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Clinical and Vaccine Immunology, June 2008, p. 916-924, Vol. 15, No. 6
1071-412X/08/$08.00+0     doi:10.1128/CVI.00056-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Efficient Testing of Large Pools of Mycobacterium tuberculosis RD1 Peptides and Identification of Major Antigens and Immunodominant Peptides Recognized by Human Th1 Cells

Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat 13110, Kuwait

Received 22 January 2008/ Returned for modification 13 March 2008/ Accepted 27 March 2008

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- 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.

Published ahead of print on 9 April 2008.

This article has been cited by other articles:

• Al-Attiyah, R., Mustafa, A. S. (2008). Characterization of Human Cellular Immune Responses to Novel Mycobacterium tuberculosis Antigens Encoded by Genomic Regions Absent in Mycobacterium bovis BCG. Infect. Immun. 76: 4190-4198 [Abstract] [Full Text]