Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera—Oguntuyo 2021

==The global COVID-19 pandemic has spurred the development of vaccines and antibody-based therapeutics, such as convalescent-phase plasma therapy, that induce or transfer neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, thorough effectiveness testing involves prolonged screening with live virus under difficult biosafety level 3 (BSL3) settings, limiting high-throughput screening of patient and vaccination sera. To overcome this hurdle, many BSL2-compatible surrogate viral neutralization tests (VNAs) exist. VNAs report data differently, making intergroup comparisons challenging. We developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus (VSV) with the Renilla luciferase gene instead of its G glycoprotein (VSV-G). This assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA correlated well with CoV2-S ELISA and live-virus neutralizations in confirmed convalescent-patient sera. Three separate groups confirmed our standardized CoV2pp VNA (120+). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of absIC80 as a more meaningful metric for assessing vaccine or convalescent-phase sera neutralization potency. Finally, we used CoV2pp to screen ultrapermissive 293T clones that stably express ACE2 or ACE2 + TMPRSS2. These and our CoV2pp can generate 150,000 standardized VNAs/week.IMPORTANCE Vaccines and antibody-based therapies like convalescent-phase plasma therapy induce or transfer neutralizing antibodies that block SARS-CoV-2 cell entrance. Virus neutralization assays (VNAs) for assessing neutralizing antibody titers (NATs) determine vaccination or treatment efficacy. Because working with live viruses needs high-level biocontainment facilities, effectiveness testing is limited. Thus, we created a standardized replication-defective pseudotyped particle system that mimics live SARS-CoV-2 entrance. This tool measures NATs, other entrance inhibitions, and virus entry methods safely and efficiently. Four independent labs worldwide verified our standardized VNA employing varied cohorts. A consistent and scalable assay is needed to compare the several vaccinations and antibody-based therapies on the market. Our efficacy indicators are generalizable.

Oguntuyo, K. Y., Stevens, C. S., Hung, C. T., Ikegame, S., Acklin, J. A., Kowdle, S. S., Carmichael, J. C., Chiu, H. P., Azarm, K. D., Haas, G. D., Amanat, F., Klingler, J., Baine, I., Arinsburg, S., Bandres, J. C., Siddiquey, M. N. A., Schilke, R. M., Woolard, M. D., Zhang, H., COVIDAR Argentina Consortium, … Lee, B. (2021). Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera. mBio, 12(1), e02492-20. https://doi.org/10.1128/mBio.02492-20