Native antimicrobial peptides are multifunctional molecules. Their main structural feature is the presence of a series of cationic amino acids that promote entry into host and bacterial cells. Some antimicrobial peptides also possess domains to inactivate bacteria-specific intracellular proteins. We showed that derivatives of pyrrhocoricin, a member of the short, proline-rich antibacterial peptide family with a bacterial DnaK protein target, can deliver fluorescein cargo into not only bacterial cells, but also into human monocytes. A newer pyrrhocoricin analog, designed to enter hard-to-penetrate bacterial and mammalian cells, is able to carry an influenza virus-derived MHC class I epitope into human fibroblasts. The Epitope-Pip-pyrr-MeArg dimer chimera activates human dendritic cells significantly better than the epitope or the antibacterial peptide derivative alone. Moreover, the chimera elicits an unusually vigorous anti-epitope CD8 + cytotoxic T lymphocyte (CTL) production when injected into mice. The same or similar pyrrhocoricin analogs are active in mouse models of bacterial infections when given intravenously, subcutaneously, as an aerosol or sprayed into the oral cavity, yet they completely lack toxicity or immunogenic properties in mammals. CTL are considered to be important effectors responsible for the clearance of viral infections and are consequently a valuable population of cells to induce by vaccination. Neither soluble proteins nor minimal CD8 + T cell peptide epitopes are potent inducers of cytotoxic responses mostly because of the lack of efficient targeting/delivery systems for these polyamides into the cytosol. Although subunit vaccines are safe, inexpensive and elicit specific immune responses, reliable and non-toxic adjuvants are still to be identified. Our hypothesis is that the pyrrhocoricin-based passive transport system is suitable to deliver various epitopic cargoes into antigen presenting cells, and the chimeras can elicit CTL production without any adjuvant. This grant application is concerned with providing proof-of-principle for this hypothesis. A chimera of the Pip-pyrr-MeArg dimer and an influenza nucleoprotein MHC class I epitope will be synthesized and will be injected into Balb/c mice in single or multiple doses. The ensuing immune response will be compared with that obtained using complete Freund's adjuvant. The general applicability of the model will be studied by repeating the experiments with a CTL epitope corresponding to the human papillomavirus (HPV)-16 E7 protein. In this case first the chimera will be studied for its ability to activate dendritic cells in vitro. Subsequently, C57BL/6 mice will be inoculated and to improve the immunogenicity another construct will also carry a short T helper cell determinant of the same protein. Our hope is that by the end of the grant period we will be able to provide a revolutionary novel vaccination strategy against viral infections.
