Geometry of a TLR2-Agonist-Based Adjuvant Can Affect the Resulting Antigen-Specific Immune Response

Targeted delivery of otherwise nonimmunogenic antigens to Toll-like receptors (TLRs) expressed on dendritic cells (DCs) has proven to be an effective means of improving immunogenicity. For this purpose, we have used a branched cationic lipopeptide, R₄Pam₂Cys, which is an agonist for TLR2 and enables electrostatic association with antigen for this purpose. Here, we compare the immunological properties of ovalbumin formulated with different geometrical configurations of R₄Pam₂Cys. Our results demonstrate that notwithstanding the presence of the same adjuvant, branched forms of R₄Pam₂Cys are more effective at inducing immune responses than are linear geometries. CD8⁺ T-cell-mediated responses are particularly improved, resulting in significantly higher levels of antigen-specific cytokine secretion and cytolysis of antigen-bearing target cells in vivo. The results correlate with the ability of branched R₄Pam₂Cys conformations to encourage higher levels of DC maturation and facilitate superior antigen uptake, leading to increased production of proinflammatory cytokines. These differences are not attributable to particle size because both branched and linear lipopeptides associate with antigen-forming complexes of similar size, but rather the ability of branched lipopeptides to induce more efficient TLR2-mediated cell signaling. Branched lipopeptides are also more resistant to trypsin-mediated proteolysis, suggesting greater stability than their linear counterparts. The branched lipopeptide facilitates presentation of antigen more efficiently to CD8⁺ T cells, resulting in rapid cell division and upregulation of early cell surface activation markers. These results as well as cognate recognition of Pam₂Cys by TLR2 indicate that the adjuvant's efficiency is also dependent on its geometry.