Polymorphisms in human immunoglobulin heavy chain genes may influence SARS-CoV-2 neutralizing antibody function

In a recent study published in Immunityresearchers isolated mAbs (monoclonal antibodies) targeted to SARS-CoV-2 spike (S) proteins in convalescent healthcare workers (HCWs), with a focus on the IGHV1-69 gene (heavy variable immunoglobulin 1-69), the gene with the greatest structure and allelic variation.

Study: Immunoglobulin germline gene polymorphisms influence SARS-CoV-2 neutralizing antibody function.  Image Credit: ktsdesign/Shutterstock
Study: Immunoglobulin germline gene polymorphisms influence SARS-CoV-2 neutralizing antibody function. Image Credit: ktsdesign/Shutterstock


The human immunoglobulin (Ig) heavy chain (HC) locus exhibits remarkable polymorphisms with a high degree of structural and allelic variation. Personal germline IGH genotypes may alter responses to vaccination and infection. However, data on allele-specific responses are limited and further research is needed to further the understanding of interindividual differences in Ab responses.

About the study

In the present study, researchers performed immunoglobulin genotyping of healthcare workers infected with anterior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to detect structural and allelic variations, particularly those affecting the ‘IGHV 1-69.

An individual with many anti-S memory B cells and high IGHV composition was chosen, from which 29 anti-SARS-CoV-2 mAbs were isolated. The effects of using IGHV were assessed by returning a robust IGHV1-69*20 using a neutralizing antibody, CAB-I47, to sequences of the IGHV1-69*20 allele and five other IGHV1-69 alleles . High-resolution cryo-electron microscopy (cryo-EM) was performed to assess the molecular interactions of the Ab-SARS-CoV-2 S receptor binding domain (RBD).

The study cohort included 14 healthcare workers (SP01 to SP14), who tested positive for SARS-CoV-2 by reverse transcription-polymerase chain reaction (RT-PCR) analysis in May 2020 for assess memory B cell responses and the evolution of anti-S antibodies. Sera and peripheral blood mononuclear cells (PBMC) were obtained 7.0 months after SARS-CoV-2 infection. Study participants achieved anti-S IgG titers and neutralized SARS-CoV-2. To assess participants’ IGHVV genotypes, IgM libraries were expressed, mass sequenced, and analyzed.

Additionally, haplotype analysis was performed to identify the alleles present on the participants’ chromosomes. SP14 (which possessed 3.0 alleles of the IGHV1-69 gene IGHV1-69*01, 02, *20) was selected for further analysis, for which the V alleles of lambda and kappa light chains, IGLV and IGKV, were selected for further analysis. been determined. IgG+ anti-S memory B cells were sorted for Ab sequence analysis.

In addition, PMBC ribonucleic acid (RNA) sequencing was performed and Ab V(D)J sites of sorted cells were amplified by RT-PCR. Sequence alignment of IGHV1-69 alleles was assessed and mAb CAB-147 (which used IGHV1-69*20) was chosen to assess the role of IGHV1-69 allele variations.

In addition, the V gene site was shifted to the IGHV1-69*20 configuration to assess the contribution of somatic hypermutations (SHMs) to CAB-I47 neutralization capacity. Structural analysis was performed and binding affinities for CAB-I47, CAB-I47gL*20, CAB-I47gL*04 and CAB-I47 F55L Fabs (fragment-antigen binding) were assessed by surface plasmon resonance analysis ( SPR).

Germline-reverted (gL) HCs were co-expressed with mature CAB-N86 and CAB-M77 LCs, and their neutralizing potencies were determined. In addition, HDX-MS (hydrogen-deuterium exchange mass spectrometry) was performed to assess the specificities of CAB-M77 and CAB-I47 targets.


CAB-I47 Ab was critically dependent on allele usage, and SARS-CoV-2 neutralization was maintained upon reverting from the variable site (V) to the IGHV1-69*20 allele, but a was lost when reverting to other IGHV1-69 alleles. Structural analysis results showed that germline-encoded genetic polymorphisms F55 and R50 in IGHV1-69 were essential for high binding affinity S RBD-Ab interactions. The mAbs CAB-I12, CAB-M77, CAB-I47, CAB-J39 and CAB-N86 potently neutralized SARS-CoV-2 using the IGHV1-69*20 allele, considered essential for Ab function .

Six alleles were detected in the participants, which were IGHV1-69 *01, *02, *04, *06, *09 and *20. Participants SP-01, -02, -06, -08 and -11 showed IGHJ heterozygosity (IGHJ6*02, -03), SP14 showed IGHD2-21 heterozygosity (IGHD2-21*01,*02) and SP-04.05 showed IGHD3-10 heterozygosity. The results of haplotype analysis confirmed high structural and allelic variations in IGHV1-69. In the study, heavy chain sequences (n=177) and paired heavy and light chain sequences (n=146) were identified.

A highly polyclonal anti-S response was observed, with 171 distinct clones from 177 HC sequences analyzed. Many mAbs used IGHV3-53 and included small HCDR3 (HC 3 complementarity determining region), indicative of class 1-like anti-SARS-CoV-2 Abs, and CAB-52, a very potent mAb neutralizing SARS-CoV-2. CoV-2, used IGHV3-30-3*01. Most of the potent SARS-CoV-2 neutralizing mAbs used the IGHV1-69*02, *20 alleles.

The average SHMs to neutralize mAbs were 10% and 6.0%, and 10% in amino acid sequence and nucleotide (nt) sequence, respectively, and the J gene lacked SHMs. SP14 induced IGHV1-69 using potent neutralizing mAbs. The neutralization potential of the ancestral SARS-CoV-2 strain of CAB-I47gL*20 (variable version of the gL gene CAB-I47 mAb) was 6.0 times lower than that of other Abs.

The gL allele-swap Abs lost their ability to neutralize SARS-CoV-2, with the exception of mAb CAB-I47gL*04. Neutralization of CAB-I47 F55L was more than 10-fold lower than mAb CAB-I47. Binding affinity was significantly lower for the CAB-147-antigen mAb fragment binding region than for CAB-147gL*20. The antigen-binding region of the CAB-I47 fragment F55L showed activation rates comparable to those of CAB-I47, but rapid deactivation rates and the antigen-binding region of the CAB-I47gL fragment* 04 showed very low binding affinity to S RBD.

From the CoV-Ab database, the team identified the neutralizing antibodies IGHV1-69-using, BD56-031, BD57-005, REGN1097740, XGv-23239, C1210, C121142 and C14646P3S41, which used IGHV1-69 *20. CAB-I47 Fab was linked to the upper RBD region and belonged to class 2 anti-RBD Abs. CAB-I47, CAB-M77 and CAB-N86 were susceptible to Zeta mutation (ancestral strain D614G including E484K). CAB-I47 Fab blocked angiotensin-converting enzyme 2 (ACE2) binding due to steric conflicts. The light chain was also involved. mAbs CV1206, CV118237 and BG1-2438 used the IGHV1-69*20 allele and were susceptible to the Zeta mutation.


Overall, the study results showed that single IGH gene polymorphisms may impact SARS-CoV-2 neutralizing Ab function.

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