Efficient testing of large pools of Mycobacterium tuberculosis RD1 peptides and identification of major antigens and immunodominant peptides recognized by human Th1 cells.
Comparative genomics has identified several regions of difference (RDs) of Mycobacterium tuberculosis that are deleted or absent in Mycobacterium bovis BCG vaccines. To determine their relevance for diagnostic and vaccine applications, it is imperative that efficient methods are developed to test the encoded proteins for immunological reactivity. In this study, we have used 220 synthetic peptides covering sequences of 12 open reading frames (ORFs) of RD1 and tested them as a single pool (RD1(pool)) with peripheral blood mononuclear cells obtained from pulmonary tuberculosis (TB) patients and M. bovis BCG-vaccinated healthy subjects in Th1 cell assays that measure antigen-induced proliferation and IFN-gamma secretion. The results showed that RD1(pool) induced strong responses in both TB patients and BCG-vaccinated healthy subjects. The subsequent testing of peptide pools of individual ORFs revealed that all ORFs induced positive responses in a portion of donors, but PPE68, CFP10, and ESAT6 induced strong responses in TB patients and PPE68 induced strong responses in BCG-vaccinated healthy subjects. In addition, HLA-DR and -DQ typing of donors and HLA-DR binding prediction analysis of proteins suggested HLA-promiscuous presentation of PPE68, CFP10, and ESAT6. Further testing of individual peptides showed that a single peptide of PPE68 (121-VLTATNFFGINTIPIALTEMDYFIR-145) was immunodominant. The search for sequence homology revealed that a part of this peptide, 124-ATNFFGINTIPIAL-137, was present in several PPE family proteins of M. tuberculosis and M. bovis BCG vaccines. Further experiments limited the promiscuous and immunodominant epitope region to the 10-amino-acid cross-reactive sequence 127-FFGINTIPIA-136.
Mustafa
Abu S
AS
Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait. abusalim@hsc.edu.kw
Al-Attiyah
Raja'a
R
Hanif
Sumaila N M
SN
Shaban
Fatema A
FA
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000276:immunology* / Q000378:metabolism; D001500:BCG Vaccine / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology* / Q000378:metabolism; D049109:Cell Proliferation; D000939:Epitopes / Q000276:immunology; D006684:HLA-DR Antigens / Q000378:metabolism; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000235:genetics / Q000276:immunology; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D016366:Open Reading Frames; D010455:Peptides / Q000276:immunology* / Q000378:metabolism; D018417:Th1 Cells / Q000276:immunology* / Q000378:metabolism; D014397:Tuberculosis, Pulmonary / Q000276:immunology*
D000942:Antigens, Bacterial; D001500:BCG Vaccine; D001426:Bacterial Proteins; D000939:Epitopes; D006684:HLA-DR Antigens; D016056:Immunodominant Epitopes; C528814:PPE68 protein, Mycobacterium tuberculosis; D010455:Peptides; D007371:Interferon-gamma