Dual TLR7 /8 Activation by Dihydroxyimidazoquinoline Adjuvant Orchestrates NF ‐κβ‐ iNOS ‐Driven Th1 Immunity and Durable Protection Against Leishmania donovani

Targeting Toll like receptors (TLRs) signalling represents a powerful approach to re-calibrate host immunity against intracellular pathogens. Among these, dual TLR7/8 agonists uniquely bridge innate and adaptive immune response through NF-κβ-associated cytokine programming and activation of innate effector pathways. Here we, identify and characterise a dihydroxyimidazoquinoline-based TLR7/8 agonist as a potent immunomodulatory adjuvant that enhances the efficacy of heat-killed Leishmania donovani antigen vaccine in BALB/c mice. Comparative immunisation with escalating adjuvant doses (10, 25 and 50 μg) demonstrated a robust, dose-dependent reduction in splenic parasite burden, accompanied by sustained elevation of Th1 cytokine (IFN-γ and TNF-α) and suppression of Th2-associated cytokine (IL-10 and IL-13) up to 16-week post-challenge. Enhanced iNOS and NF-κβ gene expression, together with elevated ROS and NO production, indicated activation of host effector pathways underlying parasite clearance. Flow cytometric analysis revealed persistent expansion of both CD4+ and CD8+ T cell subsets in the high-dose groups supporting durable adaptive immunity. The dual TLR7/8 activity of this compound mediated broad and sustained immunoregulatory signalling that surpassed resiquimod in magnitude and persistence. Collectively, this study delineated a mechanistic framework for TLR7/8 driven immunoregulation and establishes dihydroxyimidazoquinoline as a next-generation adjuvant for rational, host-directed vaccine design against intracellular pathogens such as L. donovani.