Developing a pseudo-lentivirus-based neutralization assay against SARS-CoV-2

Abstract

Abstract Background Convenient and reliable neutralization assays are vital for development of accurate diagnosis and new vaccine/drug production. Present research aimed to produce a SARS-COV-2 pseudo-lentivirus to evaluate neutralizing antibodies in the convalescent patients from Covid-19 and to assess the ability of the pseudovirus to infect different cell lines. Methods Lentix 293T cell line was employed for the transfection of the plasmid, and SARS-CoV-2 S gene was codon-optimized, synthesized, and cloned into the pcDNA3.1-SARS-CoV-2 plasmid followed by amplification and transforming into E. coli DH5α. Confirmation of the extracted plasmid was performed by gel electrophoresis. ThepcDNA3.1-SARS-CoV-2 plasmid, psPax-2 and reporter plasmid pLOX-CWgfp were transfected into Lentix 293T cells using the Turbofect transfection reagent. Western blot assay was undertaken to conform the SARS-CoV-2 S-protein transfection, and the titer of the produced SARS-CoV-2 pseudovirus was assessed by the Real-Time PCR. Sera samples of 24 convalescent patients and five samples of healthy persons (negative control) were tested by both the EUROIMMUN Anti‐SARS‐CoV‐2 QuantiVac ELISA (IgG) and the neutralization assay. Results By ELISA and neutralizing antibody assays 24 (100%) and 17 (70.83%) samples were detected as positive, respectively. Calculation of Kappa coefficient exhibited a medium correlation agreement. By Pearson correlation coefficient no significant (p=0.24) was seen between the two assays. Further, the positive predictive value for the presence of high neutralizing antibodies was 100%, whereas the negative predictive value for low neutralizing antibodies was 41.66%. Conclusion Based on WHO guidelines neutralization assays are considered as the gold standard for assessing the protective potential of antibodies induced by the SARS-CoV-2 vaccines. Given these results, by optimizing the pseudoviral production and neutralization assay, we will be able to determine a threshold between the two assays.