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-(Glu(i)-DL-Ala(m))] (EAK) with poly(L-lysine) backbone has been used. This polypeptide with free α-amino and γ-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 (α-amino group of peptide and the free γ-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.