Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

AbstractSingle-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

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Selection of Single-Domain Antibodies towards Western Equine Encephalitis Virus

Antibodies ◽

10.3390/antib7040044 ◽

2018 ◽

Vol 7 (4) ◽

pp. 44 ◽

Cited By ~ 2

Author(s):

Jinny Liu ◽

Lisa Shriver-Lake ◽

Dan Zabetakis ◽

Ellen Goldman ◽

George Anderson

Keyword(s):

Phage Display Library ◽

Peripheral Blood Lymphocytes ◽

Encephalitis Virus ◽

Single Domain ◽

Equine Encephalitis Virus ◽

Western Equine Encephalitis Virus ◽

Recognition Elements ◽

Single Domain Antibodies ◽

Western Equine Encephalitis

In this work, we describe the selection and characterization of single-domain antibodies (sdAb) towards the E2/E3E2 envelope protein of the Western equine encephalitis virus (WEEV). Our purpose was to identify novel recognition elements which could be used for the detection, diagnosis, and perhaps treatment of western equine encephalitis (WEE). To achieve this goal, we prepared an immune phage display library derived from the peripheral blood lymphocytes of a llama that had been immunized with an equine vaccine that includes killed WEEV (West Nile Innovator + VEWT). This library was panned against recombinant envelope (E2/E3E2) protein from WEEV, and seven representative sdAb from the five identified sequence families were characterized. The specificity, affinity, and melting point of each sdAb was determined, and their ability to detect the recombinant protein in a MagPlex sandwich immunoassay was confirmed. Thus, these new binders represent novel recognition elements for the E2/E3E2 proteins of WEEV that are available to the research community for further investigation into their applicability for use in the diagnosis or treatment of WEE.

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Transforming nanobodies into high precision tools for protein function analysis.

AJP Cell Physiology ◽

10.1152/ajpcell.00435.2020 ◽

2020 ◽

Author(s):

Jan Gettemans ◽

Brian De Dobbelaer

Keyword(s):

Protein Function ◽

Function Analysis ◽

High Specificity ◽

Single Domain ◽

Antigen Receptors ◽

High Level Expression ◽

Car T ◽

Experimental Approaches ◽

Single Domain Antibodies ◽

High Level

Single domain antibodies, derived from camelid heavy antibodies (nanobodies®) or shark variable new antigen receptors, have attracted increasing attention in recent years due to their extremely versatile nature and opportunities they offer for downstream modification. Discovered more than three decades ago, these 120 amino acid (~15kDa) antibody fragments are known to bind their target with high specificity and affinity . Key features of nanobodies that make them very attractive include their single domain nature, small size, affordable high level expression in prokaroytes, and their cDNAs are routinely obtained in the process of their isolation. This facilitates and stimulates new experimental approaches. Hence, it allows researchers to formulate new answers to complex biomedical questions. Through elementary PCR-based technologies and chemical modification strategies, their primary structure can be altered almost at leisure whilst retaining their specificity and biological activity, transforming them into highly tailored tools that meet the increasing demands of current day biomedical research. In this review, various aspects of camelid Nanobodies are expounded, including intracellular delivery in recombinant format for manipulation of i.e. cytoplasmic targets, their derivatization to improve nanobody orientation as a capturing device, approaches to reversibly bind their target, their potential as protein silencing devices in cells, the development of strategies to transfer nanobodies through the blood brain barrier and their application in CAR-T experimentation. We also discuss some of their disadvantages and conclude with future prospects.

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Brain Delivery of Single-Domain Antibodies: A Focus on VHH and VNAR

Pharmaceutics ◽

10.3390/pharmaceutics12100937 ◽

2020 ◽

Vol 12 (10) ◽

pp. 937 ◽

Cited By ~ 1

Author(s):

Elodie Pothin ◽

Dominique Lesuisse ◽

Pierre Lafaye

Keyword(s):

Blood Brain Barrier ◽

Heavy Chain ◽

Single Domain ◽

Brain Delivery ◽

Therapeutic Antibodies ◽

Antigen Receptors ◽

Brain Penetration ◽

Passive Immunotherapy ◽

Single Domain Antibodies ◽

The Brain

Passive immunotherapy, i.e., treatment with therapeutic antibodies, has been increasingly used over the last decade in several diseases such as cancers or inflammation. However, these proteins have some limitations that single-domain antibodies could potentially solve. One of the main issues of conventional antibodies is their limited brain penetration because of the blood–brain barrier (BBB). In this review, we aim at exploring the different options single-domain antibodies (sDAbs) such as variable domain of heavy-chain antibodies (VHHs) and variable new antigen receptors (VNARs) have already taken to reach the brain allowing them to be used as therapeutic, diagnosis or transporter tools.

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Isolation, characterization and pentamerization of α-cobrotoxin specific single-domain antibodies from a naïve phage display library: Preliminary findings for antivenom development

Toxicon ◽

10.1016/j.toxicon.2006.11.023 ◽

2007 ◽

Vol 49 (5) ◽

pp. 699-709 ◽

Cited By ~ 41

Author(s):

Christine S. Stewart ◽

C. Roger MacKenzie ◽

J. Christopher Hall

Keyword(s):

Phage Display ◽

Phage Display Library ◽

Single Domain ◽

Single Domain Antibodies

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Targeting conserved viral virulence determinants by single domain antibodies to block SARS-CoV2 infectivity

10.1101/2021.01.13.426537 ◽

2021 ◽

Author(s):

Sudhakar Singh ◽

Surbhi Dahiya ◽

Yuviana J. Singh ◽

Komal Beeton ◽

Ayush Jain ◽

...

Keyword(s):

Phage Display Library ◽

Proteolytic Processing ◽

Single Domain ◽

Virus Infectivity ◽

Virulence Determinants ◽

Characteristic Structure ◽

S Protein ◽

Viral Virulence ◽

Single Domain Antibodies ◽

Ph Range

AbstractWe selected SARS-CoV2 specific single domain antibodies (sdAbs) from a previously constructed phage display library using synthetic immunogenic peptides of the virus spike (S) protein as bait. The sdAbs targeting the cleavage site (CS) and the receptor binding domain (RBD) in S protein efficiently neutralised the infectivity of a pseudovirus expressing SARS-CoV2 S protein. Anti-CS sdAb blocked the virus infectivity by inhibiting proteolytic processing of SARS-CoV2 S protein. Both the sdAbs retained characteristic structure within the pH range of 2 to 12 and remained stable upto 65°C. Furthermore, structural disruptions induced by a high temperature in both the sdAbs were largely reversed upon their gradual cooling and the resulting products neutralised the reporter virus. Our results therefore suggest that targeting CS in addition to the RBD of S protein by sdAbs could serve as a viable option to reduce SARS-CoV2 infectivity and that proteolytic processing of the viral S protein is critical for infection.

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Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies

PLoS Pathogens ◽

10.1371/journal.ppat.1010169 ◽

2022 ◽

Vol 18 (1) ◽

pp. e1010169

Author(s):

Kwok-ho Lam ◽

Jacqueline M. Tremblay ◽

Kay Perry ◽

Konstantin Ichtchenko ◽

Charles B. Shoemaker ◽

...

Keyword(s):

Single Domain ◽

Protease Domain ◽

High Potency ◽

X Ray Crystallography ◽

Biochemical Assays ◽

Variable Heavy ◽

Botulinum Neurotoxins ◽

Single Domain Antibodies ◽

Vhh Antibodies ◽

And Function

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.

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