Theses supervised by Prof. Dr. İhsan Gürsel
22 theses · İhsan Doğramacı Bilkent University
Astımı olan pedı̇atrı̇k covıd-19 hastalarında ekstraselüler vesı̇küllerı̇n rolü
SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), has infected millions of people, and asthma was initially considered a risk factor for COVID-19. Although numerous studies on asthma and COVID-19 have been conducted, the role of plasma-derived extracellular vesicles (EVs) in COVID-19 patients with asthma remains unknown. In this study, we assessed the influence of EVs from healthy controls, severe and mild COVID-19 pediatric patients with or without asthma during acute and convalescent periods on healthy naïve CD4+T cells and monocytes. While plasma cytokines and anti-SARS-CoV-2 antibodies were similar between the groups with and without asthma, immune responses varied depending on the severity of COVID-19. In the severe acute group, whereas all cytokines increased, IFNγ, CD4+T cell counts, and monocyte numbers decreased. Stimulating healthy cells with EVs from severe acute patients led to increased PDL1 expression, Th2 and Treg cell proportions, decreased IFNγ secretion, Th1, and Th17 cell ratios. Patient EVs also reduced proinflammatory cytokine secretion from monocytes. Severe acute patient EVs caused a decline in healthy CD4+T cell and monocyte populations. Overall, our results indicate immunological responses and EV-related outcomes depending on the severity of COVID-19 rather than the presence of asthma, immunosuppression seen in severe acute COVID-19 and potential contribution of EVs to this immunosuppressive pattern in severe cases.
N. meningitidis ve SARS-CoV-2'ye karma aşı geliştirilmesi ve pediatrik hastalarda hastalık şiddeti ile ilişkili olarak MIS-C plazması ve ekstraselüler veziküllerin analizi
Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). Serogroup B remains the leading cause of IMD, representing 62% of documented serogroup cases overall, and is the most prevalent across all age groups under 65. In Türkiye, Serogroups B and W are responsible for most cases, accounting for over 75%. IMD has a case fatality rate of 10%, and 10% to 20% of survivors experience lifelong, disabling complications. Coronavirus disease 2019 (COVID-19), caused by the highly contagious SARS-CoV-2 virus, has had a devastating impact globally, leading to over 7 million deaths. It has become the most significant global health crisis since the influenza pandemic in 1918. Since the introduction of the first COVID-19 vaccine in the U.S., it is estimated that over 18 million hospitalizations and 3 million deaths have been prevented. Like flu vaccines, COVID-19 vaccines are expected to be introduced seasonally. In this thesis, we aim to develop a combination vaccine for populations where the prevalence of both COVID-19 and meningitis presents significant health risks. Our previous data indicated that our OMV-based bivalent vaccine, targeting B and W serogroups of Neisseria meningitidis, generates a broader humoral response and exhibits strong bactericidal activity. Similarly, our lab has developed a virus-like particle-based SARS-CoV-2 vaccine incorporating all four virus structural proteins, eliciting both humoral and cell-mediated immunity. Herein, we combined these two platforms and assessed their respective protective potencies in mice. Data revealed that combining the OMV and VLP vaccines with Alum adjuvant produced the most robust anti-meningococcal and anti-SARS-CoV-2 responses. The second part of the study focused on uncovering the immune parameters present in the plasma of MIS-C patients and investigating the role of extracellular vesicles in influencing disease severity. The data revealed that plasma from MIS-C patients contained significantly elevated levels of proinflammatory cytokines, including TNF-α, IFN-γ, IL-6, and IL-17. We also investigated the pathological role of extracellular vesicles (EVs) in MIS-C. Our findings showed that patient-derived EVs induced significantly higher levels of IRF9 in the THP1-dual reporter cell line. Contrary to our expectations, however, we did not observe EV-specific NF-κB induction in the THP1 cells.
Eksozomların immünomodülator etkileri: İmmün tedavide umut verici adaylar
Exosomes are the type of extracellular vesicles secreted from almost all cell types and carry numerous biological molecules such as nucleic acids, protein and lipids. They mediate many cellular processes including cellular communication and immune responses. Accumulating evidence suggests that these vesicles play a key role in the pathogenesis of inflammatory diseases, infectious diseases and many malignancies. The significant role of exosomes in cellular level also describe their intriguing potential in cancer therapeutics. The primary aim of this thesis is to identify the immunomodulatory roles of distinct exosome species and extend the knowledge of exosome utilization in immunotherapy. The exosomes purified from i) RAW264.7 (murine macrophage-like), ii) EG7 (murine T cell lymphoma), and iii) HUH7 (human hepatocellular carcinoma) had distinct characteristics as well as immunomodulatory features upon murine splenocyte stimulation either alone or in combination with poly(I:C) (a TLR3 ligand), R848(a TLR7/8 ligand), and CpG ODN(a TLR9 ligand). Strikingly, these cell line-derived exosomes displayed changing internalization kinetics by immune cells. Furthermore, the involvement of exosomes in the liver disease progression in the course of Hepatitis B virus (HBV) infection, cirrhosis and hepatocellular carcinoma (HCC) was investigated. As a result of patients' exosome stimulation assays with healthy peripheral blood mononuclear cells (PBMCs) and murine splenocytes, patient plasma-derived exosomes had inflammatory effects correlated with the severity of liver damage suggesting that these exosomes might have a pathological role in liver disease progression and/or pathogenesis. In the final part, we used a dehydration-rehydration technique enabling external loading of desired Toll-like receptors (TLR) ligand within exosome and liposome. We developed a robust therapeutic vaccine delivery system in which injection of a therapeutic vaccine to tumor burden animals enhanced anti-tumor activity and prolonged survival of HCC xenografts. In summary, this approach could broaden the immunotherapeutic utility of exosomes in the clinic.
