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Article

Redirecting a Broad-Spectrum Nanobody Against the Receptor-Binding Domain of SARS-CoV-2 to Target Omicron Variants

by
Kwanpet Intasurat
1,
Nonth Submunkongtawee
1,
Phoomintara Longsompurana
1,
Apisitt Thaiprayoon
1,
Warisara Kasemsukwimol
1,
Suwitchaya Sirimanakul
1,
Siriphan Boonsilp
2,
Supaphron Seetaha
3,
Kiattawee Choowongkomon
3 and
Dujduan Waraho-Zhmayev
1,*
1
Biological Engineering Program, Faculty of Engineering, King Mongkut’s University of Technology Thonburi, Bangkok 10140, Thailand
2
Department of Clinical Pathology, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok 10300, Thailand
3
Department of Biochemistry, Faculty of Science, Kasetsart University, 50 Pahonyothin Road, Chutuchak, Bangkok 10900, Thailand
*
Author to whom correspondence should be addressed.
Appl. Sci. 2024, 14(22), 10548; https://doi.org/10.3390/app142210548
Submission received: 16 September 2024 / Revised: 4 November 2024 / Accepted: 8 November 2024 / Published: 15 November 2024
(This article belongs to the Section Applied Biosciences and Bioengineering)
Browse Figures
Versions Notes

Abstract

:
The urgent need for an effective COVID-19 therapy has propelled the exploration of innovative strategies to combat the fast-mutating SARS-CoV-2 virus. This study attempted to develop nanobodies (Nbs) against the SARS-CoV-2 Omicron variants by redirecting the 1.29 neutralizing Nb, a receptor-binding domain (RBD)-specific Nb that can protect against various SARS-CoV-2 variants other than Omicron, to target SARS-CoV-2 Omicron subvariant BA.5, the variant used for the development of the bivalent vaccine. Error-prone libraries of the 1.29 Nb were constructed. Following two rounds of selection using the functional ligand-binding identification by Tat-based recognition of associating proteins (FLI-TRAP) technique, we rapidly identified two Nbs, namely, C11 and K9, that could target the RBD of the Omicron subvariant BA.5, XBB.1.5, and XBB.1.16 subvariants. Molecular docking provided insights into how these Nbs interact with the RBD of the BA.5 and JN.1 variants. The application of directed evolution via utilization of error-prone PCR and the synthetic E. coli applied in the FLI-TRAP selection method may be a powerful tool for facilitating simple, fast and economical selection to redirect existing antibodies and to generate antibody fragments to target proteins susceptible to autonomous mutation, not only for viral infection but also other diseases, such as cancer.

1. Introduction

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the ensuing coronavirus disease 2019 (COVID-19) precipitated a global health crisis after their origin in Wuhan, China. The severity of this pandemic is intricately linked to critical viral proteins, especially the receptor-binding domain (RBD) within spike proteins, which is critical for viral entry by binding to the human angiotensin-converting enzyme 2 (ACE-2) receptor. Amid the evolving viral landscape, the Omicron variant (PANGO lineage B.1.1.529) has emerged as a dominant variant, significantly challenging global public health strategies. Omicron’s distinctiveness stems from an extensive mutation profile: it has accumulated over 30 mutations in the spike protein [1]. Notably, this variant efficiently evades neutralizing antibodies induced by prior infections or vaccinations, heightening its transmission within populations with preexisting immunity. However, in contrast to earlier variants, the Omicron variant exhibits reduced pathogenicity due to its less efficient infection of lung cells, thereby decreasing the incidence of severe pneumonia [2]. Recent scientific revelations have underscored the significance of the Omicron subvariant BA.5 as a pivotal progression within this viral landscape. Unlike preceding Omicron subvariants, Omicron subvariant BA.5 demonstrates efficient lung cell entry caused by specific spike protein mutations, which enhance viral cell fusion and replication within lung cells [3]. This variant was then used for bivalent vaccine development by Pfizer–BioNTech and Moderna.
This study utilized nanobodies (Nbs), recombinantly produced single antigen-binding domains derived from the heavy-chain only antibodies (HCAb) present in animals of the Camelidae family, as potent tools targeting the RBD’s functional intricacies. Nbs offer higher stability and solubility, and more efficient target-binding, than conventional monoclonal antibodies (mAbs) due to their diminutive size, which confers superior tissue penetration capabilities essential in impeding viral intrusion, as well as greater accessibility to difficult-to-reach tissue areas [4,5]. Several studies have demonstrated the potentials of Nbs for COVID-19 treatment by blocking RBD-ACE2 interaction [6,7,8,9].
The functional ligand-binding identification by Tat-based recognition of associating proteins (FLI-TRAP) technique is an in vivo technique that is useful for the selection of binding proteins. It has previously been utilized for both identification and enhancement of antibody fragments (single-chain variable fragment, scFv, and single-domain antibody, sdAb, also known as Nb) specific to various targets [10,11,12,13]. Our study focused on the directed evolution of Nbs that specifically target the RBD of the Omicron subvariant BA.5, using the FLI-TRAP technique as an in vivo selection method [10]. Our goal was to create Nbs that could bind the SARS-CoV-2 Omicron subvariant BA.5 by redirecting the specificity of the existing broad-spectrum Nb, the 1.29 Nb, capable of effectively binding several SARS-CoV-2 variants of concern (VOCs) [9]. To achieve this, we employed error-prone PCR to generate Nb mutation libraries and FLI-TRAP to isolate Nbs specific to the RBD of the SARS-CoV-2 spike protein of the Omicron subvariant BA.5. After two rounds of evolution, Nbs C11 and K9 emerged as potential candidates, displaying notable binding abilities, particularly toward the RBD of Omicron subvariant BA.5 as well as other Omicron subvariants tested, including XBB.1.5 and XBB.1.16.
The combined use of error-prone PCR and the FLI-TRAP technique has shown the potential to redirect the specificity of the currently available Nb toward targets with mutations. Our tool offers a rapid and simple evolution method facilitating the creation of tailored treatments for various diseases other than viral infections. We envision that this technique could be useful for cancer-related antibody treatment, as mutations of cancer cells can render a current antibody treatment ineffective. Currently, the generation of antibodies capable of recognizing single amino acid alterations, which are frequently induced by missense mutations in cancers, is not easily achievable through conventional technologies.

