Robust neutralization assay based on SARS-CoV-2 S-protein-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells

Emerg Microbes Infect. 2020 Dec;9(1):2105-2113. doi: 10.1080/22221751.2020.1815589.

Abstract

The global pandemic of coronavirus disease 2019 (COVID-19) is a disaster for human society. A convenient and reliable neutralization assay is very important for the development of vaccines and novel drugs. In this study, a G protein-deficient vesicular stomatitis virus (VSVdG) bearing a truncated spike protein (S with C-terminal 18 amino acid truncation) was compared to that bearing the full-length spike protein of SARS-CoV-2 and showed much higher efficiency. A neutralization assay was established based on VSV-SARS-CoV-2-Sdel18 pseudovirus and hACE2-overexpressing BHK21 cells (BHK21-hACE2 cells). The experimental results can be obtained by automatically counting the number of EGFP-positive cells at 12 h after infection, making the assay convenient and high-throughput. The serum neutralizing titer measured by the VSV-SARS-CoV-2-Sdel18 pseudovirus assay has a good correlation with that measured by the wild type SARS-CoV-2 assay. Seven neutralizing monoclonal antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 S protein were obtained. This efficient and reliable pseudovirus assay model could facilitate the development of new drugs and vaccines.

Keywords: SARS-CoV-2; VSV-SARS-CoV-2; neutralization assay; pseudovirus; vesicular stomatitis virus.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibodies, Neutralizing / blood*
  • Antibodies, Neutralizing / immunology
  • Antibodies, Viral / blood*
  • Antibodies, Viral / immunology
  • Betacoronavirus / immunology*
  • COVID-19
  • Cell Line
  • Chlorocebus aethiops
  • Coronavirus Infections / diagnosis*
  • Cricetinae
  • Neutralization Tests / methods*
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • SARS-CoV-2
  • Spike Glycoprotein, Coronavirus / genetics
  • Spike Glycoprotein, Coronavirus / immunology*
  • Vero Cells
  • Vesicular stomatitis Indiana virus / genetics
  • Vesicular stomatitis Indiana virus / immunology

Substances

  • Antibodies, Monoclonal
  • Antibodies, Neutralizing
  • Antibodies, Viral
  • Spike Glycoprotein, Coronavirus
  • spike protein, SARS-CoV-2

Grants and funding

This work was supported by the National Natural Science Foundation of China [grant number 81993149041, 81702006], Science and Technology Major Project of the Fujian Province [grant number 2020YZ014001], Xiamen Science and Technology Major Project [grant number 3502Z2020YJ02] and National Key Research and Development Program of China [grant number 2016YFD0500301].