The biological activity of CpG motifs in TLR9 agonists also depends upon the 3’ and 5’ flanking bases, and several immunostimulatory sequences have been defined such as hexameric palindromes containing CpG motifs or 5’- pur-pur-CG-pyr-pyr hexameric sequences. It has been reported that the optimal CpG motif for activating human cells might be different from the effective mouse sequence (5’-GTCGTT versus 5’-GACGTT). However, by screening a large number of ODNs, Dr Antoine Carpentier found that some ODNs containing the palindromic hexamers 5’-AACGTT or 5’-GACGTC displayed optimal immunostimulatory activity on human B-lymphocytes (almost twice as immunostimulatory as 5’-GTCGTT) and were also very potent in the stimulation of murine macrophages or lymphocytes.
Nevertheless, the biological activity of a given hexamer is strongly modulated by the remaining sequences within the oligonucleotide. Inhibitory motifs have been described, such as over representation of 5’-CCG, 5’-GCGGG or 5’-ACGGG motifs or poly-G sequences in a phosphorothioate (but not phosphodiester or chimeric) backbone oligonucleotide. On the other hand, repeating several times an immunostimulating CpG motif within an ODN enhances its immunostimulatory activity. In our experience, oligonucleotides comprising several repeats of 5’-AACGTT or 5’-GACGTC motifs are among the most effective.