Lipozoma yüklenmiş immünbaskılayıcı bir oligodeoksinükleotidin bleomisin ile farede oluşturulmuş akciğer enflamasyonu ve fibrozu üzerindeki tedavi edici potansiyeli
Systemic sclerosis (SSc) is an autoimmune/autoinflammatory disease with unknown etiology. It is characterized by vascular dysfunction, inflammation and disseminated fibrosis of skin or internal organs. Although its prevalence is low, development of fibrosis on internal organs and lack of a curative treatment result in high morbidity. Current therapies targeting specific symptoms such as interstitial lung disease, Raynaud's phenomenon and pulmonary arterial hypertension are inefficient, and at best, temporarily relieves the symptoms throughout the course of the treatment. Herein, we investigated the therapeutic potential of an immunosuppressive oligodeoxynucleotide expressing TTAGGG telomeric repeats which is known as the "A151 ODN" on bleomycin-induced mouse model of systemic sclerosis. A151 ODN is the single stranded synthetic form of the telomeric repeat sequence expressed on mammalian chromosome, and it contains four repeats of "TTAGGG" motif. In order to enhance the therapeutic effectivity while protecting its digestion from nuclease activity following administration, we encapsulated A151 ODN within anionic liposomes. Since pattern recognition receptors and their signaling pathways were demonstrated to initiate inflammation in SSc, we first explored the immunosuppressive capacity of A151 ODN by analyzing in vitro cytokine productions and surface marker expression levels. Similar with the previous findings, A151 ODN was highly potent to abolish cytokine production in response to TLR9 induction. Although A151 ODN by itself was not very effective to suppress cytokine secretion following TLR1/2 and TLR4 induction, encapsulation within anionic liposomes further improved the immunosuppressive potential in response to TLR engagement. Furthermore, flow cytometry analyses revealed that A151 ODN decreased antigen presentation capacity and activation of bone-marrow derived macrophages (BMDMs) in response to TLR stimulation which was demonstrated by the reduction in levels of surface MHCII and co-stimulatory molecules as well as proteins having role on macrophage adherence and migration. A151 ODN also inhibited transcription of two major genes known to play a critical role on the development of fibrosis, TGFβ and Col1a1, from fibroblasts. Following these promising results on A151 ODN's immunosuppressive and anti-fibrotic potential, we tested its therapeutic role on bleomycin-induced lung inflammation and fibrosis on mice which reflects different phases of systemic sclerosis. First in vivo experiment that A151 ODN was used prior to bleomycin administration revealed that A151 ODN could prevent development of systemic sclerosis by reducing immune cell recruitment into alveolar space and suppressing the secretion of inflammatory cytokines. After that, we investigated if A151 ODN could abolish established lung inflammation triggered by bleomycin instillation. For that, we treated animals with an A151 ODN either in free form or encapsulated within anionic liposomes after lung inflammation was initiated following bleomycin instillation. Data indicated that A151 ODN reduced macrophage activation marker expressions, monocyte and neutrophil infiltration into alveolar space. Moreover, suppression on immune cells activation in bronchoalveolar lavage fluid (BALF) correlated with the inhibited cytokine production. As a result of reduced inflammation, pro-fibrotic gene expressions were less in A151 ODN-treated mice. Of note, liposomal encapsulation provided reduced gene expressions while failed to further enhance the immunosuppressive potential on surface marker expression or cytokine secretion of A151 ODN. Lastly, we tested whether treatment with liposome-encapsulated A151 ODN is still effective to regress fibrosis once it has been developed; therefore, we treated mice with single injection of liposomal A151 on different time points. Unfortunately, single instillation was insufficient to decrease fibrosis and macrophage activation as well as cytokine production. Taken together, our findings indicated that liposome-encapsulated A151 ODN is very potent to attenuate the lung inflammation whereas single injection was ineffective to regress established lung fibrosis.
İnsan göbek kordonu dokusundan elde edilen mezenkimal kök hücre (UCX®) eksozomlarının immünbaskılayıcı oligodeoksinükleotid A151 ile birlikte immün düzenleyici etkileri
Mesenchymal stromal or stem cells (MSCs) modulate immune responses apart from their regenerative capacities. Accumulating evidence suggests that MSCs exert their paracrine effects through extracellular vesicles known as exosomes. In this study, we utilized a particular human umbilical cord tissue-derived MSC type termed as UCX®. UCX® is superior to the gold-standard bone marrow-derived MSCs in terms of immunosuppressive properties. We aimed to characterize and employ UCX® exosomes as cell-free immunosuppressive therapeutic agents. Another aim was to compare the functionalities of exosomes either isolated from 2-dimensional (2D) cultures or isolated from 3-dimensional (3D) spheroid cultures. 3D culture provides better cell-to-cell and cell-to-matrix interactions thereby mimics the in vivo environment better. A synthetic oligodeoxynucleotide called A151 ODN, which consists of 4 repeats of the mammalian telomeric TTAGGG motif, has broad immunosuppressive effects. Delivery of A151 ODN within liposomes or exosomes protects it from degradation by nucleases and improve the desired outcome. We also aimed to combine the immunomodulatory potentials of UCX® exosomes and A151 ODN through direct loading of A151 ODN into exosomes with ~95% efficiency via a dehydration-rehydration-based lyophilization method. First, we determined the binding and internalization kinetics of exosomes with different immune cells. 3D-exosomes interacted with the target cells much faster and more efficiently. Next, we investigated how UCX® exosomes influence Toll-like receptor (TLR) signaling in mouse splenocytes and bone marrow-derived macrophages (BMDMs). 3D-exosomes compared to 2D-exosomes were more potent to suppress IFNγ, IL6, IL12, and to a lesser extent TNFα production mediated by TLR1/2, TLR4, TLR7/8 and TLR9 but not by TLR3 triggering. A151 ODN-loading to either 2D- or 3D-exosomes improved the inhibition of all the above mentioned pro-inflammatory cytokines. Especially 3D-exosomes downregulated co-stimulatory molecules CD80 and CD86 along with MHC-II on BMDMs following TLR stimulation. Macrophage polarization experiments revealed that UCX® exosomes reprogram BMDMs to produce high amounts of nitric oxide and arginase-1 which are the key immunomodulatory factors induced by myeloid-derived suppressor cells (MDSCs) to inhibit T- and NK-cell activity. Besides shifting macrophages to an MDSC-like suppressive phenotype, exosomes also supported expansion of MDSC populations in vivo upon intraperitoneal injection. Next, we tested the therapeutic efficiency of UCX® exosomes with or without A151 ODN in zymosan-induced peritonitis and dextran sodium sulfate (DSS)-induced colitis models in mice. Exosomes could not alleviate zymosan-induced peritonitis which is an acute and severe inflammation. However, 3D-exosomes and A151 ODN-loaded versions of both 2D- and 3D-exosomes remarkably prevented DSS-induced colitis progression. A151 ODN itself was also therapeutic, albeit to a lesser degree. Standalone 3D-exosomes and A151 ODN-loaded exosomes prevented weight loss and colon shortening. All treatments except for 2D-exosomes could restore DSS-induced loss of T-cell numbers and cytokine-producing capacities in mesenteric lymph nodes and spleen. All treatments except for A151 ODN prevented DSS-induced macrophage accumulation in the lymph nodes. 3D-exosomes and A151 ODN-loaded versions of both exosomes normalized serum IL6 levels while only A151 ODN-loaded 3D-exosomes could impact the cytokine production capacities of macrophages. Finally, we tested the effects of UCX® exosomes with or without A151 ODN on wound healing. In vitro, 2D- and 3D-exosomes differentially upregulated the productions of wound healing-related cytokines and growth factors such as IL1α, TGFβ and VEGFα from fibroblast and keratinocyte cell lines. In vivo, in an excisional wound healing model, free or A151 ODN-loaded exosomes did not accelerate wound closure. However, they caused systemic immunosuppression at the late stages of wound healing. Systemic outcomes include reduced inflammatory capacity of macrophages and higher granulocytic MDSC numbers in spleen. A151 ODN-loaded 3D-exosomes also reduced T-cell numbers in spleen and pro-inflammatory cytokine levels in circulation. Taken together, this study revealed that 2D- but more importantly 3D-culturing of umbilical cord MSCs result in functionally different exosomes, 3D culture-derived exosomes display higher immunosuppressive potential, A151 ODN-loading into these exosomes improves immunosuppressive capacity and A151 ODN-loaded UCX® exosomes could be a valuable therapeutic agent for inflammatory and autoimmune disorders.
