Development of a second-generation multi-epitope homologous virus-like particle (VLP)-based vaccine candidate against SARS-CoV-2 on a plant platform

The severe impact of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been mitigated using a wide range of vaccines worldwide. However, the equitable distribution of COVID-19 vaccines remains challenging, particularly in developing and underdeveloped countries. Despite the great efforts to date in both the production and administration of COVID-19 vaccines against SARS-CoV-2, developing an accessible vaccine that can cross-neutralize emerging variants remains important. Therefore, in this thesis, we aimed to produce a virus-like particle (VLP)-based vaccine candidate containing all the structural proteins of SARS-CoV-2 in plant platform, as a cost-effective and fast expression system, that may exhibit a broad range of antigenicity with the highest similarity to the structure of SARS-CoV-2, and thus may protect against emerging variants. Firstly, expression cassettes were designed for the genes encoding the structural proteins [spike (S), membrane (M), nucleocapsid (N), and envelope (E)] of SARS-CoV-2. To investigate the effect of cellular compartmentalization (endoplasmic reticulum; ER and chloroplast; chl) on the level of the expression products, different signal sequences were added. Following codon optimization, the gene cassettes were commercially synthesized. The gene cassettes were successfully cloned individually into a plant expression vector, pEAQ-HT. Each plasmid carrying the gene of interest (GOI) was transformed into Agrobacterium tumefaciens by electroporation. The leaves of approximately four-week-old Nicotiana benthamiana plants were co-infiltrated with Agrobacterium tumefaciens cells carrying these plasmids. As a control, leaves were infiltrated in parallel with pEAQ-HT-GFP containing Agrobacterium cells and visualized under ultraviolet light at 3-, 5-, and 7-days post-infiltration. The expressions of the GOIs at mRNA level were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). To demonstrate protein expression, total soluble proteins (TSPs) were isolated from the leaves and used for Western blot and ELISA analyses. Finally, VLPs were purified by double-layer sucrose cushion ultracentrifugation and analyzed by Western blot and ELISA using appropriate antibodies (anti-N and anti-S). In this thesis, mRNA expressions of all the GOIs (S, S-chl, E, M N) were confirmed in the Agrobacterium co-infiltrated leaf samples. N protein was detected in both TSPs and the purified samples by Western blot and ELISA analyses. The expression of N protein was higher in ER-targeted samples (VLP-ER) than chloroplast-targeted samples (VLP-Chl) according to the Western blot band density and ELISA as well. This study is the first demonstration of plant-based production of VLPs containing all structural proteins of SARS-CoV-2 and is considered an important step towards the development of a low-cost and scalable COVID-19 vaccine.