Toward self-adjuvanting subunit vaccines: model peptide and protein antigens incorporating covalently bound toll-like receptor-7 agonistic imidazoquinolines

Toll-like Receptor (TLR)-7 agonists show prominent Th1-biased immunostimulatory activities. A TLR7-active N(1)-(4-aminomethyl)benzyl substituted imidazoquinoline 1 served as a convenient precursor for the syntheses of isothiocyanate and maleimide derivatives for covalent attachment to free amine and thiol groups of peptides and proteins. 1 was also amenable to direct reductive amination with maltoheptaose without significant loss of activity. Covalent conjugation of the isothiocyanate derivative 2 to α-lactalbumin could be achieved under mild, non-denaturing conditions, in a controlled manner and with full preservation of antigenicity. The self-adjuvanting α-lactalbumin construct induced robust, high-affinity immunoglobulin titers in murine models. The premise of covalently decorating protein antigens with adjuvants offers the possibility of drastically reducing systemic exposure of the Adjuvant, and yet eliciting strong, Th1-biased immune responses.