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SARS-CoV-2 Neutralizing Antibody

The COVID-19 pandemic has spread over the world, and effective therapeutic and prophylactic interventions are urgently needed. Human-sourced monoclonal antibodies generated by convalescent patients’ B cells are promising therapeutic candidates. However, due to VDJ recombination and somatic hypermutation, B cells exhibit diverse B-cell repertoires, and this necessitates the analysis of one B cell at a time to obtain paired immunoglobulin heavy-light chain RNA sequences for monoclonal antibodies production. Using high-throughput single-cell RNA and VDJ sequencing, we rapidly identified multiple SARS-CoV-2 neutralizing antibodies from antigen-binding B cells from convalescent COVID-19 patients (Figure 1).
Figure 1. Efficient neutralizing antibody identification through antigen-enriched high-throughput single-cell RNA sequencing. Schematic overview of the neutralizing antibody identification process. The sequence of the mAbs could be obtained within two days using 10X Genomics 5’ VDJ sequencing.
Over 8,500 antigen-binding B cell clonotypes expressing IgG1 antibodies were identified from 60 convalescent patients. In total, we identified 14 potent neutralizing mAbs, among which the most potent mAb, BD-368-2, exhibited an IC50 of 1.2 ng/mL and 15 ng/mL against pseudotyped and authentic SARS-CoV-2 (Figure 2).
Figure 2. Affinity specificity and neutralizing abilities of the potent neutralizing mAbs. A) Neutralization potency measured by using a pseudotyped virus neutralization assay. B) Neutralization potency measured by an authentic SARS-CoV-2 plaque reduction neutralizing test (PRNT) assay. C) Characteristics of the neutralizing mAbs. IC50 and IC80 were calculated by using a four-parameter logistic curve-fitting. Kd targeting RBD was measured by using SPR with a 1:1 binding model.
To evaluate whether the identified neutralizing mAbs could serve as therapeutic interventions and prophylactic protection against SARS-COV-2 in vivo, we tested the neutralization efficacy of BD-368-2 on hACE2 transgenic mice infected with SARS-CoV-2 (Figure 3A). The results showed that BD-368-2 applied both before and after the infection could greatly improve the physiological condition of the SARS-CoV-2-infected mice (Figure 3B). Moreover, we analyzed the viral load by qRT-PCR of the lungs at 5 dpi and found that injections of BD-368-2 before the viral challenge could completely inhibit SARS-CoV-2 (Figure 3C). Furthermore, applying BD-368-2 2 h after infection could result in a 3-4 log decrease in virus copies in mice lung, indicating an effective reduction of SARS-CoV-2 replication(Figure 3C). Together, BD-368-2 exhibits high therapeutic and prophylactic efficacy in vivo.
Figure 3. BD-368-2 showed high therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2 transgenic mice. A) Experimental design for therapeutic and prophylactic testing of BD-368-2 in hACE2 transgenic mice. BD-368-2 or unrelated antibody HG1K (20 mg/kg of body weight) was intraperitoneally injected into the transgenic mice before or after SAR-CoV-2 infection. B) Body weight change (%) of the hACE2 transgenic mice recorded over 5 days (one-sided permutation test, *p<0.05). Each group contains 3 mice. C) Virus titers of lung tissue at 5 dpi. The viral loads of the lung were determined by qRT-PCR (one-tailed t-test, ***p<0.001).
Additionally, the 3.8Å Cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody’s epitope overlaps with the ACE2 binding site (Figure 4).
Figure 4. Cryo-EM structure of BD23-Fab in complex with the Spike trimer. A) Cryo-EM structure of the S trimer in complex with BD23-Fab reconstructed at 3.8 Å resolution. The three protomers in the S trimer are depicted in cyan, green, and yellow, respectively. BD23-Fab is depicted in magenta (heavy chain) and blue (light chain). B) N165 glycan in the NTD of protomer C facilitates the interaction between BD23-Fab and the RBD of protomer B. C) The crystal structure of the RBD/ACE2 complex is overlaid onto the RBD/BD23-Fab structure. BD23-Fab would collide with ACE2 and therefore block the interaction between RBD and ACE2. RBD is shown in green and white, whereas ACE2 in orange.
Moreover, we demonstrated that SARS-CoV-2 neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV neutralizing antibodies (Figure 5).
Figure 5. Characteristics of the neutralizing mAbs identified based on CDR3H structural similarity to SARS-CoV neutralizing mAbs. A) The CDR3 sequence comparison between SARS-CoV neutralizing mAb m396 and the SARS-CoV-2 neutralizing mAbs identified based on CDR3H structure similarity. B) Neutralization potency measured by using a pseudotyped virus neutralization assay. C) Characteristics of the neutralizing mAbs identified based on structure similarity. The CDR3H structure prediction was performed using FREAD. D) The crystal structure of m396-Fab/SARS-CoV-RBD.
Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B-cell sequencing.The application of high-throughput single-cell sequencing methodology in this study could also be expanded to infectious diseases other than COVID-19 and serve as a technical reserve for rapid neutralizing mAbs discovery during future pandemics.