Development & characterization of CpG adjuvanted SARS-CoV-2 Virus-Like Particle vaccine
| buir.advisor | Gürsel, İhsan | |
| dc.contributor.author | Turay, Nilsu | |
| dc.date.accessioned | 2022-08-08T10:45:34Z | |
| dc.date.available | 2022-08-08T10:45:34Z | |
| dc.date.copyright | 2022-06 | |
| dc.date.issued | 2022-06 | |
| dc.date.submitted | 2022-07-01 | |
| dc.description | Cataloged from PDF version of article. | en_US |
| dc.description | Thesis (Master's): Bilkent University, Department of Molecular Biology and Genetics, İhsan Doğramacı Bilkent University, 2022. | en_US |
| dc.description | Includes bibliographical references (leaves 87-101). | en_US |
| dc.description.abstract | 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. | en_US |
| dc.description.provenance | Submitted by Betül Özen (ozen@bilkent.edu.tr) on 2022-08-08T10:45:34Z No. of bitstreams: 1 B161059.pdf: 15252083 bytes, checksum: ecba6c5e96247bf0157ae8e64e82b07e (MD5) | en |
| dc.description.provenance | Made available in DSpace on 2022-08-08T10:45:34Z (GMT). No. of bitstreams: 1 B161059.pdf: 15252083 bytes, checksum: ecba6c5e96247bf0157ae8e64e82b07e (MD5) Previous issue date: 2022-06 | en |
| dc.description.statementofresponsibility | by İhsan Gürsel | en_US |
| dc.format.extent | by Nilsu Turay | en_US |
| dc.format.extent | xviii, 112 leaves : illustrations, charts ; 30 cm. | en_US |
| dc.identifier.itemid | B161059 | |
| dc.identifier.uri | http://hdl.handle.net/11693/110391 | |
| dc.language.iso | English | en_US |
| dc.rights | info:eu-repo/semantics/openAccess | en_US |
| dc.subject | SARS-CoV-2 | en_US |
| dc.subject | Vaccine | en_US |
| dc.subject | Virus-Like Particle | en_US |
| dc.subject | CpG ODN | en_US |
| dc.title | Development & characterization of CpG adjuvanted SARS-CoV-2 Virus-Like Particle vaccine | en_US |
| dc.title.alternative | CpG adjuvantlı SARS-COV-2 virüs benzeri parçacık aşısının geliştirilmesi ve karakterizasyonu | en_US |
| dc.type | Thesis | en_US |
| thesis.degree.discipline | Molecular Biology and Genetics | |
| thesis.degree.grantor | Bilkent University | |
| thesis.degree.level | Master's | |
| thesis.degree.name | MS (Master of Science) |