2. Results and Discussion

Selection of the target RBD variant and the prototype Nb. The Omicron variant has displaced other variants, with its rising prevalence and continued propensity for mutation [14]. In addition, the amino acid sequences of the RBD of Omicron subvariants BA.4/5 were used to design the most updated bivalent booster vaccines, BNT162b2 BA.4/5 bivalent mRNA vaccine (Pfizer–BioNTech) and SPIKEVAX Bivalent Original/Omicron BA.4–5 (Moderna). Unfortunately, the accumulated additional spike mutations may reduce the effectiveness of the vaccine, as a study found that the BA.5 bivalent booster did not provide strong neutralization against some of the Omicron sublineages, including the BA.2-derived BA.2.75.2 and the BA.5-derived BQ.1.1 and XBB.1 [15].
From a therapeutic standpoint, the search for broad-spectrum antibodies continues. Our literature review revealed that the 1.29 Nb exhibits comprehensive binding to RBD variants of SARS-CoV-2, including the VOCs Wuhan (WA1), Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2). Nonetheless, its efficacy in binding to the Omicron BA.1 variant is notably diminished compared to WA1 [9]. This poses a challenge for the development of the 1.29 Nb against the new Omicron variants. To address this challenge, we utilized the 1.29 Nb as a prototype Nb to generate a library of diverse Nbs using an error-prone PCR technique, and the library was selected against the RBD of the BA.5 variant using the FLI-TRAP technique.
Redirection of a broad-spectrum anti-RBD 1.29 Nb to the Omicron subvariant BA.5. Figure 1 illustrates the overall selection process used for identification of the 1.29 Nb mutants that could bind to the RBD of BA.5 variant. For FLI-TRAP screening, we first constructed RBD fused to mature TEM-1 β-lactamase (Bla) protein in the pDD18 plasmid [10], and the parental 1.29 Nb was then cloned with an N-terminal Tat signal peptide in the same plasmid (Figure S1). The mutant 1.29 Nb library gene created by an error-prone PCR technique utilizing Mutazyme II DNA polymerase from a GeneMorph II mutagenesis kit was subcloned by replacing the parental 1.29 Nb. For the first round of mutation, the library size and error rate were 9.8 × 105 members and 1–2 mutations per gene, respectively. For the second round of mutation, the library size and error rate were determined to be 4.2 × 105 members and 1–4 mutations per gene, respectively.
Since we wanted to identify mutant Nbs that could bind to the RBD of the BA.5 variant better than the parental 1.29 Nb, this original 1.29 Nb sequence was used to establish the library selection conditions by spot plating (Figure 2A). During this first round of mutation, 51 hits were obtained at 100 µg/mL Carb and 10−4 cell dilution. Figure 2B shows the results of the spot plating, which was performed to compare the cell resistance of Carb among 15 randomly selected clones and the parental 1.29 Nb. The sequencing of 13 of 15 colonies was successful and revealed that 10 of them had the same amino acid sequence as the 1.29 Nb. Of the three unique sequences, i.e., C3, C4, and C11, that we obtained, two contained stop codons (C3 and C4) hence were false positives. Therefore, C11 was the only unique Nb with a sequence with two nucleotide mutations resulting in R38C and V64E mutations (Figure S2). It should be noted that the cell resistance of the colonies that contained the same sequence as the 1.29 Nb could be due to natural mutations of the bacterial cells that created resistance to the antibiotic that was unrelated to the FLI-TRAP selection system [16].
In the second round, 21 hits were isolated at 200 μg/mL Carb and 10−4 cell dilution. All hits were submitted for sequencing analysis. Surprisingly, 17 sequencing results displayed the exact same sequence, which was distinctly different from that of the C11 Nb and is a full-length Nb, while the other 4 sequencing results showed a stop codon within the Nbs. The sequence of the representative clone of these 17 Nbs, the K9 Nb, is illustrated in Figure S3. It had 2, 4, and 2 mutations in frameworks 1, 2, and 3, respectively. Surprisingly, the complementarity determining region 1 (CDR1) and CDR3 of the K9 Nb were very different from those of the 1.29 and C11 Nbs; the K9 Nb had an elongated CDR3 (19 amino acids) compared to that of the 1.29 and C11 Nbs (13 amino acids). We hypothesized that such differences between the template, the C11 Nb, and the isolated Nb (the K9 Nb) could be due to a phenomenon called “replication slippage” [17]. It should be noted that the diversity and mutation rate of the library produced by the second round of mutation were assessed by submitted random clones that appeared on the plate used for library size estimation and were not subjected to FLI-TRAP selection for sequencing. The sequencing results showed that all Nbs had distinct Nucleotide sequences and that the K9 Nb should not have been a false positive from our FLI-TRAP selection. We also confirmed this by comparing the Carb resistance of cells containing RBD BA.5-Bla and K9 Nb fusion proteins in not only the E. coli strain NEB10β but also in strains MC4100 and DADE using spot plating, as shown in Figure 2C. DADE is an isogenic ΔtatABCDΔtatE derivative of MC4100; hence, it is not capable of performing Tat transport. The pDD18-Cm plasmid co-expressing RBD BA.5-Bla and ssTorA-K9-FLAG was co-transformed into MC4100 and DADE cells. Spot plating results showed a resistance of up to 50 µg/mL Carb in MC4100 cells at the single colony level (10−6), up to 50 µg/mL Carb while DADE cells showed only background growth at 10−1. This result strongly confirmed that cell resistance was related to the Tat transport of the two binding proteins. At 100 µg/mL Carb, NEB10β cells containing the K9 Nb grew at 10−6, while cells containing the 1.29 Nb grew at only 10−1, a 100,000-fold difference. Notably, the observable difference between the abilities of MC4100 and NEB10β to resist Carb could be due to many factors, for example, differences in abilities to produce recombinant protein and to resist toxicity of the arabinose used as an inducer.
