Strong immunostimulatory activity of AT-oligodeoxynucleotide requires a six-base loop with a self-stabilized 5′-C...G-3′ stem structure

Lactobacillus gasseri OLL2716 has recently been discovered as a probiotic that suppresses the growth of Helicobacter pylori and reduces gastric mucosal inflammation in humans. This has resulted in the development of a new type of probiotic yoghurt 'LG21' in Japan. In our previous study, we found an immunostimulatory AT5ACL oligodeoxynucleotide (AT-ODN) containing a unique core sequence (5′-ATTTTTAC-3′) in L. gasseri JCM1131^T. Interestingly, although the ATODN does not contain any CpG sequences, it exerts mitogenic activity in B cells and augments Th-1-typeimmune responses via Toll-like receptor 9. These findings prompted us to identify strong immunostimulatory non-CpG AT-ODNs that contain the 5′-ATTTTTAC-3′ motif in the genomic sequence of L. gasseri OLL2716. We identified 280 kinds of AT-ODNs in the L. gasseri OLL2716 genome. Mitogenicity and NF-κB gene reporting assays showed that 13 of the 280 ATODNs were strongly immunostimulatory when in the TLR9 transfectant. Of these, AT-ODNs LGAT-145 and LGAT-243 were the most potent. With respect to the induction of Th-1-type cytokines, LGAT-243 had the greatest activity and was more potent than the swine prototype, ODN D25. We further found that a six-base secondary loop structure containing a self-stabilized 5′-C...G-3′ stem sequence is important for potent immunostimulatory activity. These results show for the first time that AT-ODNs with a specific loop and stem structure are important factors for immunostimulatory activity. Finally, we found that novel strong immunostimulatory non-CpG AT-ODNs exist in the genome of probiotic lactic acid bacteria.