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Transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with BCG at birth.

Fletcher HA, Keyser A, Bowmaker M, Sayles PC, Kaplan G, Hussey G, Hill AV, Hanekom WA - BMC Med Genomics (2009)

Bottom Line: We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation.In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted.Interestingly, more leukocyte genes were down-regulated than up-regulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jenner Institute, ORCRB, University of Oxford, Churchill Hospital, Oxford, OX3 7DQ, UK. helen.fletcher@ndm.ox.ac.uk

ABSTRACT

Background: Novel tuberculosis (TB) vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG). Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult.

Methods: To better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD)-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix(R) HumanRef-8 Expression BeadChip (Illumina) to measure expression of >16,000 genes.

Results: We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, including PPAR-gamma, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs), including integrin alpha M (ITGAM), which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated.

Conclusion: Our results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.

No MeSH data available.


Related in: MedlinePlus

Comparison of expression of highly and moderately expressed genes using DNA microarray and real time RT-PCR. Gene expression shown is that in relation to media, for BCG (A), and PPD (B). Microarray analysis involved 5 infants, whereas real time RT-PCR analysis involved these 5 infants plus an additional 10 infants (total n = 15). Box plots represent median plus 95th, 75th, 50th and 25th percentiles.
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Figure 4: Comparison of expression of highly and moderately expressed genes using DNA microarray and real time RT-PCR. Gene expression shown is that in relation to media, for BCG (A), and PPD (B). Microarray analysis involved 5 infants, whereas real time RT-PCR analysis involved these 5 infants plus an additional 10 infants (total n = 15). Box plots represent median plus 95th, 75th, 50th and 25th percentiles.

Mentions: To confirm the expression patterns of genes identified using the array, real time RT-PCR for specific genes was performed using total RNA from the 5 infants used for array analysis, and using RNA from 10 additional 10-week old infants, routinely vaccinated with BCG at birth (total of 15 infants for RT-PCR analysis). PBMC from the latter infants were processed identically to PBMC from infants whose RNA was examined in the array study. Primers were designed against selected transcripts and the copy number of each gene relative to the housekeeping gene HPRT was determined. There was concordance between DNA microarray and real-time RT-PCR results regarding expression of both highly and moderately expressed genes in response to both BCG (Figure 4A) and PPD (Figure 4B). When the median array expression from the 5 infants selected for array analysis was compared with the median RT-PCR expression from the total cohort of 15 infants, a significant correlation was found following both PPD (r = 0.806, p = 0.005, Spearman's test) and BCG stimulation (r = 0.697, p = 0.025, Spearman's test). Although the magnitude of the response was greater to BCG than PPD, the variability of response to stimulation with BCG was also greater.


Transcriptional profiling of mycobacterial antigen-induced responses in infants vaccinated with BCG at birth.

Fletcher HA, Keyser A, Bowmaker M, Sayles PC, Kaplan G, Hussey G, Hill AV, Hanekom WA - BMC Med Genomics (2009)

Comparison of expression of highly and moderately expressed genes using DNA microarray and real time RT-PCR. Gene expression shown is that in relation to media, for BCG (A), and PPD (B). Microarray analysis involved 5 infants, whereas real time RT-PCR analysis involved these 5 infants plus an additional 10 infants (total n = 15). Box plots represent median plus 95th, 75th, 50th and 25th percentiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654906&req=5

Figure 4: Comparison of expression of highly and moderately expressed genes using DNA microarray and real time RT-PCR. Gene expression shown is that in relation to media, for BCG (A), and PPD (B). Microarray analysis involved 5 infants, whereas real time RT-PCR analysis involved these 5 infants plus an additional 10 infants (total n = 15). Box plots represent median plus 95th, 75th, 50th and 25th percentiles.
Mentions: To confirm the expression patterns of genes identified using the array, real time RT-PCR for specific genes was performed using total RNA from the 5 infants used for array analysis, and using RNA from 10 additional 10-week old infants, routinely vaccinated with BCG at birth (total of 15 infants for RT-PCR analysis). PBMC from the latter infants were processed identically to PBMC from infants whose RNA was examined in the array study. Primers were designed against selected transcripts and the copy number of each gene relative to the housekeeping gene HPRT was determined. There was concordance between DNA microarray and real-time RT-PCR results regarding expression of both highly and moderately expressed genes in response to both BCG (Figure 4A) and PPD (Figure 4B). When the median array expression from the 5 infants selected for array analysis was compared with the median RT-PCR expression from the total cohort of 15 infants, a significant correlation was found following both PPD (r = 0.806, p = 0.005, Spearman's test) and BCG stimulation (r = 0.697, p = 0.025, Spearman's test). Although the magnitude of the response was greater to BCG than PPD, the variability of response to stimulation with BCG was also greater.

Bottom Line: We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation.In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted.Interestingly, more leukocyte genes were down-regulated than up-regulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Jenner Institute, ORCRB, University of Oxford, Churchill Hospital, Oxford, OX3 7DQ, UK. helen.fletcher@ndm.ox.ac.uk

ABSTRACT

Background: Novel tuberculosis (TB) vaccines recently tested in humans have been designed to boost immunity induced by the current vaccine, Mycobacterium bovis Bacille Calmette-Guérin (BCG). Because BCG vaccination is used extensively in infants, this population group is likely to be the first in which efficacy trials of new vaccines will be conducted. However, our understanding of the complexity of immunity to BCG in infants is inadequate, making interpretation of vaccine-induced immune responses difficult.

Methods: To better understand BCG-induced immunity, we performed gene expression profiling in five 10-week old infants routinely vaccinated with BCG at birth. RNA was extracted from 12 hour BCG-stimulated or purified protein derivative of tuberculin (PPD)-stimulated PBMC, isolated from neonatal blood collected 10 weeks after vaccination. RNA was hybridised to the Sentrix(R) HumanRef-8 Expression BeadChip (Illumina) to measure expression of >16,000 genes.

Results: We found that ex vivo stimulation of PBMC with PPD and BCG induced largely similar gene expression profiles, except that BCG induced greater macrophage activation. The peroxisome proliferator-activated receptor (PPAR) signaling pathway, including PPAR-gamma, involved in activation of the alternative, anti-inflammatory macrophage response was down-regulated following stimulation with both antigens. In contrast, up-regulation of genes associated with the classic, pro-inflammatory macrophage response was noted. Further analysis revealed a decrease in the expression of cell adhesion molecules (CAMs), including integrin alpha M (ITGAM), which is known to be important for entry of mycobacteria into the macrophage. Interestingly, more leukocyte genes were down-regulated than up-regulated.

Conclusion: Our results suggest that a combination of suppressed and up-regulated genes may be key in determining development of protective immunity to TB induced by vaccination with BCG.

No MeSH data available.


Related in: MedlinePlus