SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Virus
2.2. Recombinant Protein Expression and Purification
2.3. Llama Immunization and Library Construction
2.4. Isolation of SARS-CoV-2 S-2P- and RBD-Specific Nanobodies
2.5. Periplasmic Extract ELISA (PEE) and Phage ELISA (rPE)
2.6. Phylogenetic Analysis and Germline Origin
2.7. Expression and Purification of Nanobodies
2.8. Binding Affinity to the Target Antigen Estimated by ELISA EC50
2.9. Pseudovirus Neutralization Test (pVNT)
2.10. Wild-Type Virus Neutralization Assays (VNA)
2.11. Competition of Nanobodies with ACE2
2.12. Interference of Nanobody Binding to Spike by Biliverdin
2.13. Efficacy of Nanobodies in a Mouse Model
2.14. In Silico Analysis
3. Results
3.1. Selection of SARS-CoV-2 S-2P and RBD-Specific Nanobodies
3.2. SARS-CoV-2 Nanobody Characterization
3.3. Screening of Neutralizing Nanobodies against SARS-CoV-2 WT Strain
3.4. Protection against SARS-CoV-2 Challenge in k18-hACE2 Mouse Model
3.5. Nanobodies to SARS-CoV-2 WT Strain Neutralize SARS-CoV-2 Variants
3.6. A cocktail of Nanobodies Enhances Neutralization Potencies against Omicron Variants
3.7. Nanobody Epitope Mapping and Binding Mode by In Silico Prediction
4. Discussion
5. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
References
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Nbs | pVNT IC50 (nM) | |||||||
---|---|---|---|---|---|---|---|---|
Alpha | Beta | Delta | Omicron B.1.1.529 | Omicron BA.2 | Omicron XBB | Omicron XBB.1.5 | ||
RBD binders | Nb-43 | - | - | - | 394.7 | 204.8 | - | - |
Nb-104 | 18.29 | - | 121.8 | - | - | - | - | |
Nb-110 | 133.9 | 324.8 | 121.8 | - | - | - | - | |
Nb-145 | 86.65 | 451.6 | 384.4 | - | - | - | - | |
Non-RBD binders | Nb-45 | 83.66 | 237.5 | 248.0 | 214.6 | 112.0 | 30.73 | 42.17 |
Nb-51 | 215.3 | 616.3 | - | - | - | 285.6 | 316.9 | |
Nb-53 | 91.2 | 112.4 | 701.8 | 322.6 | 135.3 | 145.2 | 136.7 |
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Pavan, M.F.; Bok, M.; Betanzos San Juan, R.; Malito, J.P.; Marcoppido, G.A.; Franco, D.R.; Militello, D.A.; Schammas, J.M.; Bari, S.E.; Stone, W.; et al. SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice. Viruses 2024, 16, 185. https://doi.org/10.3390/v16020185
Pavan MF, Bok M, Betanzos San Juan R, Malito JP, Marcoppido GA, Franco DR, Militello DA, Schammas JM, Bari SE, Stone W, et al. SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice. Viruses. 2024; 16(2):185. https://doi.org/10.3390/v16020185
Chicago/Turabian StylePavan, María Florencia, Marina Bok, Rafael Betanzos San Juan, Juan Pablo Malito, Gisela Ariana Marcoppido, Diego Rafael Franco, Daniela Ayelen Militello, Juan Manuel Schammas, Sara Elizabeth Bari, William Stone, and et al. 2024. "SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice" Viruses 16, no. 2: 185. https://doi.org/10.3390/v16020185