Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation

Abstract

Antibody responses develop following SARS-CoV-2 infection, but little is known about their epitope specificities, clonality, binding affinities, epitopes, and neutralizing activity. We isolated B cells specific for the SARS-CoV-2 envelope glycoprotein spike (S) from a COVID-19-infected subject 21 days after the onset of clinical disease. 45 S-specific monoclonal antibodies were generated. They had undergone minimal somatic mutation with limited clonal expansion, and three bound the receptor-binding domain (RBD). Two antibodies neutralized SARS-CoV-2. The most potent antibody bound the RBD and prevented binding to the ACE2 receptor, while the other bound outside the RBD. Thus, most anti-S antibodies that were generated in this patient during the first weeks of COVID-19 infection were non-neutralizing and target epitopes outside the RBD. Antibodies that disrupt the SARS-CoV-2 S-ACE2 interaction can potently neutralize the virus without undergoing extensive maturation. Such antibodies have potential preventive and/or therapeutic potential and can serve as templates for vaccine design.

Keywords: ACE2; B cells; COVID-19; MERS; SARS; SARS-CoV-2; antibodies; neutralization; receptor-binding domain; spike protein.

Graphical abstract

Figure 1

SARS-CoV-2 Infection Elicits Binding and Neutralizing Antibodies Directed at the Spike Protein (A–D) Total antibody binding in serum from a donor with confirmed SARS-CoV-2 infection (COVID-19⁺), from two donors collected prior to the COVID-19 pandemic with an unknown history of coronavirus infection (pre-pandemic), and from nine donors with confirmed infection by endemic corona viruses (endemic), was tested for binding to the SARS-CoV-2 S2P ectodomain (A) and the RBD (B) by ELISA. Serum from the donor in SARS-CoV-2 infection in (A) was tested for binding to the SARS-CoV-2 S2P ectodomain (C) and the RBD (D) using isotype-specific secondary antibodies by ELISA. (E) Serum from donor with confirmed SARS-CoV-2 infection, and serum from a pre-pandemic donor were evaluated for their ability to neutralize a SARS-CoV-2 pseudovirus. Data points indicate the mean, and error bars represent standard deviation of 2–3 technical replicates. Due to the limited amount of serum available, these analyses were restricted to a single analysis.

Figure 2

Early B Cell Response to SARS-CoV-2 Is Diverse and Largely Unmutated (A) Class switched (IgM⁻ IgG⁺) B cells were stained with SARS-CoV-2 S2P labeled with BV710 or PE. (B) SARS-CoV-2 S2P⁺ IgG⁺ B cells were further analyzed for binding to Alexa Fluor 647-labeled SARS-CoV-2 RBD. (C–E) Individual SARS-CoV-2 S2P⁺ IgG⁺ B cells were sorted into separate wells of a 96-well plate and sequenced using RT-PCR. VH (C), VK (D), and VL (E) gene usage of successfully sequenced S2P-specific B cells. (F and G) CDRH3 (F) and CDRL3 (G) length distributions of successfully sequenced S2P-specific B cells. (H) Number of amino acid substitutions from germline in S2P-specific heavy and light chains. See also Figure S1 and Table S1.

Figure 3

Recombinant Anti-spike mAbs Bind to a Stabilized SARS-CoV-2 Ectodomain Trimer and a Subset Cross-React with SARS-CoV S (A and B) mAbs isolated from SARS-CoV-2 S2P-specific B cells were tested for binding to SARS-CoV-2 S2P (A) and to SARS-CoV-2 RBD (B) using BLI. (C) mAbs were labeled with phycoerythrin (PE) and used to stain 293 cells transfected with wild-type SARS-Cov-2 S by flow cytometry. Heatmap shows mean fluorescence intensity of PE⁺ cells at 2.5 μg/mL. Titration curves are shown in Figure S2. Boxes with an X through them indicate antibodies that were not tested. (D) mAbs were tested for binding to SARS-CoV S2P by BLI (D). (E) Heatmap shows maximum binding response (average nm shift of the last 5 s of association phase) of binding data in (A), (B), and (D). Data presented here are representative of 2 independent experiments. See also Figure S2.

Figure 4

The RBD-Specific mAb CV30 Neutralizes SARS CoV-2 by Blocking the ACE2- SARS-CoV-2 S Interaction (A) CV1 and CV30 were serially diluted and tested for their ability to neutralize SARS-CoV-2 pseudovirus infection of 293T cells stably expressing ACE2. An ACE2-FC fusion and the anti-EBV mAb AMMO1 were included as positive and negative controls. Data points represent the mean and, error bars indicate the standard deviation of quadruplicate replicates. Data are representative of 6 independent experiments (see Table S1 for details). (B) The same mAbs were tested for neutralization of an MLV pseudovirus. Data points represent the mean, and error bars indicate the standard deviation of quadruplicate replicates. (C) Biotinylated ACE2-Fc was immobilized on streptavidin biosensors and then tested for binding to SARS-CoV-2 RBD in the absence and presence of the indicated mAbs using BLI. (D) ACE2-Fc was immobilized Protein A biosensors and binding to the indicated serial dilutions of SARS-CoV-2 RBD were measured by BLI and used to determine the dissociation constant (K_D). Red lines represent the measured data and black lines indicate the theoretical fit. (E) CV30 was immobilized onto anti-human Fc biosensors and binding to the indicated serial dilutions of SARS-CoV-2 RBD were measured by BLI and used to determine the binding constant (kD). Blue lines represent the measured data and black lines indicate the theoretical fit. Kinetic measurements from (D) and (E) are summarized in Table S2. BLI analyses are representative of 2–3 independent experiments.