Construction of SARS-CoV-2 pseudovirus and its application in measuring the neutralization activity of serum antibodies

ABSTRACT

Objective To construct SARS-CoV-2 pseudovirus, optimize its preparation protocol, and apply it to the evaluation of antibody neutralization activity. Methods The optimized sequence of spike (S) gene of SARS-CoV-2 was synthesized, the pseudovirus titers were measured, and the expressed S protein was then detected by Western blot. Finally, quantitative ELISA was used to measure the serum IgG antibody titers in recipients who had received either one or two doses of COVID-19 inactivated vaccine. Meanwhile, the sera were tested for their reactivity with the pseudovirus using neutralization tests. Results S gene was confirmed to have been successfully cloned into the vector, generating the pcDNA3.1-S plasmid. Co-transfection of pNL4-3.Luc.R-E- and pcDNA3.1-S at a ratio of 2∶1 could lead to higher packing efficacy and pseudovirus titers. Expression of the S protein was verified by Western blot. Moreover, this SARS-CoV-2 pseudovirus showed a broad host infectivity in Vero, Huh7.5, A549-hACE2 and 293T-hACE2 cells, with the highest relative luciferase unit (RLU) in 293T-hACE2. Comparing the IgG levels measured by ELISA in sera collected from COVID-19 vaccine recipients, we observed a higher titer in those who received two doses of inactivated vaccine (S/CO=10.27±3.33), measured one week after the second shot. However, the IgG level significantly dropped（S/CO=2.36±2.25）half year post-vaccination. Amongst the serum samples tested, one with an S/CO of 10.32 could successfully inhibit the infection of SARS-CoV-2 pseudovirus in 293T-hACE2 cells at a dilution of 1/1 066. Conclusion We have established a method for preparing the SARS-CoV-2 pseudovirus, which can be used for detection of the neutralizing antibodies and the evaluation of humoral immune response post-vaccination. © 2022 Editorial Office of Chinese Journal of Schistosomiasis Control. All Rights Reserved.