A high-throughput cell- and virus-free assay shows reduced neutralization of SARS-CoV-2 variants by COVID-19 convalescent plasma

Abstract

A multiplexed virus-free surrogate neutralization assay measures antibody responses against spike protein mutations in SARS-CoV-2 variants. Multiplexing SARS-CoV-2 neutralization Neutralizing antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are primarily assessed using cell-based assays requiring live virus. These assays are time-consuming and necessitate that additional biosafety precautions be taken, thus limiting their clinical use. Here, Fenwick et al. developed a cell-free surrogate neutralization assay to quantify neutralizing antibody responses. In this assay, neutralizing antibodies block the ability of fluorescent angiotensin-converting enzyme 2 (ACE2) molecules from binding to recombinant SARS-CoV-2 spike protein trimers. The assay achieved 96.7% sensitivity and 100% specificity in comparison to a gold standard, cell-based assay and could be multiplexed to quantify responses against SARS-CoV-2 variants of concern in one test. The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–specific antibodies in the serum of an individual indicates previous infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral spike protein are more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, inflexible, and potentially biohazardous. Here, we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 spike protein binding to the angiotensin-converting enzyme 2 (ACE2) receptor. This high-throughput method matches the performance of the gold standard live virus infection assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific immunoglobulin G titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 spike protein variants of concern. We used our assay to profile serum samples from 59 patients hospitalized with coronavirus disease 2019 (COVID-19). We found that although most sera had high activity against the 2019-nCoV parental spike protein and, to a lesser extent, the α (B.1.1.7) variant, only 58% of serum samples could efficiently neutralize a spike protein derivative containing mutations present in the β (B.1.351) variant. Thus, we have developed an assay that can evaluate effective neutralizing antibody responses to SARS-CoV-2 spike protein variants of concern after natural infection and that can be applied to characterize vaccine-induced antibody responses or to assess the potency of monoclonal antibodies.