Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists

Wenjun Meng,Yuuki Nishida,Tomohiko Yamazaki,Nobutaka Hanagata

doi:10.1186/1472-6750-11-88

Wenjun Meng, Yuuki Nishida + Show 2 more

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https://doi.org/10.1186/1472-6750-11-88

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Abstract

BackgroundUnmethylated cytosine-guanine (CpG) motif-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists of Toll-like receptor 9 (TLR9). ODNs with a phosphorothioate (PTO) backbone have been studied as TLR9 agonists since natural ODNs with a phosphodiester (PD) backbone are easily degraded by a serum nuclease, which makes them problematic for therapeutic applications. However, ODNs with a PTO backbone have been shown to have undesirable side effects. Thus, our goal was to develop nuclease-resistant, PD ODNs that are effective as human TLR9 (hTLR9) agonists.ResultsThe sequence of ODN2006, a CpG ODN that acts as an hTLR9 agonist, was used as the basic CpG ODN material. The 3'-end modification of ODN2006 with a PD backbone (PD-ODN2006) improved its potential as an hTLR9 agonist because of increased resistance to nucleolytic degradation. Moreover, 3'-end modification with oligonucleotides showed higher induction than modification with biotin, FITC, and amino groups. Further, enhancement of hTLR9 activity was found to be dependent on the number of CpG core motifs (GTCGTT) in the PD ODN containing the 3'-end oligonucleotides. In particular, ODN sequences consisting of two to three linked ODN2006 sequences with a PD backbone (e.g., PD-ODN2006-2006 and PD-ODN2006-2006-2006) acted as effective agonists of hTLR9 even at lower concentrations.ConclusionsThis study showed that PD-ODN2006-2006 and PD-ODN-2006-2006-2006 can be used as potentially safe agonists for hTLR9 activation instead of CpG ODNs with a PTO backbone. We propose these CpG ODNs consisting of only a PD backbone as a novel class of CpG ODN.

Highlights

Unmethylated cytosine-guanine (CpG) motif-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists of Toll-like receptor 9 (TLR9)
Because of safety considerations, the development of novel natural CpG ODNs consisting of an entirely PD backbone is desirable. These natural CpG ODNs should be able to strongly activate TLR9-dependent NF-B signaling. To develop such a CpG ODN, we examined the effects of the sugar backbone, the sequence of the CpG motif, and modification of both terminals in CpG ODNs on NF-B activation
We found that a series of linked PD sequences of ODN2006 has the potential to resist nuclease degradation and to activate NF-B

Summary

Introduction

Unmethylated cytosine-guanine (CpG) motif-containing oligodeoxynucleotides (ODNs) have been well characterized as agonists of Toll-like receptor 9 (TLR9). CpG ODNs in this class can induce high levels of interferona (IFN-a) production in plasmacytoid dendritic cells (pDCs), but are weak stimulators of TLR9-dependent NF-B signaling [11]. The class B CpG ODNs consist entirely of PTO backbones and do not form palindromic structures. These ODNs can strongly activate B cells through NF-B signaling but show no effect on IFN-a production [12,13]. A novel type of CpG ODN called the P class, which has two palindromic motifs with a PTO backbone, was reported to show higher potential to produce IFN-a and activate NF-B [15]

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