Probing the Structural Elements of Polysaccharide Adjuvants for Enhancing Respiratory Mucosal Response: From Surmounting Multi-Obstacles to Eliciting Cascade Immunity

The immunomodulatory effects and excellent tolerability of polysaccharides make them optimal candidates for pulmonary vaccine adjuvants. Yet, the structure-immunostimulatory activity relationship of polysaccharides remains unrevealed. Here, we developed nanovaccines decorated with four polysaccharides of distinct structures─hyaluronic acid (HA), pectin (PC), chondroitin sulfate (SC), and heparan sulfate (SH)─all sharing similar particle sizes and zeta potential. Polysaccharides containing sulfate groups (SC, SH) exhibited superior efficacy in overcoming natural inhalation barriers and recruiting dendritic cells (DC). DC stimulation assays revealed that HA and SH significantly upregulated the expression of costimulation signals, with IL-6 secretion rising over 8.7-fold compared to pure OVA. Fluorescence resonance energy transfer demonstrated their detachment within the lysosomal microenvironment, thereby enhancing antigen cross-presentation. However, in vivo findings only showed that SH upregulated CCR7 chemokine and swiftly migrated to lymph nodes. Molecular docking and Western blot analyses further elucidated the involvement of the TLR─MyD88─TRAF6─NF-κB/MAPK/IRF-7 signaling pathways. Notably, SH-modified nanovaccines induced a more robust cellular and humoral immune response with the potential for immune memory. This study confirms that sulfate groups in polysaccharides enhance immune activation and that combining sulfate with acetyl groups offers a promising adjuvant configuration for augmenting mucosal, cellular, and humoral immunity.