İki adet nadir görülen primer immün yetmezliklerin doğal ve edinsel bağışıklık cevaplarının karakterize edilmesi: CTPS1 ve CD55
Primary Immune deficiencies (PIDs) are disorders of immune system caused by mutated genes. There are approximately 350 different disorders and each day novel ones are being defined. They can be categorized based on part of immune system harboring mutation; that is, they can be divided into disorders of innate and adaptive immune system. Each of them represents itself distinctly. In that perspective, studies based on characterization of PIDs enable us to comprehend how immune system works. Herein, we characterized innate and adaptive immune responses of two rare immune deficiencies: CTPS1 and CD55 which are novel examples of disorders of adaptive and innate immune system, respectively. CTPS1 is an enzyme functioning in de novo synthesis of nucleotide, CTP. Defective CTPS1 enzyme impairs lymphocytes to proliferate, however, other aspects of this deficiency still remain elusive. Since patients are prone to viral infections, we first explored functionality of cytotoxic T-cells through assessing STAT1 phosphorylation levels and expression of activation markers. Even though flow cytometry analyses revealed that CTPS1 deficient CD8+ T-cells had normal phospho-STAT1 levels, degranulation marker confined to surface of CD8+ T-cells were found to be elevated. Next, we investigated CD4+ T-cells with cytokines that are crucial for differentiation and fate. We detected that patient CD4+ T-cells had low phospho-STAT3 and phospho-STAT5 levels. Then, we checked the cytokine production profiles of CD4+ T-cells. Data indicated that percentages of IL-17a and IL-10 secreting cells are reduced in patient whereas Th1 and Th2 signatures were similar to healthy controls. Moreover, IFN-gamma levels of PBMCs upon TLR3, TLR7 and TLR9 ligand stimulations were found to be similar to healthy responses. Notably, patient had slightly reduced TLR7 and IFI16-STING mediated type II IFN secretion. We further showed that CTPS1 PBMCs had normal IL-12 levels, implying that the reduction in IFN-gamma was not due to either dysfunction of innate immune cells or by aberrant APC function. Surprisingly, patient PBMCs had higher number of granulocytes and flow cytometry analyses revealed that these granulocytes were CD14- CD15+ low-density granulocytes. This prompted us to assess the NETotic tendencies of CTPS1 neutrophils and we observed via microscopic and spectrofluorometric investigations that they underwent spontaneous NETosis. In the second part of this study, we worked with CD55 deficient patient PBMCs. CD55 is a complement regulatory protein and it inhibits formation of C3-convertase in classical and alternative complement pathways. Thus, patients suffer from aberrant complement activation in its absence as well as severe bowel inflammation and recurrent infections along with nutrient loss leading to malnutrition and growth deprivation. Eculizumab therapy was initiated to these patients in order to neutralize their pathologic C5 levels. We attempted to investigate effect of CD55 deficiency on PRR-complement cross-talk, recurrent infections and checked the contribution of Eculizumab therapy to their immune status. PBMCs of 4 patients, i) before (BT) and ii) after (AT) a single dose of Eculizumab administration were isolated. BT PBMCs had significantly reduced IFN-alpha and IP-10 secretions upon endosomal (TLR3, TLR7 and TLR9) TLRs and nucleic acid sensors (STING, DAI, RIG-I & MAVS) stimulations. Moreover, single Eculizumab therapy did not alter this innate immune dysfunction. Furthermore, we assessed levels of TNF-alpha, IL-6 productions from PBMCs stimulated with same ligands and observed that IL-6 but not TNF-alpha was reduced after PRR stimulations. Next, immunomodulatory effects of CD55 EVs before and after Eculizumab therapy was sought. ELISA results demonstrated that AT EV incubation on CD55-/- PBMCs lead to reduced TNF-alpha, IL-1alpha and IFN-alpha production. Meanwhile, AT EVs increased IL-10 production from patient PBMCs. Lastly, we assessed cytokine levels after healthy PBMCs were incubated with BT and AT EVs and found that PBMCs that incubated with BT EVs had elevated levels of IP-10 cytokine. When taken together, progression of PIDs might have been contributed by extracellular vesicles.
İdrarda bulunan eksozomların incelenmesi ile prostat kanserinin miRNA tabanlı tanımlanması
Prostate cancer is one of the most incident cancer subtypes with high mortality rate. Currently, diagnosis of prostate cancer is based on rectal examination, Prostate Specific Antigen (PSA) testing and biopsy. Normal range of PSA is defined as 0-4 ng/ml and individuals with higher PSA levels are considered as potential prostate cancer patients. However, PSA fluctuates as a result of many factors and it is shown to increase with age. Thus, PSA testing causes significantly high false positive results and many healthy men have biopsy unnecessarily due to high PSA levels or they even get overtreated. This situation has huge psychological as well as financial effects on these people. Herein, we investigated the diagnostic potential of urinary exosomal microRNAs (miRNA) in prostate cancer. Rather than investigation of cellular miRNAs, we focused on exosomal miRNAs because of high integrity of exosomes and their abundance in many biofluids including urine. Development of a sensitive diagnostic method from urine would be advantageous because accurate diagnosis of prostate cancer would be possible by a non-invasive procedure. miRNAs are the small non-coding RNAs and they suppress expression of target genes via degradation of mRNA or post-translational regulation. miRNAs have high potential as cancer biomarkers because they can act as tumor suppressor and repress oncogenic gene expression or function as oncogenic miRNA and suppress tumor suppressor gene expression. For this reason, we identified several candidate exosomal tumor suppressor and oncogenic miRNAs and continued our study with the most potent ones. At the beginning of the study, we validated that we efficiently isolated exosomes via several techniques such as flow cytometry, Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and Transmission Electron Microscopy (TEM). Then, we studied differential expression of candidate miRNAs in prostate cancer PC-3 cell line. Similar to our literature search findings, we found that expression levels of miR-107, miR-139, miR-145 and miR-204 were significantly lower in PC-3 exosomes in comparison to healthy urinary exosomes. On the other hand, oncomiRs; miR-21-5p, miR-375-5p and miR-574-3p were upregulated in PC-3 cell line exosomes. Of note, expression of another candidate miRNA; miR-30a was almost the same in PC-3 exosomes with healthy controls. We used these results as preliminary data and collected urine specimens from 17 prostate cancer patients. We firstly analyzed their PSA level-age and PSA Level- Gleason Score correlations. Our analyses revealed that PSA level was increasing with age and PSA was not correlated with Gleason Score. We concluded that PSA was being affected by several reasons in addition to tumor formation and it was not correlated with disease progression. Again, this was a finding which supported that a more sensitive diagnosis method than PSA testing was necessary for