Oral administration of second-generation immunomodulatory oligonucleotides induces mucosal Th1 immune responses and adjuvant activity.

CpG DNA induces potent Th1 immune responses through Toll-like receptor 9. In the present study, we used oligonucleotides consisting of a novel 3'-3'-linked structure and synthetic stimulatory motifs, referred as second-generation immunomodulatory oligonucleotides (IMOs). The stimulatory motifs included: CpR, YpG, or R'pG (R = 2'-deoxy-7-deazaguanosine, Y = 2'-deoxy-5-hydroxy-cytidine, and R' = 1-[2'-deoxy-beta-d-ribofuranosyl]-2-oxo-7-deaza-8-methyl-purine). We evaluated the stability of orally administered IMOs in the gastrointestinal (GI) environment and their ability to induce mucosal immune responses in mice, and compared these characteristics with those of a conventional CpG DNA. The IMOs were significantly more stable than CpG DNA following oral administration, and IMOs induced stronger local and systemic immune responses as determined by MIP-1beta, MCP-1, IP-10, and IL-12 production. Mice orally immunized with ovalbumin (OVA) and IMO had higher levels of IgG2a antibodies in serum and IgA antibodies in intestinal mucosa than did mice immunized with OVA and CpG DNA. These studies demonstrate that IMOs are more stable than CpG DNA in the GI tract and can induce more potent mucosal Th1 adjuvant responses. IMOs may prove to be effective oral adjuvants, able to promote strong systemic and mucosal immune responses to oral vaccines and antigens for therapeutic and prophylactic applications.