Competitive SARS-CoV-2 Serology Reveals Most Antibodies Targeting the Spike Receptor-Binding Domain Compete for ACE2 Binding

Abstract

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Here, we present an efficient, competitive serological assay that can simultaneously determine an individual's seroreactivity against the SARS-CoV-2 Spike protein and determine the proportion of anti-Spike antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. In this approach based on the use of enzyme-linked immunosorbent assays (ELISA), we present natively folded viral Spike protein receptor-binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then competed with soluble ACE2-Fc, or with a higher-affinity variant thereof, to determine the proportion of ACE2 blocking anti-RBD antibodies. Assessment of sera from 144 SARS-CoV-2 patients ultimately revealed that a remarkably consistent and high proportion of antibodies in the anti-RBD pool targeted the epitope responsible for ACE2 engagement (83% ± 11%; 50% to 107% signal inhibition in our largest cohort), further underscoring the importance of tailoring vaccines to promote the development of such antibodies.IMPORTANCE With the emergence and continued spread of the SARS-CoV-2 virus, and of the associated disease, coronavirus disease 2019 (COVID-19), there is an urgent need for improved understanding of how the body mounts an immune response to the virus. Here, we developed a competitive SARS-CoV-2 serological assay that can simultaneously determine whether an individual has developed antibodies against the SARS-CoV-2 Spike protein receptor-binding domain (RBD) and measure the proportion of these antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. Using this assay and 144 SARS-CoV-2 patient serum samples, we found that a majority of anti-RBD antibodies compete for ACE2 binding. These results not only highlight the need to design vaccines to generate such blocking antibodies but also demonstrate the utility of this assay to rapidly screen patient sera for potentially neutralizing antibodies.

Keywords: COVID-19; SARS-CoV-2; angiotensin-converting enzyme 2; immunoserology; neutralizing antibodies; receptor-binding domain; serology.

FIG 1

Natively presented SARS-CoV-2 Spike protein antigens effectively detect anti-Spike antibodies. (A) Schematic of NeutrAvidin/biotinylated antigen serology ELISA setup and detection strategy using protein L-HRP (PL-HRP). (B to G) Data represent ELISA results for the indicated antigens presented via NeutrAvidin (NAV, B to E) or passively adsorbed to the plate (F and G). Sera from three patients (P1, P2, and P5) and two healthy controls (C1 and C2) were tested. Antigen coating solutions were 20 nM. Each sample was run with two technical replicates. Dots indicate the mean signal of technical replicates from each of two (n = 2) independent experiments. RT, room temperature; NFM, nonfat milk.

FIG 2

ACE2-Fc competes with patient antibodies for RBD binding. (A) Schematic of ACE2-Fc competitive serology ELISA. (B) Competition ELISA (100 nM ACE2-Fc) results from four patients (P1 to P4) and one healthy control (C1) using RBD-biotin as the capture antigen. (C to F) Competition ELISA results using the indicated antigens for eight patients (P2 to P9) and one healthy control (C2). All sera were diluted 1:50 for analysis, and bound antibodies were detected with protein L-HRP. Each sample was run with two technical replicates. Dots indicate mean signal of technical replicates from two (n = 2) independent experiments. Bars show the means of results from these two experiments. (G) Percent inhibition of signal seen with competition. Dots represent means ± SD (n = 2).

FIG 3

A high-affinity ACE2-Fc variant enhances competition with patient antibodies. (A) Competition ELISA results from 36 patients obtained using a 100 nM concentration of either wild-type (WT) or high-affinity (HA) ACE2-Fc and RBD-biotin as the capture antigen. All sera were diluted 1:50 for analysis, and bound antibodies were detected with anti-Fab-HRP. Each sample was run once with two technical replicates. Dots indicate signal of each technical replicate. Bars show the means of results from these two replicates. (B and C) Correlation of direct anti-RBD signal and percent signal inhibition with competition using either WT ACE2-Fc (B) or HA ACE2-Fc (C). Patients with direct anti-RBD signal values of <0.2 were excluded from percent decrease analysis (2/36). (D) Compiled percent inhibition data for each ACE2-Fc variant. Lines connect values representing results from the same patient.

FIG 4

Patients produced a consistent proportion of competitive anti-RBD antibodies. (A) Compiled percent signal inhibition with RBD-biotin or FL Spike-biotin as the capture antigen in competition assay using 100 nM HA ACE2-Fc. All sera were diluted 1:50 for analysis, and bound antibodies were detected with anti-Fab-HRP. Each sample was run once with two technical replicates. Dots represent mean values obtained from these two replicates. Patients with direct anti-RBD signal values of <0.2 were excluded from percent inhibition analysis (22/99). (B) Correlation of direct anti-RBD signal and signal decrease with HA ACE2-Fc competition. (C and D) Correlation of signal decrease with either HA ACE2-Fc (C) or direct anti-RBD signal (D) with NT50 values published for these patients by Robbiani et al. (13).