prostate cancer. When we detected expression levels of candidate miRNAs in urinary exosomes of prostate cancer patients and healthy controls, we observed that miR-107, miR-139, miR-145 and miR-204 were downregulated whereas miR-375-5p was upregulated in patients' urinary exosomes. For miR-21, there was a very slight upregulation and miR-30a-5p and miR-574-3p levels were almost the same with healthy controls. Further, we wondered the diagnostic potential of these miRNAs in our patient cohort and we performed Receiver Operator Characteristic (ROC) Curve analysis for them. When they were used as combination, our miRNAs had 77% (AUC=0.7731) accuracy in distinguishing patients from healthy controls. After that, we examined miRNA dysregulations in patients with different PSA levels with the hypothesis that they may have different miRNA expression profiles. We saw that expression of miRNAs in exosomes patients with PSA<10 ng/ml and PSA=10-15 ng/ml were very similar with our expectations; downregulation of tumor suppressor and upregulation in oncogenic miRNAs whereas patients with PSA>15 ng/ml had a unique profile and all miRNAs were downregulated. Due to sample size limitations, we only tested diagnostic potential of candidate miRNAs in exosomes of patients with PSA<10 ng/ml and observed that AUC was 0.8500 so we could discriminate patients and healthy controls with 85% accuracy by using our candidate miRNA panel in testing. Taken together, our findings indicated that candidate urinary exosomal tumor suppressor and oncogenic miRNAs which we suggested in thesis are very potent in diagnosis of prostate cancer with differential expressions in prostate cancer patients with different PSA levels and this study opens the way for development of a non-invasive prostate cancer diagnosis method. Keywords: Exosome, miRNA, urine biomarker, prostate cancer, diagnosis
Birincil bağışıklık yetersizliği ve omurilik yaralanmasında bağışıklık bozukluğu durumunun araştırılması
Primary Immunodeficiencies (PIDs) are raised by immune impairment causing disorders. In the patients carrying deficiency in any part of immune system, various defects and symptoms are observed. Generally, abnormality against microbial agents are seen in patients and different therapies are applied depending on defect. That's why it is crucial to characterize how immune system works in PIDs for better understating of disease and treatment options. In this study, we investigated two different mutations (CD27 and STAT1 GOF) and their effects on innate and adaptive arm of immune system. A trans-membrane receptor, CD27, is a one of the leukocyte differentiation markers for T, B and NK cells. It is involved in the activation of these cells by binding to the co-stimulatory molecule CD70 and mediates cell survival. In CD27 deficiency, loss of T cell dependent B cell responses and antibody production along with T cell dysfunction are mainly seen. Herein, immunological phenotypes of two patients with genetic CD27 deficiency were described. CD27 expression of the PBMCs, CD3+ T and CD19+ B cells obtained from the patients were absent in patients. Frequency of CD19+/CD24hi/CD38hi transitional B cells were higher compared to healthy controls. Reversed CD4+/CD8+ T cell ratio was observed in one patient. After IFN-β stimulation, the phosphorylation level of STAT1 in CD4+ T cells was increased in CD27-/- patients as compared to healthy group. There was no difference in phospho-STAT3 levels between CD27-/- and healthy donor CD4+ T cells upon IL-6 stimulation. However, decreased STAT5 phosphorylation levels were observed upon IL-2 stimulation of CD4+ T cells of CD27-/-. When PBMCs from healthy and CD27 deficient patients were stimulated with PMA/Ionomycin and analyzed for cytokine secretions, increased IFN-𝛾, IL-4 and IL-17a secretion were investigated. Through activation of different TLR and inflammasome pathways, normal IFN-𝛾 and IL-1β responses were observed while increased TNF-α and IL-10 was present in patients. Results suggest that patients' both humoral and cellular immune responses could be impaired in CD27 deficiency. STAT1 is a member of the STAT protein family whose mutations can cause gain-of-function (GOF) or loss of function (LOF). Recurrent widespread infections are common in both mutations. In this study, the responses of innate and adaptive immune systems of 6 patients from different hospitals known to have STAT1 GOF were examined. To determine the STAT1 phosphorylation and dephosphorylation levels, cells were treated with IFN-β and CD4+ T cell phosphorylation levels were analyzed by flow cytometry. Th1 and Th17 responses were examined since imbalanced Th1 and Th17 response is known in STAT1 GOF patients. To identify defects in innate immunity, cytokine ELISA was performed through different PRR ligands. For neutrophil activities, cells were examined as untreated and treated form by microscopically and fluorometrically. As a result, it was found that p-STAT1 levels and p-STAT1 positive CD4+ T cells of all patients were higher than healthy controls which approves STAT1 GOF mutation in patients. Although the STAT1 phosphorylation level was high, it was determined that IFN-𝛾 released due to Th1 response was low in patients. Th17 response were low in patients which is supported by current infection of Chronic Mucocutaneous Candidiasis. Type II interferon responses of the patients to the ligands of the endosomal and inflammatory pathways were lower than healthy controls while normal IL-12 secretion was observed. Some patients had increased NETotic activities at the basal levels supported by spontaneous NET formation, while some patients were not able to induce NETosis through PMA stimulation. Impaired immune status was observed in STAT1 GOF patients as well. Spinal cord injury (SCI) is damage occurring in the spinal cord and nerves within the spinal canal that causes completely or partially loss of sensation around injury site. Since uncontrolled immune response occurs at the post-traumatic injury site in SCI patients and this situation increases the damage caused by the lesion, it is important to examine the immune system responses in these patients. In this study, we tried to determine how innate and adaptive immune system responses of SCI patients with and without infection differ. Percentages of T, B, NK cells and pDCs were determined from whole blood. Reversed CD4/CD8 ratio was observed in two patients. Increased Treg and pDCs cell percentage was seen in patients while B and NK cell percentages are different from patient to patient. Released IFN- 𝛾, IL-17 and IL-10 levels were determined by flow cytometry upon PMA/Ionomycin. Normal Th17, increased Th1 and Treg was investigated in SCI patients. NETotic activities were examined microscopically and fluorometrically and via ROS production. Although NETotic activities of neutrophils of all patients were similar in healthy subjects, fluorometric results showed that some patients had more NETotic activities compared to healthy controls. It was also found that patients produced lower amounts of ROS as a result of incubation with PMA. These results suggest that SCI patients become more susceptible to infection due to the low response to the pathogen despite increased neutrophil activity and the immunosuppressive effect of the amount of IL-10 caused by excess Treg activity.
