Development of short non-CpG phosphodiester oligonucleotides as immune stimulatory agents

We have previously reported that DNA isolated from Mycobacterium phlei (M. phlei) stimulates the synthesis of cytokines by monocytes and macrophages independently of the presence of unmethylated CpG motifs. Oligonucleotides as small as five to six bases isolated from M. phlei DNA have been found to induce cytokine synthesis. In the present study, we have investigated the potential for such CpG-lacking DNA to act as an immune stimulant. A series of six base length phosphodiester oligonucleotides derived from the genome of M. phlei were synthesised and tested for their ability to induce the synthesis of cytokines by murine, non-human primate (rhesus macaques and chimpanzee) and human peripheral blood mononuclear cells. The results show that phosphodiester oligonucleotides with a 5'GGGxGG3' sequence where x is A, C, G or T have the ability to induce the synthesis of IL-1beta, IL-6, IL10 or IL-12 by non-human primate and human PBMC, murine cells being unresponsive. The phosphodiester 5'GGGxGG3' oligonucleotides were shown to be stable in human serum, with a half-life of approximately 72 h. The addition of aluminium hydroxide to these 5'GGGxGG3' oligonucleotides potentiated, in a concentration-dependent manner, the synthesis of IL-12 by human peripheral blood mononuclear cells. These phosphodiester six base length non-CpG motif oligonucleotides may have potential as immunopotentiators for vaccines.