Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera
Authors: Oguntuyo Kasopefoluwa Y. , Stevens Christian S. , Hung Chuan-Tien , Ikegame Satoshi , Acklin Joshua A. , Kowdle Shreyas S. , Carmichael Jillian C. , Chiu Hsin-Ping , Azarm Kristopher D. , Haas Griffin D. , Amanat Fatima , Klingler Jéromine , Baine Ian , Arinsburg Suzanne , Bandres Juan C. , Siddiquey Mohammed N.A. , Schilke Robert M. , Woolard Matthew D. , Zhang Hongbo ,   COVIDAR Argentina Consortium, Duty Andrew J. , Kraus Thomas A. , Moran Thomas M. , Tortorella Domenico , Lim Jean K. , Gamarnik Andrea V. , Hioe Catarina E. , Zolla-Pazner Susan , Ivanov Stanimir S. , Kamil Jeremy P. , Krammer Florian , Lee Benhur 
Published: August 2020

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVAG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

Importance

Vaccines and antibody-based therapeutics like convalescent plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) is an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.