Birincil bağışıklık yetersizliği hastalarının doğal ve edinsel bağışıklık yanıtların karakterizasyonu
The immune system dysregulations led to primary immune deficiencies (PIDs). Immune deficiencies can be divided into two groups, primary and secondary immune deficiencies. The reasons for primary immune deficiencies could be inherited immune dysfunctions originated from autosomal recessive or autosomal dominant mutations. Combinations of ongoing infections, lymphoproliferation, atopies, malignancies, autoimmunities and granulomatous processes are seen in primary immune deficiency disorders. To establish distinctive therapies, characterization of immune deficiencies along with the understanding the course of impaired mechanisms are very critical to offer robust means of cure. In this study, the effects of different mutations (RLTPR, RLTPR-TLR1, and CTLA-4) on innate and adaptive immune systems were investigated. RLTPR (CARMIL2) is a cytosolic scaffold protein which facilitates CD28 co-stimulation for T-cell activation. RLTPR facilitates recruitment of CARMA1, a cytosolic adaptor, along with the BCL10 and MALT1 which form CBM complex to CD28 site to activate NF-κB signaling pathway. Besides facilitating CD28 co-stimulation, RLTPR takes place in cell shape control, phagocytosis and endocytosis movements by promoting actin polymerization. Reduced CD4+ T-cells, memory B-cells and antibody response along with the impaired B-cell receptor mediated NF-κB activation were mainly observed in RLTPR deficient patients. Moreover, polarity and migration of the T-cells were affected by RLTPR deficiency in patients. Herein, immunological phenotypes of RLTPR deficient patient with and without CMV infection were described. As expected, Treg counts of the patient were reduced as compared to healthy controls both with and without infection. Although Treg cell counts of patient were decreased, IL-10 secretion upon cytosolic nucleic acid ligands was increased in infection due to ongoing defense and homeostasis. Under CMV infection, patient showed elevated IL-1β and TNF-α responses upon activation of TLRs and cytosolic nucleic acid sensors. However, without any viral infections, TLR7 and TLR8 mediated IL-1β and TNF-α responses were impaired in patient. Moreover, under CMV infection, TLR and cytosolic nucleic acid sensors mediated antiviral IFN-α, IFN-γ and IL-12 responses were substantially increased compared to healthy subjects. Unexpectedly, when patient PBMCs were assessed following infection, we detect that IFN-α and IFN-γ levels of the patient in response to endosomal TLRs and cytosolic nucleic acids stimulations were reduced. Throughout the course of viral infection due to ongoing defense by innate immune cells, one would predict to detect higher type I and II IFN responses. When infection was cleared with medical treatment, it is observed that not only endosomal TLRs but also the cytosolic nucleic acid sensors were impaired in RLTPR patient which makes the patient vulnerable to viral infections. Thereafter, altered immune responses of RLTPR-TLR1 deficient patient was investigated. Decreased number of Treg cells from whole blood of the patient was confirmed. IL-1β response from PBMCs of patient was elevated by stimulation with the TLR and cytosolic nucleic acid ligands. Especially, TLR2-6 response was elevated which could be the result of TLR1 deficiency because immune response could be compensated when TLR1 is deficient but TLR6 is not. Also, remaining improved proinflammatory cytokine response to different PRR ligands could be reasoned by ongoing infection. IFN-α secretion was increased by endosomal TLR and cytosolic nucleic acid ligands while IL-12 secretion of patient showed ligand specific modulated responses which suggest an ongoing infection. As expected there was no detectable TLR1 mediated IL-12 response due to TLR1 deficiency. However, TLR2-6 mediated IL-12 secretion was elevated in patient compared to healthy subjects which could be regarded as a compensatory response against TLR1 deficiency. RLTPR-TLR1 deficient patient PBMCs elicited elevated IL-10 response to TLR and cytosolic nucleic acid ligand triggering even though Treg cells were reduced., implying that other suppressor cells could be involved in this response. CTLA-4, a negative regulator of T-cells, is expressed on activated T-cells and Treg cells. CTLA-4 binds to CD80/86 molecules on APCs which prevents CD28 co-stimulation. Without CD28 co-stimulation, the T-cell cannot be activated. Patients with insufficient CTLA-4 receptors could have an increased number of Treg cells with reduced function. CTLA-4 haploinsufficiency leads to hyperactive T-cells, enhanced autoreactive B-cells and reduced numbers of circulating B-cells. In this work, immune responses of four CTLA-4 patients were also studied. Patient #1 showed higher CD4+/CD8+ ratio, elevated LDG frequency, as well as reduced TCR expression on CD3+ cells and elevated pAKT protein levels. Patient #2 had increased LDG count, reduced pDC and Treg population in whole blood. Similar blood cell profile to Patient #2, Patient #3 additionally had increased monocyte percentage and lower CD4+/CD8+ ratio which could indicate an ongoing infection. Patient #4 showed decreased pDC, Treg counts, increased levels of PD-L1 on CD8+ cells, Treg cells and B-cells, reduced TCR expression on CD3+ T-cells, increased pAKT and p4EBP1 levels which all may contribute to compensate the autoimmune status of the CTLA-4 mutation. B-cell percentages and CTLA-4 expression levels of all patients were not altered while mTOR, pmTOR, STAT3, pSTAT3, AKT, p4EBP1 and HIf-1α expression levels were impaired in all patients. Collectively, our findings imply that the complexity of the dysregulation of these deficiencies, and point-out to an unappreciated immune functional status of these patients. We propose that investigation of the innate immune arm of these individuals which were perceived as solely related to and impacting only adaptive immune system is necessary to offer more robust therapies to these patients.
Çocukluk çağı idiyopatik nefrotik sendrom hastalarının dolaşımda bulunan hücre dışı keseciklerinin foksiyonel ve moleküler özelliklerinin karakterizasyonu
Nephrotic syndrome (NS) is one of the most common causes of glomerular disease in children and is characterized by the triad of proteinuria, hypoalbuminemia, and edema. The major molecular event in the pathogenesis of NS is the disruption of the glomerular filtration barrier, which is primarily driven by podocyte injury. The most common clinical presentation of NS in children is steroid-sensitive nephrotic syndrome (SSNS), characterized by complete remission within 4 weeks of steroid therapy and no apparent glomerular change in the light microscopic evaluation of kidney biopsies, thereby named as Minimal Change Disease (MCD). Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (SSNS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with SSNS in relapse and remission, 10 healthy controls and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry and western blotting. The major proteins from the plasma EVs were identified via mass spectrometry. A Gene Ontology classification analysis and ingenuity pathway analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and the particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively express proteins which involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared to remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho p38 (p-p38) and decreased levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the nephrotic syndrome phenotype in podocytes in vitro.
SARSCOV-2 pandemisine karşı virüs benzeri parçacık temelli, alum absorbe ve K3 CPG olıgodeoksinükleotid adjuvantlı aşı adayının faz 1/2 klinik deneyler için üretimi ve incelenmesi
Emergence of COVID-19 pandemic has been met by an exceptionally fast response from vaccine makers around the globe. Vaccines that elicit excellent immunological responses against SARS-CoV-2 are now widely utilized. Existing platforms include mRNA-lipid nanoparticle-based vaccines, adenovirus vectored vaccines, various inactivated virus vaccines and subunit vaccines. We have previously described a novel virus-like particle (VLP) platform expressing the hexaproline prefusion stabilized Spike protein along with the nucleocapsid, membrane and envelope structural proteins. In mice, ferrets and rats, VLPs adjuvanted with K3 CpG Oligodeoxynucleotide (ODN) and adsorbed onto 2% Aluminum Hydroxide (Alum), induced robust humoral and cellular immune response against Spike and Nucleocapsid proteins. Herein, we have expanded our work to manufacture the virus like particles in a GMP compliant facility intended for testing in phase I/II clinical trials. The technology transfer comprises i) VLP production from suspension adapted HEK293 cells, ii) purification with multimodal fast protein liquid chromatography (FPLC) and iii) concentration and diafiltration using tangential flow filtration (TFF). We have successfully scaled up our production from 50 mL of HEK293 cell culture to 5 L bioreactor, achieving yields reaching up to 40 mg VLPs per L of cell culture. Furthermore, several methods were developed to determine protein identity, purity, functionality, stability and immunopotency of VLP vaccine that was finally formulated with Alum + CpG ODN. Moreover, we investigated the immunogenicity of VLPs decorated either with Wuhan (Hu-1) or with Alpha (B.1.1.7) variant Spike against receptor binding domains (RBD) specific to other variants of concern (VoC). Although our vaccine platform, could further benefit from process optimization to improve VLP yield, this study presents the first pilot scale production and purification of variant specific hexaproline prefusion stabilized SARS-CoV-2 VLPs. VLP preparations complying with our quality control parameters were released for fill and finish and were used for subsequent Phase 1 (NCT04818281) and Phase 2 clinical trials (NCT04962893).
