Structure-Function and Treg induction of Methylated CpG ODNs from Bifidobacterium longum susp. infantis in an Allergic Murine Model

In our previous studies, methylated CpG oligodeoxynucleotides (ODN) derived from Bifidobacterium longum subsp. infantis have demonstrated immunomodulatory activity through the induction of regulatory T cells (Tregs). To define the structural determinants underlying this effect, we synthesized four CpG ODNs varying in methylation status, CpG motif placement, and backbone length. These include: 1) ODN-A (2m-V1), a 20-nucleotide CpG oligodeoxynucleotide incorporating two 5-methylcytosines at positions 4 and 12 within centrally placed CpG motifs; 2) ODN-B (um-V2), a 20-nucleotide CpG oligodeoxynucleotide with a backbone structure identical to ODN-A but unmethylated; 3) ODN-C (2m´-V3), a 20-nucleotide CpG oligodeoxynucleotide with a backbone structure identical to ODN-A, with two 5-methylcytosines shifted at positions 7 and 15; 4) ODN-D (3m-V4), a 27-nucleotide CpG oligodeoxynucleotide with an extended backbone structure, with three 5-methylcytosines at positions 3, 11 and 19.Using a murine model of OVA-induced allergy, we show that methylated ODN-A (2m-V1) and ODN-D (3m-V4) markedly reduced serum anti-OVA IgE, clinical symptoms, eosinophilic infiltration, and Th2/Th17 responses, while promoting splenic Treg expansion and IL-10 production. In contrast, unmethylated ODN-B (um-V2) and a positionally altered methylated ODN-C (2m’-V3) failed to suppress allergic inflammation, reversely enhanced Th2/Th17 response, and induced in vitro robust TLR7/8/9 expression in native splenocytes. These findings suggest that both methylation and motif architecture critically influence the immunologic profile of CpG ODNs. Our results provide mechanistic insights into CpG ODN structure–function relationships and support the therapeutic potential of select methylated sequences for restoring immune tolerance in allergic disease.