Whole Blood Assays to Identify Th1 Cell Antigens and Peptides Encoded by <i>Mycobacterium tuberculosis</i>-Specific RD1 Genes

Abstract

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. Materials and 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-γ (IFN-γ) 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-γ responses with whole blood from TB patients. However, the positive IFN-γ 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-γ responses, suggesting that these peptides represented the immunodominant Th1 cell epitopes of RD1 ORF6 and ORF7 in the patients tested. Conclusion: 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.

Characterization of Human Cellular Immune Responses to NovelMycobacterium tuberculosisAntigens Encoded by Genomic Regions Absent inMycobacterium bovisBCG

“…These results suggest that only RD1 contains immunodominant antigens. It has been shown that the RD1 genomic region of M. tuberculosis has three major T-cell antigens, i.e., PPE68, ESAT6, and CFP10 (18,28). When tested alone, ESAT6 and CFP10 induce strong antigen-induced proliferation and IFN-␥ responses in TB patients but weak responses in M. bovis BCG-vaccinated healthy subjects residing in Kuwait, which could be attributed to previous exposure to M. tuberculosis (2,3,4,26,32), whereas, PPE68 induces strong responses in both TB patients and M. bovis BCG-vaccinated healthy subjects (32).…”

HLA-Promiscuous Th1-Cell Reactivity of MPT64 (Rv1980c), a Major Secreted Antigen of <i>Mycobacterium tuberculosis</i>, in Healthy Subjects

“…The complex mycobacterial antigens used in this study were irradiated whole-cell M. tuberculosis H37Rv [18] , MTCF enriched for secreted antigens and purified M. tuberculosis cell walls [19] .…”

Comparative Evaluation of MPT83 (Rv2873) for T Helper-1 Cell Reactivity and Identification of HLA-Promiscuous Peptides in Mycobacterium bovis BCG-Vaccinated Healthy Subjects

“…The complex mycobacterial antigens used in this study were irradiated whole-cell M. tuberculosis H37Ra and M. bovis BCG (29,50), M. tuberculosis culture filtrate (MT-CF) enriched for secreted antigens, and purified M. tuberculosis cell walls (MT-CW) (48). MT-CF and MT-CW were kindly provided by J. T. Belisle (Colorado State University, Fort Collins, CO).…”