Impact of Secondary Structure of Toll-Like Receptor 9 Agonists on Interferon Alpha Induction (original) (raw)
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Proceedings of the National Academy of Sciences, 2005
Bacterial DNA and synthetic oligomers containing CpG dinucleotides activate the immune system through Toll-like receptor (TLR) 9. Here, we compare the immunostimulatory activity of three immunomers with different nucleotide sequences containing a synthetic cytosine-phosphate-2′-deoxy-7-deazaguanosine dinucleotide (CpR), called immunomodulatory oligonucleotides (IMOs), in mouse, human, and monkey systems. IMOs induced IL-12 and IFN-γ secretion more than a control non-CpG IMO in mice. All three IMOs activated HEK293 cells expressing TLR9 but not TLR3, -7, or -8. IMOs induced human B-cell proliferation and enhanced expression of CD86 and CD69 surface markers on B cells. The three IMOs induced CD86 expression on human plasmacytoid dendritic cells, but only IMOs that contained a 5′-terminal TCR nucleotide sequence induced IFN-α secretion. A sequence that forms a duplex structure also was required for IFN-α induction in human peripheral blood mononuclear cell cultures. IMOs induced chemok...
Journal of Medicinal Chemistry, 2009
Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides act as agonists of TLR9 and induce Th1-type immune responses. In the present study, we synthesized CpG containing ODNs in which C or G was substituted with 2 0-O-methylribonucleotides, 5-methyl-dC, or 2 0-O-methyl-5-methyl-C and studied their immune stimulatory activity alone and in combination with TLR agonists. In mouse and human primary cell-based assays, modified ODNs did not stimulate immune responses but inhibited TLR9 agonist-induced immune stimulatory activity. In mice, modified ODNs did not induce cytokines but inhibited immune responses induced by agonists of TLR7 and TLR9. Modified ODNs did not inhibit endosomal TLR3-or cell-surface TLR4-agonist-induced cytokines. This study demonstrates that ODNs incorporated with chemical modifications in CpG dinucleotides do not induce immune stimulatory activity but act as antagonists of TLR7 and TLR9 in vitro and in vivo. These types of modifications are commonly employed in antisense sequences and thereby may affect the intended mechanism of action.
Oligodeoxyribonucleotide-Based Antagonists for Toll-Like Receptors 7 and 9
Journal of Medicinal Chemistry, 2009
Oligodeoxyribonucleotides containing unmethylated CpG motifs act as TLR9 agonists. In this study, we evaluated oligonucleotides containing an unmethylated CpG motif in which two nucleotides adjacent to the CpG dinucleotide were substituted with 2′-O-methylribonucleotides, resulting in TLR7 and TLR9 antagonists. In mouse and human cell cultures, antagonists did not stimulate immune activation but inhibited TLR7 and TLR9 agonist-induced activity. In mice, antagonists inhibited immune responses induced by TLR9 agonists for up to several days, and the inhibition was dose-dependent. Antagonists also inhibited immune responses induced by an RNA-based TLR7/8 agonist but not TLRs 2, 3, 4, or 5 agonists in mice. Additionally, antagonist inhibited TLR9 agonist-induced IL-6 in lupus-prone MRL/lpr mouse spleen cell cultures. These results indicate that antagonists described herein can suppress immune responses induced by TLR7 and TLR9 agonists. Antagonists may be suitable candidates for treating inflammatory and autoimmune diseases where inappropriate or uncontrolled TLR activation has been implicated.
Synthesis and immunological activities of novel agonists of toll-like receptor 9
Cellular Immunology, 2010
Novel agonists of TLR9 with two 5 0 -ends and synthetic immune stimulatory motifs, referred to as immune modulatory oligonucleotides (IMOs) are potent agonists of TLR9. In the present study, we have designed and synthesized 15 novel IMOs by incorporating specific chemical modifications and studied their immune response profiles both in vitro and in vivo. Analysis of the immunostimulatory profiles of these IMOs in human and NHP cell-based assays suggest that changes in the number of synthetic immunostimulatory motifs gave only a subtle change in immune stimulation of pDCs as indicated by IFN-a production and pDC maturation while the addition of self-complementary sequences produced more dramatic changes in both pDC and B cell stimulation. All IMOs induced cytokine production in vivo immediately after administration in mice. Representative compounds were also compared for the ability to stimulate cytokine production in vivo (IFN-a and IP-10) in rhesus macaques after intra-muscular administration.
