Immunostimulatory CpG Oligonucleotides as an Adjuvant with Recombinant DNA Vaccine Encoding Hepatitis C Virus Core Protein

The immunogenicity and protective efficacy of DNA vaccines have been amply demonstrated in numerous animal models against infectious diseases. In order to increase the potency of DNA vaccines, we have compared the immune response of conventional adjuvants such as aluminum phosphates, Dendrosome, CpG motif and mixture of aluminum phosphate and CpG motif. Female BALB/c mice were immunized with 10, 25 and 50 microgram of HCV core pcDNA3 plasmid mixed with the adjuvants. Each dose of recombinant pcDNA3 and different adjuvant were used as an immunogen in three IM injection periods. Blood samples were collected at four different times. The data indicate that the antibody response achieved following DNA immunization can be enhanced by CpG motif as molecular adjuvant

Single Stranded DNA Immune Modulators with Unmethylated CpG Motifs: Structure and Molecular Recognition by Toll-Like Receptor 9

Single stranded microbial DNA fragments with unmethylated deoxycytidylyldeoxyguanosine dinucleotide (CpG) motifs are interpreted as danger signals by the innate immune system via recognition by the Toll-like Receptor 9 (TLR9). Their synthetic analogues, Oligodeoxynucleotides (ODN) comprise a promising class of immune modulators with potential applications in the treatment of multiple diseases, such as cancer, autoimmune diseases or allergy. ODN molecules contain a core hexamer sequence, which is species specific consisting of GACGTT and AACGT for mouse and GTCGTT in humans. Assessment of structural features of different type of ODNs is highly challenging. NMR spectroscopic insights were gained for a short, single CpG motif containing ODN 1668. The structural basis of ODN recognition by TLR9 recently started to unravel as crystal structures of TLR9 orthologues in complex with ODN 1668 were solved. Systematic investigations of ODN sequences revealed that ODNs with a single CpG motif are capable of activating mouse TLR9, but two closely positioned CpG motifs are necessary for activation of human TLR9. Furthermore, longer ODNs with TCC and TCG sequences at the 5’ end were shown to activate TLR9 with higher efficiency. It was revealed that 5’-xCx motif containing short ODNs (sODN) are able to augment the immune response of short, single CpG containing ODNs, which are incapable of activating of TLR9 alone. All these observations pointed to the existence of a second binding site on TLR9, which was characterized in crystal structures that delivered further insights of the nucleic acid recognition of the innate immune system by TLR9.

Unmethylated CpG motif-containing genomic DNA fragment of Bacillus calmette-guerin promotes macrophage functions through TLR9-mediated activation of NF-κB and MAPKs signaling pathways

The potency of synthetic CpG-oligo-deoxynucleotides (CpG-ODNs) adjuvants in modulating the immune cell functions through the TLR pathway has been tested and reported previously. However, the cellular signaling involved in the stimulation of macrophages by natural, CpG motif-containing adjuvant and the effector functions modulated by such stimulation has not been well studied. Here, we used in vitro and ex vivo murine macrophage assay systems, and mouse model of in vivo stimulation to explore the signaling pathway and the effector functions mediated by BC01. Results show that BC01 can induce the production of TNF-α and MCP-1 in macrophages by up-regulating the activation of NF-κB and MAPKs signaling pathway, and elevated the expression of MHC-II, CD40, CD80, and CD86. Upon stimulation with BC01, the peritoneal macrophages isolated from TLR9−/− mice produced significantly low levels of pro-inflammatory cytokines, attenuated the activation of NF-κB and MAPKs signaling pathways, and showed reduced phagocytosis. Following in vivo stimulation with BC01, the TLR9−/− mice produced significantly lower levels of pro-inflammatory cytokines in the serum and lymph nodes showed reduced cell proliferation. These results indicate that BC01 is an efficient agonist of TLR9 that can significantly enhance the host-protective immune functions of macrophages.

Phosphodiester backbone of the CpG motif within immunostimulatory oligodeoxynucleotides augments activation of Toll-like receptor 9

Toll-like receptor 9 (TLR9) stimulatory CpG-containing oligodeoxynucleotides (ODNs) with phosphorothioate backbones have successfully replaced the naturally occurring agonists of TLR9 in drug development due to their increased stability. Replacing the nonbridging oxygen with a sulfur atom in the phosphate linkage of ODNs has been accepted as having a minor impact on the chemical and physical properties of the agonists. Here, we report that the TLR9 binding site exhibits a strong bias in favor of a phosphodiester backbone over the phosphorothioate backbone of the CpG motif. Furthermore, we show that while single point mutations of W47, W96 and K690 within the TLR9 binding site retains full TLR9 activation by phosphodiester-based ODNs, activation by phosphorothioate-based ODNs is strongly impaired. The substitution of a phosphorothioate linkage for a phosphodiester linkage of just the CpG motif considerably improves the activation potency of a phosphorothioate-based oligonucleotide for human B-cells and plasmacytoid dendritic cells, as well as for mouse bone marrow-derived dendritic cells and macrophages. Our results highlight the functional significance of the phosphodiester linkage of a CpG dinucleotide for binding, which is important in designing improved immunostimulatory TLR9 agonists.