Marle, Guido vanChen, Michelle2023-06-232023-06-232023-06Chen, M. (2023). Developing SINrep5 as a viral vector platform to test SARS-CoV-2 vaccine candidates (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.https://hdl.handle.net/1880/116646https://dx.doi.org/10.11575/PRISM/41489The COVID-19 pandemic, caused by SARS-CoV-2, serves as a stark reminder of the importance of vaccines and immunization in the arms race against novel viral agents. SARS-CoV-2 vaccines target the mutation-prone Spike Protein, leading to the rise of viral variants, and warranting additional research into the potential of diversifying vaccine target candidates, as well as vaccine options. One particular vaccine of interest is the adenovirus vector-based vaccine, which has raised apprehension since its use against SARS-CoV-2. Public hesitation and unclear human-use risks surrounding adenovirus vector vaccines have warranted exploration into other vectors—not just as ready-for-use vaccines, but also as expression platforms for vaccine study. SINrep5, derived from the Sindbis alphavirus, is one expression platform that has been studied for its unique protein expression abilities. A workflow was developed to use SINrep5 as an expression platform for SARS-CoV-2 proteins of interest—Nucleocapsid-N, Envelope-E, and Membrane-M. The N gene was subcloned into a mammalian SINrep5 plasmid (pmSINrep5) to test the vector’s protein expression in vitro. Vector transfections were optimized for protein expression and Virus-Like Particles (VLP) production. Techniques to purify, concentrate, and quantify VLP titres were also optimized. C57Bl/6 Mice were received VLP vaccines and humoral immunity was assessed. An efficient cloning system was developed for the SINrep5 mammalian expression plasmid (pmSINrep5) construct. Applying the system to N, E, and M resulted in successful pmSINrep5-N, pmSINrep5-E, and pmSINrep5-M creation. Transfections optimized using pmSINrep5-eGFP achieved high protein expression in nearly all transfected cells. Applied to the other pmSINrep5 constructs, low E and M Protein expression was detected, likely due to altered subgenomic promoter activity and cytotoxicity. Conversely, high N Protein expression was detected from Western Blot analysis. Transfections were able to produce mSINrep5(SV)-eGFP and SV-N VLPs. Purification was achieved by adjusting the optimized transfection protocol to eliminate contaminating proteins, and SV VLP quantification was subsequently optimized in a two-pronged approach involving VLP infection titration and RT-qPCR. The development of a working pipeline to efficiently construct and test the SINrep5 viral vector expression vector highlights its utility in studying viral proteins and antigens, diversifying the tools already proffered by alphaviruses in immunotherapy research.enUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.SindbisSINrep5VirusCOVID-19SARS-CoV-2AlphavirusCoronavirusVaccineVLPViral VectorMicrobiologyVirologymaster thesis