Development of a pseudovirus-based assay for analysis of neutralizing activity against SARS-CoV-2

Official URL: https://risc01.sabanciuniv.edu/record=b2708520_(Table of contents)

As the COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread around the globe, effective vaccination protocols are actively being implemented. The delayed deployment of effective vaccines, especially in the developing world and the lack of an effective antiviral treatment inadvertently increased the interest in approaches such as the use of convalescent plasma (CP) or monoclonal antibody products. Initial clinical evaluation revealed that critical factors determining the outcome of CP or antibody-based therapies need to be defined clearly if clinical efficacy is to be expected. Measurement of neutralizing activity against SARSCoV-2 using wildtype virus presents a reliable functional assay but the availability of suitable BSL3 facilities for virus culture restricts its applicability. Instead, the use of pseudovirus particles containing elements from the SARS-CoV-2 virus is widely applied to determine the activity of CP or other neutralizing agents. In this thesis, we present our approach to production of lentiviral particles pseudotyped with the SARS-CoV-2 Spike (S), Membrane (M), Envelope (E) and Nucleocapsid (N) proteins for use in neutralization. Furthermore, we evaluate different transgenes such as the Green Fluorescent Protein (GFP) or Secreted Alkaline Phosphatase (SEAP) as reporter genes in an attempt to develop faster and more practical analysis of multiple samplesin a short time. Our findings show that the use of optimized SEAP-based methods can increase the availability of neutralizing assays by overcoming the need for advanced equipment and increase the processing speed of multiple samples in a given lab.