title: Generation of SARS-CoV-2 Coronavirus Protein Antigens for Development of Diagnostics and Novel Vaccine Approaches
Creators: name: Burrows, Emily
Publisher: name: University of Kent
Year: 2022

description: The SARS-CoV-2 coronavirus pandemic that is reported to have originated in Wuhan in China in late 2019 has since rapidly spread globally resulting in millions of cases and hundreds of thousands of deaths. A rapid global response to this included nationwide lockdowns in countries with massive economic and social implications alongside an escalating case and death rate. In order to identify/diagnose infected individuals and those that have previously been infected to help in the management and treatment of the disease, there has been a concentrated effort to develop diagnostics for SARS-CoV-2 and antibodies raised against this. There has also been a simultaneous drive to develop vaccines to protect against SARS-CoV-2 infection. Although there are multiple approaches to the design of diagnostics and vaccines, each requires the rapid supply of SARS-CoV-2 protein antigens for us in diagnostics or vaccines or to use in assays in their development. The novel SARS-CoV-2 coronavirus genome encodes a number of structural proteins, including the Spike protein that facilitates binding of the virus to the ACE2 receptor during viral cell entry. The aim of this project was to generate coronavirus antigens for the development of novel vaccines and diagnostic tools, particularly of the Spike protein. This was undertaken by investigating recombinant expression of SARS-CoV-2 Spike protein production via the comparison of using different CHO cell line host expression systems, the comparison of various ER signal peptides, the development of stable CHO cell pools expressing the spike protein and measuring patient antibody responses to different mutant spike proteins. SARS-CoV-2 Spike DNA constructs were generated via molecular cloning and various mutants of the Spike generated. Antibody responses were measured using ELISA assays. The various Spike DNA constructs were successfully constructed into commercial vectors as well as the B.1.1.7 and B.1.351 variants as they emerged during the progress of the pandemic. CHO-S cells were found to be more effective than CHO-K1 cells for spike protein production. The native Spike CHO optimised ER signal peptide was the most effective signal peptide of those evaluated giving the highest expression of Spike protein and higher mRNA transcript amounts. Indeed, secreted protein yield, as assessed by western blot, was highest using constructs that gave the highest mRNA transcript amounts. When generating CHO cells stably expressing Spike protein using a glutamine synthetase selection system, 25 M MSX levels gave rise to higher productivity in the stable cell pools than 50 M MSX and ELISA with the recombinant Spike proteins showed a concentration-response relationship with the serum of Wuhan infected patients against various spike variants. Overall, this project was able to successfully develop coronavirus Spike protein antigens and improve the Spike protein production process via varying the CHO host cell line, the ER signal peptide selection, development of stably Spike expressing cell pools and modifying the MSX concentration used during stable cell development, collectively resulting in higher Spike protein production.