Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo

Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides withi n specific sequence contexts (CpG motifs) are detected, like bacterial or v iral DNA, as a danger signal by the vertebrate immune system, CpG ODN synth esized with a nuclease-resistant phosphorothioate backbone have been shown to be potent Th1-directed adjuvants in mice, but these motifs have been rel atively inactive on primate leukocytes in vitro, Moreover, in vitro assays that predict in vivo adjuvant activity for primates have not been reported. In the present study we tested a panel of CPG ODN for their in vitro and i n vivo immune effects in mice and identified in vitro activation of B and N K cells as excellent predictors of in vivo adjuvant activity. Therefore, we tested >250 phosphorothioate ODN for their capacity to stimulate prolifera tion and CD86 expression of human B cells and to induce lytic activity and CD69 expression of human NK cells. These studies revealed that the sequence , number, and spacing of individual CpG motifs contribute to the immunostim ulatory activity of a CpG phosphorothioate ODN, An ODN with a TpC dinucleot ide at the 5' end followed by three 6 mer CpG motifs (5'-GTCGTT-3') separat ed by TpT dinucleotides consistently showed the highest activity for human, chimpanzee, and rhesus monkey leukocytes. Chimpanzees or monkeys vaccinate d once against hepatitis B with this CpG ODN adjuvant developed 15 times hi gher anti-hepatitis B Ab titers than those receiving vaccine alone. In conc lusion, we report an optimal human CpG motif for phosphorothioate ODN that is a candidate human vaccine adjuvant.