Browsing by Subject "Vaccine carrier"

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Open Access
The development of prophylactic and therapeutic vaccine using cell derived extracellular vesicles
(2022-08) Yıldırım, Muzaffer
The primary aim of this thesis is to extend the breadth of in vivo application of externally loaded cell-line derived and tumor derived exosomes as a prophylactic and therapeutic carrier against cancer treatment. Exosomes with a size between 30 to 150 nm are small extracellular vesicles secreted by all types of mammalian cells. They mediate a novel mode of intercellular communication through their bioactive cargos such as lipids, nucleic acids, metabolites, and proteins, which can be delivered to the target cells. Exosomes have successfully served as immunotherapeutic nanocarriers in cancer treatment using their natural delivery capabilities. Furthermore, they are attractive as a delivery system because of their stability in circulation, biocompatibility, and low toxicity. In the first part of this thesis, we used exosomes as a nanocarrier system to develop cancer vaccines in a therapeutic murine melanoma cancer treatment. We show that lyophilization of exosomes together with the CpG ODN, model antigen OVA and lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution is successfully accomplished. We analyzed the effect of the lyophilization on a cell line-derived exosomes and we characterized the exosomes by using bead-based technique via flow cytometry, qNano, Scaning electron microscopy, and western blotting. We showed that lyophilization does not harm exosomes’ vesicular integrity and fundamental biological features. Furthermore, we tested the biodistribution and activating capacity of encapsulated exosomes in mouse PEC, mesenteric lymph node, and spleen cells. We found out that loaded exosomes are mostly taken up by antigen-presenting cells. Also, we showed that loading CpG ODN into exosomes significantly improves APC activation markers in macrophages, B cells, and DCs and induced significantly higher IFNγ production from mouse mLN and splenocytes. Finally, we tested the therapeutic utility of the CpG ODN, OVA and αGC encapsulating exosome in the B16F10-OVA melanoma tumor-bearing mice. We found out that therapeutic vaccination with triple (CpG ODN, OVA, and αGC) ligand encapsulating exosomes suppressed the progression of established melanoma tumors in mice. Moreover, our triple ligand loaded exosomes triggered Th-1 biased anti-IgG OVA immunity and converted immune cells in tumor microenvironment to the tumor-suppressing phenotype. In the second part of this thesis, we used tumor-derived exosomes (TEXs) as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells, TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). We showed that engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic breast cancer model, we found out that administration of 4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regressed tumor growth in 4T1 tumor-bearing mice. As a result, this work implicated that an exosome based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. The last part of this thesis, we studied the therapeutic potential of cell line-derived exosomes loaded with superparamagnetic iron oxide nanoparticles(SPION) and immunostimulatory ligands. We showed that loading SPION enhanced the in vitro delivery of exosomes within immune cells. Also, we found out that spleen cells incubated with exosomes encapsulating with SPION and CpG ODN induced significantly higher levels of IL-12 and IFNγ. Finally, we tested our exosomal vaccine candidate in human hepatocellular carcinoma tumor model in athymic mice. We showed that TLR3 and TLR9 ligands encapsulated with SPION loaded exosomes induced pronounced innate immune activation and regressed tumors and improve survival rate of treated mice.
Open Access
Immunotherapeutic action of extracellular vesicles and effects of TLR signaling to immune dysfunction of SCI patients
(2017-03) Güçlüler, Gözde
The primary aim of this thesis is to extend the breadth of in vivo application of externally loaded exosomes as prophylactic or therapeutic carriers against disease treatment. Exosomes are secreted from all cells and could be purified from all bodily fluids; however, engineering of exosomes to carry specific ligands post-purification is a daunting task. Herein, we show that lyophilization of exosomes together with the biological cargo alone or in combination of CpG ODN motifs, model protein antigen ovalbumin or lipidic ligand alpha-galactosylceramide (αGC) followed by controlled reconstitution successfully internalizes these cargos within exosomes. Furthermore, the bioactivity of the loaded ligand(s) surpasses the unloaded free ligand activities. When tested in vivo, exosome incorporated ligand(s) proved to be significantly effective against model tumors such as E.G7 thymoma or established melanoma models. The mechanism behind this elevated immune activity is the ability of exosomes to be delivered to target cells and boost immune antigen dependent immune activation. Our in vitro findings revealed that encapsulation of CpG ODN into exosomes enhances immunostimulatory activity of CpG ODN than free form as evidenced by superior levels of cytokines like IL6, IL12 and Type-I and II interferons. This magnified immune activity might be partly due the increased APC activation observed as elevated CD86/MHCII surface marker expression. Immunization of C57/Bl6 mice with exosomal CpG ODN plus OVA induced strong Th1-biased anti-OVA response. Following thymoma induction in naive and OVAimmunized animals, >85% of exosomal vaccine treated mice cleared tumors whereas almost all naive animals were positive for tumor. This