## 4 Materials and Methods
### Prediction of the Interactions Between the RBD of BA.5 Variant and Nbs  
The crystal structures of the RBD of BA.5 variant (PDB ID: 7 ZXU) and the original 1.29 Nb (PDB ID: 7R4Q) were obtained from the PDB Protein Data Bank ([[https://www.rcsb.org/](https://www.rcsb.org/)]([https://www.rcsb.org/](https://www.rcsb.org/)), accessed on 25 February 2024). The structure of the C11 Nb was prepared using site-directed mutagenesis at R38C and V64E of the parental 1.29 Nb via Discovery Studio software version 21.1. The K9 Nb (as its CDRs differ significantly from the 1.29 Nb) and RBD of JN.1 variant were modeled on the AlphaFold2 online server ([[https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb](https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb)]([https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb](https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb)), accessed on 26 February 2024) [50; 51] (see sequence alignment in Figure S3). To prepare the optimal initial structure of the RBD and Nbs, energy minimization was performed using the AMBER ff14 SB force field [52]. This involved docking the RBD of BA.5 variant with all 3 Nbs using a blind docking method through HDOCK ([[http://hdock.phys.hust.edu.cn/](http://hdock.phys.hust.edu.cn/)]([http://hdock.phys.hust.edu.cn/](http://hdock.phys.hust.edu.cn/)), accessed on 26 February 2024) [22; 23]. Subsequently, RBD-Nb complexes were energetically minimized by the AMBER ff14 SB force field, aiming for the complexes with the lowest binding energy. This process aligned the torsion angles of complementary amino acid side chains between the RBD of BA.5 variant and Nbs, ensuring uniformity in the lowest binding energy and preventing any atomic misalignment. HDOCK was employed to perform the redocking of the optimized RBD and Nb, yielding the optimal docking score and interaction [23]. In the final step, the binding affinities of the RBD-Nb complexes were calculated by rescoring using AutoDock Vina [53]. **Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app142210548/s1](https://www.mdpi.com/article/10.3390/app142210548/s1)]([https://www.mdpi.com/article/10.3390/app142210548/s1](https://www.mdpi.com/article/10.3390/app142210548/s1)), Table S1: PCR program setup; Table S2: Plasmids used in this study; Figure S1: The engineered plasmids in FLI-TRAP format; Figure S2: Amino acid sequences of RBD-specific Nbs; Figure S3: Amino acid sequence comparisons of the 1.29, C11, and K9 Nbs; Figure S4: Nb purification; Figure S5: Protein-protein interactions of the RBD of BA.5 with various Nbs; Figure S6: A comparison of protein-protein interactions of the RBD of BA.5 with Nbs and the RBD of JN.1 with Nbs; Figure S7: The structure and surface hydrophobicity of the RBD of SARS-CoV-2 BA.5 and Nbs; Figure S8: Protein-protein interactions of the RBD of JN.1 with various Nbs; Figure S9: Amino acid sequences of the Receptor Binding Domain of SARS-CoV-2 variants used in this study. **Author Contributions:** Conceptualization, K.I., A.T., N.S. and D.W.-Z. ; methodology, K.I., A.T., P.L., N.S., W.K., S.S. ([PERSON]) and D.W.-Z. ; validation, K.I., P.L., W.K. and S.S. ([PERSON]); formal analysis, K.I., A.T., P.L., N.S. and D.W.-Z. ; investigation, K.I., A.T., P.L., W.K., N.S. and S.S. ([PERSON]); resources, K.C. and D.W.-Z. ; data curation, K.I., A.T., P.L. and N.S. ; writing--original draft preparation, K.I., A.T., P.L. and D.W.-Z. ; writing--review and editing, K.I., P.L., K.C., S.B. and D.W.-Z. ; visualization, K.I., P.L. and N.S. ; supervision, D.W.-Z. ; project administration, D.W.-Z. ; funding acquisition, K.I., S.B. and D.W.-Z. All authors have read and agreed to the published version of the manuscript. **Funding:** This research project is supported by the National Research Council of Thailand: Fiscal year 2022 (to D.W.-Z. and S.B.