Differences in Nb production of the two systems. For FLI-TRAP selection, Nbs were produced from the pDD18-Cm plasmid, which is derived from the pBAD18-Cm plasmid of E. coli NEB10β [10]. For in vitro analysis, the pET vector system was employed, as high recombinant protein expression can be achieved via the T7 promoter system present in the pET vectors using E. coli BL21(DE3) [18]. Western blot analysis (Figure 3) using Mouse Anti-DDDDK tag Monoclonal Antibody, HRP Conjugated, Clone M2 (1:3000; Abcam Cat# ab49763, RRID:AB_869428)to detect the FLAG epitope tag present in both systems revealed distinct bands corresponding to the estimated sizes of the 1.29 (17 kDa), C11 (17 kDa), and K9 (20 kDa) Nbs based on the identified amino acid sequences. Interestingly, differences in the solubility of the K9 Nb expressed in the two systems were quite apparent. At a low expression level using pDD18-Cm, the K9 Nb showed higher solubility than the 1.29 and C11 Nbs (approximately twice that of the 1.29 Nb). However, at a high expression level using pET28a, the solubility of the K9 Nb was much lower than that of the 1.29 Nb expressed using the same system (~100 fold that of the 1.29 Nb). Nonetheless, for the in vitro analysis, Nbs were expressed using pET28a since the overall yield of Nbs was higher when expressed using this system. Figure S4 illustrates the Coomassie analysis of the purified Nbs, which simply involved batch-mode Ni–NTA affinity purification by gravity flow. It should be noted that the C11 nanobody (Nb) contains the R38C mutation, meaning that the extra cysteine in one nanobody might form a covalent disulfide bond with the extra cysteine in a second nanobody, resulting in a homodimer. The formation of this bond would depend on the expression environment. For example, an oxidizing environment, such as the E. coli periplasm, would be required to efficiently form this bond. However, the strain used to produce the nanobody, BL21(DE3), has a strongly reducing cytoplasm that generally disfavors cysteine oxidation and disulfide bond formation. Thus, this linkage likely forms at some point after the protein is released from the cells, resulting from spontaneous oxidation in air. Nonetheless, a molecular-weight cutoff (MWCO) column was used for buffer exchange, concentrating the protein and removing high-molecular-weight contaminants or multimeric Nbs.
Evaluation of the antigen-binding activity of the Nbs isolated using FLI-TRAP. The abilities of the 1.29, C11, and K9 Nbs to bind to a range of RBDs of the Omicron subvariant BA.5 and a few other Omicron variants were examined using enzyme-linked immunosorbent assay (ELISA) and microscale thermophoresis (MST). Redirecting the 1.29 Nb toward the RBD of Omicron subvariant BA.5 revealed a significant increase in binding activities for the C11 (OD450 = 1.5544 ± 0.0619) and K9 Nbs (OD450 = 1.6508 ± 0.0392) compared to the 1.29 Nb (OD450 = 0.2906 ± 0.0312) (Figure 4). Additionally, the binding activities of all Nbs against the Delta and BA.1 variants were also evaluated, as the 1.29 Nb has been reported to bind strongly to Delta but not to BA.1 [9]. The results confirmed that the 1.29 Nb remained functional when expressed in this study since it produced a high ELISA signal to Delta but not to BA.1, as expected.
Expanding our analysis to include various Omicron variants, the C11 Nb consistently demonstrated binding across multiple Omicron variants other than Omicron BA.5, surpassing the binding potency observed for the 1.29 Nb. In contrast, the binding capacity of the K9 Nb varied among the Omicron variants, suggesting potential limitations in binding to variants other than Omicron subvariant BA.5.
In this study, the MST technique was used to determine the equilibrium dissociation constants (KD) of the Nbs (Table 1). This technique has been used in previous studies to identify the binding affinity between human ACE-2 and the RBDs of many variants, including the Omicron B.1.1.529 variant [19]. Notably, the C11 and K9 Nbs had ~4.3-fold and 46.5-fold higher binding affinities for Omicron BA.5 compared with the parental 1.29 Nb (KD values of 326 and 29.9, respectively, versus 1390 nM). The C11 Nb also bound Omicron XBB.1.5and XBB.1.16, with affinities in the sub-micromolar range (KD values of 569 and 251 nM, respectively).
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 hydropathicity (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].
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 Yan et al. [22]. Recently, Longsompurana et al. [23] 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%. This computational tool was successfully utilized to design novel Nbs targeting the RBD of many VOCs of SARS-CoV-2 [23]. The binding postures of the 1.29, C11, and K9 Nbs on the receptor-binding motif (RBM) of the RBD of BA.5 variant are depicted. While the 1.29 Nb exhibited a similar posture to the C11 Nb, the C11 Nb demonstrated a slightly superior binding affinity (−10.05 kcal/mol) than the 1.29 Nb (−9.58 kcal/mol). This phenomenon suggests that the two mutated residues of the C11 Nb might influence conformational changes, leading to an increase in its binding affinity. Both Figure 6 and Figure S5 confirm that the 1.29 and C11 Nbs interacted through similar contributing contact residues on CDR1–3. Although the R38C and V64E mutation residues of the C11 Nb did not interact with the RBD of BA.5 variant, as they were not located in the CDR binding sites, the C11 Nb had more salt bridge interactions, resulting in a stronger affinity (Figure S6). An additional cysteine residue introduced by replication slippage in Framework 2 may promote nanobody multimerization, particularly notable in the VHH subfamily 3, which has contributed to further classification of VHH gene subfamilies in the dromedary genome [24]. In the conserved framework region responsible for VH/VL interactions, typical hydrophobic amino acids are replaced with hydrophilic hallmarks, enhancing solvent accessibility [25]. The R38C mutation in Framework 2 packs closely against a conserved hydrophobic core, while the unique hydrophilic hallmarks are oriented outward. This structural configuration introduces steric hindrance, restricting R38C oxidation or covalent bonding with other cysteines. Typically, nanobody multimerization is achieved via genetic fusion or domain linking [26]. However, connecting the N/C termini directly to the scaffold may weaken binding interactions, potentially diminishing affinity and stability. Notably, serine-to-cysteine mutations at four framework positions have promoted dimerization without compromising antigen binding [27]. Thus, the R38C mutation in Framework 2 is less likely to cause nanobody multimerization due to steric hindrance, and it retains binding affinity by packing closely against the hydrophobic core without interfering with antigen binding.
