In a recent study posted to the bioRxiv* pre-print server, researchers developed a novel high-throughput method to obtain monoclonal antibodies (mAbs) from convalescent coronavirus disease 2019 (COVID-19) individuals infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ancestral strain Wuhan-Hu-1 (WA1) and the Beta or Gamma variants of concern (VOCs).

They sequenced and functionally tested these cloning-free, recombinant mAbs. It is critical to characterize the total-binding antibody repertoire, including B cell repertoires, to understand the similarities and differences in the immune responses induced by each SARS-CoV-2 VOC, and leverage the same to appraise strategies for next-generation COVID-19 booster vaccines and an effective pan-coronavirus vaccine.

Background

SARS-CoV-2 VOCs, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) have acquired enhanced transmissibility and immune evasion capabilities under selection pressure. Previous studies of the SARS-CoV-2 spike (S)-specific antibody repertoire have revealed divergence toward using specific human immunoglobulin heavy-chain-variable (V_H) genes, including IGHV3-53, IGHV3-30, IGHV3-66, and IGHV1-2.

The commonly observed convergent V(D)J rearrangements, also called public clones, have been identified in many individuals; likewise, researchers have identified several neutralizing antibody (nAb) classes derived from the same genetic elements.

Although some nAbs can maintain potency through specific mutations that alter their binding conformation, many contact the variable K417 and E484 amino acid residues and lose their neutralizing potential against the VOCs. However, nAbs constitute a minority of the total-binding antibody repertoire, which researchers have not comprehensively examined in the VOC infections compared to WA1.

About the study

In the present study, researchers collected serum or peripheral blood mononuclear cells (PBMC) from WA1-, Beta-, or Gamma-infected individuals, 17-38 days after the symptom onset to compare the elicited antibody and B cell responses.

They utilized a Meso Scale Discovery electrochemiluminescence immunoassay (MSD-ECLIA) to measure the serum binding titers to the stabilized S trimer (S-2P) of the WA1, Alpha, Beta, Gamma, and Delta variants and the receptor-binding domain (RBD) of all the other variants except Delta.

The team used a surface plasmon resonance (SPR)-based competition assay to characterize the epitopes targeted by serum antibodies. Further, they individually blocked specific RBD epitopes on S-2P using structurally validated mAbs and measured the residual polyclonal serum binding activity compared to the unblocked trimer.

The team selected three individuals with the highest binding antibody titers for an in-depth characterization of the antibody repertoire and the identification of the mAb-binding patterns. Of these, two individuals (SAV1 and SAV3) were Beta-infected, and the third (A49) was Gamma-infected. They used S-2P, RBD, or N-terminal domain (NTD) probes to sort cross-reactive WA1*Beta*B cells from SAV1, SAV3, and A49 for the analysis.

The researchers used the rapid assembly, transfection, and production of immunoglobulins (RAPT-Ig) method for the high-throughput discovery of mAbs from single-sorted B cells. They screened the RATP-Ig supernatants by an enzyme-linked immunosorbent assay (ELISA) for binding to the S, RBD, and NTD-derived from the WA1, Beta, and Gamma variants.

In all, they recovered the paired heavy and light chain sequences from 70% of cells, and in 47% of wells with a single sorted B cell, they produced at least one antigen binding to IgG.

Study findings

MSD-ECLIA results showed that all convalescent individuals had antibodies against the homologous S and cross-reactive antibodies against the S from other variants. Individuals with the highest serum binding titers (SAV1, SAV3, and A49) could cross-neutralize the WA1, Beta, Gamma, and Delta VOC, albeit with lower potency. Moreover, they yielded high levels of cross-reactive antibodies to the S, NTD, and RBD.

Against the homologous S, SPR revealed a pattern of binding pattern/reactivity comparable between individuals infected either with WA1 or Beta, suggesting a similar immunodominance hierarchy across all the SARS-CoV-2 variants. Likewise, when mapped against the S of WA1, Beta, or Delta, there was no competition at the epitope level in sera from the Beta- or Gamma-infected individuals. However, one WA1-infected individual produced sufficiently high binding titers against the variant S to enable epitope mapping by competition.

The cluster of differentiation 4 (CD4) and CD8 T cell responses to WA1 S were similar in the Beta- and Gamma-infected individuals compared to the WA1-infected individuals. Overall, the RAPT-Ig method reliably predicted mAb functionality, and 94% of functional interactions were reproducible. Notably, two antibodies, SAV1-109.1 and SAV1-168.1, targeted the epitope of mAb CR3022 on the RBD to produce broadly-reactive nAbs against several sarbecoviruses.

The authors also observed an unanticipated excess of somatic hypermutations (SHM) in the B cells isolated from Beta-infected individuals, raising the possibility that the Beta VOC is distinct from other SARS-CoV-2 variants in inducing nAbs that wane slowly but are boostable by additional vaccine doses.

Conclusions

The study data revealed distinct convergence that defined multiple aspects of the humoral immune response to different SARS-CoV-2 variants. The convergent V_H gene usage and epitope specificities elicited by the primary exposure to SARS-CoV-2 partially rationalize why the key elements for protection against all the SARS-CoV-2 variants are likely to remain the same.

Therefore, despite the emergence of immune-evading variants limiting the impact of nAbs, first-generation vaccines using the WA1 S protein can generate cross-reactive immune responses against novel SARS-CoV-2 variants. Overall, the study highlights that the human immune system can consistently combat all the SARS-CoV-2 variants.

*Important notice

bioRxiv 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

Neha Mathur

Neha is a digital marketing professional based in Gurugram, India. She has a Master’s degree from the University of Rajasthan with a specialization in Biotechnology in 2008. She has experience in pre-clinical research as part of her research project in The Department of Toxicology at the prestigious Central Drug Research Institute (CDRI), Lucknow, India. She also holds a certification in C++ programming.