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

Abstract

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies in the serum of an individual indicates prior 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 IgG 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.

Development and utilization of a surrogate SARS-CoV-2 viral neutralization assay to assess mRNA vaccine responses

“…However, some research use only kits for measuring RBD-ACE2r inhibiting antibodies are designed with plate bound RBD and soluble human ACE2r, without obvious explanation for this "reversed" orientation in regard to in vivo biophysics (e.g., cell surface bound ACE2r and soluble RBD). Recent work by Fenwick et al [25] in hospitalized COVID-19 patients using a soluble ACE2r-based binding format similar to the present study found strong correlations with PRNT, but did not study vaccinated individuals. The present data extend accumulating evidence that SARS-CoV-2 neutralizing capacity serum can be accomplished with competition assays based on soluble phase ACE2r attachment to immobilized spike or its RBD, and further demonstrate the utility of the approach in monitoring vaccine responses.…”

“…In summary, we developed surrogate neutralization assay that significantly (p < 0.0001) correlates (rs=0.83) with IC50 measurements using live viral PRNT. The assay is similar in principle to methodology recently reported by Fenwick et al [25], but can be run with standard microtiter plate technology (vs.…”

“…In summary, we developed surrogate neutralization assay that significantly (p < 0.0001) correlates (rs=0.83) with IC50 measurements using live viral PRNT. The assay is similar in principle to methodology recently reported by Fenwick et al [25], but can be run with standard microtiter plate technology (vs. fluorescent microbeads) using commercially available reagents, and may provide an economical approach to evaluating vaccine responses and assessing immunity among large populations. Our initial data using the assay document heterogenous levels of surrogate neutralizing activity among vaccinated subjects and marked declines over a 6 month period.…”

Rapid test to assess the escape of SARS-CoV-2 variants of concern

“…Our work is motivated by the urgent need for a rapid and easy-to-use assay that supplements conventional antibody neutralization tests that are labor-intensive, costly, require highly trained personnel, and are thus inaccessible in many regions around the world. While other assays have been developed that measure the ability of nAbs to block ACE2-RBD binding ( 33 , 34 , 37 ), to the best of our knowledge, CoVariant-SCAN is the first test that can detect nAbs against several SARS-CoV-2 variants simultaneously within 1 hour. Furthermore, because the CoVariant-SCAN is built upon a “nonfouling” polymer coating that eliminates nearly all nonspecific binding, the assay can be conducted directly from undiluted plasma ( 38 , 61 , 62 ).…”

A Rapid Bead-Based Assay For Screening Of SARS-CoV-2 Neutralising Antibodies

“…In this approach, ACE2-GFP immobilized beads were used to bind the recombinant S1-RBD in a solution that is similar to the ELISA-based surrogate neutralizing antibody testing approach commercially available consisting of non-fluorescent h ACE2 and HRP based colorimetric detection (Fenwick et al, 2021). However, we have evaluated the best-known GFP immobilization approach, GFP nanobody, the single-chain VHH antibody domain of specific binding activity against GFP and its fusion proteins (Muyldermans et al, 2009) for direct visualization.…”