Studying adjuvant properties of cell-penetrating cationic dendrimeric peptides

The adjuvant agents represent important components of vaccines, including medications for specific immunotherapy, which improve their efficacy and safety. Current scope of vaccines development includes approaches based on inclusion of adjuvant into the immunogenic molecule. Such covalent adjuvants vary greatly in chemical structure, and are found among particular peptides. Peptides have distinct advantages over substances of other chemical classes, e.g., high efficiency and biodegradability, whereas dendrimeric cationic peptides are often capable of enhanced translocation ability when compared to linear peptides. In this work, the ability to enhance IgG induction was studied for two cationic dendrimeric cell-penetrating peptides with previously reported transmembrane activity, i.e., LTP and SA-40. The peptides were obtained by solidphase synthesis and characterized by mass-spectrometry and zone capillary electrophoresis. To study adjuvant activity, the peptides were conjugated with recombinant protein Bet v 1, a major birch pollen allergen, produced as biotechnology product, using E. coli. Chemical conjugation was carried out with Michael reaction after maleimide functionalization of the protein. The conjugates were purified by gel chromatography and dialysis, followed by estimation of conjugation efficiency with SDS-PAGE. Conjugates and pure rBet v 1 were used to immunize BALB/c mice to compare IgG levels to rBet v 1 after 4 injections without using other adjuvants. Mouse blood sera were studied with ELISA for IgG levels against rBet v 1, being the target criterion of the study, as well as against the conjugates and free LTP and SA-40. It was found that both conjugates significantly increased the IgG level to the initial protein, while one of them, LTP-rBet v 1, also induced serum reactivity both to itself and to free LTP. The increase in IgG level to rBet v 1 was approximately four-fold in the case of LTP-rBet v 1, and three-fold with SA-40-rBet v 1. Based on the results of this study, it was concluded that both LTP and SA-40 possess adjuvant activity. We believe that LTP and SA-40 structures can be used as reference agents in development of new peptide adjuvants, and SA-40 itself may be used as a covalent adjuvant. Moreover, LTP may be considered a prospective non-covalent adjuvant.