THE DEVELOPMENT OF NEUTRALIZATION ASSAY BASED ON VESICULAR STOMATITIS VIRUS PSEUDOVIRUS BEARING SARS-COV-2 SPIKE

• Main Author: Arcelia, Chatleen
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/73712
• Institution: Institut Teknologi Bandung

Neutralization assay can be used to evaluate the efficacy of COVID-19 vaccine in preventing SARSCoV-2 infection through the detection of neutralizing antibodies (NAb) that are formed after vaccination. However, the existing method involves native SARS-CoV-2 virus which must be handled at BSL-3. Through pseudovirus-based neutralization assay, the same assay can be done in BSL-2 by utilizing a SARS-CoV-2 spike bearing pseudovirus. This research was conducted to develop a neutralization assay based on vesicular stomatitis virus (VSV) pseudovirus possessing wild-type SARS-CoV-2 spike (rVSV-?G*Spike). The experiment was initiated by transformation of the plasmids into Escherichia coli DH5? and isolation of the plasmids from the transformants. Confirmation of the isolated plasmids was done through migration and restriction analysis which showed that the plasmids was confirmed. Prior to rVSV-?G*Spike production, production of recombinant VSV (rVSV?G*G) was carried out first through co-transfection of 6 plasmids in HEK293T cells. Production of rVSV-?G*Spike was done by transfection of plasmid carrying the wild-type SARS-CoV-2 spike protein and infection of rVSV-?G*G into HEK293T cells. Analysis of rVSV-?G*G and rVSV-?G*Spike production was performed through luciferase expression assay. This study used pseudolentivirus that had been constructed in previous study as a positive control for the luciferase expression assay and the neutralization assay. The results of the luciferase expression assay showed RLU value of 1,025 upon rVSV-?G*G infection in HEK293T-ACE2. Meanwhile, rVSV-?G*Spike infection showed an RLU value that was close to the negative control. Therefore, it was suspected that the pseudovirus was not produced or had a very low titer. The neutralization assay was performed using neutralizing antibody (NAb) against SARS-CoV-2. Production of NAb was conducted by co-transfection of plasmids into HEK293T cells and the NAb concentration was 2.72 ± 0.17 ?g/mL. The neutralization assay showed decrease in RLU along with the increase NAb. However, both rVSV-?G*G and rVSV?G*Spike showed very low RLU values at various NAb amounts and did not respond to the NAb. In conclusion, the pseudovirus VSV-based SARS-CoV-2 neutralization system was not successfully developed and require further optimization during pseudovirus production.