The identification of a novel class of shark‐derived single domain antibodies, named vnarbodies that show picomolar affinities binding to the receptor binding domain (RBD) of Wuhan and Alpha, Beta, Kappa, Delta, Delta‐plus, and Lambda variants, is reported. Vnarbody 20G6 and 17F6 have broad neutralizing activities against all these SARS‐CoV‐2 viruses as well as other sarbecoviruses, including Pangolin coronavirus and Bat coronavirus. Intranasal administration of 20G6 effectively protects mice from the challenges of SARS‐CoV‐2 Wuhan and Beta variants. 20G6 and 17F6 contain a unique “WXGY” motif in the complementary determining region 3 that binds to a hidden epitope on RBD, which is highly conserved in sarbecoviruses through a novel β‐sheet interaction. It is found that the S375F mutation on Omicron RBD disrupts the structure of β‐strand, thus impair the binding with 20G6. The study demonstrates that shark‐derived vnarbodies offer a prophylactic and therapeutic option against most SARS‐CoV‐2 variants and provide insights into antibody evasion by the Omicron variant.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/smtd.202200387

A Class of Shark‐Derived Single‐Domain Antibodies can Broadly Neutralize SARS‐Related Coronaviruses and the Structural Basis of Neutralization and Omicron Escape

Phage display enables to screen probes such as peptide, antibody and nanobody with high specificity and affinity to the target, providing excellent probes for various analytical or biosensing platforms. Furthermore, by employing techniques like as genetic engineering and chemical modification, it is possible to conveniently functionalize these ligands for analytical chemistry with selectivity and sensitivity. This review initially presents the fundamental aspects of phage biology that are commonly employed in phage display vectors, elaborates the construction process of phage display peptide library and phage display antibody library, and evaluates different biopanning strategies. Then from the viewpoint of analytes, this paper summarizes phage display based biosensing or bioanalysis of a wide range of target molecules. This paper presents knowledge insights and research foundations for the identification of additional phage display probes and their subsequent application in biosensing or bioanalysis with excellent performance. 

Phage display based biosensing: Recent advances and challenges

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is the major target for antibody therapeutics. Shark-derived variable domains of new antigen receptors (VNARs) are the smallest antibody fragments with flexible paratopes that can recognize protein motifs inaccessible to classical antibodies. This study reported four VNARs binders (JM-2, JM-5, JM-17, and JM-18) isolated from Chiloscyllium plagiosum immunized with SARS-CoV-2 RBD. Biolayer interferometry showed that the VNARs bound to the RBD with an affinity KD ranging from 38.5 to 2720 nM, and their Fc fusions had over ten times improved affinity. Gel filtration chromatography revealed that JM-2-Fc, JM-5-Fc, and JM-18-Fc could form stable complexes with RBD in solution. In addition, five bi-paratopic VNARs, named JM-2-5, JM-2-17, JM-2-18, JM-5-18, and JM-17-18, were constructed by fusing two VNARs targeting distinct RBD epitopes based on epitope grouping results. All these bi-paratopic VNARs except for JM-5-18 showed higher RBD binding affinities than its component VNARs, and their Fc fusions exhibited further enhanced binding affinities, with JM-2-5-Fc, JM-2-17-Fc, JM-2-18-Fc, and JM-5-18-Fc having KD values lower than 1 pM. Among these Fc fusions of bi-paratopic VNARs, JM-2-5-Fc, JM-2-17-Fc, and JM-2-18-Fc could block the angiotensin-converting enzyme 2 (ACE2) binding to the RBD of SARS-CoV-2 wildtype, Delta, Omicron, and SARS-CoV, with inhibition rates of 48.9~84.3%. Therefore, these high-affinity VNAR binders showed promise as detectors and therapeutics of COVID-19.

/pdf/screening-and-characterization-of-shark-derived-vnars-l4z1dn44.pdf

Screening and Characterization of Shark-Derived VNARs against SARS-CoV-2 Spike RBD Protein

The new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) has significantly altered people’s way of life. Despite widespread knowledge of vaccination, mask use, and avoidance of close contact, COVID-19 is still spreading around the world. Numerous research teams are examining the SARS-CoV-2 infection process to discover strategies to identify, prevent, and treat COVID-19 to limit the spread of this chronic coronavirus illness and restore lives to normalcy. Nanobodies have advantages over polyclonal and monoclonal antibodies (Ab) and Ab fragments, including reduced size, high stability, simplicity in manufacture, compatibility with genetic engineering methods, and lack of solubility and aggregation issues. Recent studies have shown that nanobodies that target the SARS-CoV-2 receptor-binding domain and disrupt ACE2 interactions are helpful in the prevention and treatment of SARS-CoV-2-infected animal models, despite the lack of evidence in human patients. The creation and evaluation of nanobodies, as well as their diagnostic and therapeutic applications against COVID-19, are discussed in this paper.

https://pubs.acs.org/doi/pdf/10.1021/acsptsci.3c00042

Emerging Landscape of Nanobodies and Their Neutralizing Applications against SARS-CoV-2 Virus

SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other β coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1096/fj.202202099RR

Identification of nurse shark <scp>VNAR</scp> single‐domain antibodies targeting the spike <scp>S2</scp> subunit of <scp>SARS‐CoV</scp>‐2

The continuously evolving Omicron subvariants has diminished the effectiveness of almost all RBD-targeted antibodies in neutralizing these subvariants. The development of broad-spectrum neutralizing antibodies is desired for addressing both current and future variants. Here, we identified a shark-derived nanobody, 79C11, that can neutralize all Omicron subvariants tested so far, including BA.1 to JN.1 and KP.2, and exhibits comparable neutralizing potency against SARS-CoV-1 and pangolin coronavirus. Intranasal instillation of 79C11 can effectively prevent the infection of Omicron subvariant XBB in vivo. The designs of multivalent forms of 79C11 further enhance binding and neutralizing activity. Epitope mapping and structure simulation reveal that this nanobody binds to a highly conserved HR1 region in S2 domain of the spikes from all sarbecoviruses, suggesting that a universal vaccine may be designed to target this region for eliciting broadly neutralizing antibody response. This nanobody can also be developed as an intranasally administered prophylactic agent for preventing the infection of current and likely future SARS-CoV-2 variants, as well as other animal derived sarbecoviruses that may infect humans.

A shark-derived broadly neutralizing nanobody targeting a highly conserved epitope on the S2 domain of sarbecoviruses

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A Class of Shark‐Derived Single‐Domain Antibodies can Broadly Neutralize SARS‐Related Coronaviruses and the Structural Basis of Neutralization and Omicron Escape

A shark-derived broadly neutralizing nanobody targeting a highly conserved epitope on the S2 domain of sarbecoviruses