Investigation of pre-clinical and clinical results against SARS-CoV-2 wild-type and alpha variants combination for VLP-58-1023-AL-K3 vaccine
The SARS-CoV-2 virus, which first appeared in Wuhan, China and had a pandemic effect at the end of 2019, has urgently created need of suitable vaccine candidates' development with its quick spread. Scientists in different laboratories from all over the world, including Turkey, have started the studies of vaccine development with different methods based on the basic proteins of the SARS-CoV-2 virus. Most of these vaccines are based on the Spike protein, which allows the virus to enter the host and survive. Although the Spike protein, which has high antigenic properties and contains the RBD region to bind ACE2 receptor in the host, is effective in the formation of neutralizing antibodies in the person, the virus essentially has 4 structural proteins, not only spike. Our vaccine strategy containing virus-like particles (VLP) was based on the self-assembly property of Spike, Membrane, Envelope and Nucleocapsid proteins, and these protein structures were transformed into plasmids with his-tag labels, and the products were collected in mammalian cells in vitro by the transfection method. VLPs were purified by affinity chromatography and formulated with 2 different adjuvants which are Alum and CpG. Since the virus, which has changed rapidly in the last 2 years, is open to new mutations over spreading, it is very important that our vaccine should be editable and adaptable to new virus variants. Due to the alpha variant effect in the world, which was first seen in the UK and has become widespread all over the world recent years, WT VLP formed with a more stable Spike protein containing a thermostable sub-proline (6p; HexaPro) mutation was used in the first dose of our vaccine, but UK VLP containing alpha variant mutations was used in the second dose. Alum and CpG adjuvants were used together for both injection formulations. Our vaccine has successfully completed Phase 2 studies. As a result of these studies, approximately 115 vaccinated volunteers were followed up for 90 days and immunological analyzes were performed with samples taken at certain day intervals (Day 21, Day 35, Day49 and Day90). As a result of these analyzes, moderate and high levels of neutralizing and non-neutralizing antibody responses were observed in many patients, and a humoral immune response was induced until day 90. In addition, it was observed that the cellular responses of the volunteers are progressed in tendency with the Th1 cell response.
Hücre kökenli hücre dışı kesecikler kullanılarak profilaktik ve terapötik aşı geliştirilmesi
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.
SARS-CoV-2 VLP aşısına karşı geliştirilen hümoral ve hücresel immün yanıtların klinik öncesi ve klinik çalışmalarda incelenmesi
COVID-19 pandemic, caused by SARS-CoV-2, emerged in China in late 2019, and as of March 2020 has become a serious concern for the whole world due to severe progression of the disease especially in the elderly and high transmission rate of the virus. The ravages of pandemic on health sector, economy and social life accelerated the vaccine race as vaccination is the most critical measure to hamper the pace of the pandemic and the most important step for acquiring herd immunity. Various vaccine studies are registered by WHO most of which target Spike protein or a certain region of Spike protein, such as receptor binding domain (RBD) for inducing immune reactions. Alternatively, the use of virus-like particle (VLP) technology for vaccine development broadens the magnitude of immune responses as the four main structural proteins of the virus, Spike, Nucleocapsid, Membrane and Envelope, are incorporated. Therefore, we developed a VLP vaccine against SARS-CoV-2 and adjuvanted it using Alum and K3 CpG ODN. Herein, we investigated the humoral and cellular immune responses induced by SARS-CoV-2 VLP vaccine in pre-clinical and clinical studies. The results from pre-clinical experiments conducted on vaccinated BALB/c mice indicated that VLP vaccine triggered effective antibody response against Spike, Nucleocapsid, Wild Type RBD, Alpha RBD and Delta RBD proteins as shown by ELISA. Additionally, it was proven that the diminishing antibody responses can be boosted and a prolonged immune response was achieved by a third dose of VLP vaccine. For the clinical studies, the humoral and cellular immune responses of Phase II clinical trial volunteers were analyzed by employing ELISA and CBA methods respectively. The results obtained from ELISA demonstrated that the Phase II volunteers developed effective antibody titers against Spike, Nucleocapsid, Wild Type RBD, Alpha RBD and Delta RBD proteins. Analysis of the cellular immune responses by CBA showed that VLP vaccine induced a Th1-skewed immune reaction with coexistence of Th2-, Th17- and Treg related responses owing to its formulation with K3 CpG ODN and Alum. Taken together, our results indicated that SARS-CoV-2 VLP vaccine triggered effective cellular and humoral immune reactions against Spike and Nucleocapsid along with RBD variants which proved the cross-protective response achieved. Elicitation of multi-functional immune response and adoptability of the VLP technology to newly emerging variants makes VLP vaccine a promising candidate for the future booster injections.
Farkli adjuvantlar ve li̇pozom kompleksasyonuyla geliştirilmiş SARS-CoV-2 VLP aşinin farelerdeki̇ bağişiklik gücünün değerlendi̇ri̇lmesi̇
SARS-CoV-2, emerging in December 2019 in Wuhan, China, led to a swift global pandemic declaration in March 2020, prompting widespread vaccine development. Most vaccines target specific regions, mainly the spike protein. In our lab, we have employed an innovative virus- like particle (VLP) vaccine approach encompassing all four structural proteins of the virus: spike, nucleocapsid, membrane, and envelope. Our study utilizes sterically stabilized cationic liposomes (SSCL) to encapsulate VLPs expressing the Delta variant spike protein, along with various adjuvants: CpG ODN, poly(I:C), and 2'3'-cGAMP. We characterized liposomes using tunable resistive pulse sensing for size and concentration. In C57BL/6 mice, we administered primary and two booster injections on Day 0, Day 15, and Day 73, respectively, collecting blood samples at intervals (Day 14, Day 28, Day 42, Day 72, and Day 90). To assess vaccine impact on mouse humoral immunity, we conducted ELISAs for total IgG, IgG1, and IgG2c antibodies against recombinant Spike and the receptor-binding domain (RBD). IgG titers increased until Day 42, remained stable or slightly decreased on Day 72, and significantly rose on Day 90. We calculated IgG2c/IgG1 ratios, reflecting Th1 immune responses, revealing enhanced cellular immunity potential in groups with adjuvants compared to the VLP-only group. This study underscores the effectiveness of our VLP vaccine strategy in stimulating robust immune responses and opens avenues for further research and development.
