Our results may have important implications for the design of new adjuvants and vaccines. DNA vaccines have attracted considerable enthusiasm as an approach for protecting against a variety of infectious diseases (Wang et al., 2001; Yang et al., 2004) , but there are safety concerns about the insertional mutagenic potential of DNA vaccines and/or their potential to trigger pathogenic anti-DNA autoimmune antibody responses (Schalk et al., 2006) . The potency of DNA vaccines appears to derive from their ability to stimulate the TBK1 and innate cytosolic DNA-sensing pathways . Thus, our demonstration that a synthetic nonself non-DNA molecule such as c-di-GMP can stimulate an in vitro and in vivo innate and adaptive immune response (Fig. 6 ) similar to that induced by DNA, without similar autoimmune or mutagenic risks, suggests that c-di-GMP might have valuable application as a small-molecule adjuvant. Understanding the molecular basis of c-di-GMP signaling in mammalian cells will be a crucial step toward achieving this aim.