Die Rolle von RNA/DNA-Hybriden in der angeborenen Immunantwort und das Potential von cholesterolkonjugierter RNA als Adjuvans

Die Rolle von RNA/DNA-Hybriden in der angeborenen Immunantwort Das angeborene Immunsystem detektiert RNA und DNA über verschiedene pattern recognition receptors (PRR), zu welchen unter anderem die Toll-like Rezeptoren (TLR) gehören. RNA/DNA-Hybride treten im Verlauf viraler Infektionen, während...

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Bibliographic Details
Main Author: Obermann, Hannah-Lena
Contributors: Bauer, Stefan (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2014
Online Access:PDF Full Text
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Table of Contents: RNA/DNA-hybrids in innate immunity The innate immune system senses viral and bacterial nucleic acids in the cytoplasm or in endosomal compartments via different pattern recognition receptors (PRR) such as Toll-like receptors (TLR). The repertoire of pathogen-associated molecular patterns (PAMP) is still not exhausted. Since RNA/DNA-hybrids are formed transiently during viral infection, cell endogenous processes and may lead to autoimmune diseases, the immunstimulatory capacity of RNA/DNA-hybrids needs to be further investigated. Analysis of HCMV- and HIV-derived synthetic RNA/DNA-hybrids showed an efficient induction of IFNα-secretion in human PBMC as well as in murine Flt3L-DC. Additionally, murine Flt3L-DC produced TNFα, IL-6 and IP-10 upon RNA/DNA-hybrid stimulation. Cytokine induction occured in a TLR9-dependent manner since TLR9-deficient Flt3L-DC did not respond with cytokine production to RNA/DNA-hybrids. Direct interaction of RNA/DNA-hybrids with TLR9 was demonstrated by an ELISA-based binding assay. Furthermore, intact RNA/DNA-hybrids could be detected within transfected Flt3L-DC with the RNA/DNA-hybrid-specific antibody S9.6. The influence of RNA/DNA-hybrids within viral, bacterial or mitochondrial ge-nomes could not be analysed in detail, because their immunostimulatory potential was probably masked by high amounts of genomic DNA. Infection of Flt3L-DC with Mo-MuLV lead to RNA/DNA-hybrid formation and accumulation within the cells cytoplasm and in vesicular structures. However, Mo-MuLV negatively influenced CpG2216-mediated IFN-signalling independent of Siglec-H or DAP12 in Flt3L-DC and therefore avoided antiviral immune responses. Cholesterol-coupled dsRNA40 serves as a potent adjuvant delivery system Vaccination is indispensable to prevent infectious diseases and therefore development of safe and potent vaccine adjuvants is still highly important. Adjuvants are included in vaccines to enhance immune responses to an antigen. In general, TLR-ligands possess the potential to be utilized as adjuvants. Different TLR are expressed within diverse subtypes of immune cells and induce various cytokines. The cytokine milieu mainly influences the characteristics of an vaccine. RNA40, which is a well established HIV-derived TLR7-ligand, has to be complexed to a transfection reagent for cellular uptake and for preventing enzymatic degradation. In this study a very potent adjuvant delivery system for TLR7 has been developed. Double-stranded RNA40 was coupled to cholesterol to force cellular uptake. Cholesterol occurs naturally within cell membranes for which reason undesired side-effect should be prevented. Cellular uptake of cholesterol coupled dsRNA40 by Flt3L-DC was analyzed by flow cytometry and was as effective as uptake of dsRNA40 complexed to Dotap. Interestingly, stimulation of Flt3L-DC with dsRNA40 coupled to cholesterol lead to an increased secretion of IFNα, TNFα, IL-6 and IL-10 in comparison to complexation of dsRNA40 to Dotap. Immune stimulation by cholesterol itself could be excluded. Overall, these in vitro experiments showed an effective activation of murine Flt3L-DC by dsRNA40-Chol in a TLR7-dependent manner.