data suggests that CpG ODN encapsulation into exosomes improve immunostimulatory activity, provide better anti-OVA immunity thereby contribute effective tumor clearance in mice. A second aim of this thesis was to establish that it is feasible to load exosomes with more than two ligands. Next, invariant natural killer T (iNKT) cell ligand αGC was included within exosomes as the third element next to OVA and CpG ODN. Initial in vivo studies revealed that exosomes containing αGC were significantly more potent in inducing antigen dependent immune responses in comparison to free form of CpG ODN, OVA and αGC. In therapeutic tumor vaccine model, two exosome injections (@d: 9 and d: 15) were done to B16-OVA tumor bearing animals and tumor regression was followed. Mice that had triple exosomal ligands significantly reduced tumors compared to mice treated with non-exosomal ligands. This study confirmed that exosomes with triple ligands could be effectively control established tumor development. In this thesis, the elucidation of the involvement of extracellular vesicles (EVs) on the pathogenesis of autoimmune/autoinflammatory diseases was studied. The underlying mechanism in BD pathogenesis is still unclear. We found that one of the human cathelicidin group members, antimicrobial peptide LL37 along with EVs were elevated in active BD patients` plasmas. Strikingly, majority of plasma LL37 was associated with circulating EVs. We found that there was a strong correlation between i) LL37 level, ii) EV #/ml plasma and iii) cytokine production. In the last part of this thesis, one of the possible mechanisms of immune dysfunction contributing to severe neurological deterioration of chronic spinal cord injured (SCI) patients was unearthed. We aimed to investigate whether there is a correlation between susceptibility to infections of chronic SCI patients within the context of impaired innate recognition of pathogen associated molecular patterns (PAMPs). Our data implicated that although there was no dysfunction of B cell, or CD4+ Treg activity, but sensing TLR7 and TLR9 ligands by monocytes and pDCs were ablated in patients with SCI, leading to lower IFNγ and IP10 production along with costimulatory molecule expression, that could explain the immunological dysfunction in patient with SCI contributing to persistent complications.
Open Access
Therapeutic and diagnostic applications of extracellular vesicles
(2016-03) Kahraman, Tamer
Extracellular vesicles (EV), consisting of exosomes and microvesicles, are secreted biological nanovesicles and assumed plethora of physiological functions ranging from transport of cargo, regulating distant cell communication, and altering immune response. Accumulating evidence suggests that extracellular vesicles may participate in disease pathogenesis of inflammatory diseases. Moreover, accumulating evidence suggests that EVs are promising nanocarrier capable of modulating immune response. This thesis aims to harness EVs in immunotherapeutic and diagnostic applications. Behçet’s Disease (BD) activity is manifested with sustained, over exuberant immune activation, yet the underlying mechanisms leading to active BD state is poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 and EVs are elevated in BD plasma. Our data suggested that majority of LL37 is associated with EVs. This association drives plasma EVs to immune cells, enhancing pathologic and sustained immune response, thereby leading to aggravating BD pathology. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. Findings of this study could be of clinical interest in the management of BD, implicating that LL37/EV association as one of the major contributors of BD pathogenesis and might be used as a diagnostic readout to stratify the severity of BD patients. EVs, more specifically exosomes, suggested as new tools for biomedical applications such as drug/vaccine carrier vesicles. However, efforts to engineer cells to express desired cargo in/on these secreted exosomes or induce physical complexation with candidate bioactive agents or even use of membrane-breaching techniques such as electroporation to load exosomes with desirable cargo showed limited in vivo performance. Here we developed a mild and simple technique enabling external loading of any type of desired bioactive molecule within exosomes at high yield. Using this approach, we exploited therapeutic potential of exosomes encapsulating CpG ODN together with a protein antigen as a vaccine cancer for preventive tumor therapy. CpG ODN loaded within exosomes displayed pronounced in-vitro activity as evidenced by up to 6-fold higher IL6 and IL12 secretion from splenocytes as well as increased IFNα secretion from pDCs. Exosomes protected CpG ODN from digestion by DNase-I up to 90%. In order to demonstrate improved in-vivo activity, exosomes co-encapsulating CpG ODN and ovalbumin were tested as a potential vaccine vector against EG-7 thymoma. Animals that received Exo(CpG ODN+OVA) vaccine led to a magnified and persistent Th1-biased antiOVA IgG responses that was sufficient to fully protect mice from EG-7 derived tumor challenge even after 24 weeks post-booster injection as opposed to free vaccine combination. Our results suggest that EVs could be of clinical interest in both prognosis and management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. In addition, our studies related with exosomes present a platform that opens a new avenue to personalized cell-free therapeutic intervention and could be developed to harbor other therapeutically important molecules ranging from plasmid to mRNA or si/miRNA for more effective therapeutic modality development in the clinic against debilitating diseases ranging from cancer to infectious diseases