Synthesis and in vitro T-cell immunogenicity of conjugates with dual specificity: attachment of epitope peptides of 16 and 38 kDa proteins from Mycobacterium tuberculosis to branched polypeptide.

T-cell epitope containing peptides covalently attached to macromolecular carriers can be considered as synthetic immunogens for the development of skin-test diagnostics and of vaccines. As a carrier, an amphoteric branched chain polypeptide, poly[Lys-(Glui-DL-Alam)] (EAK) with poly(L-lysine) backbone has been used. This polypeptide with free alpha-amino and gamma-carboxyl groups at the end of the side chains was conjugated with peptides representing two immunodominant regions of the 16 and 38 kDa proteins of Mycobacterium tuberculosis, respectively. Peptide C91SEFAYGSFVRTVSLPVGADE110 was elongated by Cys at the N-terminal and attached to the carrier containing protected SH groups to form disulfide bridges. Peptide 65FNLWGPAFHERYPNVTITA83 was conjugated to the 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) modified and acetylated EAK by introducing amide bond between the free alpha-amino group of peptide and the free gamma-COOH group of Glu at the terminal position of the branches. This strategy lead to chemically well-defined synthetic immunogens that contain two different epitopes in multiple copies covalently linked to a synthetic branched polypeptide carrier. In vitro T-cell immunogenicity of a prototype conjugate was studied using T-cell hybridomas, lymph node cells from 38 kDa protein immunized mice, and human peripheral blood mononuclear cell (PBMC) cultures from sensitized individuals. These data document that the specific T-cell stimulatory effect of each mycobacterial epitope was maintained in this conjugate. Taken together, these findings suggest that it is feasible to use a biodegradable polymeric polypeptide for producing macromolecular bioconjugates for the stimulation of T-cell responses.