A Rapid and Efficient Screening System for Neutralizing Antibodies and Its Application for SARS-CoV-2

Abstract

After the pandemic of COVID-19, neutralizing antibodies (NAbs) against SARS-CoV-2 have been developed for the prophylactic and therapeutic purposes. However, few methodologies are described in detail on how to rapidly and efficiently generate effective NAbs to SARS-CoV-2. Here, we integrated and optimized a strategically screening method for NAbs, which has enabled us to obtain SARS-CoV-2 receptor-binding domain (RBD) specific NAbs within 6 days, followed by additional 9 days for antibody production and function analysis. Using this method, we obtained 198 specific Abs against SARS-CoV-2 RBD from the blood samples of COVID-19 convalescent patients, and 96 of them showed neutralizing activity. At least 20% of these NAbs exhibited advanced neutralizing potency and high affinity, with the top two NAbs showing half-maximal inhibitory concentration (IC₅₀) to block authentic SARS-CoV-2 at 9.88 and 11.13 ng/ml, respectively. Altogether, our study provides an effective methodology with high applicable value for discovering potential preventative and therapeutic NAbs for the emerging infectious diseases.

Keywords: SARS-CoV-2; methodology; neutralizing antibodies; receptor-binding domain (RBD); spike protein.

Figure 1

Schematic overview of the rapid and efficient NAbs screening system. Rapid NAbs screening workflows and timelines are shown, representing the multiple processes conducted in order. The PBMC were isolated from collected convalescent patients’ blood, and the RBD-specific memory B cells in the PBMC were sorted as single cells via flow cytometry (day 1). Then, the IgG heavy and light chains of mAb genes were amplified by RT-PCR on the same day. 2^nd PCR products were cloned into the linear expression cassettes (day 2). HEK293T cells expressed antibodies by transient transfection with equal amounts of paired heavy and light chain linear expression cassettes and culture for 48 hours. The supernatants were used to detect the antigen reactivity of antibodies by ELISA in 384-well plates (day 4). The neutralizing activities of antibodies were measured with pseudovirus bearing SARS-CoV-2 S in 96-well plates (day 6). Plasmids expressing potential neutralizing Abs were transfected into Exi293F cells for the large-scale production of Ab proteins. The cell supernatants of Exi293F cells were collected, and the antibody proteins were purified by protein G beads. Antigen reactivity, neutralizing activity, and binding affinity were further accessed via ELISA, competitive ELISA, and surface plasmon resonance (SPR). The figure was created with Biorender.com.

Figure 2

Analyses of plasma response to SARS-CoV-2 and the RBD-specific memory B cells. (A) The heatmap depicts the specificity of convalescent patients’ plasma against S1 or RBD of SARS-CoV-2, SARS-CoV, and MERS-CoV, with serial dilutions, measured by ELISA. The plasma of healthy donors was used as a negative control. All results were derived from at least two independent experiments. (B) Gating strategy for SARS-CoV-2 RBD-specific IgG⁺ mB cells in the PBMC of COVID-19 convalescent patients. Living CD19⁺IgD^-IgG⁺ cells were gated, and B cells binding SARS-CoV-2 RBD were selected for single-cell sorting. (C) FACS analysis of RBD-specific memory B cells in CD19⁺IgD^-IgG⁺ mB cells from the PBMC of three batches of convalescent patient samples. Plots show CD19⁺IgD^-IgG⁺ populations using the gating strategy described in (B).

Figure 3

Identification of RBD specific monoclonal antibodies from convalescent COVID-19 patients. (A) Screening of SARS-CoV-2 potential neutralizing Abs. The heatmap reveals that the binding ability of 198 Ab supernatants produced by HEK239T cells transfected with linear Ab gene expression cassette. The binding activity of mAbs against SARS-CoV-2 S1 and RBD were tested by ELISA. mAbs were ranked as the binding ability of RBD in each batch, and the brightness of blue represents the binding ability.The OD was measured at 405 nm. The neutralizing activity of mAbs was discriminated according to the neutralizing value. The neutralizing capability was identified by SARS-CoV-2 pseudovirus neutralization assay. The Green columns represent potential neutralizing Abs with the inhibition rate >75%, while white indicate the other Abs. The mAbs were ranked as same as the above. For each evaluated antibody, at least two independent measurements were performed. (B) Frequencies of variable region of the heavy chain (VH) gene clusters for potential neutralizing and non-neutralizing antibodies. Clonal sequences groups were collapsed and treated as one sample for calculation of the frequencies. (C) Frequency of various the heavy chain complementarity determining region 3 (CDRH3) length of in potential neutralizing and non-neutralizing antibodies.

Figure 4

Functional characterization of NAbs against authentic SARS-CoV-2. (A) The neutralization activities of mAbs against authentic SARS-CoV-2 virus (nCoV-SH01), analyzed by the Cytopathic effects (CPE) test. Serial dilutions of each mAbs were tested, initial concentration is 0.75 mg/ml. CPE results was summarized, where “-” indicates no cytopathy, “+” indicates <25% cytopathy, “++” indicates 25-50% cytopathy, “+++” indicates 50-75% cytopathy, and “++++” indicates 100% cytopathy. 13G9 was marked with “*” to indicate that it was obtained by the method previously described (28). (B) IC₅₀ of 58G6 and 510A5 against the authentic SARS-CoV-2 virus, determined in Vero-E6 cells by RT-qPCR. Dashed lines indicated a 50% inhibition rate of viral infection. Data for each NAb were obtained from a representative neutralization experiment, with three replicates. Data are presented as mean ± SEM.

Figure 5

The binding kinetics of the top 20 NAbs with SARS-CoV-2 S-RBD measured by SPR. The purified S-RBD NAbs were captured on the CM5 chip followed by the injection of various concentrations of soluble SARS-CoV-2 S-RBD. In terms of assessment of ACE2 affinity, S-RBD was coated on the CM5 chip. The various concentrations of ACE2 were injected to the coated chip. The color lines represent experimentally derived curves, while the black lines represent fitted curves based on the experimental data. Data are representative of at least 2 independent experiments.