The binding affinity of the K9 Nb was −19.82 kcal/mol, significantly higher than those of the 1.29 and C11 Nbs. This difference is attributed to the numerous changes in the amino acid residues within the K9 Nb’s CDR1–3, contributing to interactions with the RBM of the RBD of BA.5 variant. The optimum posture of the K9 Nb shows many interface residues and a substantial interface area (Figures S6 and S7) including aliphatic and aromatic residues that played a crucial role in these interactions. The insertion mutation in the K9 Nb, specifically the extension of the CDR3 region with a tyrosine-rich sequence, was a significant factor for RBM interaction (Figure 6 and Figure S7). Remarkably, the amino acid residues on the CDRs of the K9 Nb, such as Val31, Ser99, Thr102, Pro112, and Tyr113, significantly increased hydrogen bond interactions with the RBM of the RBD of BA.5 variant (Figure S5). Moreover, more non-bonded contacts between the RBD of BA.5 variant and the K9 Nb (207 non-bonded contacts) are predicted than between the RBD of BA.5 variant and the 1.29 Nb (149 non-bonded contacts). This phenomenon aligns with a previous study that suggested that aromatic and non-polar amino acids on Nbs at the interaction sites could be essential for establishing complementary binding to the RBD of BA.5 variant, driven by the hydrophobic characteristics of these residues, enabling favorable interactions with the hydrophobic regions present on the RBM that also contain many non-polar and aromatic amino acids (Figure S7) [23]. Interestingly, the optimal interaction posture of the K9 Nb on the RBD of BA.5 variant illustrated that the residues Asp52, Val31, Val32, Glu44, and Tyr114 strongly interacted with the key amino acids of the RBD of BA.5 variant via hydrogen bonds and hydrophobic interactions. Particularly, Glu44 exhibited a strong interaction through a salt bridge with Arg498, a key amino acid of the RBD of BA.5 variant. These findings support the experimental results that the K9 Nb binds to the RBD of BA.5 variant with high binding affinity.
Prediction of the binding affinity of Nbs when interacting with the RBD of JN.1 variant. In late 2023, JN.1 (BA.2.86.1.1), a descendant of the Omicron subvariant BA.2.86, emerged and rapidly became predominant in France. This variant exhibits alterations in the spike protein at a specific location, thereby enhancing its ability to evade immunity compared with BA.2.86 [28]. As of February 23, 2024, the GISAID database shows that JN.1 variant has become the predominant strain, with 86.0–96.0% GRA in the top 10 countries in the past 4 weeks [14]. We were interested in exploring the potency of the 1.29, C11, and K9 Nbs and their potential interaction strengths with the RBD of JN.1 variant by prediction using molecular docking. Both the 1.29 and C11 Nbs could interact with the RBM of the RBD of JN.1 variant in a manner similar to that of BA.5 variant, by adopting the same interaction posture. Figure 7 illustrates the posture of the 1.29 and C11 Nbs, which allowed for interactions between CDR1 and CDR3 with the RBM of JN.1 variant (as shown in Figure S8). Moreover, both Nbs could bind to the RBD of JN.1 variant with higher interface residues and interface area than for BA.5 variant (Figure S6), resulting in more interactions and a higher binding affinity. Figure 7 confirms that both the 1.29 and C11 Nbs demonstrated a higher binding affinity to JN.1 variant (−13.32 and −10.75 kcal/mol, respectively) than to BA.5 variant. This can be attributed to the presence of E484K, a key mutation amino acid in JN.1 variant, which introduced a positive charge. This positive charge facilitated a strong salt bridge interaction (Figures S7 and S9) with Asp45, thereby affecting their structural fit. The E484K mutation has been reported to reduce neutralization [29]. It should be noted that this mutation is present in the Beta and Gamma variants and that the 1.29 Nb was reported to be able to bind to these variants [9]. Additionally, both the 1.29 and C11 Nbs had a lower number of hydrogen bond interactions while exhibiting a higher number of non-contact interactions with JN.1 variant compared to BA.5 variant (Figure S6).
Interestingly, Figure 7 illustrates that K9 could interact with the RBD of JN.1 variant with a maximum binding affinity of −16.57 kcal/mol. Similar to the RBD of BA.5 variant, the K9 Nb did not facilitate the direct CDR–RBM interaction. However, not only a large number of polar amino acids in framework 3 but also four amino acids in the CDR2 of the K9 Nb could interact with the RBD of JN.1 variant (Figure S8). Due to the interaction posture of the K9 Nb, the JN.1–K9 interaction exhibited a higher number of interface residues than BA.5–Nbs, JN.1–1.29, and JN.1–C11. The interface area of JN.1 variant was notably larger than that of BA.5 variant. In contrast, the interface area of the K9 Nb on JN.1 variant was smaller than that of BA.5 variant (Figure S6). The K9 Nb provided 2 salt bridge interactions, similar to BA.5–K9. Remarkably, polar side chain amino acids, including Gln13, Ser17, Arg19, Thr58, Arg67, Thr69, Asp73, Asn84, and Ser85, interacted extensively with the RBD of JN.1 variant via hydrogen bond interactions, exceeding those observed in BA.5–K9 (Figures S6 and S8). Moreover, this prediction illustrated that the polar amino acids on the K9 Nb obviously interacted with key polar amino acids of JN.1 variant, including Ser17(K9)–Asn405(JN.1), Arg72(K9)–Lys460(JN.1), Asn57(K9)–Asn477(JN.1), and Asp73(K9)–Lys460(JN.1).

