Browsing by Subject "Immunological adjuvants."

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Investigation of improved immunostimulatory activity of D and K type CpG ODNs in liposomes
(2013) Dereli, İhsan
CpG ODNs are potent immunotherapeutic agents. In human, two major classes of CpG ODNs were shown to induce differential immune activation. D ODNs are strong IFNα inducers, thus promising antiviral agents, whereas K ODNs are effective against bacterial infections. However, their effects cannot be combined. When K and D type ODNs are used simultaneously, K ODN cancels D specific effect, a phenomenon known as K and D ODN dichotomy. The prime reason for this counter acting K ODN action was subcellular compartmentalization of K type CpG ODNs upon internalization. Besides, CpG ODNs have labile nature. When investigated in clinical trials, these nucleic acid based ligands are eliminated upon administration and displayed limited bio-availability due to nuclease digestion. Hence, efforts to protect in vivo performance, and increase stability and accumulation near target cells became a crucial task. Liposome technology offers a simple and mild approach to harbor these ODNs within membrane bilayers and protect them. We also reasoned that, if we use liposomes that alter subcellular fate of K and D ODNs, we can retain both K and D effect when liposomal ODNs are co-administered and the breadth of immunotherapeutic spectrum could be improved. This thesis was designed to understand and characterize different types of CpG ODNs loaded into different liposomes and aimed to determine their activities in different in vitro and in vivo settings. Our results revealed that when two different classes of clinically important CpG ODNs were encapsulated within proper liposome types, it is possible to recapitulate both K and D type ODN effect in PBMCs. Furthermore, in a vaccine model against H. felis, although initially did not induce significantly higher anti H.felis immunity, liposomal CpG ODNs improved persisting antibody levels for extended periods compared to free counterparts. Collectively, our results demonstrate that this platform allows more effective in vivo utilization of CpG ODNs and can be formulated to develop more efficient means to combat several health problems, ranging from cancer to allergy.
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Self-nanoparticle forming immunostimulatory DNA : structure-function relationship studies
(2009) Mammadov, Rashad
Toll-like receptors (TLRs) are one the most critical and widely studied members of the family of pattern recognition receptors expressed on innate immune cells. They recognize microbial signatures, such as bacterial/viral DNA, LPS, from gram negative bacteria, peptidoglycan from gram positive bacteria, zymosan from yeast, lipopeptides or profilin protein from parasites, and even single or double stranded RNA of viruses. Among several members of TLR family, TLR9, that recognizes microbial unmethylated dinucleotide motifs on DNA initiate a robust Th1- biased inflammatory response. Synthetic oligodeoxynucleotides expressing unmethylated CpG motifs, mimic bacterial DNA effect and can be harnessed for the treatment of health problems ranging from infectious diseases to cancer, or to allergy/asthma as well as stand alone immunoprotective agents and also as a vaccine adjuvants that improve protection against pathogens. To date, various classes of CpG ODNs have been identified and were shown to induce differential immune activation in mice and man. Distinct structure-function relationship analyses revealed that these single-stranded linear ODNs alter the immune milieu as they are formulated to form complex multimeric DNA aggregates. Recently, Guanosine-rich D type CpG ODNs has been reported to form complex aggregates, that are differentially regulating immune cells to mount an anti-viral immunity. However, the clinical trials of this type are hampered mainly due to batch to batch variation during large-scale synthesis. To the best of our knowledge, there is no report on self-nanoparticle forming DNA except G-rich sequences. This thesis project was designed to generate stable, selfnanoparticle forming, G-run free, CpG expressing ODNs. In this thesis, we designed a new generation CpG ODN, then characterized their structural and immunological properties. Our results suggest that dendrimeric structure confers higher immunostimulatory potential unparallel to conventional ODNs. Following four hours of in vivo ODN administration into mice indicated that nanoparticle-forming CpG ODNs initiated substantially high spleen and peritoneal exudate cell activation as evidenced by IFNγ and IL-12 production from culture medium. In order to shed light on the uptake and binding mechanisms, blocking experiments revealed that at least one type of scavenger receptor is critical for nanoparticle ODN internalization. Collectively, these data suggested that the improved stability to nucleases along with significantly higher binding to immune cells (no additional ODN formulation is required) seem to be the critical factors contributing to the nanoparticle CpG ODN mediated immune activation. The in vitro and in vivo performances implicated that these next generation immune stimulatory DNA molecules are promising candidates for various clinical applications.