title: Immunomodulatory oligonucleotides containing a cytosine-phosphate-2'-deoxy-7-deazaguanosine motif as potent toll-like receptor 9 agonists.
abstract: 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-gamma 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-alpha secretion. A sequence that forms a duplex structure also was required for IFN-alpha induction in human peripheral blood mononuclear cell cultures. IMOs induced chemokine and cytokine gene expression in human peripheral blood mononuclear cells. In monkeys, all three IMOs induced transient changes in peripheral blood leukocytes and lymphocytes and activated B and T lymphocytes. All three IMOs induced IFN-alpha in vivo in monkeys; the IMO sequence that forms a stable secondary structure induced the highest levels of IFN-alpha. These studies are, to our knowledge, the first comprehensive studies to compare the activity of IMOs containing synthetic stimulatory CpR dinucleotides in mouse, monkey, and human systems. These results suggest that IMOs induce strong and rapid immunostimulation and that the CpR dinucleotide is recognized by TLR9, leading to immune-cell activation and cytokine secretion in vitro and in vivo.
authors.affiliation: Hybridon, Inc., 345 Vassar Street, Cambridge, MA 02139, USA.
authors.forename: Ekambar R
authors.initials: ER
authors.lastname: Kandimalla
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Bhagat
authors.forename: Yukui
authors.initials: Y
authors.lastname: Li
authors.forename: Dong
authors.initials: D
authors.lastname: Yu
authors.forename: Daqing
authors.initials: D
authors.lastname: Wang
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Cong
authors.forename: Sam S
authors.initials: SS
authors.lastname: Song
authors.forename: Jimmy X
authors.initials: JX
authors.lastname: Tang
authors.forename: Tim
authors.initials: T
authors.lastname: Sullivan
authors.forename: Sudhir
authors.initials: S
authors.lastname: Agrawal
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism; D051940:B7-2 Antigen; D001483:Base Sequence; D002460:Cell Line; D049109:Cell Proliferation; D018925:Chemokines / Q000378:metabolism; D018899:CpG Islands; D016207:Cytokines / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000819:agonists*; D003713:Dendritic Cells / Q000166:cytology; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D000882:Haplorhini; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D037181:Lectins, C-Type; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000819:agonists* / Q000378:metabolism; D013997:Time Factors; D051196:Toll-Like Receptor 3; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D015854:Up-Regulation; D000099192:CD69 Antigens
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D051940:B7-2 Antigen; D018925:Chemokines; D016207:Cytokines; D004268:DNA-Binding Proteins; D003849:Deoxyguanosine; D007371:Interferon-gamma; D018664:Interleukin-12; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D009711:Nucleotides; D009841:Oligonucleotides; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D051196:Toll-Like Receptor 3; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D000099192:CD69 Antigens; C497964:CD86 protein, human; C497965:Cd86 protein, mouse; C495344:TLR3 protein, human; C495371:TLR9 protein, human; C495370:Tlr9 protein, mouse; C045550:7-deaza-2'-deoxyguanosine