Efficient Testing of Pools of Large Numbers of Peptides covering 12 Open Reading Frames of M. Tuberculosis RD1 and Identification of Major Antigens and Immunodominant Peptides Recognized by HumanTh1 Cells

Department of Microbiology, Faculty of Medicine, Kuwait University, PO Box 24923, Safat 13110, Kuwait

^* To whom correspondence should be addressed. Email: abusalim{at}hsc.kuniv.edu.kw.

	Abstract

Comparative genomics has identified several RDs of Mycobacterium tuberculosis deleted/absent in M. 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 (PBMC) obtained from pulmonary TB patients and M. bovis BCG-vaccinated healthy subjects in Th1 cell assays, i. e. 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 proportion of donors, but PPE68, CFP10 and ESAT6 induced strong responses in TB patients and PPE68 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, i.e. 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 10 aa crossreactive sequence 127-FFGINTIPIA-136.