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. 2023 Jan 10;56(1):193-206.e7.
doi: 10.1016/j.immuni.2022.12.005. Epub 2022 Dec 12.

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies

Pradeepa Pushparaj  1 , Andrea Nicoletto  1 , Daniel J Sheward  1 , Hrishikesh Das  2 , Xaquin Castro Dopico  1 , Laura Perez Vidakovics  1 , Leo Hanke  1 , Mark Chernyshev  1 , Sanjana Narang  1 , Sungyong Kim  1 , Julian Fischbach  1 , Simon Ekström  3 , Gerald McInerney  1 , B Martin Hällberg  2 , Ben Murrell  1 , Martin Corcoran  1 , Gunilla B Karlsson Hedestam  4
Affiliations

Immunoglobulin germline gene polymorphisms influence the function of SARS-CoV-2 neutralizing antibodies

Pradeepa Pushparaj et al. Immunity. .

Abstract

The human immunoglobulin heavy-chain (IGH) locus is exceptionally polymorphic, with high levels of allelic and structural variation. Thus, germline IGH genotypes are personal, which may influence responses to infection and vaccination. For an improved understanding of inter-individual differences in antibody responses, we isolated SARS-CoV-2 spike-specific monoclonal antibodies from convalescent health care workers, focusing on the IGHV1-69 gene, which has the highest level of allelic variation of all IGHV genes. The IGHV1-69∗20-using CAB-I47 antibody and two similar antibodies isolated from an independent donor were critically dependent on allele usage. Neutralization was retained when reverting the V region to the germline IGHV1-69∗20 allele but lost when reverting to other IGHV1-69 alleles. Structural data confirmed that two germline-encoded polymorphisms, R50 and F55, in the IGHV1-69 gene were required for high-affinity receptor-binding domain interaction. These results demonstrate that polymorphisms in IGH genes can influence the function of SARS-CoV-2 neutralizing antibodies.

Keywords: SARS-CoV-2; copy-number variation; immunoglobulin allelic diversity; neutralizing antibody; single-nucleotide polymorphism.

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Conflict of interest statement

Declaration of interests M. Corcoran and G.B.K.H. are founders of ImmuneDiscover Sweden.

Figures

None
Graphical abstract
Figure 1
Figure 1
Study design and demonstration of high IGHV1-69 diversity among the donors (A) Schematic representation of the sampling of 14 SARS-CoV-2 RT-PCR+ study participants followed by IGH genotyping and memory B cell sorting 7 months after the infection. (B) Spike-specific IgG in serum samples from the 14 study participants. (C) Serum ID50 neutralization values against the ancestral virus in the serum samples. (D) Amino acid sequence alignment of functional human IGHV1-69 alleles. (E) A summary plot showing which IGHV1-69 alleles were present in each study participant. (F) Schematic illustration of IGHV1-69 alleles present on each chromosome of eight study participants. Neutralization measurements were repeated twice. See also Figure S1.
Figure 2
Figure 2
Spike-specific memory B cell sorting yields several IGHV1-69∗20-using neutralizing mAbs (A) Dendrogram showing IGHV, IGKV, and IGLV alleles present in SP14. (B) Panels showing the gating strategy used to sort single S-specific CD20+CD27+IgG+ B cells. (C) Comparison of IGHV allele usage in the total and spike-specific IgG repertoires of SP14. (D) Number of HC sequences from sorted B cells and clonal lineages derived from the total HC sequences. (E) Pie chart showing the proportion of neutralizing (blue) and non-neutralizing (orange) antibodies among the 29 isolated mAbs. (F) Genetic and functional properties of the 15 neutralizing mAbs isolated from SP14. Neutralization measurements were repeated twice. See also Figure S2.
Figure 3
Figure 3
IGHV1-69 allele usage influences CAB-I47 neutralizing activity (A) Amino acid sequence alignment of the IGHV1-69∗20 germline allele with the V gene region of CAB-I47. (B) The design of CAB-I47 germline-reverted HC sequences and swaps to the IGHV1-69 germline alleles ∗20, ∗01, ∗02, ∗04, ∗06, or ∗10, which were paired with the mature CAB-I47 LC for functional testing (left). Neutralizing activity of the variant mAbs against the ancestral SARS-CoV-2 strain is shown as curves (middle) and IC50 values (μg/mL) (right). (C) Design of the mature CAB-I47 with the HCDR2 F55L substitution, paired with the CAB-I47 mature LC (top panel). Neutralization activity of CAB-I47 and CAB-I47 F55L against the ancestral SARS-CoV-2 strain (bottom left) and IC50 values (μg/mL) (bottom right). Neutralization measurements were repeated twice. See also Figure S3.
Figure 4
Figure 4
IGHV1-69∗20-using neutralizing mAbs isolated in an independent donor (A) Dendrogram showing IGHV alleles present in SP13. (B) Serum ID50 neutralization values against ancestral SARS-CoV-2 at pre- and post-vaccination time points. (C) Panels showing the gating strategy used to isolate single spike-specific CD20+CD27+ IgG B cells. (D) Comparison of IGHV allele usage in the total and spike-specific IgG repertoire of SP13. (E) Pie chart showing the number of HCs of the total IGHV1-69-using HCs using a given IGHV1-69 allele from the sorted B cells. (F) Genetic and functional properties of the selected neutralizing mAbs. (G) Neutralization of the ancestral SARS-CoV-2 strain by the mature CAB-M77 and CAB-N86 mAbs with and without the F55L mutation. (H) Neutralizing activity by CAB-M77 and CAB-N86 with their HC V gene regions reverted to the IGHV1-69 germline alleles ∗20, ∗01, ∗02, ∗04, ∗06, or ∗10, tested against the ancestral SARS-CoV-2 strain. Neutralization measurements were repeated twice. See also Figure S4.
Figure 5
Figure 5
Cryo-EM analysis of the spike-CAB-I47 Fab complex reveals its binding mode (A) Cryo-EM reconstruction of the spike-CAB-I47 Fab complex in 1-up conformation, three CAB-I47 Fabs occupy the RBDs (left). The cryo-EM map (gray transparent) overlayed with atomic model, trimeric spike with three the CAB-I47 Fab variable domains (middle). Atomic model of RBD- the CAB-I47 Fab (variable domains) with the HCDR1, HCDR2, and HCDR3 color-coded (right). (B) Molecular details of the CAB-I47 Fab and RBD interaction with residues important for the interaction labeled. (C) The IGHV1-69∗20 F55 residue (shown in red) located in a hydrophobic pocket of the RBD. The RBD is shown in surface representation and colored according to relative hydrophobicity with various RBD residues in the binding area are labeled. See also Figure S5.
Figure 6
Figure 6
Substitution of the germline-encoded R50 residue abolishes the activity of CAB-I47 (A) Cartoon and stick representation of the IGHV1-69∗20 R50 residue interactions with G482 and E484 on the RBD, color coding as in Figure 5 (left). Neutralization of the ancestral SARS-CoV-2 strain by the mature CAB-I47 and versions containing the F55L mutation, the R50G mutation or both, and ELISA binding by the same antibodies against RBD (right). (B) Summary of variant amino acid positions in different IGHV1-69 alleles with the presence of R50 and F55 highlighted in red. Gray background indicates alleles that were evaluated in this study. Neutralization measurements were repeated twice. See also Figure S6.
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