## 2 Results and Discussion
Different superscript letters (a, b, c) indicate significant differences (\(p<0.05\)) between groups assessed by ANOVA followed by [PERSON]’s test for multiple comparisons. Data are the average of biological replicates, N = 3; mean \(\pm\) SD. Figure 3: Nb production. Western blot analysis of the Nbs expressed in the two systems, as indicated. **Physicochemical properties of Nbs. ** Figure 5A depicts the molecular assessment findings indicating that, compared to the 1.29 Nb, the sequence similarities of the C11 and K9 NBs were 98.35% and 73.33%, respectively. This result is attributed to the numbers of mutations in the C11 and K9 Nbs, which were 2 and 37 residues, respectively (Figure 5A). Figure 5B illustrates the structural alignment between the 1.29 and C11 Nbs, which exhibited very high similarity due to the limited number of mutated residues in the C11 Nb, which were R38C and V64E located in frameworks 2 and 3, respectively. These mutations slightly affected the conformation of the C11 Nb. Many differences in structure between the 1.29 and K9 Nbs were due to 38 mutation residues distributed across both frameworks and CDR1-3. Particularly, CDR3 of the K9 Nb revealed insertion mutations resulting in the extension of CDR3. The calculated physicochemical properties are illustrated in Table 2. Generally, since the C11 Nb differed from the parental 1.29 Nb only by two residues, their physicochemical properties were quite similar. Nonetheless, the analysis revealed that the negative charge-rich residues present in the C11 and K9 Nbs gave rise to their lower negative charge at biological pH and lower pI values. The grand average of hydroptability (GRAVY) score indicates the hydrophobic property of each Nb [20]. All Nbs had negative GRAVY scores, which means they were hydrophilic. The K9 Nb had the largest negative GRAVY score due to many charged residues present in the protein. The various aliphatic residues of the K9 Nb contributed to the increase in the aliphatic index; hence, it could be more thermally stable than the 1.29 Nb. Although the K9 Nb had a higher instability index than the 1.29 Nb, its value was still well below 40. Therefore, it was considered a stable protein [21]. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**RBD Variants**} & \multicolumn{3}{c}{**K\({}_{\text{D}}\) (nM)**} \\ \cline{2-4}  & **1.29** & **C11** & **K9** \\ \hline
**Delta** & 19.3, 95\% CI [6.4, 58.2] & 878, 95\% CI [109, 7097] & 1510, 95\% CI [880, 2600] \\
**BA.5** & 1390, 95\% CI [60, 3430] & 326, 95\% CI [90, 1185] & 29.9, 95\% CI [23, 37.5] \\
**XBB.1.5** & 4090, 95\% CI [940, 17,870] & 569, 95\% CI [155, 2081] & 1150, 95\% CI [570, 9150] \\
**XBB.1.16** & 448, 95\% CI [255, 788] & 251, 95\% CI [109, 578] & 213, 95\% CI [126, 360] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: K\({}_{\text{D}}\) values of the parental and isolated Nbs toward various RBD variants. Figure 5: **A comparison of the amino acid and structural similarity of Nbs. ** (**A**) Amino acid sequence similarity among all Nbs. Structural similarity of Nbs; alignment of the 1.29 Nb compared to (**B**) the C11 Nb and (**C**) the K9 Nb. The purple, pink, and yellow colors represent 1.29 Nb, C11 Nb, and K9 Nb, respectively. **Prediction of RBD BA.5-Nb interactions. ** To help understanding how these Nbs may bind to the RBD of BA.5 variant the HDOCK program was used to predict the interactions between the RBD of BA.5 variant and all Nbs--the 1.29, C11, and K9 Nbs as illustrated in Figure 6. HDOCK ranks poses based on predicted binding affinity, with the top pose indicating the most favorable interaction in terms of orientation and affinity, as previously described by [PERSON] et al. (2020). Recently, [PERSON] et al. (2020) validated HDOCK's performance with nanobody/RBD complexes from the Protein Data Bank, demonstrating HDOCK's high accuracy in generating poses with the lowest RMSD, closely resembling native crystal structures with an accuracy of over 83%.