DEVELOPMENT OF PSEUDOLENTIVIRUS NEUTRALIZATION ASSAY WITH SPIKE VARIANTS OF SARS-COV-2

• Main Author: Firdaus Syuaib, Afina
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/71024
• Institution: Institut Teknologi Bandung

Spike protein is the main target of neutralizing antibodies because it is located on the surface of the virus and plays a role in the infection of SARS-CoV-2. Variants of SARS-CoV-2 that have substitutions in the spike protein can evade neutralizing antibodies and cause a decrease in vaccine efficacy. Neutralization assay is a method to evaluate the protective efficacy of antibodies in the sera of vaccinated individuals. SARS-CoV-2 is a biosafety level-3 virus, thus, pseudolentivirus can be used as a safer alternative to carrying out neutralization assay. This study aims to develop a pseudolentivirus-based neutralization assay with SARS-CoV-2 spike from various variants. This research was initiated by isolating and confirming the plasmid coding for pseudolentivirus. Pseudolentivirus production was carried out by plasmid co-transfection of HEK 293T cells. Determination of pseudolentivirus titers through the luciferase expression test showed that pseudolentivirus titers of alpha, beta, gamma, delta, and omicron variants were lower than those of the wild type. To test this pseudolentivirus neutralization system, a recombinant neutralizing antibody (NAb) was used at various concentrations. Production of NAb was carried out by plasmid co-transfection into HEK 293T cells and the NAb titer was 2.54 ± 0.28 ?g/mL compared to the standard curve of Human Anti-Spike S1 using the indirect ELISA method. Neutralization assay against monoclonal neutralizing antibody (NAb) was successfully carried out with NT50 NAb values against SARS-CoV-2 wild type, alpha, beta, gamma, delta, and omicron were 0.29; 0.58; 0.66; 0.31; 0.56; and 1.16 ?g/mL respectively. Serum neutralization assay showed differences in the % infection of the SARS-CoV-2 variants. This pseudolentivirus neutralization system can be used to measure the neutralizing activity of NAb and human sera against SARS-CoV-2 variants.