Biochemical and biophysical research communications, 2013
Toll-like receptor 9 (TLR9) recognizes a synthetic ligand, oligodeoxynucleotide (ODN) containing cytosine-phosphate-guanine (CpG). Activation of TLR9 by CpG ODN induces a signal transduction cascade that plays a pivotal role in first-line immune defense in the human body. The three-dimensional structure of TLR9 has not yet been reported, and the ligand-binding mechanism of TLR9 is still poorly understood; therefore, the mechanism of human TLR9 (hTLR9) ligand binding needs to be elucidated. In this study, we constructed several hTLR9 mutants, including truncated mutants and single mutants in the predicted CpG ODN-binding site. We used these mutants to analyze the role of potential important regions of hTLR9 in receptor signaling induced by phosphorothioate (PTO)-modified CpG ODN and CpG ODNs only consist entirely of a phosphodiester (PD) backbone, CpG ODN2006x3-PD that we developed. We found truncated mutants of hTLR9 lost the signaling activity, indicating that both the C- and N-ter...
Design and Structural Requirements of the Potent and Safe TLR-9 Agonistic Immunomodulator MGN1703
Nucleic acid therapeutics, 2015
Single-stranded oligodeoxynucleotides (ODN), containing nonmethylated cytosine-guanine motifs (CpG ODN), are recognized by the innate immune system as "danger signals." CpG ODN are efficacious immunomodulators but require phosphorothioate (PT) or other backbone modifications for metabolic stability, which cause toxicities in mice and primates. We therefore designed a covalently closed DNA molecule (dSLIM(®)) where two single-stranded loops containing CG motifs are connected through a double-stranded stem in the absence of any nonnatural DNA component. The most promising immunomodulator, MGN1703, comprises two loops of 30 nucleotides containing three CG motifs each, and a connecting stem stem of 28 base pairs. MGN1703 stimulates cytokine secretion [interferon (IFN)-α, IFN-γ, interleukin (IL)-12, IL-6, and IL-2] and activates immune cells by increased expression of CD80, CD40, human leukocyte antigen (HLA)-DR and ICAM-1. Efficacy of immunomodulation strictly depends on the d...
Immunology, 2008
Synthetic oligodeoxynucleotides containing unmethylated CpG sequences (CpG-ODNs) stimulate Toll-like receptor-9 (TLR-9), thereby activating innate immunity. Stimulatory CpG-ODNs have been shown to be valuable in modifying immune responses in allergy, infection and cancer. Recently, it has been reported that the stimulation of TLR-9 by endogenous DNA might contribute to the pathogenesis of autoimmune diseases. We here report the identification of a suppressive, guanosine-rich ODN (G-ODN) that inhibited the activation of TLR-9 by stimulatory CpG-ODNs. The G-ODN was suppressive in murine macrophages and dendritic cells as well as in human plasmacytoid dendritic cells in vitro. G-ODN blocked the secretion of tumour necrosis factor-a (TNF-a) and interleukin-12p40 and interfered with the up-regulation of major histocompatibility complex (MHC) class II and costimulatory molecules. G-ODN was inhibitory even at a molar ratio of 1 : 10 (G-ODN:CpG-ODN) and when administered up to 7 hr after stimulation with CpG. G-ODN specifically inhibited TLR-9 but not other TLRs. Inhibition was dependent on a string of five guanosines. G-ODN was also inhibitory in an in vivo model of CpG/galactosamin (GalN) lethal shock. G-ODN interfered with upstream TLR-9 signalling. However, by extensive analysis we can exclude that G-ODN acts at the stage of cellular uptake. G-ODN therefore represents a class of suppressive ODNs that could be of therapeutic use in situations with pathologic TLR-9 activation, as has been proposed for certain autoimmune diseases.
Ligand-induced conformational changes allosterically activate Toll-like receptor 9
Nature Immunology, 2007
Microbial and synthetic DNA rich in CpG dinucleotides stimulates Toll-like receptor 9 (TLR9), whereas DNA lacking CpG either is inert or can inhibit TLR9 activation. The molecular mechanisms by which TLR9 becomes activated or is inhibited are not well understood. Here we show that TLR9 bound to stimulatory and inhibitory DNA; however, only stimulatory DNA led to substantial conformational changes in the TLR9 ectodomain. In the steady state, 'inactive' TLR9 homodimers formed in an inactivated conformation. Binding of DNA containing CpG, but not of DNA lacking CpG, to TLR9 dimers resulted in allosteric changes in the TLR9 cytoplasmic signaling domains. In endosomes, conformational changes induced by DNA containing CpG resulted in close apposition of the cytoplasmic signaling domains, a change that is probably required for the recruitment of signaling adaptor molecules. Our results indicate that the formation of TLR9 dimers is not sufficient for its activation but instead that TLR9 activation is regulated by conformational changes induced by DNA containing CpG.
PloS one, 2014
CpG-oligodeoxynucleotides (CpG-ODN) are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9) is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18-24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund's adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody re...