Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow Virometry
Nanobodies, or single-domain antibodies (V<sub>H</sub>Hs) from camelid heavy-chain-only antibodies, offer significant advantages in therapeutic and diagnostic applications due to their small size and ability to bind cryptic protein epitopes inaccessible to conventional antibodies. In thi...
Saved in:
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-04-01
|
| Series: | Viruses |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1999-4915/17/4/571 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850180360442216448 |
|---|---|
| author | Mariam Maltseva Martin A. Rossotti Jamshid Tanha Marc-André Langlois |
| author_facet | Mariam Maltseva Martin A. Rossotti Jamshid Tanha Marc-André Langlois |
| author_sort | Mariam Maltseva |
| collection | DOAJ |
| description | Nanobodies, or single-domain antibodies (V<sub>H</sub>Hs) from camelid heavy-chain-only antibodies, offer significant advantages in therapeutic and diagnostic applications due to their small size and ability to bind cryptic protein epitopes inaccessible to conventional antibodies. In this study, we examined nanobodies specific to regions of the SARS-CoV-2 spike glycoprotein, including the receptor-binding domain (RBD), N-terminal domain (NTD), and subunit 2 (S2). Using flow virometry, a high-throughput technique for viral quantification, we achieved the efficient detection of pseudotyped viruses expressing the spike glycoprotein. RBD-targeting nanobodies showed the most effective staining, followed by NTD-targeting ones, while S2-specific nanobodies exhibited limited resolution. The simple genetic structure of nanobodies enables the creation of multimeric formats, improving binding specificity and avidity. Bivalent V<sub>H</sub>H-Fc constructs (V<sub>H</sub>Hs fused to the Fc region of human IgG) outperformed monovalent formats in resolving viral particles from background noise. However, S2-specific monovalent V<sub>H</sub>Hs demonstrated improved staining efficiency, suggesting their smaller size better accesses restricted antigenic sites. Furthermore, direct staining of cell supernatants was possible without virus purification. This versatile nanobody platform, initially developed for antiviral therapy against SARS-CoV-2, can be readily adapted for flow virometry applications and other diagnostic assays. |
| format | Article |
| id | doaj-art-0be1daab78eb4eec8f0dababf7da28d6 |
| institution | OA Journals |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj-art-0be1daab78eb4eec8f0dababf7da28d62025-08-20T02:18:11ZengMDPI AGViruses1999-49152025-04-0117457110.3390/v17040571Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow VirometryMariam Maltseva0Martin A. Rossotti1Jamshid Tanha2Marc-André Langlois3Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, CanadaHuman Health Therapeutics Research Centre, Life Sciences Division, National Research Council Canada, Ottawa, ON K1N 1J1, CanadaDepartment of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, CanadaDepartment of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, CanadaNanobodies, or single-domain antibodies (V<sub>H</sub>Hs) from camelid heavy-chain-only antibodies, offer significant advantages in therapeutic and diagnostic applications due to their small size and ability to bind cryptic protein epitopes inaccessible to conventional antibodies. In this study, we examined nanobodies specific to regions of the SARS-CoV-2 spike glycoprotein, including the receptor-binding domain (RBD), N-terminal domain (NTD), and subunit 2 (S2). Using flow virometry, a high-throughput technique for viral quantification, we achieved the efficient detection of pseudotyped viruses expressing the spike glycoprotein. RBD-targeting nanobodies showed the most effective staining, followed by NTD-targeting ones, while S2-specific nanobodies exhibited limited resolution. The simple genetic structure of nanobodies enables the creation of multimeric formats, improving binding specificity and avidity. Bivalent V<sub>H</sub>H-Fc constructs (V<sub>H</sub>Hs fused to the Fc region of human IgG) outperformed monovalent formats in resolving viral particles from background noise. However, S2-specific monovalent V<sub>H</sub>Hs demonstrated improved staining efficiency, suggesting their smaller size better accesses restricted antigenic sites. Furthermore, direct staining of cell supernatants was possible without virus purification. This versatile nanobody platform, initially developed for antiviral therapy against SARS-CoV-2, can be readily adapted for flow virometry applications and other diagnostic assays.https://www.mdpi.com/1999-4915/17/4/571flow virometrynanobodiesdiagnosticsantiviralsSARS-CoV-2 |
| spellingShingle | Mariam Maltseva Martin A. Rossotti Jamshid Tanha Marc-André Langlois Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow Virometry Viruses flow virometry nanobodies diagnostics antivirals SARS-CoV-2 |
| title | Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow Virometry |
| title_full | Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow Virometry |
| title_fullStr | Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow Virometry |
| title_full_unstemmed | Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow Virometry |
| title_short | Characterization of Nanobody Binding to Distinct Regions of the SARS-CoV-2 Spike Protein by Flow Virometry |
| title_sort | characterization of nanobody binding to distinct regions of the sars cov 2 spike protein by flow virometry |
| topic | flow virometry nanobodies diagnostics antivirals SARS-CoV-2 |
| url | https://www.mdpi.com/1999-4915/17/4/571 |
| work_keys_str_mv | AT mariammaltseva characterizationofnanobodybindingtodistinctregionsofthesarscov2spikeproteinbyflowvirometry AT martinarossotti characterizationofnanobodybindingtodistinctregionsofthesarscov2spikeproteinbyflowvirometry AT jamshidtanha characterizationofnanobodybindingtodistinctregionsofthesarscov2spikeproteinbyflowvirometry AT marcandrelanglois characterizationofnanobodybindingtodistinctregionsofthesarscov2spikeproteinbyflowvirometry |