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Clinical and Vaccine Immunology, October 2006, p. 1143-1154, Vol. 13, No. 10
1071-412X/06/$08.00+0     doi:10.1128/CVI.00127-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Specific and Randomly Derived Immunoactive Peptide Mimotopes of Mycobacterial Antigens

Bose Institute, Department of Microbiology, P1/12 C.I.T. Scheme VIIM, Kolkata 700054, India

Received 22 March 2006/ Returned for modification 4 May 2006/ Accepted 7 August 2006

The mycobacterial cell surface contains complex nonprotein antigens that are highly immunoactive in nature. However, these antigens are chemically heterogeneous and structurally complex, thereby limiting their applications. To identify their peptide mimotopes, phage-displayed peptide libraries Ph.D.-7 and Ph.D.-12 were panned on either defined template, monoclonal antibody (MAb) CS-35 against lipoarabinomannan (LAM), or a polyclonal rabbit immune serum reactive against whole cells of Mycobacterium bovis BCG. Panning on anti-LAM MAb CS-35 yielded two confirmed mimotopes of LAM, a 7-mer and a 12-mer, whereas panning on polyclonal serum yielded a large repertoire of mimotopes reactive against sera from BCG-immunized rabbits, one of which turned out to have the same sequence as the 7-mer LAM mimotope. The dissociation constant of the interaction between MAb CS-35 and a synthetic peptide corresponding to the 7-mer LAM mimotope was determined to be 7.55 µM. Dot blot assays were performed with peptides corresponding to the two LAM mimotopes to evaluate their diagnostic potential. Both peptides gave discernibly higher signals with a panel of tuberculosis (TB) patient sera than with sera from healthy controls. The peptides were also found to stimulate the release of tumor necrosis factor alpha and interleukin-12 cytokines in the J774A.1 cell line and primary bone marrow-derived macrophages, indicating that they may have immunomodulatory potential. The present study demonstrates that peptide mimotopes of known and unknown mycobacterial antigens could be isolated by using subtractive phage display techniques and that these peptides could have potential applications in areas such as TB diagnostics and immunotherapy.

Published ahead of print on 17 August 2006.