AAC Accepts, published online ahead of print on 13 October 2008

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Antimicrob. Agents Chemother. doi:10.1128/AAC.00701-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Impact of secondary structure of Toll-like receptor 9 agonists on interferon- induction

Dong Yu, Mallikarjuna R. Putta, Bhagat Lakshmi, Meiru Dai, Daqing Wang, Anthony F. Trombino, Tim Sullivan, Ekambar R. Kandimalla, and Sudhir Agrawal^*

Idera Pharmaceuticals, 167 Sidney Street, Cambridge, MA 02139, USA

^* To whom correspondence should be addressed. Email: sagrawal{at}iderapharma.com.

Oligodeoxynucleotides containing a CpG motif and double- or multi-stranded structure-forming sequences act as agonists of Toll-like receptor 9 (TLR9) and induce high levels of 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 non-human 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 formation of inter-molecular 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 inter-molecular duplexes. All three agonists induced production of Th1-type cytokines and chemokines, including high levels of IFN-, in human PBMC and pDC cultures. Subcutaneous administration of the two inter-molecular duplex-forming agonists, but not the intra-molecular duplex-forming agonist, induced cytokine secretion in mice. In non-human primates, the two agonists that formed inter-molecular duplexes induced IFN- and IP-10 secretion. On the contrary, the agonist that formed an intra-molecular duplex induced only low levels of cytokines in non-human 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 inter-molecular duplexes induce potent immune responses in vivo.