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.

Challenges and opportunities for antiviral monoclonal antibodies as COVID-19 therapy

“…For example mAbs C121, C144, and C135 isolated by the Nussenzweig group have respective IC 50 values of 1.64, 2.55, and 2.98 ng/mL against SARS-CoV-2 [ 99 ]. This in turn translates to exceptional neutralization potency against SARS-COV-2 [ 115 ]: many of them possess IC 50 below 15 ng/mL against pseudoviruses, with comparable potencies against live virus ( Table 1 ).…”

Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2

“…SARS-CoV-2 antibodies have been derived from several sources, including B-cells of convalescent patients 7,12,13,17 and people with prior coronavirus infections, 14,15 animal immunization, 10,18 and synthetic libraries or de novo design. 8,9,11,16,19 Most of the antibodies reported to date potently target the receptor-binding domain (RBD) in the trimeric Spike protein on the surface of SARS-CoV-2, 7,13,[22][23][24] which is highly immunogenic and is the key protein that mediates cellular entry via interaction with the host angiotensin-converting enzyme II (ACE2) receptor. 25 However, given the widespread global impact of this pandemic and limitations in biologic manufacturing capacities, means to further increase the potency of these antibodies and thereby decrease the dose required will be critical in meeting the global demand for therapeutics.…”

Comparative Assessment of Sera from Individuals after S-Gene RNA-Based SARS-CoV-2 Vaccination with Spike-Protein-Based and Nucleocapsid-Based Serological Assays

“…Although the viral nucleocapsid as well as the antigens of the viral open reading frames (ORFs) 8 and 3b were shown to elicit the strongest specific antibody responses after SARS-CoV-2-infections [ 16 ], serological assays targeting the spike protein and the nucleocapsid protein have been most frequently designed. Serological response against the spike protein has been considered as particularly interesting due to reported interaction with ACE2 (angiotensin-converting enzyme 2) binding [ 17 ]. Further, spike-protein-specific antibodies have been reported to persist longer than nucleocapsid-specific antibodies [ 18 ], which decrease within weeks to months [ 19 ].…”