Synthetic oligodeoxyribonucleotides (ODNs) containing CpG (unmethylated deoxycytidylyl-deoxyguanosine dinucleotide) motifs activate endosomal TLR9. The nucleotide sequence, length, and dimerization properties of ODNs modulate their activation of TLR9. We performed a systematic investigation of the sequence motifs of B-class and C-class phosphodiester ODNs to identify the sequence properties that govern TLR9 activation. ODNs shorter than 21 nt and with the adenosine adjacent to the cytidine-guanosine (CG) dinucleotide motif led to a significant loss of the propensity to activate TLR9. The distance between the stimulatory CpG motifs within the ODN fine-tunes the activation of B cells. The minimal ODNs that activate human TLR9 comprise 2 CG dinucleotides separated by 6–10 nt, where the first CpG motif is preceded by the 5′-thymidine and the elongated poly-thymidine tail at the 3′ end of the ODN. The minimal sequence provides insight into the molecular mechanism of TLR9 ligand recognition. On the basis of sequence requirements, we conclude that two binding sites with different affinities for CG are formed in the human TLR9 dimer, with a very stringent binding site interacting with the 5′ CpG motif.

Toll-like receptors (TLRs) play a key role in the recognition of pathogen-associated molecular patterns, including immunostimulatory nucleic acids (INAs). INAs are recognized by TLRs in endosomes, leading to the activation of signalling pathways that activate the innate immune response. This feature makes INAs and their synthetic analogues useful as adjuvants in vaccines and in cancer treatment. We tested a delivery system for the improvement of the therapeutic effect of INAs which consists of a conjugate between transferrin (Tf) and poly-L-lysine (PLL). Tf is a ligand of the transferrin receptor (TfR) and is internalized via receptor-mediated endocytosis, while PLL binds negatively charged INAs. The TfPLL conjugate protected TLR3 ligand polyinosinic:polycytidylic acid [poly(I:C)] from RNase degradation and enhanced the uptake of poly(I:C) in HeLa cells. Co-localization between TfPLL-bound poly(I:C) and lysosomes demonstrated delivery into the endosomal pathway. Time dependence of the production of IL-6 in the primary cell line showed that TfPLL conjugate enabled a gradual release of poly(I:C) and stronger activation of TLR3 receptor in comparison with poly(I:C) alone. Only 3 h of stimulation by poly(I:C) + TfPLL complexes initiated a strong immune response in contrast to poly(I:C) alone. The poly(I:C) + TfPLL complexes have potential use for development of advanced vaccine adjuvants and targeted cancer immune therapy in cells that express higher levels of TfR.