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Cataloged from PDF version of article. Thesis (Master's): Bilkent University, Department of Molecular Biology and Genetics, İhsan Doğramacı Bilkent University, 2022. Includes bibliographical references (leaves 87-101). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared in late 2019, led to countless cases and deaths and eventually regarded as one of the severe pandemics of modern times. To reduce hospitalization and mortality rates against COVID-19 an immediate need for effective vaccines has emerged. SARS-CoV-2 contains 4 structural proteins; Nucleocapsid (N), Envelope (E), Membrane (M) and Spike (S). Numerous vaccine platforms targeting Spike antigen were developed and tested on healthy volunteers. Among vaccine platforms, Virus-Like Particles (VLPs) capable of mimicking the structure, morphology and conformation of authentic and variant viruses offer a selective advantage for a better protection. VLPs, due to multivalent antigen display capabilities and ease of expressing different Spike variants can elicit cellular, as well as humoral immune responses and were expected to be superior to that of other COVID-19 vaccine platforms. Herein, we produced, purified and characterized the first mammalian derived, 4 structural protein containing SARS-CoV-2 VLP vaccine. Various animal models were used to assess the immunogenicity and immuno-protective potential of the candidate VLP vaccine that was formulated with two different CpG ODN adjuvants. Following cloning of four viral structural proteins to two separate dual expression plasmids and transiently transfecting them to adherent HEK293-FT cells, VLP yields were monitored daily from cell culture supernatant. Presence of SARS-CoV-2 structural proteins on VLPs were verified via immunoblotting. Furthermore, the size of VLPs was investigated and was shown to resemble to that of authentic SARS-CoV-2. To assess in vivo immunogenicity of the adjuvanted VLPs, BALB/c mice were subcutaneously (sc) immunized and end point titer ELISA was studied from sera. For the toxicity assay of the candidate VLP vaccine, the selected highest human dose was sc injected to female Sprague Dawley rats. Lastly, to show the immunoprotective effect of the VLP vaccine, challenge studies were conducted with transgenic K18-hACE2. Results demonstrated that VLPs induce robust antiviral humoral and Th1-biased cellular responses when adjuvanted with CpG ODNs. Furthermore, VLP immunization did not lead to any significant systemic effect during repeated dose toxicity studies on rats. A robust immuno-protection against the live SARS-CoV-2 virus was observed with the challenge experiments. Collectively, the results provide the rationale for using this vaccine candidate in human clinical trials. In the second part of the thesis, SARS-CoV-2 recombinant RBD and Nucleocapsid proteins were produced and purified via affinity chromatography. After 6X Histidinetagged RBD and N were produced in HEK293-FT cells, supernatant passed through the immobilized metal affinity chromatography (IMAC) column. Quality of the recombinant proteins was investigated using silver staining and western blotting. Afterward, immunized mice sera were used to find an optimal coating concentration of the purified recombinant proteins. Finally, purified recombinant proteins were used in in-house ELISA experiments. by İhsan Gürsel M.S.

Background Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2. Methods VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. Results Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. Conclusion These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (NS), 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 steroid-sensitive NS 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 blot analysis. Major proteins from plasma EVs were identified via mass spectrometry. 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 particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively expressed proteins that involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared with 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 and decreased the levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the NS phenotype in podocytes in vitro.

Tumor-derived exosomes (TEXs) could be harnessed 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. Engineering TEXs to harbor immunostimulatory molecules still remains a challenge. Previously, we demonstrated that nucleic acid ligand encapsulated liposomes could trigger synergistic strong humoral, and cell mediated immune responses and provokes tumor regression to that of their standalone counterparts. 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). 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 cancer model, administration of4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regress tumor growth in 4T1 tumor-bearing mice. Taken together this work implicated that an exosome-based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. This approach offers a personalized tumor therapy strategy that could be implemented in the clinic.

Background Vaccines that incorporate multiple SARS‐CoV‐2 antigens can further broaden the breadth of virus‐specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS‐CoV‐2 VLP vaccine that incorporates the four structural proteins of SARS‐CoV‐2. Methods VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable‐resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. Results Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS‐CoV‐2. Alum adsorbed, K3‐CpG ODN‐adjuvanted VLPs elicited high titer anti‐S, anti‐RBD, anti‐N IgG, triggered multifunctional Th1‐biased T‐cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. Conclusion These data suggest that VLPs expressing all four structural protein antigens of SARS‐CoV‐2 are immunogenic and can protect animals from developing COVID‐19 infection following vaccination., SARS‐CoV‐2 VLP vaccine that incorporates the four structural proteins of SARS‐CoV‐2 is reproducibly produced in suspension adapted HEK293 cells. Alum adsorbed, K3‐CpG ODN‐adjuvanted VLPs elicit high titer anti‐S, anti‐RBD, anti‐N IgG, and neutralizing antibodies in mice, rats, and ferrets. The VLP vaccine supports multifunctional Th1‐biased T‐cell responses and demonstrate immunoprotective activity against live SARS‐CoV‐2 challenge in vaccinated mice.