SARS-CoV-2 VLP aşısının tekli, ikili veya üçlü patojenlere özgü moleküler örüntülerden oluşan destekleyici maddelerle karıştırılarak elde edilmiş formüllerin hayvanlarda kullanımının incelenmesi
COVID-19 was first identified in Wuhan, a Chinese city, and it has been a part of our lives since late December 2019. The WHO declared this disease, caused by SARS-CoV-2, a pandemic due to its high transmission rate, a status that remained in effect until May 2023. The escalating death toll and virus spread expedited vaccine development efforts. Various platforms for vaccine development have been utilized, including inactivated virus, mRNA, and subunit vaccines. Given the virus's propensity for mutations due to its high transmission rate, the chosen platform must be easy to produce, effective, and safe for immunocompromised individuals. One such platform is virus-like particles (VLPs). VLP vaccines consist of viral proteins without genetic material and are known for their ease of production. A VLP vaccine, adjuvanted with Alum and K-type CpG ODN against SARS-CoV-2, is tested in Phase II clinical trials. In addition to CpG ODN, this study aimed to evaluate the efficacy of other adjuvants and their combinations to explore potential clinical advantages. The additional adjuvants included poly(I:C) and a member of cyclic dinucleotides, 2,3-cGAMP, which are Toll-like receptor 3 (TLR3) and STING agonists, respectively. Nine groups of male mice (C57BL/6, 6–8 weeks old) received intraperitoneal injections three times with a mixture of VLP vaccine using different combinations of adjuvants, including CpG ODN, CDN, and poly(I:C). Blood samples were collected from the tail vein two weeks after each injection. Antibody ELISA was employed to assess humoral immunity against each combination of vaccines, with plates coated using in-house recombinant 6P-Spike and commercial recombinant RBD. ELISA results indicated that VLPs combined with CpG plus poly(I:C) and VLPs combined with all three adjuvants induced high levels of total IgG against RecS. Two weeks after the third injection, the animals' spleens were isolated, and cytokine profiles were assessed through an antigen recall assay. As a result, only one group exhibited the expected cytokine profile: VLPs combined with the triple adjuvant combination. This profile demonstrated the requisite Th1-biased cytokine response necessary for vaccine development. This study suggests that the triple adjuvant combination holds promise as a vaccine adjuvant for addressing future pandemics.
Lipozoma yüklenmiş D-tipi ve K-tipi ODN'lerin sinerjik immun aktivasyonu
Liposomes are one of the best candidates for the encapsulation of labile bioactive agents due to their safety and high entrapment efficiency. In human, two structurally distinct classes of CpG ODN are capable of activating different signaling pathways, leading to differential immune activation. While K-type ODN triggers plasmacytoid dendritic cells (pDCs) to mature and produce TNFα, D-type ODN leads to IRF-7 dependent IFNα secretion. Strikingly, when K-and D-type ODN are co-incubated in their free forms, K-ODN masks the D-ODN specific immune activation. Identifying proper delivery vehicles that provide both ODN types to display their superior features upon stimulation is of great clinical importance. In this study, first we investigated the synergistic effects of K- and D-ODN upon encapsulating them within five different liposome types. Then with the selected potential liposome combinations, we identified synergistic activation capacities both on human PBMCs and on mice splenocytes. In PBMC cytokine results revealed that D-ODN loaded in all five liposome types stimulated more IFNα than free D-ODN. Similarly, liposomal K-ODN triggered more TNFα than free K-ODN type. While incubation of free K and D- type ODN as expected, abrogated D-specific IFNα production from PBMC, simultaneous incubation with neutral or anionic D-ODN loaded liposomes plus cationic liposomes loaded with K-ODN significantly increased K-specific as well as D-specific effect rather than masking it (i.e. more production of TNFα and IFNα specific for K and D, respectively). This improved synergistic immune activity for both D and K ODN observed with ND+CK combination in 100% of individuals (TNFa) and 90% of individuals for IFNa. Additionally, intracellular cytokine staining findings supported improved TNFα and IFNα, from pDC population of PBMCs. Co-stimulatory molecule expressions and APC activation also significantly upregulated compared with free treatment. In mice contrary to ND+CK combination, sterically stabilized cationic liposome encapsulated K-ODN combined with i) neutral, ii) anionic, iii) cationic or iv) stealth encapsulated D-ODN increased IL6, IL12 and IFNγ levels, when stimulated simultaneously. Moreover, ex vivo experiments showed that cellular uptake and pro-inflammatory cytokine gene expressions significantly increased with combined liposomal formulations. This study established that by selecting proper liposome type(s) we reverse antagonistic action of K-ODN on D-ODN and induce a synergistic effect leading to a more robust immunostimulatory activity in both human and mice. This approach could broaden the immunotherapeutic application of these two important CpG ODN classes in clinic.
T lenfosit farklılaşması üzerine cGAMP`ın rolünün incelenmesi
STING is the pivotal mediator for the recognition of host and pathogenic cytosolic dsDNA as well as cyclic di-nucleotides metabolites from microbes. STING can either recognize DNA itself or sense the presence of cGAMP, which is converted from ATP and GTP upon DNA binding to cGAS enzyme. Not only strategy against intracellular pathogens makes STING an ideal target, but also the recognition of DNA from host cells has a significant role in tumor immunity. Previous studies demonstrated that DNA released from cancerous cells are internalized by innate immune cells such as macrophages and dendritic cells in tumor microenvironment and trigger the production of IFN-β and other pro-inflammatory cytokines including IL-6, TNF-α, and IL-12 through STING triggered signaling pathway. These cytokines then enhance cytotoxic activity of CD8+ T cells by further increasing IFNγ production. Since enhanced T cell immunity is the hallmark of vaccine adjuvants, cyclic di-nucleotides such as cGAMP become an important and effective vaccine adjuvants against intracellular pathogens and malignant cells. Although STING activating cyclic di-nucleotides are envisioned as novel and potent vaccine adjuvants, more thorough research is needed to unearth the mechanism of action of STING on different immune cells. Therefore, it will pave the way for the initiation of successful human trials. The important criteria while developing vaccine adjuvant are the magnitude, and the quality of an immune response and its toxic side effects. To identify these, members of the both innate and adaptive immune system should be taken into account. However, previous studies merely focus on the function and effect of cGAMP in innate immune cells such as macrophages, monocytes and dendritic cells. However, to date there is no explicit study investigating the effect of STING signaling cascade on T-cells. In the light of these findings, we aimed to investigate the direct effect and function of cGAMP on T lymphocytes. Since there were not any preliminary data, we firstly stimulated Pan T cells with cGAMP alone or together with various TLR ligands and then, checked the cytokine profiles and the viability of cells. Surprisingly, 2.5μg/ml dose of cGAMP had a toxic effect on T cell but not on bone marrow derived dendritic cells and macrophages. While cGAMP triggered cell death, interestingly IL-17 secretion from both CD4+ and CD8+ T cells was dramatically increased. Beside, cGAMP stimulation drastically increased CD4+/CD8+ T cells ratio of Pan T cells population. Next, we sought to identify the source of IL-17. The IL17 inductive capacity of cGAMP was investigated on purified CD4+ T cells from mice. Unexpectedly, data revealed that cGAMP elicited apoptosis of CD4+ T cells. Moreover, there was no significant induction of IL-17 secretion. Next, we aimed to find a condition that will reduce the toxic effect of cGAMP, while maintaining IL-17 secretion. When Pan T cells were stimulated with cGAMP and R848 (a TLR7 ligand), the toxic action of cGAMP decreased while IL-17 secretion was enhanced. Lastly, the potency of T cells stimulated with cGAMP was investigated. According to our results, macrophages were activated in the presence of conditioned medium obtained from T cells stimulated with cGAMP. When taken together our findings point out that STING dependent direct activation of T-cells via cGAMP and its subsequent effect on macrophages might be utilized as an immunotherapeutic approach where IL17 induction is important and could be harnessed as vaccine adjuvants against mucosal infections or against cancer. Keywords: cGAMP, STING, T lymphocytes, apoptosis, immune response, adjuvant
