Effects of a Hexameric Deoxyriboguanosine Run Conjugation into CpG Oligodeoxynucleotides on Their Immunostimulatory Potentials

Abstract

CpG oligodeoxynucleotides (ODNs) are promising immunomodulatory agents for treating human diseases and vaccine development. Phosphodiester CpG ODNs were demonstrated to have poor immunostimulatory potentials for cytokine production. However, the conjugation of consecutive deoxyriboguanosine residues, called a dG run, at the 3′ terminus of phosphodiester CpG ODNs significantly enhanced TNF-α and IL-12 production from mouse splenic dendritic cells (DCs). The optimal induction of cytokine production was achieved by the addition of a hexameric dG (dG6) run. In contrast, the existence of a dG6 run either at the 5′ terminus of phosphodiester CpG ODNs or at the 3′ terminus of phosphorothioate CpG ODNs diminished CpG-mediated cytokine induction, suggesting that the effects of a dG run depend on its location and the chemical property of the ODN backbone, respectively. In addition, we provided the evidence that the conjugation of a dG6 run caused the structural transformation of CpG ODNs, which facilitates their targeting into mouse APCs such as splenic DCs, B cells, and peritoneal macrophages with a scavenger receptor type A ligand specificity. Among primary APCs, DCs were the most potent for CpG ODN-mediated IL-12 production. Furthermore, we demonstrated that the conjugation of a dG6 run into the 3′ terminus of phosphodiester CpG ODNs was crucial for their ability to generate Th1 immunity in vivo. Thus, the conjugation of a dG6 run into phosphodiester CpG ODNs would be an alternative way to optimize their immunostimulatory potentials in vitro and in vivo.

The effect of CpG-oligodeoxynucleotides with different backbone structures and 3' hexameric deoxyriboguanosine run conjugation on the treatment of asthma in mice*1

“…This finding contradicts that of our previous in vivo study, which found that 3′ dG6-run conjugation showed an enhanced immunostimulatory effect for both PE and PS CpG when delivered at the time of sensitization (4). However, it concurs with those findings of previous studies (6,11). In in vitro studies, it has been reported that the conjugation of poly-G to PS-CpG may act like an neutralizing motif (CpG-N) and the conjugation of a 3′ dG6-run to PS-CpG was not found to potentiate TNFαand IL-12 production by splenic dendritic cells; rather it had an inhibitory effect (6,11,17).…”

“…We have shown that the failure of PE-CpG to inhibit asthmatic responses could be overcome by 3′ dG6-run conjugation when PE-CpG-dG6 is injected at the time of sensitization (4). Runs of dG residues can form a four stranded conformation, which is called the G-quartet, which confer nuclease resistance and facilitate binding to the scavenger receptors of macrophages (11,12). Accordingly, 3′ dG6- run may extend the half-life of PE-CpG and allow the efficient targeting of PE-CpG by APCs, presumably via a scavenger receptor (4).…”

The Roles of Toll-Like Receptor 9, MyD88, and DNA-Dependent Protein Kinase Catalytic Subunit in the Effects of Two Distinct CpG DNAs on Dendritic Cell Subsets

“…It is also possible that differential TLR9 responses are caused by coligation of other transmembrane proteins. In this context, it is noteworthy that scavenger receptor A (SR-A) can bind to the polyG stretch and that SR-A ligands can inhibit the binding of CpG DNAs containing the polyG stretch to splenic CD11c ϩ DCs (30). However, it remains unknown whether SR-A is involved in DC responses caused by A/D-type CpG DNAs.…”

Necessity of Oligonucleotide Aggregation for Toll-like Receptor 9 Activation

“…However, there are ample reports showing that ODNs with a single ISS sequence can display potent immunostimulatory activity. With some exceptions, these ODNs usually have either a guanine stretch at the 3Ј-end of the molecule or a chemically modified backbone such as phosphorothioate (21,(43)(44)(45)(46)(47)(48). Single-stranded guanine-rich ODNs are known to have a propensity to form quadruplex structures and higher aggregates that are often essential for their biologic activities (48 -51).…”