Conjugated NOD1-Centered Dual Agonists Exhibit Modest In Vitro Immunomodulation but Potent In Vivo Adjuvanticity

Immunomodulators that simultaneously engage multiple pattern recognition receptors (PRRs) represent a promising strategy for shaping immune responses. Here we report the design and synthesis of covalently linked dual PRR agonists composed of a NOD1-selective ligand conjugated to agonists of TLR4, TLR7, or RIG-I. The resulting chimeric molecules were evaluated for receptor-specific agonist activation, in vitro immunomodulatory activity in human peripheral blood mononuclear cells, and in vivo adjuvant properties. In most cases, conjugation attenuated receptor agonist activity and reduced cytokine responses in vitro compared with mixtures of the corresponding unconjugated agonists. Despite modest in vitro activity, the conjugates, particularly the dual NOD1/TLR7 agonist, displayed robust adjuvant effects in a murine vaccination model. These results highlight the distinct immune signatures of conjugated NOD1-based dual agonists and support their development as next-generation vaccine adjuvants.