Production of pseudovirus for use in SARS-CoV-2 neutralization antibody tests

SARS-CoV-2 enters cells using the Spike protein, which is the target of neutralizing antibodies. Therefore, the assays that measure how antibodies affects Spike-mediated viral infection are important for studying immunity. Since SARS-CoV-2 is a biosafety level 3 virus, the safe way to perform such tests is to Spike pseudotyping biosafety level 2 viral particles. Such pseudo-typing has now been defined for single-cycle lentiviral, retroviral, and VSV particles. However, the reagents and protocols used in these procedures must be developed. In this study, pseudoviruses containing human immunodeficiency virus core (HIV-1), GFP protein reporter and spike (S) protein of SARS-CoV-2; using the transfection procedure of the HEK293T cell line, were produced in BSL-2 laboratories for use in neutralizing antibody tests. Pseudoviruses were generated by isolation of plasmids from stable plasmid cultures, transformation into competent bacterial cells, and transfection of isolated pDNAs into HEK293T, a specialized cell line for transfection. Plasmid #39196 (pHIV-Luc-ZsGreen), plasmid #12263 (pCMV-delta R8.2) and plasmid #149543 (pGBW-m4137384) were used in triple plasmid transfection. The success of pseudotyping was assessed by GFP irradiation, as the pHIV-Luc-ZsGreen plasmid integrates the gene coding for GFP. GFP irradiation was detected in the cell line at 48 hours of transfection. Pseudoviruses could be produced with a safe and easily obtainable method to be used in neutralizing antibody tests performed after vaccine administration. Keywords: COVID-19, SARS-CoV-2, pseudovirus, spike protein, neutralizing antibodies