Oligosaccharide adjuvant-deferred antigen endocytosis promotes cross-presentation

Antigen cross-presentation by dendritic cells (DCs) is essential for inducing cytotoxic immune responses, which are crucial for eliminating infected or malignant cells. Although microbial signal processing reveals that the temporal relationship between DC activation and antigen uptake dictates cross-presentation efficiency, these two fundamental events are poorly coordinated in current vaccine design. Here, we report an oligosaccharide (OG) adjuvant that induces rapid DC activation but deferred antigen uptake, by sequentially activating two separate pathogen-recognition receptors on the same cells. Specifically, we synthesize a series of glucomannan hexasaccharides with varying degrees of acetylation (acOG6) and identify a low-acetylated candidate (0.6; acOG6-L) that could rapidly activate DCs into a pro-inflammatory state through toll-like receptor 2 (TLR2) signaling while, after 6 h, triggering cluster of differentiation 14 (CD14)-mediated antigen internalization. Molecular dynamics (MD) simulations provide structural insight into how acetylation affects the assembly of acOG6-L and its interaction with TLR2 pockets. This temporal deferment enables the trafficking of ovalbumin (OVA), which is readily conjugated with acOG6-L, into endosomal compartments, where the recruited nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) contributes to the alkalization of the antigen-containing phagosome - a critical step for promoting cross-presentation. Comprehensive in vitro and in vivo tests validate that OVA-acOG6 elicits enhanced antigen-specific CD8⁺ T cell -mediated cellular immunity against tumor cells in a subcutaneous B16 ovalbumin-expressing (B16-OVA) melanoma model. This work demonstrates that temporally coordinating cellular activation and antigen uptake by an acetyl hexasaccharide effectively enhances antigen cross-presentation.