Peptide Conjugation at the 5′-End of Oligodeoxynucleotides Abrogates Toll-Like Receptor 9-Mediated Immune Stimulatory Activity

Abstract

Bacterial and synthetic DNA containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). Our earlier studies showed that 5'-accessibility of synthetic oligodeoxynucleotides containing CpG motif (ODN) is required for TLR9-mediated immune stimulatory activity. Blocking the 5'-end of ODN through conjugation to a variety of moieties reduces immune stimulatory activity (Bioconjugate Chem. 2002, 13, 966-974). In the present study, we conjugated a model peptide, a 28-amino-acid-long beta-amyloid peptide, to either the 5'- or the 3'-end of an ODN via C3 and C6 alkyl linkers. We compared the immune stimulatory activity of the resulting conjugates with that of a parent ODN without conjugation in TLR9-transfected cells, mouse spleen cell cultures, and in vivo in mice. ODN with the peptide conjugated at the 3'-end via C3 and C6 linkers had immune stimulatory activity similar to that of the parent ODN in both in vitro and in vivo in mice. On the contrary, conjugation of peptide at the 5'-end of the ODN significantly abrogated immune stimulatory activity. In conclusion, the results presented here demonstrate that peptide/protein conjugation to ODN is optimal at the 3'-end with either C3 or C6 linker and conjugation at the 5'-end leads to significant loss of TLR9-mediated immune stimulation.

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

“…This enhancement can be explained by the increased resistance of the ODNs to nuclease degradation. Although it has been reported that free 5' ends in CpG ODNs with a PTO backbone are required for the activation of TLR9 [28,29], loss of activity in 5'-modified PD-ODN2006 in this study was attributed to degradation. When we modified PD-ODN2006 with 14-mer oligonucleotides at the 5'-end, there was still some potential to activate NF-κB, with some bands still present after serum degradation.…”

Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose

“…Together, these results indicate that CpG-B conjugation to NPs led to a higher frequency of activated cross-presenting DCs in the draining LNs, as well as a higher fraction of DCs able to present OVA in a Th1 and CD8 + T-cell-promoting context, for a given dose compared with free CpG-B. Notably, it was previously shown that modifications and conjugation of peptides to the 5′ end of CpG had a tendency to decrease their immunostimulatory effect (33,34). Our 5′ end conjugation strategy between NP and CpG, which allows the latter to be released in the reductive endosomal environment, did not negatively influence the activity of the bound CpG, which were even more stimulatory when conjugated than their free counterparts.…”

TLR9 Agonist Protects Mice from Radiation-Induced Gastrointestinal Syndrome

“…In this study we have used a novel TLR9 agonist containing synthetic immunomodulatory CpR (R = 2′-deoxy-7-dezaguanosine) dinucleotide and 3′-3′-attached novel structures that has been shown to induce potent TLR9-mediated immune responses [18], [19], [20], [21]. The presence of 3′-3′-attached structure provides higher metabolic stability and also optimal 5′-end presentation required for TLR9 recognition [18], [22], [23], [24]. These novel TLR9 agonists have been shown to induce potent Th1-type immune responses and a broad spectrum of antitumor activity in a number of tumor models [18], [25], [26], [27], [28].…”