Whole blood assays to identify Th1 cell antigens and peptides encoded by Mycobacterium tuberculosis-specific RD1 genes.
OBJECTIVE
To identify Th1 cell-stimulating antigens/peptides encoded by the genes predicted in the Mycobacterium tuberculosis-specific genomic region of difference (RD)1, deleted in Mycobacterium bovis Bacille Calmette-Guérin(BCG), by using synthetic peptides and whole blood from tuberculosis (TB) patients.


METHODS
Heparinized peripheral blood was obtained from culture-proven pulmonary TB patients (n = 16) attending the Chest Disease Hospital, Kuwait. Whole blood was diluted with tissue culture medium RPMI-1640 and tested for Th1 cell stimulation using antigen-induced proliferation and interferon-gamma (IFN-gamma) secretion assays. The antigens included a peptide pool of 220 peptides covering the sequence of 12 open reading frames (ORFs) of RD1 (RD1(mix)), peptide pools of RD1 ORF5 (ORF5(mix)), ORF6 (ORF6(mix)) and ORF7 (ORF7(mix)), and individual peptides of ORF6 (P6.1-P6.6) and ORF7 (P7.1-P7.6). M. tuberculosis culture filtrate, cell walls and whole-cell M. bovis BCG were used as complex mycobacterial antigens. The results obtained with different antigens and peptides were statistically analyzed for significant differences using Z test.


RESULTS
The complex mycobacterial antigens (culture filtrate, cell walls and M.bovis BCG) and RD1(mix) induced comparable (p > 0.05) positive antigen-induced proliferation and IFN-gamma responses with whole blood from TB patients. However, the positive IFN-gamma responses induced by ORF6(mix) and ORF7(mix) were higher than ORF5(mix). Among the individual peptides, P6.4 and P7.1 of ORF6 and ORF7, respectively, induced the highest IFN-gamma responses, suggesting that these peptides represented the immunodominant Th1 cell epitopes of RD1 ORF6 and ORF7 in the patients tested.


CONCLUSIONS
The whole blood assays with synthetic peptides are useful to identify Th1 cell antigens/peptides encoded by genes located in M. tuberculosis-specific genomic regions.
Hanif
Shumaila N M
SN
Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait. shumaila_nida@yahoo.co.in
El-Shammy
Abdulsalam M
AM
Al-Attiyah
Rajaa
R
Mustafa
Abu S
AS
D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D001681:Biological Assay; D005798:Genes, Bacterial; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000276:immunology; D010455:Peptides / Q000235:genetics*; D010865:Pilot Projects; D018417:Th1 Cells / Q000276:immunology*
D000942:Antigens, Bacterial; D010455:Peptides; D007371:Interferon-gamma