Impact of Secondary Structure of Toll-Like Receptor 9 Agonists on Interferon Alpha Induction

Abstract

Oligodeoxynucleotides containing a CpG motif and double-or multistranded structure-forming sequences act as agonists of Toll-like receptor 9 (TLR9) and induce high levels of interferon alpha (IFN-␣) in addition to other Th1-type cytokines. In the present study, we evaluated three highly effective IFN-␣-inducing agonists of TLR9 to determine the type of duplex structures formed and the agonist's ability to induce immune responses, including IFN-␣ induction, in human cell-based assays and in vivo in mice and nonhuman primates. Thermal melting studies showed that two of the agonists evaluated had a single melting transition with similar hyperchromicity in both heating and cooling cycles, suggesting the formation of intermolecular duplexes. A third agonist showed a biphasic melting transition in the heating cycle and a monophasic melting transition with lower hyperchromicity during the cooling cycle, suggesting the formation of both intra-and intermolecular duplexes. All three agonists induced the production of Th1-type cytokines and chemokines, including high levels of IFN-␣, in human peripheral blood mononuclear cell and plasmacytoid dendritic cell cultures. Subcutaneous administration of the two intermolecular duplex-forming agonists, but not the intramolecular duplex-forming agonist, induced cytokine secretion in mice. In nonhuman primates, the two agonists that formed intermolecular duplexes induced IFN-␣ and IP-10 secretion. On the contrary, the agonist that formed an intramolecular duplex induced only low levels of cytokines in nonhuman primates, suggesting that this type of structure formation is less immunostimulatory in vivo than the other structure. Taken together, the present results suggest that oligonucleotide-based agonists of TLR9 that form intermolecular duplexes induce potent immune responses in vivo.The vertebrate immune system recognizes highly conserved molecular patterns that are present in pathogens through a number of pattern recognition receptors. Toll-like receptors (TLRs) are among the well-characterized pattern recognition receptors. At least 10 TLRs have been identified in humans, and one of them, TLR9, is the receptor for bacterial and synthetic DNA containing unmethylated CpG motifs (4). TLR9 is expressed predominantly in B cells and plasmacytoid dendritic cells (pDCs) in humans. Activation of these two cell types by synthetic oligonucleotides containing unmethylated CpG motifs via TLR9 results in a Th1-type immune response which includes the secretion of interferon alpha (IFN-␣), IFN-␥, interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-␣), and IL-6 with an increase in the levels of costimulatory surface molecules (1,11,12,17,19,28). A number of TLR9 agonists are currently being evaluated in clinical trials as therapies for various diseases, including cancers, infectious diseases, allergy, and asthma, and as vaccine adjuvants (1, 10).The immune response profiles induced via TLR9 stimulation depend on the stimulatory motif and secondary structure present in the oligonucleotides (...

Minimal Sequence Requirements for Oligodeoxyribonucleotides Activating Human TLR9

“…4B, 4C). To summarize, the minimal sequence of minH that activates TLR9 is TCG[T] 6-10 CG[T] [10][11][12][13][14][15][16][17][18][19] , with the length of the ODN ranging from 21 to 30 nt.…”

“…A-, C-, and P-class (17,18) CpG ODNs form intra-and intermolecular duplexes. These multi-ODN structures are suggested to take a different route of endocytosis and, therefore, induce the synthesis of type I IFNs (17,19). In addition to unmethylated CpG motifs within the ODN, other factors determine the immune response of TLR9-expressing cells.…”

“…The impact of the nucleotide context and the distance between the CpG motifs, as well as the effect of ODN duplex formation on TLR9 activity, were determined. Finally, we established the minimal ODNs defined by the sequence TCG[T] 6-10 CG[T] [9][10][11][12][13][14][15][16][17][18][19] and a total length of .21 nt. These minimal ODNs induce production of TNF-a in human PBMCs.…”

Polyvalent Immunostimulatory Nanoagents with Self‐Assembled CpG Oligonucleotide‐Conjugated Gold Nanoparticles

“…[10] For example, sequences and modifications of synthetic CpG ODNs have been designed to achieve optimal stability and immunostimulatory activities. [11] Replacement of the native phosphodiester (PO) bond with a nuclease-resistant phosphorothioate (PS) backbone can greatly increase the stability of synthetic CpG ODNs in vivo. [1,12] Moreover, a variety of transfection agents have been utilized in an attempt to increase cellular uptake and reduce the cellular toxicity of CpG ODNs.…”

Species-Specific Minimal Sequence Motif for Oligodeoxyribonucleotides Activating Mouse TLR9

“…The A, C, and P class (23,24) CpG ODNs form intra-and intermolecular duplexes. These multi-ODN structures are suggested to take different routes of endocytosis and therefore induce synthesis of type I IFNs (23,25,26). For activation of mouse TLR9 with A class ODNs, endosomal DNase II has to cleave the A class ODNs, which consequently leads to the formation of short ODN fragments resembling B type ODN (25).…”