Development of an automated, high-throughput SARS-CoV-2 neutralization assay based on a pseudotyped virus using a vesicular stomatitis virus (VSV) vector

Emerg Microbes Infect. 2023 Dec;12(2):e2261566. doi: 10.1080/22221751.2023.2261566. Epub 2023 Sep 28.

Abstract

ABSTRACTThe global outbreak of COVID-19 has caused a severe threat to human health; therefore, simple, high-throughput neutralization assays are desirable for developing vaccines and drugs against COVID-19. In this study, a high-titre SARS-CoV-2 pseudovirus was successfully packaged by truncating the C-terminus of the SARS-CoV-2 spike protein by 21 amino acids and infecting 293 T cells that had been stably transfected with the angiotensin-converting enzyme 2 (ACE2) receptor and furin (named AF cells), to establish a simple, high-throughput, and automated 384-well plate neutralization assay. The method was optimized for cell amount, virus inoculation, incubation time, and detection time. The automated assay showed good sensitivity, accuracy, reproducibility, Z' factor, and a good correlation with the live virus neutralization assay. The high-throughput approach would make it available for the SARS-CoV-2 neutralization test in large-scale clinical trials and seroepidemiological surveys which would aid the accelerated vaccine development and evaluation.

Keywords: SARS-CoV-2; VSV; high-throughput; neutralization assay; pseudovirus.

MeSH terms

  • Animals
  • Antibodies, Neutralizing
  • Antibodies, Viral
  • COVID-19*
  • Humans
  • Neutralization Tests / methods
  • Reproducibility of Results
  • SARS-CoV-2 / genetics
  • Spike Glycoprotein, Coronavirus
  • Vesicular Stomatitis*
  • Vesicular stomatitis Indiana virus / genetics
  • Viral Pseudotyping

Substances

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

Grants and funding

This work was supported by Beijing Municipal Science and Technology Project [grant no Z211100002521018], the General Program of the National Natural Science Foundation of China [grant no 82172244 & 32070678], National Key R&D Program of China [grant no 2021YFC2302500, 2021YFC2301700, and 2021YFC0863300] and Changping laboratory.