Somatic hypermutations found to enhance humoral immunity against SARS-CoV-2 variants

In a recent study posted to the Research Square* preprint server, researchers explored the molecular basis for increased immunity against heterologous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains after repeated antigen exposures.

Background

SARS-CoV-2 Omicron variant spike (S) protein contains several mutations that confer evasive immune properties. Studies have reported that repeated exposure to SARS-CoV-2 antigens by natural infection or vaccination induces robust humoral immune responses against Omicron; however, the molecular pathways underlying such immune responses have not been characterized completely.

About the study

In the present study, researchers elucidated molecular pathways responsible for enhancing humoral responses generated by repeated SARS-CoV-2 antigen exposure against heterologous SARS-CoV-2 strains.

Peripheral blood mononuclear cells (PBMCs) were obtained from coronavirus disease 2019 (COVID-19) convalescents with SARS-CoV-2 Delta strain breakthrough infections on discharge. The cluster of differentiation 19+ (CD19+) CD27+ memory B lymphocytes (MBCs) binding to the homologous Delta strain receptor-binding domain (RBD)/S protein subunit 1 (S1) were isolated by fluorescence-activated cell sorting (FACS) and were subjected to single-cell sequencing.

Antibody isotype composition and functional subtypes in B lymphocytes among the Delta breakthrough infection patients and unvaccinated COVID-19 patients were compared. Antibodies induced by Delta breakthrough infections were further investigated using neutralizing monoclonal antibodies (mAbs).

Only clonotypes that contained immunoglobulin G1 (IgG1)-expressing B lymphocytes somatic hypermutation (SHM) rates >2% were analyzed. SARS-CoV-2 authentic viruses or pseudoviruses were used to test the neutralization potential of 24 mAbs [with cross-reaction to the wildtype (WT) and ≥5 SARS-CoV-2 strains such as Alpha, Beta, Gamma, Kappa, Mu, and Lambda].

Out of 24 mAbs, 14 and four mAbs demonstrated Omicron RBD binding and cross-reactivity with SARS-CoV-1 S. In addition, biolayer interferometry (BLI) assays were performed. Further, the genetic and structural basis of the two most potent broadly neutralizing antibodies (bnAbs) viz. YB13-292 and YB9-258 broad neutralizing activities were explored. In addition, cryo-electron microscopy (EM) was performed to model the antibody-epitope interactions and understand the neutralization pathways.

Results

FACS sorting demonstrated that MBCs accounted for most (88.8%) of the B lymphocytes. Four main types of B lymphocytes were observed: plasmablasts, naïve B lymphocytes, switched MBCs, and non-switched MBCs. Of note, MBCs induced by Delta breakthrough infections generated antibodies with extensive SHMs, which enhanced bnAb activity against heterologous SARS-CoV-2 strains such as the WT, Beta, and Delta strains, including the Omicron variant.

The YB13-292 and YB9-258 and bnAbs encoded by the VH3-21 and VH3-53 genes were identified as class 2 and class 1 S RBD antibodies, respectively. The structural analysis showed that the two antibodies featured either SHM residues or an unusual SNIL insertion in the heavy chain complementarity determining region 2 (HCDR2) epitope loop, which were extensively involved in epitope recognition, widened the breadth of neutralization and enhanced resistance to RBD mutations at positions such as 452, 417, 501, and 484.

Greater high-SHM antibody (SHM ≥5) titers were observed among patients with Delta breakthrough infections (68%) compared to those in unvaccinated patients (60%) and CoV-AbDab (40%) (a collection of 3,984 SARS-CoV-2-specific mAbs). An 11% increase in the proportion of isotype-switched antibodies and two-fold higher Ig heavy constant gamma 1(IGHG1) genetic expression was observed among individuals with breakthrough infections.

Six mAbs demonstrated potent WT, Beta, and Delta neutralization with half-maximal inhibitory concentration (IC₅₀) values <0.05 mg/ml, and the YB13-281 antibody was also bound to SARS-CoV-1 S. YB13-208, and YB9-120 demonstrated high binding affinities for SARS-CoV-2 strains (including Omicron).

BLI assays showed that Delta and Omicron S mutations did not substantially affect YB13-292 and YB9-258 binding. In the cryo-EM analysis, the YB9-258 could bind with an S RBD the up and down conformation. The R1-32 and YB9-258 antibodies could simultaneously bind to S RBDs, permitting the formation of 3:3:3 YB9-258:R1-32:S protomers.

Modeling demonstrated no clashing between angiotensin-converting enzyme 2 (ACE2) and YB13-292 and on RBD binding, indicating that the antibody could inhibit SARS-CoV-2 fusion with host cells without ACE2 blockade. 

Conclusion

Overall, the study findings showed that antibodies with high SHM levels induced by breakthrough SARS-CoV-2 infections enhance humoral (antibody-mediated) immune responses against heterologous SARS-CoV-2 strains, and repeated exposure to SARS-CoV-2 antigens induces antibodies that are resistant to mutations in the SARS-CoV-2 S RBD of several SARS-CoV-2 variants.

*Important notice

Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.