Evaluation of T-cell responses to peptides and lipopeptides with MHC classI binding motifs derived from the amino acid sequence of the19-kDa lipoprotein of Mycobacterium tuberculosis

Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycoba cterium tuberculosis were identified by the use of lipopeptides and their c ytokine profile studied. Selection of candidate CTL epitopes was based on s ynthetic peptides derived from the amino acid sequence of the 19-kDa lipopr otein showing major histocompatibility complex class I (MHC-I) binding moti fs (H-2D(b) and H-2L(d)). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studie s were performed with peptides, in which the anchor amino acid of the H-2D( b) MHC-I motif was replaced by alanine. Three out of five peptides with H-2 D(b) or H-2Ld binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D(b) molecules on RMA-S cells. Replaceme nt of the anchor amino acid residues of the H-2D(b) MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed m ore to binding of peptide to H-2D(b) molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with inco mplete Freund's adjuvant (IFA), induced CTL responses in C57BL/6 (H-2(b)) m ice. These CTLs could recognize the naturally processed antigen, i.e. the 1 9-kDa antigen protein produced and processed by the EX-19 cell line. The ca pacity of the various lipopeptides to induce CTL correlated well with the a bility of the (lipo)peptide to up-regulate and to stabilize H-2D(b) molecul es. Lipopeptide LP19c primed spleen cells showed a T helper type one profil e after in vitro stimulation with P19c and P19d 19 kDa peptides. The approa ch to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subun it tuberculosis vaccines. (C) 2000 Elsevier Science Ltd. All rights reserve d.