C-Class CpG ODN: sequence requirements and characterization of immunostimulatory activities on mRNA level

Abstract

Synthetic oligodeoxynucleotides (ODN) containing unmethylated deoxycytosine–deoxyguanosine (CpG) motifs are very potent inducers of the innate immune system, mimicking the effects of bacterial DNA. CpG ODN are recognized by Toll-like receptor 9 (TLR9). Three classes of TLR9 agonists have been described: B-Class CpG ODN that induce strong B- and NK-cell activation and A-Class ODN that induce very high levels of IFN- α by plasmacytoid dendritic cells. The recently described C-Class ODN combine most efficiently properties of A- and B-Class ODN in that they induce strong B-cell activation comparable to B-Class ODN together with IFN- α secretion comparable to A-Class ODN. Here, we investigate sequence requirements of C-Class ODN regarding optimal IFN- α secretion. Sequence as well as backbone modifications like 2′- O-methyl modifications especially in the 5′ part of the ODN influence IFN- α-producing capacity. Kinetic studies on mRNA level for CD69, IFN- γ, IP-10 and IL-18 by semi-quantitative PCR demonstrated differences in mRNA transcription for some cytokines suggesting different regulatory mechanisms for different ODN classes. High amounts of IP-10 mRNA and protein as well as up-regulation of IL-18 mRNA were observed especially for the A- and C-Classes. According to these data, C-Class ODN can be described as strong Th1 inducers with the stimulation of type I and II interferon as well as IP-10 production and strong NK activation. These characteristics can be availed to induce potent anti-tumor or anti-viral effects. Consequently, C-Class CpG ODN represent ideal drug candidates for anti-viral and/or anti-tumor therapy.