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. 2023 Mar 1:3:1128253.
doi: 10.3389/fviro.2023.1128253.

Frequent use of IGHV3-30-3 in SARS-CoV-2 neutralizing antibody responses

Pradeepa Pushparaj  1 , Andrea Nicoletto  1 , Xaquin Castro Dopico  1 , Daniel J Sheward  1 , Sungyong Kim  1 , Simon Ekström  2 , Ben Murrell  1 , Martin Corcoran  1 , Gunilla B Karlsson Hedestam  1
Affiliations

Frequent use of IGHV3-30-3 in SARS-CoV-2 neutralizing antibody responses

Pradeepa Pushparaj et al. Front Virol. .

Abstract

The antibody response to SARS-CoV-2 shows biased immunoglobulin heavy chain variable (IGHV) gene usage, allowing definition of genetic signatures for some classes of neutralizing antibodies. We investigated IGHV gene usage frequencies by sorting spike-specific single memory B cells from individuals infected with SARS-CoV-2 early in the pandemic. From two study participants and 703 spikespecific B cells, the most used genes were IGHV1-69, IGHV3-30-3, and IGHV3-30. Here, we focused on the IGHV3-30 group of genes and an IGHV3-30-3-using ultrapotent neutralizing monoclonal antibody, CAB-F52, which displayed broad neutralizing activity also in its germline-reverted form. IGHV3-30-3 is encoded by a region of the IGH locus that is highly variable at both the allelic and structural levels. Using personalized IG genotyping, we found that 4 of 14 study participants lacked the IGHV3-30-3 gene on both chromosomes, raising the question if other, highly similar IGHV genes could substitute for IGHV3-30-3 in persons lacking this gene. In the context of CAB-F52, we found that none of the tested IGHV3-33 alleles, but several IGHV3-30 alleles could substitute for IGHV3-30-3, suggesting functional redundancy between the highly homologous IGHV3-30 and IGHV3-30-3 genes for this antibody.

Keywords: SARS-CoV-2; allelic diversity; antibodies; copy number variation; immunoglobulin germline genes.

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Conflict of interest statement

Conflict of interest MC and GKH are founders of ImmuneDiscover Sweden. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1. Spike HCs frequently use IGHV3-30 group of genes.
(A) A schematic representation of the SARS-CoV-2 cohort consisting of 14 individuals. PBMCs, sera and RNA samples were isolated from each of these individuals. The genotyping of 14 study participants was done using the RNA sample. Two of the 14 individuals’ PBMCs were used to isolated S-specific HCs and subsequent antibody expression. (B) A bar plot showing the IGHV gene usage of S-specific HCs with IGHV3-30 group highlighted in olive green. (C) Titration curves showing percent neutralization of CAB-F52 and REGN10987 against VoCs. (D) IC50 neutralization values of CAB-F52 and REGN10987 against the VoCs. (E) A table showing the genetic properties, specificity, and neutralization activity of CAB-F52 and several IGHV3-30-3-using mAbs from the CoVAbDab.
Figure 2
Figure 2. CAB-F52 targets RBD and retains neutralization activity upon germline reversion
(A) Epitope mapping of CAB-F52 using HDX-MS. (A) A heat map showing the most probable (blue) and least probable (red) interaction sites of CAB-F52 on the RBD (B) A three-dimensional image of the targeted epitope (C) Amino acid sequence alignment of CAB-F52 VH region and germline allele IGHV3-30-3*01 allele. (D) Cartoon showing the gLCAB-F52 HC design (E) Titration curves showing percent neutralization of CAB-F52 and gLCAB-F52 against VoCs. (F) IC50 neutralization values of CAB-F52 and gLCAB-F52 against several VoCs.
Figure 3
Figure 3. Haplotype and genotype analysis reveals variation and IGHV3-30-3 deletion
(A) Illustration of IGHV3-30, IGHV3-30-3, IGHV3-33 alleles presence on each chromosome of the haplotypable study participants. (B) A pie chart showing frequencies of individuals with the IGHV3-30-3 gene present (green) or absent (gray). (C) Combined IGHV3-30/IGHV3-30-3 frequencies in IgM libraries of individuals with the IGHV3-30-3 present (green) or absent (gray). ** refers to the significant difference between the groups indicated by p value ≤ 0.01.
Figure 4
Figure 4. IGHV3-30 group of genes are highly variable and can impact CAB-F52 function.
(A) Alignment of all the amino acid sequences of IGHV3-30, IGHV3-30-3 and IGHV3-33 alleles present in the extended group of 46 cases. (B) Allelic content in the 46 cases: IGHV3-30 (top graph), IGHV3-30-3 (middle graph) and IGHV3-33 (lower graph). The IGHV3-30*04/IGHV3-30-3*03 (dark green) is shown in both the IGHV3-30-3 and IGHV3-30 plots since they cannot be distinguished (C) Cartoons showing the design of the germline-reverted CAB-F52 HC variants. (D) Titration curves showing percentage neutralization of the allele-swapped CAB-F52 mAb variants. (E) IC50 neutralization values of the allele-swapped CAB-F52 mAb variants.
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