Induction of sustained antibody responses by subcutaneous administration of immunostimulatory Takumi-shaped CpG DNA hydrogel loaded with Japanese cedar pollen antigen Cryj1 in mice

The development of effective vaccine delivery systems capable of eliciting sustained antigen-specific immune responses represents a significant challenge in immunotherapy. Our previous studies have demonstrated that Takumi-shaped DNA nanostructures are a suitable unit for DNA hydrogels with a prolonged retention property. To examine the potential of Takumi-shaped DNA hydrogels to induce sustained antigen-specific immune responses, Japanese cedar pollen Cryj1 was selected as a model antigen, extracted, and purified. The properties of Cryj1-loaded Takumi-shaped CpG DNA hydrogels were then examined to determine their suitability for this application. Takumi-shaped DNA nanostructures containing CpG or GpC motifs were designed, and Takumi-shaped CpG or GpC DNA hydrogels were prepared. Treatment with CpG DNA hydrogels induced a concentration-dependent increase in TNF-α secretion from RAW264.7 cells and a significant increase in IL-6 secretion from DC2.4 cells (approximately 2,000 pg/mL) compared to controls. In contrast, GpC DNA hydrogels induced significantly lower cytokine release, with TNF-α levels below 1,000 pg/mL. The rheological properties of Takumi-shaped CpG DNA hydrogels exhibited minimal changes when the CpG motif was replaced with a GpC motif, or when Cryj1 was loaded. The administration of Cryj1-loaded CpG DNA hydrogels to mice induced Cryj1-specific IgG1 and IgG2a antibody responses that persisted for 8 weeks, peaking at week 4. These levels were significantly higher than those induced by Cryj1 alone, a mixture of Cryj1 and Takumi-shaped CpG units, or Cryj1-loaded GpC DNA hydrogels. These results indicate that the Takumi-shaped CpG DNA hydrogels are an effective platform for vaccine delivery systems.