Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polyclonal B-cell (3,4,(7)(8)(9)11). Recent work from our laboratory suggested that this B-cell stimulation was mediated by a 6-base nt motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (12). This motif is expressed nearly 20 times more frequently in bacterial than vertebrate DNA (12)(13)(14).In addition to B-cell activation, the innate immune response to bacterial infection is characterized by the production of immunomodulatory cytokines (15,16). These include interleukin (IL) 6, which contributes to T-and B-cell activation (17-20), IFN-y, which (25)(26)(27).The concentration of cytokine in culture supernatants was determined by using a modification of the assay system described above. Supernatants were serially diluted in PBS/1% bovine serum albumin and incubated on anti-cytokine-coated microtiter plates for 2 hr. Biotinylated anti-cytokine antibody followed by avidin-conjugated alkaline phosphatase were used in a colorimetric assay to detect the presence of bound cytokine (27).Cell Purification. Mononuclear cells were incubated with biotinylated anti-CD4, anti-CD8, anti-CD11, anti-CD14, or

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DNA technology has been harnessed to produce a variety of plasmid-based vaccines designed to prevent viral, bacterial and parasitic infections. The rapid adoption and implementation of this novel vaccine strategy carries with it important safety and efficacy concerns. This review will focus on whether DNA vaccines (1) are likely to induce systemic or organ-specific autoimmune disease, (2) have the potential to induce tolerance rather than immunity, and (3) are as effective in individuals with depressed immune function as they are in healthy adults.

Synthetic oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs stimulate an innate immune response characterized by the production of polyreactive immunoglobulin M antibodies and immunomodulatory cytokines. This immune response has been shown to protect mice from challenge by Listeria monocytogenes andFrancisella tularensis for up to 2 weeks. By repeatedly administering CpG ODN two to four times/month, we found that this protection could be maintained indefinitely. Protection was associated with a significant increase in the number of spleen cells that could be triggered by subsequent pathogen exposure to secrete gamma interferon and interleukin-6 in vivo (P < 0.01). ODN-treated animals remained healthy and developed neither macroscopic nor microscopic evidence of tissue damage or inflammation. Thus, repeated administration of CpG ODN may provide a safe means of conferring long-term protection against infectious pathogens.

Synthetic immunostimulatory nucleic acids such as CpG DNA are being harnessed therapeutically as vaccine adjuvants, anticancer or antiallergic agents. Efforts to identify nucleic acid-based agents capable of more specifically modulating the immune system are being developed. The current study identifies a novel class of single-stranded oligoribonucleotides (ORN) containing unmethylated CpG motifs and a poly(G) run at the 3′ end (CpG ORN) that directly stimulate human CD14+CD11c+ monocytes but not dendritic cells or B cells. CpG ORN activate NF-κB and p38 MAPK, resulting in IL-6 and IL-12 production and costimulatory molecule up-regulation but not IFNα. Methylation of cytosine at the 5′ portion in core CpG motif abrogates such activation. TLR3, 7, 8, or 9 alone did not confer response to CpG ORN, in contrast to previously reported respective nucleic acid ligands. These data suggest that CpG ORN represent a novel class of synthetic immunostimulatory nucleic acids with distinct target cells, receptors, and functions from that of previously known immunomodulatory nucleic acids.