3. Conclusions

Nbs hold promise across various fields such as basic research, therapeutics, diagnostics, and biotechnology [30,31,32]. Several selection technologies that have been used for mAbs or other types of antibody fragments have been applied to identify Nbs specific to a desired target from a library [33]. The aim of this study is to present a combination of methods that can be utilized to alter an existing antigen-specific Nb to bind with the target harboring mutations. In our study, we were able to redirect the binding affinity of the 1.29 Nb toward the RBD of BA.5 variant by employing error-prone PCR and FLI-TRAP techniques. The protein sequence of SARS-CoV-2 spike RBD (amino acids R319–F541 of the S protein) from the Omicron subvariant BA.5 is based on wild-type SARS-CoV-2 with 17 mutations (see Figure S9), the same mutations identified in the RBD domain of the BA.4 and BA.5.2 variants. Unsurprisingly, even the broad-spectrum 1.29 Nb, which was reported to bind some earlier VOCs, such as the Alpha, Gamma, Beta, and Delta variants (containing 1, 3, 3, and 2 mutations in the RBD region, respectively), was unable to bind BA.5 variant. Our methods identified 2 Nbs, C11 and K9, that demonstrated significantly improved binding to the RBD of the Omicron subvariant BA.5 compared to the prototype 1.29 Nb.
In the case of the K9 Nb, we reduced the amount of template from 5 ng to 1 ng, hoping to induce a higher mutation rate. Interestingly, FLI-TRAP was able to identify such an Nb with a unique sequence. Instead of adding point mutations to the template, the C11 Nb, the K9 Nb contains very different CDR1 and CRD3 sequences, as well as elongation in the CDR3. We hypothesize that replication slippage occurred during the mutation process, which could have caused insertion or deletion. Replication slippage refers to occasional slips or stutters of DNA polymerase during replication, leading to the insertion or deletion of nucleotides in the newly synthesized DNA strand. Hence, this phenomenon is a natural source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. An in vitro study of this phenomenon was conducted on different thermostable DNA polymerases, such as those from Thermus aquaticus (Taq pol), Thermococcus litoralis (Vent® pol), and Pyrococcus furiosus (Pfu pol) [17]. The manufacturer’s manual for the GeneMorph II Random Mutagenesis Kit indicates that it employs Mutazyme II polymerase, which consists of 2 error-prone DNA polymerases, i.e., Mutazyme I DNA polymerase and an engineered Taq DNA polymerase variant. In fact, the manual also reported 0.7% insertion and 4.8% deletion rates when using Mutazyme II polymerase. When designing mutations or generating diversity in the Nb sequence, especially through techniques like error-prone PCR or site-directed mutagenesis, DNA polymerase may occasionally insert or delete nucleotides, resulting in sequences with altered lengths or structures [34,35]. CDRs often exhibit higher variability and sometimes possess repetitive motifs or sequences prone to replication slippage [34,35].
Antigen-binding assays, ELISA and MST, indicated significantly higher affinities of both the C11 and K9 Nbs to the Omicron BA.5 variant compared to the 1.29 Nb. It should be noted that RBD variants used for these assays were commercially available and produced in mammalian cells so they should possess native structure. A previous study demonstrated that RBD produced in E. coli produced significantly lower ELISA signals if the antibody binds near a glycosite [36]. Molecular docking was utilized to predict the binding patterns and residues involved in these interactions and to elucidate the structural modifications induced by mutations on Nbs and their effects on binding affinity, supporting the experimental findings: 1) Nb-antigen binding through FLI-TRAP resulting in cell resistance to carbenicillin as shown by the spot-plate method, 2) in vitro binding activity as shown by ELISA, and 3) determination of Nb’s affinity for RBDs using MST. Even though two mutations of C11 are not on the CDRs, they provide structural modification such that more salt bridges can form between BA.5-C11. A previous study reported that salt bridges and hydrogen bonding interactions significantly enhanced the stability of the binding between synthetic Nbs and the flexible RBD loop [37]. As for K9 Nb, various point mutations as well as large differences in CDR1 and CDR3, facilitated interactions with more contact residues, enabling specific residues to interact with key amino acids of the RBD of BA.5 variant. Furthermore, prediction using molecular docking revealed that the K9 Nb binds to the RBD of JN.1 variant with the highest affinity. As SARS-CoV-2 is still rapidly evolving, as of August 20, 2024, the SARS-CoV-2 Omicron variants KP.2, KP.2.3, KP.3, KP.3.1.1, and LB.1, all descendants of the JN.1 variant, have a high prevalence in the United States. [38]
It should be noted that the binding of Nb to RBD is complex. It is mediated by a large number of interactions that remodel the way protein complexes react to molecular forces in cellular environments. Cofas-Vargas et al. [39]. demonstrated the mechanical stability of mutated nanobodies that interact with the RBD of the Delta and XBB.1.5 variants under simulated biophysical conditions, using molecular dynamics simulations. They reported that the transition from the bound to unbound state occurs without unfolding, driven by a balance of forces that both strengthen and weaken the protein interface. Many of these forces were nonpolar, highlighting the importance of hydrophobic interactions in maintaining the stability of the complex. Their finding strongly supports our results, which also show that nonpolar and aromatic amino acids at the contact surface between the RBD and the nanobody play a key role in stabilizing the interaction. Further study should be performed to investigate interactions between the obtained Nbs and RBD variants under biophysical conditions, similar to the study performed by Ray et al. [40], using spike protein expressed on a mammalian cell line.
In this study, we demonstrated the use of directed evolution by error-prone PCR to create mutants and the FLI-TRAP method to facilitate the isolation of Nbs that bind to a closely related specific target by addressing a viral variant with several mutations. The FLI-TRAP selection method offers a dual advantage, as the selection is based on the Nb’s function, thus assessing its solubility and binding affinity simultaneously, guaranteeing that both characteristics are preserved. Recently, another clever method called ‘autonomous hypermutation yeast surface display’ (AHEAD) was developed for the molecular evolution of Nbs [41]. It utilizes yeast cells to mimic vertebrate somatic hypermutation by combining orthogonal DNA replication (OrthoRep) with yeast surface display (YSD) for rapid antibody evolution. The process involves an orthogonal error-prone DNA polymerase that replicates a special cytosolic plasmid (p1) encoding antibody fragments, thus allowing yeast cells to autonomously diversify YSD Nbs as they are cultured. High-affinity, high-quality Nbs were isolated through several cycles of yeast growth and fluorescence-activated cell sorting (FACS)-based selection. The main advantage of the AHEAD method is that the yeast cells can self-diversify Nb sequences. Since in vitro library construction is not required, several Nbs can be easily evolved at the same time. However, the selection method requires a flow cytometer with sorting capabilities, which is expensive and requires skilled operators [42]. Although our method requires in vitro mutated library construction, the in vivo selection method is straight forward, low-cost, and can be performed in most molecular biology laboratories. We expect that our strategy will be valuable in developing Nbs for various applications.
Unfortunately, the SARS-CoV-2 virus has proven to be difficult to combat due to its very fast mutation rate. Future uses of FLI-TRAP may involve facilitation of the redirection of the scFv of existing antibodies to target proteins that are susceptible to autonomous mutation, such as cancer targets. Some cancer-targeted antibody drugs are no longer potent to a specific cancer due to mutation of the cancer drug target, such as epidermal growth factor receptor (EGFR). An S492R mutation in the extracellular domain of EGFR was observed in a colorectal cancer cell line, rendering it resistant to cetuximab. This mutation hinders the antibody’s ability to bind to the receptor and has also been identified in some patients experiencing relapse after cetuximab treatment [43]. The extensive array of mutations found in various cancers represents an unexplored pool of targets that could prove valuable for therapeutic interventions with highly specific reagents tailored to each mutation, paving the way toward precision medicine.

4. Materials and Methods

4.1. Bacterial Strains and Growth Conditions

E. coli strain NEB10β (New England Biolabs, Ipswich, MA, USA) was used for plasmid and library construction as well as Nb selection experiments. Spot plating for identification of library selection conditions and selective plating for FLI-TRAP selection were performed using NEB10β, as described by Waraho and DeLisa [10,11]. Wild-type E. coli strain MC4100 [44] and DADE [45], the isogenic ΔtatABCDΔtatE derivative of MC4100, were used in a spot plating assay to confirm the Tat transport. Bacterial cells co-transformed with pDD322-Kan TatABC and pDD18-Cm RBD-Bla::ssTorA-1.29 Nb-FLAG or pDD18-Cm RBD-Bla::ssTorA-C11 Nb-FLAG were used for identification of library selection conditions by spot plating, while pDD18-Cm RBD-Bla::ssTorA-Nb library-FLAG was used for library selection by spreading cells on selective plates. The introduction of the pDD322-Kan TatABC vector was supposed to increase the number of Tat channels present on the cell membrane, thereby enhancing the export of the protein complex [10]. Cells were grown overnight at 37 °C in LB medium supplemented with 25 µg/mL chloramphenicol (Cm) and 50 µg/mL Kanamycin (Kan). The next day, overnight cells of each sample were normalized in fresh LB to OD600 = 2.5. For spot plating, 5 µL of serially diluted cells were spotted onto LB agar plates supplemented with 1.0% arabinose and varying amounts of Carb (0–200 µg/mL). For Nb library selection, 100 µL of dilution 10-fold higher than the condition identified for library selection [11] was spread onto LB agar plates supplemented with 1.0% arabinose and varying amounts of Carb (50–200 µg/mL), as indicated. Plated bacteria were incubated at 25 °C for 48 h. For Nb purification, pET-28a Nb-FLAG-His plasmids were transformed into E. coli strain BL21(DE3) (Novagen, Madison, WI, USA). Overnight cultures were subcultured in 1 L of LB medium supplemented with 50 µg/mL Kan, and protein expression was induced when OD600 reached 0.6–0.7 with isopropyl β-D-1-thiogalactopyranoside (1 mM) at 25 °C with 200 RPM agitation for 16 h. The details of strains and plasmids used in this study are included in Table S2.

