Modern vaccines exhibit limited immunogenicity and therefore require adjuvants to elicit strong and durable immune responses. Adjuvants act by stimulating the innate immune system through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I-like receptors, which are predominantly expressed on antigen-presenting cells (APCs). Immune responses can also be enhanced through activation of invariant natural killer T cells via the CD1d receptor. Together, these receptors regulate the magnitude and quality of the resulting adaptive immune response. Although activation of a single receptor generally induces only a moderate response, simultaneous activation of multiple receptors, particularly on the same APC, can both amplify and shape immunity. This can be achieved by covalently conjugating two appropriate innate immune receptor ligands into a single molecular entity.
The aim of this doctoral dissertation was to design, synthesize, and immunologically characterize covalently conjugated innate immune agonists as potential vaccine adjuvants. First, we prepared a series of dual PRR agonists consisting of a selective NOD1 agonist covalently linked to agonists of TLR4, TLR7, or RIG-I and evaluated their biological activity. Although these conjugates displayed only modest in vitro activity, the NOD1/TLR7 dual agonist exhibited pronounced adjuvant effects in a murine model by inducing a strong antigen-specific immune response. We also synthesized two series of conjugated NOD2/TLR4 agonists using structurally distinct TLR4 agonists connected through either flexible or rigid linkers. Overall, these conjugates showed reduced receptor activation and weaker in vitro immune responses compared with the parent agonists or their unconjugated mixtures. Nevertheless, they provided valuable insight into the influence of agonist selection, linker architecture, and physicochemical properties on biological activity and kinetic solubility. Finally, we developed strategies for functionalizing CD1d ligands with TLR7 agonists and prepared suitable intermediates for conjugation through either amide bond formation or cycloaddition chemistry.
Overall, this dissertation demonstrates the potential of covalently conjugated agonists as tailor-made adjuvants capable of modulating both the strength and direction of immune responses, providing a foundation for the development of safer and more effective next-generation vaccines.
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