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Unlocking the Secrets of SARS-CoV-2 Immunity: A Deep Dive into Serum Antibodies and B Cell Repertoires

Jan 29, 2025 | Science, Services

Project Summary

We’re excited to share our latest findings on the complex interplay between serum antibodies and B cell repertoires in individuals with SARS-CoV-2 immunity. Our study offers new insights into the development of high-affinity antibodies against emerging variants and the potential implications for Long COVID.

The ongoing global health threat posed by SARS-CoV-2 and its rapidly evolving variants has necessitated a continuous effort to develop effective monoclonal antibody therapeutics. While early antibodies showed promise, the emergence of escape variants has rendered many of them ineffective against current Omicron subvariants

To address this challenge, we employed our Alicanto technology, a proteogenomics approach that combines next-generation sequencing of immunoglobulin transcripts with mass spectrometry analysis of antibody proteins. This method allowed us to deeply characterize anti-SARS-CoV-2 serum antibodies and compare them to B cell repertoires.  For the full story, check out our preprint Serum proteomics reveals high-affinity and convergent antibodies by tracking SARS-CoV2 hybrid immunity to emerging variants of concern.

Methods:

Patient Screening

In this study, we focused on three donors:

  • Two who were vaccinated and later experienced Omicron breakthrough infections

  • One who was naturally infected with SARS-CoV-2 and subsequently vaccinated

All donors had mild symptoms, and we collected whole blood samples at least four months after their most recent boost or breakthrough infection. The whole blood was processed into plasma and B cells.

Serum antibody analysis

Total IgG was purified from plasma, followed by affinity purification using either recombinant Spike trimer or the receptor binding domain (RBD) of the Omicron (B.1.1.529) virus. Antigen-purified antibodies were analyzed by nano-LC-MS/MS.

B cell sequencing

B cells expressing Spike trimer-reactive B-cell receptors (BCRs) were isolated using magnetic beads coupled to antigen. Single B cell libraries were generated using the 10X Genomics Chromium to retain heavy and light chain pairing.

Alicanto analysis

The Alicanto software matched the mass spectra to the BCR repertoire and scored each antibody clone based on proteomic and genomic evidence. A set of 24 antibodies was selected to broadly sample each individual’s immune response.

Candidate testing

Selected antibodies were recombinantly expressed as IgG1. Each antibody was tested for binding to RBD, the N-terminal domain (NTD), or the Spike trimer of Omicron B.1.1.529 for coarse-grain epitope mapping. In addition, each antibody was tested against the RBD of three SARS-CoV-2 variants, wildtype, B1.1.529 and an Omicron variant that emerged after the study had begun. Finally, each antibody was tested for neutralization of RBD to angiotension-converting enzyme-2 (ACE2) receptor in vitro.

Results:

ELISA tables showing domain binding, variant binding, and neutralization of serum antibodies.

Summary of reactivity and neutralization of the tested candidates. Candidates are grouped by donor. The left table (green) shows coarse grain epitope mapping to two domains on the Spike protein, RBD and NTD. The center table (orange) shows reactivity to three SARS-CoV-2 variants, wild-type, Omicron B.1.1.529, and BQ.1.1. The right table (blue) shows in vitro neutralization of ACE2 binding to RBD.

Key Findings

  • High-Affinity Antibodies: We identified serum antibodies with subnanomolar affinity to the receptor-binding domain (RBD) of the Omicron subvariant B.1.1.529. Remarkably, these antibodies also showed binding activity to the more recent BQ.1.1 subvariant.

  • Convergent Immune Response: Our donors exhibited a convergent immune response, with serum antibodies and B cell sequences showing similarities to previously reported anti-SARS-CoV-2 antibodies.

  • Epitope Coverage: Recombinantly expressed antibodies from our panel demonstrated binding to both the RBD and N-terminal domain of the spike protein, suggesting broad epitope coverage.

  • Cross-Reactivity: We observed antibodies retaining binding activity to the BQ.1.1 variant, despite it emerging more than five months after our donors’ infections. This finding highlights the potential for broad protection against emerging variants.

  • Serum-B Cell Dynamics: Our analysis revealed that serum antibodies represent only a small subset of the anti-SARS-CoV-2 antibodies present in the B cell repertoire, underscoring the complex dynamics between B cells and circulating antibodies.

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