Lack of SARS-CoV-2 Neutralizing Antibodies Discovered in Single-Cell Antibody Study
Summary
Their study revealed that the diversity of antibody sequences in RenMab mice, coupled with the consistency in B-cell immune responses between RenMab mice and humans, enabled the screening of fully human virus-neutralizing antibodies. After identifying 215 clonotypes with binding affinities primarily targeting S2, only two of them exhibited neutralizing activity against SARS-CoV-2. Furthermore, 5' single-cell RNA sequencing showed that the sorted splenic B cells consisted mostly of plasmablasts, germinal center-dependent memory B-cells, and GC B-cells, among which plasmablasts and GC-dependent memory B-cells were deemed the most plausible sources of virus-specific antibodies. Their high-throughput single cell-based antibody discovery approach highlighted the challenges involved in developing S2-binding neutralizing antibodies against SARS-CoV-2 and also suggested a novel direction for the enrichment of antigen-specific B-cells.
Research Criteria
Amidst the ongoing coronavirus disease 2019 pandemic, the pressing need for effective antibody-based therapies persists. The high degree of similarity between the S2 subunit of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-CoV S proteins propelled researchers to investigate cross-neutralizing antibodies targeting S2, an uncharted territory. Consequently, RenMab mice were inoculated with the full-length S protein to establish a high-throughput antibody discovery method based on single-cell sequencing technology, aiming at extracting SARS-CoV-2 S-targeted neutralizing antibodies and cross-neutralizing antibodies targeting the S2 region of SARS-CoV-2/SARS-CoV S.
Fig.1 Schematic overview of each step in the study. (Chai, 2022)
Sample Type
B cells from mouse spleen.
Result—Identification of Sorted Splenic B Cells by scRNA-seq
To understand which cell subtypes are more likely to produce antigen-reactive antibodies, they determined the expression profile of sorted splenic B cells using scRNA-seq. 3267 cells from two projects were obtained after quality control. These cells were classified into 5 clusters. Notably, cells from both projects exhibited similar global cell composition, although the absolute number of cells in each cell cluster of BBGTG3 was different from that in the BBCTG6 project, indicating that activated B cells with specific immune responses to SARS-CoV-2 were consistent with those obtained from the mouse model that cross-immunized with SARS-CoV-2 and SARS-CoV.
Fig.2 Analysis of single cell transcriptome data in RenMab mice. (Chai, 2022)
Result—Comparison of Antibody Sequences in Different Cell Clusters
They hypothesized that GC-dependent memory B-cells, GC B-cells, and plasmablasts were the primary cell clusters to produce antibodies based on the cell types they clustered from the scRNA-seq data. By combining analysis of scRNA-seq and scBCR-seq data, they indicated that plasmablasts and GC-dependent memory B-cells have higher probabilities of producing antigen-specific antibodies than B-cells, and surface marker Sdc1 combined with Cd38 could be used for more effective antigen-specific B-cell enrichment.
Fig.3 Comparison of plasmablasts, GC-dependent memory B-cells, and GC B-cells at the BCR level. (Chai, 2022)
Result—Pseudovirus Neutralization Screening Among the Positive Antibodies
Finally, the authors undertook an investigation into the neutralizing efficacy of 215 SARS-CoV-2 S-binding antibodies within the context of a pseudovirus neutralization system. The results of the assay revealed that two RBD-binding antibodies displayed a comparatively low neutralizing capacity, as demonstrated by the half-maximal inhibitory concentration (IC50) values. Additionally, a subset of 16 antibodies, which tested positive but failed to bind to epitopes, were found to possess no discernible neutralizing abilities. This outcome was unexpected, as there were no known S2-targeted neutralizing antibodies against SARS-CoV-2, despite the fact that the majority of antibodies isolated from immunized RenMab mice displayed binding affinities for the S2 region.
Fig.4 Neutralization potency of 199 purified antibodies on SARS-CoV-2 pseudovirus. (Chai, 2022)
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Reference
- Chai, M.Y.; et al. A high-throughput single cell-based antibody discovery approach against the full-length SARS-CoV-2 spike protein suggests a lack of neutralizing antibodies targeting the highly conserved S2 domain. Brief in Bioinformatics. 2022, 23(3): bbac070.
