title: DNA-polymer conjugates for immune stimulation through Toll-like receptor 9 mediated pathways.
abstract: Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide motifs are agonists of Toll-like receptor 9 and are currently being investigated for use as vaccine adjuvants through the promotion of type I immunity. Several classes of ODN have been developed which differ in their propensity to aggregate, which in turn alters cytokine profiles and cellular subsets activated. Although aggregation state is correlated with the change in cytokine response, it is unknown if this results from a change in the number of ODNs available for binding and/or the possible engagement of multiple TLR9 molecules. Here, we examined the role of ligand valency on the activation of TLR9 through the synthesis of ODN-poly(acrylic acid) (PAA) conjugates. The compositions and size of the conjugates were characterized by UV-vis spectroscopy, proton nuclear magnetic resonance, gel permeation chromatography and dynamic light scattering. Enzyme-linked immunosorbent assays of cytokine secretion by murine-like macrophages indicate that these ODN-PAA polymer conjugates show enhanced immunostimulation at 100-fold lower concentrations than those required for ODN alone, for both TNF-α and IL-6 release, and are more potent than any other previously reported multivalent ODN constructs. Increasing valency was shown to significantly enhance cytokine expression, particularly for IL-6. Knockdown by siRNA demonstrates that these polymer conjugates are specific to TLR9. Our results define valency as a critical design parameter and polymer conjugation as an advantageous strategy for producing ODN immunomodulatory agents.
authors: Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA.
authors: Eric A
authors: EA
authors: Levenson
authors: Department of Materials Science and Engineering, University of Delaware, Newark, DE 19716, USA; Biomedical Engineering, University of Delaware, Newark, DE 19716, USA; The Delaware Biotechnology Institute, University of Delaware, Newark, DE 19716, USA. Electronic address: kiick@udel.edu.
authors: Kristi L
authors: KL
authors: Kiick
mesh_terms: D000180:Acrylic Resins / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D016207:Cytokines / Q000378:metabolism; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D008807:Mice, Inbred BALB C; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051217:Toll-Like Receptor 9 / Q000378:metabolism*
chemical_list: D000180:Acrylic Resins; D016207:Cytokines; D009838:Oligodeoxyribonucleotides; D034741:RNA, Small Interfering; D051217:Toll-Like Receptor 9; C006903:carbopol 940; D004247:DNA
keywords: DNA; Immune response; Immunomodulation; Immunostimulation; Toll-like receptor 9 agonist