Development of a Nanostructured RNA/DNA Assembly as an Adjuvant Targeting Toll-Like Receptor 7/8

Adjuvants are essential for efficiently inducing an antigen-specific immune response in vaccine therapy. Single-stranded RNA (ssRNA) containing guanosine- and uridine-rich sequences is recognized by Toll-like receptor (TLR)7 and/or TLR8 and induces strong immune responses; thus, the application of ssRNA as an adjuvant is desirable. The development of a ssRNA-based adjuvant, however, requires the efficient delivery of ssRNA into the endosomes of antigen-presenting cells, where the TLRs exist. To achieve this, we developed a nanostructured RNA/DNA assembly using DNA nanotechnology, which can be efficiently recognized by antigen-presenting cells. The nanostructured RNA/DNA assembly, named tetrapodRD3, was designed using a 40-mer phosphorothioate-stabilized RNA and three 40-mer phosphodiester DNAs. TetrapodRD3 was more stable than ssRNA under serum conditions. The secreted alkaline phosphatase assay using HEK-Blue hTLR cells showed that tetrapodRD3 triggered human TLR8-specific responses. Fluorescently labeled tetrapodRD3 was efficiently taken up by murine dendritic DC2.4 cells and induced a high level of tumor necrosis factor-alpha release from the cells. Antigen presentation by the major histocompatibility complex class I on bone marrow-derived dendritic cells was significantly increased by the addition of an antigen along with tetrapodRD3. These results indicate that tetrapodRD3 constructed using DNA nanotechnology can be a useful adjuvant targeting human TLR8.