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. 2024 Mar 23;13(4):272.
doi: 10.3390/pathogens13040272.

Identification of an IGHV3-53-Encoded RBD-Targeting Cross-Neutralizing Antibody from an Early COVID-19 Convalescent

Yuanyuan Hu  1   2 , Caiqin Hu  3 , Shuo Wang  2 , Li Ren  2 , Yanling Hao  2 , Zheng Wang  2 , Ying Liu  2 , Junwei Su  3 , Biao Zhu  3 , Dan Li  2 , Yiming Shao  2   3 , Hao Liang  1
Affiliations

Identification of an IGHV3-53-Encoded RBD-Targeting Cross-Neutralizing Antibody from an Early COVID-19 Convalescent

Yuanyuan Hu et al. Pathogens. .

Abstract

Since November 2021, Omicron has emerged as the dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, and its sublineages continue to appear one after another, significantly reducing the effectiveness of existing therapeutic neutralizing antibodies (NAbs). It is urgent to develop effective NAbs against circulating Omicron variants. Here, we isolated receptor binding domain (RBD)-specific single memory B cells via flow cytometry from a COVID-19 convalescent. The antibody variable region genes of the heavy chain (VHs) and light chain (VLs) were amplified and cloned into expression vectors. After antibody expression, ELISA screening and neutralizing activity detection, we obtained an IGHV3-53-encoded RBD-targeting cross-neutralizing antibody D6, whose VL originated from the IGKV1-9*01 germlines. D6 could potently neutralize circulating Omicron variants (BA.1, BA.2, BA.4/5 and BF.7), with IC50 values of less than 0.04 μg/mL, and the neutralizing ability against XBB was reduced but still effective. The KD values of D6 binding with RBD of the prototype and BA.1 were both less than 1.0 × 10-12 M. The protein structure of the D6-RBD model indicates that D6 interacts with the RBD external subdomain and belongs to the RBD-1 community. The sufficient contact and deep interaction of D6 HCDR3 and LCDR3 with RBD may be the crucial reason for its cross-neutralizing activity. The sorting and analysis of mAb D6 will provide important information for the development of anti-COVID-19 reagents.

Keywords: Omicron subvariants; RBD; SARS-CoV-2; neutralizing antibody.

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Conflict of interest statement

No potential conflicts of interest are reported by the authors.

Figures

Figure 1
Figure 1
Flow cytometry graph. Single SARS-CoV-2 RBD-specific B cells were sorted using a FACS Aria SORP. One hundred and fourteen memory B cells which bound with RBD-PE were sorted into a 96-well plate containing lysis buffer. The colors represent cell concentration, and the cell concentration decreases from red, yellow, green, to blue.
Figure 2
Figure 2
Sequence characteristics of antibodies isolated from donor YYQ. The gene family usages of VH (A) and VL (B) for the 57 antibodies. (C) Rates of nucleotide substitutions in VH and VL for the 57 antibodies. (D) The amino acid lengths of the CDR3 loop of VH and VL for the 57 antibodies.
Figure 3
Figure 3
Amino acid sequence comparisons of the mAbs heavy chain and light chain with alignment to their respective germline chain. The framework region (FR) and complementarity determining region (CDR) were determined using the IMGT/V-QUEST program. The symbol ‘.’ denotes the same amino acid. Different colors represent different amino acids, which are automatically generated by Bioedit 7.0.5.3 software.
Figure 4
Figure 4
Binding activity and binding kinetics of D6 with SARS−CoV−2 RBD. Binding activity of D6 with RBD of the prototype (A), Delta (B) and BA.2 (C) were measured by ELISA experiments. Binding kinetics of D6 with RBD of the prototype (D) and BA.1 (E) were measured by BLI. Denatured mAb D6 was detected by PAGE electrophoresis (F).
Figure 5
Figure 5
Neutralization activities of D6 against SARS−CoV−2 variants pseudoviruses, including the prototype (A), BA.1 (B), BA.2 (C), BA.4/5 (D), BF.7 (E), XBB (F) and EG.5 (G). Heat map of neutralization activities (IC50 values) of D6 against SARS-CoV-2 variant pseudoviruses (H).
Figure 6
Figure 6
Neutralization activities of D6 against authentic SARS−CoV−2 variants, including the prototype (A), BA.1 (B), BA.5 (C). Heat map of neutralization activities (IC50 values) of D6 against authentic SARS-CoV-2 variants (D).
Figure 7
Figure 7
The structure of D6 and CC12.1 binding to SARS-CoV-2 RBD. (A) Diagram comparing the structures of D6 Fab and CC12.1 Fab. D6 Fab has a high structural similarity to CC12.1 Fab. The D6 heavy chain is shown in dark red, the light chain in blue. The CC12.1 heavy chain is shown in cyan, the light chain in magentas. (B) The structure of D6 Fab-RBD complex. The D6 heavy chain is shown in dark red, the light chain in blue. RBD is coloured in green. (C) The structure of D6 Fab is superimposed onto the CC12.1-RBD complex (PDB ID: 6XC2) by PyMOL2.4.1 software, with the CC12.1 structure as the reference. The RBD is coloured in green. (D) The CDRs of D6 and CC12.1 are displayed when bound to RBD. RBD is shown in green spheres. The HCDRs and LCDRs of D6 are shown in dark red and blue, respectively. The HCDRs and LCDRs of CC12.1 are shown in cyan and magenta, respectively.
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