccine against adult pulmonary TB. Characterisation of different immune responses to M. tuberculosis and M. bovis BCG would increase understanding of pathology following M. tuberculosis infection or reactivation, and would facilitate the rational design of a new vaccine. Gene expression profiling was conducted on samples from diluted whole blood cultures from three healthy donors following incubation with live mycobacteria for six days. Approximately 8,000 gene entities were at least two-fold up- or down- regulated by the mycobacteria, and both mycobacteria induced similar expression changes in approximately 2,300 genes. Strikingly, many genes exhibited qualitatively different expression patterns, with over 1,000 genes up-regulated in response to M. bovis BCG but not changed by M. tuberculosis. Gene Ontology analysis revealed that the genes which failed to upregulate in M. tuberculosis-infected cultures included a large proportion of genes with lysosomal function. The inhibited up-regulation of expression of IFN-γ-inducible protein 30, acid phosphatase 2, cathepsin B and GM2 ganglioside activator was verified in samples from six biologically independent donors by qRT-PCR. The failure to up-regulate these genes in response to M. tuberculosis may constitute an immune evasion mechanism, preventing intracellular killing and antigen presentation. Blood from three healthy BCG-vaccinated donors was diluted with growth medium and incubated alone or with live M. tuberculosis (H37Rv), M. bovis BCG for 6 days. RNA samples were pooled before hybridisation.
Project description:This experiment explored the transcriptional response of human peripheral blood mononuclear cells (PBMC) isolated from BCG-vaccinated individuals following 6 days of in vitro stimulation with 2x10^5 cfu of different Bacillus Calmette-Guérin (BCG) strains or 100 ng/ml Mycobacterium tuberculosis-derived purified protein derivative (PPD). The BCG strains used were BCG Russia (Russian BCG-I sub-strain), BCG Japan (Tokyo 172 sub-strain), BCG Denmark (Danish 1331 sub-strain) & BCG Pasteur.
Project description:Analysis of PBMC from 10 week old infants vaccinated with BCG at birth. During follow-up 26 infants developed tuberculosis (TB) (non-protected by BCG) and 20 infants did not develop TB despite documented exposure (protected by BCG). PBMC were stimulated with media only or reconstituted BCG for 4 and 12 hours. Results provide insight into the mechanisms behind the failure of BCG to protect against disease.