SARS-CoV-2 Neutralization Assay System using Pseudo-lentivirus

Anastasia Armimi (School of Life Sciences and Technology, Institut Teknologi Bandung, Jl. Ganesa No.10, Lb. Siliwangi, Bandung 40132)
Afina Firdaus Syuaib (School of Pharmacy, Institut Teknologi Bandung, Jl. Ganesa No.10, Lb. Siliwangi, Bandung 40132)
Katherine Vanya (School of Life Sciences and Technology, Institut Teknologi Bandung, Jl. Ganesa No.10, Lb. Siliwangi, Bandung 40132)
Marselina Irasonia Tan (School of Life Sciences and Technology, Institut Teknologi Bandung, Jl. Ganesa No.10, Lb. Siliwangi, Bandung 40132)
Dessy Natalia (Biochemistry Research Group, Faculty of Mathematics and Natural Science, Institut Teknologi Bandung, Jl. Ganesa No.10, Lb. Siliwangi, Bandung 40132)
David Virya Chen (Laboratory of Virus Control, Center for Infectious Disease Education and Research, Osaka University, 1-1 Yamadaoka, Osaka 565-0871)
Chikako Ono (Laboratory of Virus Control, Center for Infectious Disease Education and Research, Osaka University, 1-1 Yamadaoka, Osaka 565-0871)
Yoshiharu Matsuura (Laboratory of Virus Control, Center for Infectious Disease Education and Research, Osaka University, 1-1 Yamadaoka, Osaka 565-0871)
Anita Artarini (School of Pharmacy, Institut Teknologi Bandung, Jl. Ganesa No.10, Lb. Siliwangi, Bandung 40132)
Ernawati Arifin Giri-Rachman (School of Life Sciences and Technology, Institut Teknologi Bandung, Jl. Ganesa No.10, Lb. Siliwangi, Bandung 40132)

The Indonesian Biomedical Journal
Vol 15, No 2 (2023)

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects humans' lower respiratory tracts and causes coronavirus disease-2019 (COVID-19). Neutralizing antibodies is one of the adaptive immune system responses that can reduce SARS-CoV-2 infection. This study aimed to develop a SARS-CoV-2 neutralization assay system using pseudo-lentivirus.METHODS: The plasmid used for pseudo-lentivirus production was characterized using restriction analysis. The gene encoding for SARS-CoV-2 spike protein was confirmed using sequencing. The transfection pseudo-lentivirus optimal condition was determined by choosing the transfection reagents and adding centrifugation step. Optimal pseudo-lentivirus infection was analysed using fluorescent assay and luciferase assay. The optimal condition of pseudo-lentivirus infection was determined by the target cell type and the number of pseudo-lentiviruses used for neutralization test. SARS-CoV-2 pseudo-lentivirus was used to detect neutralizing antibodies from serum samples.RESULTS: The plasmid used for pseudo-lentivirus production was characterized and confirmed to have no mutations. Lipofectamine 2000 reagent generated pseudo-lentivirus with a higher ability to infect target cells, as indicated by a percentage green fluorescent protein (GFP) of 12.68%. Pseudo-lentivirus centrifuged obtained more stable results in luciferase expression. Optimal pseudo-lentivirus infection conditions were obtained using puromycin-selected HEK 293T-ACE2 cells as target cells. The number of pseudo-lentiviruses used in the neutralization assay system was multiplicity of infection (MOI) 0.075. Serum A samples with a 1:10 dilution had the highest neutralizing antibody activity.CONCLUSION: This study shows that SARS-CoV-2 neutralization assay system using pseudo-lentivirus successfully detected neutralizing antibodies in human serum, which were indicated by a decrease in the percentage of pseudo-lentivirus infections.KEYWORDS: COVID-19, neutralizing antibody, neutralization assay, pseudo-lentivirus, SARS-COV-2