Tek veya ikili patojen ilişkili moleküler kalıplarla yüklü lipozomal aşıların in vivo uygulamaları
Nucleic acid-based pattern recognition receptor (PRR) agonists are promising adjuvants and immunotherapeutic agents. Combination of PRR ligands potentiates immune response by providing synergistic immune activity via triggering different signaling pathways and may impact antigen dependent T-cell immune memory. However, the duration of short circulation due to nuclease attacks is hampering their clinical performance. Liposomes enable protein and nucleic acid based compounds to have high encapsulation efficiency. Herein, we aimed to develop liposomal carrier systems that co-encapsulating single TLR9 or combinations with TLR3 or STING ligands and assess their potential as adjuvants and immunostimulatory agents in in vivo applications. Liposomal dual nucleic acid formulations induced synergistic innate immune activation, enhanced cytokine production along with internalization capacity of ligands. In anti-cancer vaccine study, CpG ODN and poly(I:C) co-encapsulation significantly increased OVA-specific Th1-biased immune even after eight months post-booster injection. Challenge with OVA-expressing tumor cell line, E.G7, demonstrated that mice immunized with liposomes co-encapsulating CpG ODN and poly(I:C) had significantly slower tumor progression dependent on OVA-specific cytotoxic memory T-cells. In our second in vivo application, liposomal CDN and TLR9 therapy led to 80% remission of established melanoma tumor. Increased IgG2c/IgG1 ratio in mice treated with liposomal formulations indicating the development of antigen specific Th1-biased immunity was observed. Furthermore, along with the treatment, IFN-γ producing CD8+ T-cells significantly increased and M2-type macrophages decreased at the tumor bed. In conclusion, co-encapsulating dual ligands into liposomes enhanced the anti-tumor activity of single ligands. In the third part, immunization with CpG ODN loaded liposomal formulations together with antigens increased antigen-specific humoral response against FMDV and Helicobacter. In addition, the liposomal CpG ODN reduced bacterial gastric colonization by antigen-dependent Th1 and Th17 immune responses after helicobacter challenging.
Hücredışı keseciklerin immünoterapötik etkinlikleri ve TLR yolaklarının omurilik hasarlı hastalarda görülen immün disfonksiyona etkileri
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 OVA-immunized 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 co-stimulatory molecule expression, that could explain the immunological dysfunction in patient with SCI contributing to persistent complications.
VLP-58-1023-AL-K3-PII aşısının alfa varyantı için klinik öncesi ve faz II klinik çalışmalarının araştırılması
In the late December of 2019, SARS-CoV-2, a new coronavirus, was discovered in Wuhan, China and described as the causative agent of Coronavirus Disease 2019 (COVID-19). The disease has spread rapidly across the world due to its high transmissibility and has been declared a pandemic by the World Health Organization (WHO). The development of an effective vaccine has become the most significant issue to constrain the pandemic. Several COVID-19 vaccines have been authorized for human use and others are in clinical trials. Although SARS-CoV-2 encodes four structural proteins, which are Spike (S), Nucleocapsid (N), Membrane (M) and Envelope (E), most of the current vaccines used only Spike as antigen in order to generate antibodies for preventing the virus entry and replication. However, concerns have raised about Spike- based vaccines with the emerging of variants as they can moderately escape from neutralizing antibodies. For these purposes, we developed Virus-like particle (VLP) vaccine which displays hexaproline prefusion-stabilized spike (S-6p), N, M, E proteins, and adjuvanted with Alum and K3-CpG ODN. Rather than using wild type, we preferred to use the sequence of Alpha variant because of its high mortality risk and selection advantages. At the beginning of the study, we designed three different vaccine formulations and based on the results of humoral immune response in mice we determined the optimal formulation and dosage for human use. Our pre-clinical studies revealed that the best vaccine combination was high dose antigen and low dose adjuvants. Next, we wondered whether a 3rd dose has an impact on long-lasting immunity or enhancing immunogenicity in mice so that its applicability to humans could be determined. It was found that 3rd dose injection increased the antibody levels much higher than 2nd dose administration and prevented humoral immunity from decreasing after a certain amount of time. Further, both humoral and cellular immunity were studied with serum and PBMC samples from 117 volunteers who participated in the Phase II clinical trial. All IgG ELISA experiments indicated that VLP-58-1023-AL-K3-PII vaccine induced great amount of humoral immune responses against S,N proteins and WT, Alpha, Delta RBDs. In terms of T cell responses, it is known that Alum-induced robust Th2 response can be redirected to the Th1 axis with the use of CpG ODN. So, we investigated whether Th1 or Th2 type of cell response was dominant after vaccination. All cytokine levels specific to SARS-CoV-2 peptides demonstrated that the vaccine elicited Th1-biased responses. Taken together, this study revealed that VLP-58-1023- AL-K3-PII vaccine for Alpha variant successfully elicited both humoral and cellular immune responses, its effectiveness against other variants was indicated and the efficiency of vaccine could be increased with the administration of 3rd dose, in terms of ensuring long-lasting immunity. Keywords: SARS-CoV-2, Virus-like particles, vaccines, CpG ODN, Alum, humoral immunity, cellular immunity
Meningokokkal dış membran vezikül aşısının geliştirilmesi ve preklinik karakterizasyonu
Invasive meningococcal disease (IMD) is caused by Neisseria meningitidis, with the main serogroups responsible for the disease being A, B, C, W, X and Y. To date, several vaccines targeting N.meningitidis have been developed albeit with a short-lived protection. Given that MenW and MenB are the most common causes of IMD in Europe, Turkey, and Middle East, we aimed to develop an outer membrane vesicle (OMV) based bivalent vaccine as the heterologous antigen source. Herein, we compared the immunogenicity, and breadth of serum bactericidal assays (SBA) based protective coverage of OMV vaccine to X serotype with existing commercial meningococcal conjugate and polysaccharide (PS) vaccines in a murine model. BALB/c mice were immunized with preclinical batches of the W+B OMV vaccine, either adjuvanted with Alum, CpG ODN or their combinations and compared with a MenACYW conjugate vaccine (NimenrixTM, Pfizer) and a MenB OMV-based vaccine (Bexsero®, GSK). The immune responses were assessed through ELISA and SBA. Antibody responses and SBA titers were significantly higher in the W+B OMV vaccine when adjuvanted with Alum or CpG ODN, as compared to the control groups. Moreover, the SBA titers were not only significantly higher than those achieved with available conjugated ACYW vaccines but also on par with the 4CMenB vaccines. In conclusion, the W+B OMV vaccine demonstrated the capacity to elicit robust antibody responses, surpassing or matching the levels induced by licensed meningococcal vaccines. Consequently, the W+B OMV vaccine could potentially serve as a viable alternative or supplement to existing meningococcal vaccines.