Investigation of toll-like receptor 9 ligand recognition and function via multivalent oligodeoxynucleotide-polymer conjugates
| Author(s) | Levenson, Eric Andrew | |
| Date Accessioned | 2014-11-24T14:29:06Z | |
| Date Available | 2014-11-24T14:29:06Z | |
| Publication Date | 2014 | |
| Abstract | Toll-like receptor 9 (TLR9) recognizes DNA encoding unmethylated CpG motifs, and upon activation, promotes signaling through NF-? in B-cells for adaptive immunity or IRF3/7 plasmacytoid dendritic cells (pDC) for antiviral responses. Oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotide motifs are currently being investigated for use as vaccine adjuvants to promote these respective responses. ODN classes differ in their propensity to aggregate, which in turn, alter cytokine profiles and immune cell subsets activated. Here, we examined the role of ligand valency on the activation of TLR9 through the synthesis of ODN-poly(acrylic acid) (PAA) conjugates. The composition and size of the conjugates were characterized by UV-Vis spectroscopy, 1H NMR, gel permeation chromatography, and dynamic light scattering. ELISA-based assays of cytokine secretion by murine-like macrophages indicate that these ODN-PAA, show enhanced immunostimulation at 100-fold lower concentrations than those required for ODN alone, for both TNF-? and IL-6 release. To further define the role of ligand number and density, we directly labeled ODN-PAA conjugates with the fluorophore Alexa 647. IL-10 expression was shown to correlate linearly with ODN spatial density. TNF-? expression was the opposite with increased ODN presentation density decreasing expression, however, scaffold dependent responses were observed. Additionally, a critical valency was required for the fastest kinetics and degree of cytokine expression. Cellular internalization is shown to be a rapid, clathrin-mediated process (flow cytometry) with enhanced accumulation in LAMP-1 staining compartments compared to labeled ODN or scaffold (confocal microscopy). Our results define valency and density as critical design parameters and polymer conjugation as a potentially advantageous strategy for ODN immunomodulatory agents. To explore whether agonistic enhancements of ODN-PAA could be extended to antagonistic ODN sequences, another series of ON-PAA conjugates were synthesized. Here, our results demonstrate polymer conjugation of an antagonistic phosphodiester-based oligodeoxyribonucleic (ODN) sequence (4380) enhances intracellular delivery (over unconjugated ODN) with a ~15X reduction in IC 50 values, similar to the phosphorothioate-based derivative. Additionally, intracellular delivery and activity are influenced by valency with the most efficient conjugate showing IC50 values roughly 2-fold lower than the corresponding phosphorothioate sequence. These results suggest antagonistic ODN-polymer conjugates as a potential strategy for autoimmune disease therapy. | en_US |
| Advisor | Zhuang, Zhihao | |
| Advisor | Kiick, Kristi L. | |
| Degree | Ph.D. | |
| Department | University of Delaware, Department of Chemistry and Biochemistry | |
| Unique Identifier | 896823238 | |
| URL | http://udspace.udel.edu/handle/19716/13397 | |
| URL | 896823238 | |
| Publisher | University of Delaware | en_US |
| URI | http://search.proquest.com/docview/1564746990?accountid=10457 | |
| dc.subject.lcsh | Oligonucleotides. | |
| dc.subject.lcsh | Deoxyribose. | |
| dc.subject.lcsh | Polymers. | |
| dc.subject.lcsh | Ligands. | |
| dc.subject.lcsh | Valence (Theoretical chemistry) | |
| Title | Investigation of toll-like receptor 9 ligand recognition and function via multivalent oligodeoxynucleotide-polymer conjugates | en_US |
| Type | Thesis | en_US |