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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 JCM1131T. Interestingly, although the AT‐ODN does not contain any CpG sequences, it exerts mitogenic activity in B cells and augments Th‐1‐type immune 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 AT‐ODNs 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.