Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein

Kim, Woo Sung; Chae, Hee Do; Jung, Inji; Lee, Won Kyu; Lee, Woo Jun; Lee, Jisun; Gong, Yang; Lee, Dohyun; Kim, Byeong-Won; Kim, J H; Hwang, Jaehyeon; Kweon, Dae-Hyuk; Jung, Sang Taek; Na, Jung-Hyun

doi:10.1016/j.fsi.2023.108807

Cited by 6 publications

(5 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Antibody titers increased in both sharks after a minimum of four doses and an 8-week immunization period. A recently published study on immunization of banded houndshark with SARS-CoV-2 RBD showed an increase in IgNAR antibody titers 11 weeks after three immunizations at 3-week intervals ( Kim et al, 2023 ). In contrast, it takes 5–6 months for antibody titers to rise in other shark families, such as nurse sharks, horn sharks, banded wobbegong sharks, and bamboo sharks ( Dooley and Flajnik, 2005 ; Camacho-Villegas et al, 2018 ; Leow et al, 2018 ; Wei et al, 2021 ; Gasperin-Bulbarela et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica)

Takeda,

Ozawa,

Zenke

et al. 2023

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

The VNAR (Variable New Antigen Receptor) is the smallest single-domain antibody derived from the variable domain of IgNAR of cartilaginous fishes. Despite its biomedical and diagnostic potential, research on VNAR has been limited due to the difficulties in obtaining and maintaining immune animals and the lack of research tools. In this study, we investigated the Japanese topeshark as a promising immune animal for the development of VNAR. This shark is an underutilized fishery resource readily available in East Asia coastal waters and can be safely handled without sharp teeth or venomous stingers. The administration of Venus fluorescent protein to Japanese topesharks markedly increased antigen-specific IgM and IgNAR antibodies in the blood. Both the phage-display library and the yeast-display library were constructed using RNA from immunized shark splenocytes. Each library was enriched by biopanning, and multiple antigen-specific VNARs were acquired. The obtained antibodies had affinities of 1 × 10−8 M order and showed high plasticity, retaining their binding activity even after high-temperature or reducing-agent treatment. The dissociation rate of a low-affinity VNAR was significantly improved via dimerization. These results demonstrate the potential utility of the Japanese topeshark for the development of VNAR. Furthermore, we conducted deep sequencing analysis to reveal the quantitative changes in the CDR3-coding sequences, revealing distinct enrichment bias between libraries. VNARs that were primarily enriched in the phage display had CDR3 coding sequences with fewer E. coli rare codons, suggesting translation machinery on the selection and enrichment process during biopanning.

show abstract

Section: Discussionmentioning

confidence: 99%

VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica)

Takeda,

Ozawa,

Zenke

et al. 2023

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

show abstract

“…Analysis of IgNARs from the banded houndshark (Triakis scyllium), production of a semi-synthetic VNAR library, and physicochemical analysis of isolated VNARs have been reported [20][21][22]. Kim et al immunized banded houndsharks with SARS-CoV-2 spike RBDs to produce antigen-specific VNARs [23]. We immunized Japanese topesharks with the Venus protein and reported an increase in the Venus-specific IgNAR antibody titer [24].…”

Section: Introductionmentioning

confidence: 88%

Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica)

Nakada-Masuta,

Takeda,

Uchida

2023

Marine Drugs

View full text Add to dashboard Cite

Diverse candidate antibodies are needed to successfully identify therapeutic and diagnostic applications. The variable domain of IgNAR (VNAR), a shark single-domain antibody, has attracted attention owing to its favorable physicochemical properties. The phage display method used to screen for optimal VNARs loses sequence diversity because of the bias caused by the differential ease of protein expression in Escherichia coli. Here, we investigated a VNAR selection method that combined panning with various selection pressures and next-generation sequencing (NGS) analyses to obtain additional candidates. Drawing inspiration from the physiological conditions of sharks and the physicochemical properties of VNARs, we examined the effects of NaCl and urea concentrations, low temperature, and preheating at the binding step of panning. VNAR phage libraries generated from Japanese topeshark (Hemitriakis japanica) were enriched under these conditions. We then performed NGS analysis and attempted to select clones that were specifically enriched under each panning condition. The identified VNARs exhibited higher reactivity than those obtained by panning without selection pressure. Additionally, they possess physicochemical properties that reflect their respective selection pressures. These results can greatly enhance our understanding of VNAR properties and offer guidance for the screening of high-quality VNAR clones that are present at low frequencies.