4.2. Plasmid Construction

The protein sequence of the 1.29 Nb was obtained from Casanovas et al. [9]. The protein sequence of the SARS-CoV-2 spike RBD (amino acids R319–F541 of the S protein) from the Omicron subvariant BA.5 is based on wild-type SARS-CoV-2 (YP_009724390.1) with G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, and Y505H mutations. The DNA sequences of both proteins were synthesized with codon optimization for E. coli expression by GenScript, Inc. (Piscataway, NJ, USA). Both genes were then cloned into the pDD18-Cm bicistronic plasmid to create pDD18-Cm RBD-Bla::ssTorA-1.29 Nb-FLAG. The sequences of all constructs were verified using Sanger method (U2Bio Co., Ltd., Seoul, South Korea).
Error-prone mutations were introduced to the 1.29 or C11 Nb genes by PCR amplification using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) with the 1.29 Nb XbaI forward primer (5′-GCGATGTCTAGACAGGTGCAACTGGTTGAG-3′) and the 1.29 Nb SalI reverse primer (5′-GCGATGGTCGACGCTGCTAACGGTCACTTG-3′). For the library for the first round of mutation, PCRs were performed for 35 cycles with 5 ng pDD18-Cm RBD-Bla::ssTorA-1.29 Nb-FLAG (containing the 1.29 Nb template) in each reaction. The resulting PCR products were cloned into the XbaI and SalI cut sites of pDD18-Cm RBD-Bla::ssTorA-1.29 Nb-FLAG. Ligation mixtures were transformed into NEB10β cells using electroporation. Cells were retrieved from an electroporation cuvette by resuspending in 1 mL LB medium. Cells from 10 cuvettes were pooled together into a 250-mL shake flask and allowed to recover at 37 °C with 200 RPM agitation for 1 h. To determine the library size, spread plate method was used. Briefly, 100 µL of 10−2–10−4 serially diluted cells were spread onto LB agar plates supplemented with 25 µg/mL Cm and incubated at 37 °C for 24 h. After incubation, the numbers of colonies appeared on the plates containing colonies between 20–300 CFU/mL were counted and used for library size calculation. To evaluate the library quality, 30 colonies were randomly picked, miniprepped and submitted to sequencing analysis by Sanger method (U2Bio Co., Ltd.) with sequencing primer, ssTorA for (5′-AACAATAACGATCTCTTTCAGGC-3′), which binds to the Tat signal peptide of TorA at the C-terminus of Nbs. The error rate determination was reported by counting the number of DNA point mutations observed in each sequenced Nb gene obtained from the library prior to subjected to FLI-TRAP isolation. A similar strategy was used to construct the library for the second round of mutation; however, to increase the mutation rate, 35 cycles of PCR were performed with 1 ng pDD18-Cm RBD-Bla::ssTorA-C11 Nb-FLAG instead.
For cytoplasmic expression of Nbs without the ssTorA signal peptide, the 1.29, C11, and K9 Nb genes were PCR amplified from pDD18-Cm and cloned into the pET-28a anti-GCN4-FLAG-His [46] plasmid between the NcoI and SalI cut sites using the 1.29 Nb NcoI forward primer (5′-GCGATGCCATGGGCCAGGTGCAACTGGTTGAG-3′) and the 1.29 Nb SalI reverse primer (5′-GCGATGGTCGACGCTGCTAACGGTCACTTG-3′), resulting in pET-28a Nb-FLAG-His.

4.3. Western Blot Analysis

The induced cells were harvested by centrifugation and normalized to OD600 = 75 then resuspended with 300 µL phosphate-buffered saline (PBS)., The samples were sonicated on ice using Sonifier® SFX150 cell disruptors and homogenizers (Branson, Danbury, CT, USA) 3 times at 30 s intervals with 45% amplitude, 50% duty cycle and centrifugation at 10,000 RCF for 10 min. The supernatant containing soluble proteins was collected as the whole-cell lysate. The protein samples were mixed with 6× loading dye containing a reducing agent (β-mercaptoethanol) and incubated at 100 °C for 10 min to denature the proteins. For samples from pDD18-Cm plasmids, 5 µg (total protein) of the samples was loaded onto 10% polyacrylamide gels (TGX FastCast Acrylamide Solutions, Bio-Rad, Hercules, CA, USA), and western blotting was performed according to standard protocols. The PVDF membranes were probed with Mouse Anti-DDDDK tag Monoclonal Antibody, HRP Conjugated, Clone M2 (1:3000; Abcam Cat# ab49763, RRID:AB_869428) to detect FLAG-tagged Nbs. Gel images were captured by the Gel Doc EZ Gel Documentation System (Bio-Rad), and densitometry analysis was performed using Image Lab software version 5.0 (Bio-Rad).

4.4. Nb Purification

For batch-mode Nb purification, BL21(DE3) cells producing the 1.29, C11, and K9 Nbs were pelleted and resuspended in binding buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, 0.5% v/v Triton-X100, pH 8), sonicated on ice using Sonifier® SFX150 cell disruptors and homogenizers (Branson) 3 times at 30 s intervals with 45% amplitude, 50% duty cycle. The samples were then centrifuged and loaded onto Econo-column® chromatography columns (Bio-Rad) packed with Ni–NTA beads (Macherey-Nagel, Düren, Germany) that were equilibrated by washing 3 times with 10 mL of His-flow buffer prior to sample loading. Binding was allowed to continue at 4 °C for 1 h; subsequently, the lysate was allowed to flow through via gravity. The column was washed 3 times with 20 mL washing buffer (His-flow buffer with 20 mM imidazole). Purified Nbs were eluted using elution buffer (His-flow buffer with 120 mM imidazole). After purification, the elutions were pooled, buffer-exchanged with PBS, partially purified to remove high molecular weight impurities, and concentrated using 30 kDa and 3 kDa MWCO columns (Amicon Ultra-0.5 centrifugal filter unit, Merck Millipore, Burlington, MA, USA). The concentration of each concentrated and purified Nb was measured using a Bradford assay (Quick Start Bradford, Bio-Rad). To assess protein purity, 5 µL of elutions and 2 µL of concentrates of each sample were mixed with 6× loading dye containing a reducing agent (β-mercaptoethanol) and incubated at 100 °C for 10 min to denature the proteins. The protein samples were loaded onto 10% polyacrylamide gels (TGX FastCast Acrylamide Solutions, Bio-Rad) and subjected to gel electrophoresis followed by staining with InstantBlue® Coomassie Protein Stain (Abcam, Cambridge, UK) for 10 min. Gel imaging was conducted using the Gel Doc EZ Gel Documentation System (Bio-Rad).

