Gene Expression
Nebulas
Gene Expression NebulasSummary: The COVID-19 pandemic prompted an unprecedented effort to develop effective countermeasures against SARS-CoV-2. While efficacious vaccines and certain therapeutic monoclonal antibodies are available, here, we report the development, cryo-EM structures and functional analyses of distinct potent monoclonal antibodies (mAbs) that neutralize SARS-CoV-2 and its variant B.1.351. We established a platform for rapid identification of highly potent and specific SARS-CoV-2-neutralizing antibodies by high-throughput B cell receptor single cell sequencing of spike receptor binding domain immunized animals. We identified two highly potent and specific SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity. We also generated a bispecific antibody of these two lead clones. The lead monospecific and bispecific antibodies showed strong neutralization ability against prototypical SARS-CoV-2 and the highly contagious South African variant B.1.351 that post a further risk of reducing the efficacy of currently available therapeutic antibodies and vaccines. The lead mAbs showed potent in vivo efficacy against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We solved five cryo-EM structures at ~3 resolution of these neutralizing antibodies in complex with the ectodomain of the prefusion spike trimer, and revealed the molecular epitopes, binding patterns and conformations between the antibodies and spike RBD, which are distinct from existing antibodies. Our recently developed antibodies expand the repertoire of the toolbox of COVID-19 countermeasures against the SARS-CoV-2 pathogen and its emerging variants.
Overall Design: 10x 5' single-cell VDJ sequencing of CD138+ B cells from mice (C57BL/6 and BALBc) immunized with SARS-CoV-2 RBD. From C57BL/6 mice: 1 sample from spleen+LN, 1 sample from bone marrow. From Balbc mice: 2 samples (pooled spleen+LN+bone marrow).
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| Extract Protocol: | The enriched CD138+ plasma cells and progenitor B cells were loaded on a 10X Chromium Next GEM Chip G. The target cell number was 10,000 cells per sample. Single-cell lysis and RNA first-strand synthesis were performed using Chromium Next GEM Single Cell 5’ Gel Bead V3.1 according to the manufacturer’s protocol. |
| Library Construction Protocol: | The following RNA and V(D)J library preparation was performed according to the manufacturer’s protocol (Chromium Next GEM Single Cell V(D)J reagent kit, mouse BCR). The resulting VDJ-enriched libraries were sequenced following the reading mode recommended by 10x Genomics. Sequencing was performed on a NovaSeq targeted for 10,000 reads/cell, with a total of 100 million reads. |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | PAIRED |
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| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-Specific: | - |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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