1086 Sequencing antibodies from de novo to identify functional and neutralizing antibodies in human plasma after SARS-CoV-2 vaccination
abstract · Regular and Young Investigator Award Abstracts · 2024
| Date | 2024-11-01 |
| Type | abstract |
| Venue | Regular and Young Investigator Award Abstracts |
| Publisher | BMJ Publishing Group Ltd |
| Contribution | downstream-application |
| DOI | 10.1136/jitc-2024-sitc2024.1086 |
| Citations (OpenAlex) | 0 |
Abstract
Background Approximately 90% of human antibody discovery related to SARS-CoV was achieved through B-cell sequencing only approaches, as evidenced by a survey of the CoV-AbDab database. Although successful, this approach often fails to match B-cells to the circulating IgG population, leaving valuable candidates unidentified. The peripheral B-cell repertoire may not adequately cover all the circulating IgG for human IgG sequencing. Combining proteomics and B-cell repertoire analysis can confirm the presence of specific antibodies in circulation. De novo pAb sequencing offers the most promising approach to identify novel antibody candidates from the entire circulating antibody repertoire. Methods The proteomics dataset was initially analyzed conventionally and compared to the IgSeq/B-cell database for the same individual and the public dataset CoV-AbDab. In addition, a de novo analysis was performed on the proteomics dataset. Some recombinant antibodies were generated from this analysis and subsequently tested for antigen binding and neutralization using ELISA, SPR, and pseudovirus neutralization assays. In addition to the short-read extension approach, we used other methods, including protein separation and peptide digestion under non-reduced conditions. Results Following de novo sequencing and IgSeq, as well as heavy and light chain pairings, we yielded twelve antibodies, which are combinations of six heavy and eight light chains. All six heavy chains contain different CDR3 regions. Four of these six chains are absent from the IgSeq data and were discovered exclusively through de novo sequencing. Seven of the 12 recombinant antibodies exhibited high affinities towards the RBD. Six recombinant antibodies have affinities similar or better against the RBD than the natural polyclonal anti-RBD antibody. Notably, the four strongest binders were obtained through de novo sequencing. Additionally, all tested recombinant antibodies displayed varying degrees of neutralization activity. Conclusions This present study highlights the significance of de novo sequencing in comprehensively understanding the final products of the humoral immune response. The findings illustrate the limitations of traditional B-cell sequencing approaches and underscore the value of de novo sequencing in uncovering missed valuable antibody candidates. The methodological improvements and stringent validation measures implemented in this study provide solid evidence for the accuracy and effectiveness of the proposed approach. The recombinant proteins we generate possess traits comparable to or surpassing the naturally occurring polyclonal antibodies. Despite the challenges encountered in de novo sequencing of polyclonal antibodies, this study successfully demonstrates the feasibility and potential of this method to contribute to the discovery of novel antibody reagents and therapeutics. Ethics Approval All specimens were collected by Discovery Life Sciences with proper consent. The samples were provided with de-identified data, but included basic demographics (age, sex, and ethnicity).
Methods and tools
- Polyclonal IgG neutralizing antibody discovery: Sequencing the circulating antibody repertoire itself rather than the B cells that make it. Human plasma-derived polyclonal IgG is sequenced by combining mass spectrometry with B-cell sequencing, applied to the IgG response to the Moderna Spikevax COVID-19 vaccine. Twelve recombinant antibodies were generated from the natural polyclonal response; six of them, including four derived by de novo protein sequencing, bound at least as well as the original polyclonal antibody, and all six neutralised the target antigen. The argument for the approach is that sequencing peripheral B cells may not represent the complete receptor repertoire, so the circulating IgG pool has to be examined directly. Notable as antibody discovery carried out end to end by a commercial de novo sequencing service.