ABSTRACT The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-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 intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA ( n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC 50 ), absIC 80 , and absIC 90 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 the absIC 80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week.
【저자키워드】 COVID-19, SARS-CoV-2, Neutralizing antibodies, convalescent-phase plasma, viral neutralization assay, TMPRSS2, 【초록키워드】 coronavirus disease, neutralizing antibody, Coronavirus disease 2019, Efficacy, ACE2, Vaccine, coronavirus, pandemic, therapy, Neutralizing antibodies, antibody, neutralization, spike glycoprotein, severe acute respiratory syndrome Coronavirus, viral entry, ELISA, Particle, enzyme-linked immunosorbent assay, Viral, Screen, sera, Patient, plasma, respiratory, group, vesicular stomatitis virus, Live virus, Neutralizing titer, Combination, biosafety level, level 3, vesicular stomatitis, Support, acute respiratory syndrome, acute respiratory syndrome coronavirus, acute respiratory syndrome coronavirus 2, positive correlation, NAbs, Variability, neutralization potency, effort, cohorts, myriad, inhibitory concentration, 293T, Express, BSL2, stomatitis, BSL3, clone, VSVΔG, pseudotyped, CoV2pp, G glycoprotein, intergroup comparisons, Renilla luciferase gene, limitations, limit, Biosafety Level 3, independent, enzyme-linked immunosorbent, highlight, Postinfection, produced, identify, develop, inhibit, generate, overcome, conditions, intergroup comparison, virus neutralization assay, 【제목키워드】 titer,