4.5. Analysis of Nb–Antigen Binding Using ELISA

ELISA was performed according to standard protocols. Briefly, ELISA plates (SpectraPlateTM) were coated overnight with 4 µg/mL purified RBD variants (Sino Biological, Beijing, China) diluted in ELISA coating buffer (50 mM carbonate-bicarbonate buffer, pH 9.6) at 4 °C. The plates were washed with washing buffer (PBS containing 0.05% Tween20) and blocked overnight with 5% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad) at 4 °C. The next morning, the plates were washed, and 50 µL of 40 µg/mL purified Nbs were added to each well in triplicate and incubated at 37 °C for 1 h. Following the incubation, the plates were washed and incubated with Mouse Anti-DDDDK tag Monoclonal Antibody, HRP Conjugated, Clone M2 (1:3000; Abcam Cat# ab49763, RRID:AB_869428) at 37 °C for 1 h. Finally, after 3 washes, 100 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) (Abcam) was added to each well. The reaction was quenched using 1 M H2SO4, and the absorbance of the wells was measured at 450 nm (Infinite M200, Tecan Austria GmbH, Grödig, Austria).

4.6. Analysis of Nb–Antigen Binding Using MST

MST was used to determine the affinity (KD) between the 1.29, C11, and K9 Nbs and the RBD of the SARS-CoV-2 variants (Delta, BA.5, XBB.1.5, and XBB.1.16; Sino Biological). First, 3 µM of purified Nbs were labeled with RED-NHS dye (Monolith Protein Labeling Kit, NanoTemper Technologies, Munich, Germany). Labeled Nbs were then mixed with 10 uM RBD proteins in a serial 16-step and 2-fold dilution in PBS containing 0.01% Pluronic® F-127. The mixtures were separately loaded into 16 premium glass capillaries (NanoTemper Technologies) and placed in the reaction chamber. The measurements were performed using a Monolith NT.115 instrument (NanoTemper Technologies) at 25 °C using 30% excitation power. The KD values were calculated using the MO Affinity Analysis software version 3 (NanoTemper Technologies).

4.7. Calculation of Physicochemical Properties of Nbs

Sequence similarity was calculated by the Clustal Omega Multiple Sequence Alignment (MSA) online server (https://www.ebi.ac.uk/jdispatcher/msa/clustalo, accessed on 23 February 2024). The calculated physicochemical characteristics of the 1.29, C11, and K9 Nbs, such as amino acid contact surface, chemical interaction, and overall physical properties, were assessed using the PDBsum server (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html, accessed on 25 February 2024) [47]. The goal of this evaluation was to analyze the intermolecular interactions between the Nbs and RBDs. The number of amino acids, GRAVY score, aliphatic index, and instability index were predicted using the ProtParam (ExPASy) tool (https://web.expasy.org/protparam/, accessed on 25 February 2024) [48]. The Protein–Sol web server (https://protein-sol.manchester.ac.uk/, accessed on 25 February 2024) was employed to predict protein solubility. PROTEIN CALCULATOR v3.4 (https://protcalc.sourceforge.net/, accessed on 25 February 2024) [49] was utilized to calculate the molecular weight, pI value, charge at pH 7.4, and molar extinction coefficient.

4.8. 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: 7ZXU) and the original 1.29 Nb (PDB ID: 7R4Q) were obtained from the PDB Protein Data Bank (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, 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 ff14SB 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/, accessed on 26 February 2024) [22,23]. Subsequently, RBD–Nb complexes were energetically minimized by the AMBER ff14SB 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, 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. (Suwitchaya Sirimanakul) and D.W.-Z.; validation, K.I., P.L., W.K. and S.S. (Supaphron Seetaha); 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. (Suwitchaya Sirimanakul); 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.), King Mongkut’s University of Technology Thonburi (KMUTT), Thailand Science Research and Innovation (TSRI), and the National Science, Research and Innovation Fund (NSRF) Fiscal year 2024 Grant number FRB670016/0164 and Fiscal year 2025 under the project Innovation and Advanced Technology for Future Construction with Sustainability (to D.W.-Z.), the Graduate Development Scholarship 2022, National Research Council of Thailand under grant number 173926 (to K.I.), and a Petchra Pra Jom Klao Master’s Degree Scholarship from King Mongkut’s University of Technology Thonburi under grant number 16/2563 (to K.I.).

Institutional Review Board Statement

The study was approved by the Institutional Biosafety Committee of King Mongkut’s University of Technology (IBC-2023-017).

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this article (and its Supplementary Information).