show abstract

“…In the most recent work on anti–SARS-CoV-2 VNARs, Kim et al. ( 46 ) report for the first time the immunization of banded houndshark ( Triakis scyllium ) for the isolation of VNAR-based therapeutics. Recombinant SARS-CoV-2 wild-type RBD protein was used to immunize the shark and construct a phage display immune library, which was then panned against the RBD as the target antigen during three rounds of selection.…”

Section: Broadly Neutralizing Shark-derived Single-domain Antibodies ...mentioning

confidence: 99%

“…This can be clearly seen in the number of publications to date on camelid-derived antibodies against SARS-CoV-2, compared with the publications on isolated VNARs against this virus. This greater inclination toward the study of camelid nanobodies is attributed to the limited availability of shark models, due to the difficulty of maintaining most reported shark species for VNAR isolation, in captivity (because of their large size and aggressive behavior) ( 46 , 54 ).…”

Section: Conclusion and Prospectsmentioning

confidence: 99%

“…It has been reported that VNARs can be isolated from libraries of immune ( 30 , 47 , 48 ), naïve ( 45 , 55 ), synthetic ( 41 ), or semi-synthetic origin ( 56 ) ( Figure 1 ). The diversity of the VNAR libraries screened in the reviewed works was 50% from natural immune sources ( 38 , 39 , 43 , 46 ), 37.5% from synthetic origin ( 34 , 35 , 40 ), and 12.5% from natural naïve (non-immunized) source ( 44 ). Whereas the immune libraries screened in the reviewed papers were constructed using similar protocols for immunization and amplification of the shark immune repertoire, the synthetic libraries screened were constructed by different methodologies.…”

Section: Conclusion and Prospectsmentioning

confidence: 99%

See 1 more Smart Citation

Unleashing the power of shark variable single domains (VNARs): broadly neutralizing tools for combating SARS-CoV-2

Cabanillas-Bernal,

Valdovinos-Navarro,

Cervantes-Luevano

et al. 2023

Front. Immunol.

View full text Add to dashboard Cite

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) generated a joint global effort to develop vaccines and other treatments that could mitigate the negative effects and the rapid spread of the virus. Single-domain antibodies derived from various sources, including cartilaginous fish, camelids, and humans, have gained attention as promising therapeutic tools against coronavirus disease 2019. Shark-derived variable new antigen receptors (VNARs) have emerged as the smallest naturally occurring antigen-binding molecules. Here, we compile and review recent published studies on VNARs with the capacity to recognize and/or neutralize SARS-CoV-2. We found a close balance between the use of natural immune libraries and synthetic VNAR libraries for the screening against SARS-CoV-2, with phage display being the preferred display technology for the selection of VNARs against this virus. In addition, we discuss potential modifications and engineering strategies employed to improve the neutralization potential of VNARs, such as exploring fusion with the Fc domain of human Immunoglobulin G (IgG) to increase avidity and therapeutic potential. This research highlights the potential of VNARs as powerful molecular tools in the fight against infectious diseases.

show abstract

Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein

Cited by 6 publications

References 24 publications

VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica)

VNAR development through antigen immunization of Japanese topeshark (Hemitriakis japanica)

Novel Approach for Obtaining Variable Domain of New Antigen Receptor with Different Physicochemical Properties from Japanese Topeshark (Hemitriakis japanica)

Unleashing the power of shark variable single domains (VNARs): broadly neutralizing tools for combating SARS-CoV-2

Contact Info

Product

Resources

About