Tuning Th1 Immunity through a TLR7/8 Agonist HYBRID2-Formalin-Killed <i>Leishmania donovani</i> Antigen Immunomodulatory System in Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a fatal parasitic disease for which safe and effective immunotherapeutic strategies remain limited. Modulation of innate immune signaling using small-molecule Toll-like receptor (TLR) agonists represents a promising approach to enhance host-directed antiparasitic responses. In this study, we investigated the immunomodulatory efficacy of a synthetic TLR7/8 agonist, HYBRID2, formulated with formalin-killed <i>Leishmania donovani</i> antigen (FK), in a BALB/c mouse model of VL. Immunized mice challenged with <i>L. donovani</i> exhibited a marked reduction in splenic parasite burden following FK + HYBRID2 administration. Mechanistic evaluation of splenocytes revealed enhanced redox activity, with significantly elevated reactive oxygen species and nitric oxide production, accompanied by up-regulation of inducible nitric oxide synthase and NF-κB expression. Cytokine analysis demonstrated a pronounced Th1-biased immune response, characterized by increased INF-γ and TNF-α levels. Comparative assessment showed that FK + HYBRID2 elicited superior immunostimulatory and antileishmanial effects relative to FKAlone or FK combined with Resiquimod. Collectively, these findings establish HYBRID2 as a potent innate immune modulator enhancing redox responses and NF-κB activation alongside Th1 polarization, highlighting its potential utility as an immunomodulatory adjuvant platform against intracellular parasitic infections.