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. The FLI-TRAP selection system. Schematic representation of the overall process used in this study. IM: inner membrane; TatABC: Twin-arginine translocation (Tat) ABC complex; TEM-1 Bla: Temoniera β-lactamase; Nb: nanobody; Carb: carbenicillin; RBD: receptor binding domain; FLI-TRAP: Tat-based recognition of associating proteins.
Figure 1. The FLI-TRAP selection system. Schematic representation of the overall process used in this study. IM: inner membrane; TatABC: Twin-arginine translocation (Tat) ABC complex; TEM-1 Bla: Temoniera β-lactamase; Nb: nanobody; Carb: carbenicillin; RBD: receptor binding domain; FLI-TRAP: Tat-based recognition of associating proteins.
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Figure 2. Redirecting the 1.29 Nb to bind the RBD of BA.5 variant using FLI-TRAP. (A) Spot titers of serially diluted NEB10β cells used for the determination of library selection conditions using FLI-TRAP. (B) Spot titers of serially diluted NEB10β cells co-expressing TatABC with RBD BA.5-Bla and the 1.29 Nb or library-selected clones C1–C15 derived from the 1.29 Nb. (C) Spot titers of serially diluted NEB10β, MC4100, and DADE cells co-expressing TatABC with RBD BA.5-Bla and the 1.29 or K9 Nbs.
Figure 2. Redirecting the 1.29 Nb to bind the RBD of BA.5 variant using FLI-TRAP. (A) Spot titers of serially diluted NEB10β cells used for the determination of library selection conditions using FLI-TRAP. (B) Spot titers of serially diluted NEB10β cells co-expressing TatABC with RBD BA.5-Bla and the 1.29 Nb or library-selected clones C1–C15 derived from the 1.29 Nb. (C) Spot titers of serially diluted NEB10β, MC4100, and DADE cells co-expressing TatABC with RBD BA.5-Bla and the 1.29 or K9 Nbs.
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Figure 3. Nb production. Western blot analysis of the Nbs expressed in the two systems, as indicated.
Figure 3. Nb production. Western blot analysis of the Nbs expressed in the two systems, as indicated.
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Figure 4. Binding activities of parental and evolved Nbs to various RBD variants observed using ELISA. Binding activity from normalized purified Nbs was measured by ELISA with microtiter plates coated with commercial RBD variants produced in HEK293 cells. Detection was with Mouse Anti-DDDDK tag Monoclonal Antibody, HRP Conjugated, Clone M2 (1:3000; Abcam Cat# ab49763, RRID:AB_869428). Different superscript letters (a, b, c) indicate significant differences (p < 0.05) between groups assessed by ANOVA followed by Duncan’s test for multiple comparisons. Data are the average of biological replicates, N = 3; mean ± SD.
Figure 4. Binding activities of parental and evolved Nbs to various RBD variants observed using ELISA. Binding activity from normalized purified Nbs was measured by ELISA with microtiter plates coated with commercial RBD variants produced in HEK293 cells. Detection was with Mouse Anti-DDDDK tag Monoclonal Antibody, HRP Conjugated, Clone M2 (1:3000; Abcam Cat# ab49763, RRID:AB_869428). Different superscript letters (a, b, c) indicate significant differences (p < 0.05) between groups assessed by ANOVA followed by Duncan’s test for multiple comparisons. Data are the average of biological replicates, N = 3; mean ± SD.
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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.
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.
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Figure 6. A comparison of amino acid interactions between the RBD of BA.5 variant and Nbs. (A) The RBD of BA.5 variant with the 1.29 Nb, (B) the RBD of BA.5 variant with the C11 Nb, and (C) the RBD of BA.5 variant with the K9 Nb.
Figure 6. A comparison of amino acid interactions between the RBD of BA.5 variant and Nbs. (A) The RBD of BA.5 variant with the 1.29 Nb, (B) the RBD of BA.5 variant with the C11 Nb, and (C) the RBD of BA.5 variant with the K9 Nb.
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Figure 7. A comparison of amino acid interactions between the RBD of JN.1 variant and Nbs. (A) The RBD of JN.1 variant with the 1.29 Nb, (B) the RBD of JN.1 variant with the C11 Nb, and (C) the RBD of JN.1 variant with the K9 Nb.
Figure 7. A comparison of amino acid interactions between the RBD of JN.1 variant and Nbs. (A) The RBD of JN.1 variant with the 1.29 Nb, (B) the RBD of JN.1 variant with the C11 Nb, and (C) the RBD of JN.1 variant with the K9 Nb.
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Table 1. KD values of the parental and isolated Nbs toward various RBD variants.
Table 1. KD values of the parental and isolated Nbs toward various RBD variants.
RBD VariantsKD (nM)
1.29C11K9
Delta19.3, 95% CI [6.4, 58.2]878, 95% CI [109, 7097]1510, 95% CI [880, 2600]
BA.51390, 95% CI [60, 3430]326, 95% CI [90, 1185]29.9, 95% CI [23.9, 37.5]
XBB.1.54090, 95% CI [940, 17,870]569, 95% CI [155, 2081]1150, 95% CI [570, 9150]
XBB.1.16448, 95% CI [255, 788]251, 95% CI [109, 578]213, 95% CI [126, 360]
Table 2. Summary of the physicochemical properties of Nbs.
Table 2. Summary of the physicochemical properties of Nbs.
Physicochemical PropertiesNb
1.29C11K9
Number of amino acids121121126
Molecular weight (g·mol−1)12,802.92612,779.8613,673.942
Charge at pH 7.4−1.4−3.4−3.4
pI5.314.774.93
GRAVY−0.378−0.378−0.513
Solubility0.6150.70.628
Aliphatic index62.8962.8965.79
Instability index18.5718.5724.99
Molar extinction (M−1·cm−1)23,02023,02025,580
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Intasurat, K.; Submunkongtawee, N.; Longsompurana, P.; Thaiprayoon, A.; Kasemsukwimol, W.; Sirimanakul, S.; Boonsilp, S.; Seetaha, S.; Choowongkomon, K.; Waraho-Zhmayev, D. Redirecting a Broad-Spectrum Nanobody Against the Receptor-Binding Domain of SARS-CoV-2 to Target Omicron Variants. Appl. Sci. 2024, 14, 10548. https://doi.org/10.3390/app142210548

AMA Style

Intasurat K, Submunkongtawee N, Longsompurana P, Thaiprayoon A, Kasemsukwimol W, Sirimanakul S, Boonsilp S, Seetaha S, Choowongkomon K, Waraho-Zhmayev D. Redirecting a Broad-Spectrum Nanobody Against the Receptor-Binding Domain of SARS-CoV-2 to Target Omicron Variants. Applied Sciences. 2024; 14(22):10548. https://doi.org/10.3390/app142210548

Chicago/Turabian Style

Intasurat, Kwanpet, Nonth Submunkongtawee, Phoomintara Longsompurana, Apisitt Thaiprayoon, Warisara Kasemsukwimol, Suwitchaya Sirimanakul, Siriphan Boonsilp, Supaphron Seetaha, Kiattawee Choowongkomon, and Dujduan Waraho-Zhmayev. 2024. "Redirecting a Broad-Spectrum Nanobody Against the Receptor-Binding Domain of SARS-CoV-2 to Target Omicron Variants" Applied Sciences 14, no. 22: 10548. https://doi.org/10.3390/app142210548

APA Style

Intasurat, K., Submunkongtawee, N., Longsompurana, P., Thaiprayoon, A., Kasemsukwimol, W., Sirimanakul, S., Boonsilp, S., Seetaha, S., Choowongkomon, K., & Waraho-Zhmayev, D. (2024). Redirecting a Broad-Spectrum Nanobody Against the Receptor-Binding Domain of SARS-CoV-2 to Target Omicron Variants. Applied Sciences, 14(22), 10548. https://doi.org/10.3390/app142210548

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