HLA-DR binding prediction and experimental evaluation of T-cell epitopes of mycolyl transferase 85B (Ag85B), a major secreted antigen of Mycobacterium tuberculosis.
OBJECTIVE
To identify T-cell epitopes of Ag85B by analysis of its sequence for prediction to bind HLA-DR alleles and evaluate the predicted peptides for recognition by T cells in antigen-induced proliferation assays.


METHODS
The complete sequence of Ag85B was analyzed for HLA-DR binding prediction to 51 HLA-DR alleles by using a virtual matrix-based prediction program (ProPred). Synthetic peptides covering the sequence of mature Ag85B were also analyzed for binding to HLA-DR alleles, and evaluated for recognition in antigen-induced proliferation assays with Ag85B-specific T-cell lines established from the peripheral blood mononuclear cells of 10 HLA-DR-heterogeneous tuberculosis patients.


RESULTS
The ProPred analysis of the full-length Ag85B (325 aa), signal peptide (40 aa) and the mature protein (285 aa) predicted their binding to 100, 76 and 98% of the 51 HLA-DR alleles, respectively. The analysis of 31 synthetic peptides for binding to HLA-DR alleles showed that 4 of them could bind >50% HLA-DR alleles, and were considered promiscuous. Testing of Ag85B-specific T-cell lines with synthetic peptides showed that all of the T-cell lines responded to one or more peptides of Ag85B, and 9 of the 10 cell lines responded to one or more of the four peptides considered promiscuous for binding to HLA-DR alleles.


CONCLUSIONS
The ProPred program was useful in predicting the HLA-DR alleles binding regions of Ag85B and identifying the promiscuous peptides recognized by T cells.
Mustafa
Abu S
AS
Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, Kuwait. abusalim@hsc.edu.kw
Abal
Adnan T
AT
Shaban
Fatma
F
El-Shamy
Abdulsalam M
AM
Amoudy
Hanady A
HA
D000217:Acyltransferases / Q000378:metabolism*; D000483:Alleles; D000942:Antigens, Bacterial / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D018984:Epitopes, T-Lymphocyte; D006684:HLA-DR Antigens; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D013601:T-Lymphocytes / Q000276:immunology*
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D018984:Epitopes, T-Lymphocyte; D006684:HLA-DR Antigens; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis