Human-IgG-Neutralizing Monoclonal Antibodies Block the SARS-CoV-2 Infection

Abstract

Coronavirus disease 2019 (COVID-19) has become a worldwide threat to humans, and neutralizing antibodies have therapeutic potential. We have purified more than 1,000 memory B cells specific to SARS-CoV-2 S1 or its RBD (receptor binding domain) and obtain 729 paired heavy- and light-chain fragments. Among these, 178 antibodies test positive for antigen binding, and the majority of the top 17 binders with EC₅₀ below 1 nM are RBD binders. Furthermore, we identify 11 neutralizing antibodies, eight of which show IC₅₀ within 10 nM, and the best one, 414-1, with IC₅₀ of 1.75 nM. Through epitope mapping, we find three main epitopes in RBD recognized by these antibodies, and epitope-B antibody 553-15 could substantially enhance the neutralizing abilities of most of the other antibodies. We also find that 515-5 could cross neutralize the SARS-CoV pseudovirus. Altogether, our study provides 11 potent human neutralizing antibodies for COVID-19 as therapeutic candidates.

Keywords: COVID-19; RBD; SARS-CoV-2; coronavirus; cross-neutralizing antibody; epitope; human antibodies; infection; neutralizing antibodies; spike protein.

Graphical abstract

Figure 1

Serological Responses of 11 Convalescent COVID19 Patients (A and B) Spike protein binding (A) and pseudoviral neutralizing tests (B) of donor plasma. RBD and S1 were coated at 1 μg/mL for binding ELISA. Plasma samples of heathy donors were used as controls. The mean values and standard deviations of two technical replicates are shown in a pseudo-typed viral neutralization assay.

Figure 2

The Identification and Characterization of Spike Protein-Specific Monoclonal Antibodies (A) Characteristics of antibodies binding with RBD and S-ECD. RBD and S-ECD dual binders, red dots; binders for S-ECD only, purple; binders for RBD only, green; and weaker binders, black. Authentic neutralizing antibodies are represented by bigger dots. (B) Maximum-likelihood phylogenetic tree analysis of the heavy chains of all sequenced monoclonal antibodies. Different colors indicate antibodies identified from individual patients. (C) Summary of the performance of the top 29 monoclonal antibodies in the indicated assays.

Figure 3

Identification and Epitope Mapping of Neutralizing Antibodies (A) Summary of the indicated characteristics of all authentic viral neutralizing antibodies. Column 1, IC₅₀ of authentic SARS-CoV-2 neutralization; column 2, epitope-mapping results (note that although 553-60 showed an identical pattern to 553-49 in the mutagenesis assay, it partially completed with 414-1, and therefore was classified as epitope C/A); column 3, IC₅₀ of pseudoviral neutralizing results; column 4, cocktail IC₅₀ of pseudoviral neutralizing results of the indicated antibodies together with 553-15 (note that 505-3 was used as representative for 515-1 and 505-5); columns 5 and 6, binding ELISA and blocking ELISA results; columns 7 and 8, FCA results of the binding and ACE2 competition to freshly expressed membrane-bound S protein (color shading in the heatmaps represents magnitude of intensities—light green, weak; darker green, strong); and columns 9 and 10, results of ELISA binding to RBD or S-ECD. Red highlights the antibodies with similar CDR3 sequences. (B) Left, neutralization results of 414-1 against authentic virus (SARS-CoV-2-SH01) using Vero-E6 (n = 2). Right, BLI examination of 414-1 and RBD binding affinity. (C) Left, positions of the 15 designed single amino acid replacements in the non-ACE2 binding surface of RBD, purple; middle-left, ACE2 binding surface, yellow; middle-right, amino acid replacements that affected epitope-B antibody 553-15 binding to RBD, green; right, amino acid replacements that affected the RBD bindings of 553-49 and 553-60, blue. Proposed epitopes A, B, and C were circled.

Figure 4

Cross Reactivity and Neutralization with SARS-CoV Cross-reactivity analysis of the indicated antibodies against S proteins of SARS-CoV and MERS-CoV. Flow cytometry analyses were performed using HEK293T cells expressing the S proteins of SARS-CoV-2, SARS-CoV, and MERS-CoV; 50 nM of the indicated antibodies were used. Non-transfected HEK293T were used as controls. (A) Heatmap summary of the results. (B) Flow cytometry analyses of the three cross-reactive antibodies. (C) Binding curves of the three cross-reactive antibodies. (D) Cross neutralization of 515-5 to pseudoviruses of SARS-CoV-2 and SARS-CoV. Data were obtained from a representative experiment containing two replicates. Data are represented as mean ± SD, and n = 2.