Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera

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, Jeromine; Baine, Ian; Arinsburg, Suzanne; Bandres, Juan C.; Siddiquey, Mohammed N.A.; Schilke, Robert M.; Woolard, Matthew D.; Zhang, Hongbo; 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

The on-going coronavirus disease 2019 (COVID-19) pandemic has mobilized a global effort to develop vaccines and therapeutics that inhibit viral entry by inducing or transferring antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). Phase I/II vaccine clinical trials, monoclonal antibodies, and convalescent sera have all shown promise. However, these efforts often require extensive screening with the live virus under onerous high biocontainment conditions (BSL-3). Virus neutralization assays (VNAs) remain the gold standard for evaluating the anti-viral potency of antibodies and entry inhibitors. The proliferation of pseudotyped virus systems that can be used in BSL-2 compatible VNAs is a positive development. Yet, there is marked variability between VNAs and how the findings are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVΔG based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale. We used our CoV2pp to interrogate the role of exogenous and endogenous proteases in CoV-2-S mediated entry and standardized our VNA based on that understanding. Our CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in a validated set of patient sera. Our system was subsequently validated by three independent groups as an “out-of-the-box” VNA. More than 120 patient sera were screened, and we report descriptive statistics for absolute (abs) IC50, IC80, and IC90 values from all positive patient sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 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.