scholarly journals A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

2020 ◽  
Author(s):  
Dianfan Li ◽  
Tingting Li ◽  
Hongmin Cai ◽  
Hebang Yao ◽  
Bingjie Zhou ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
In Vitro Selection ◽  
Binding Domain ◽  
Host Cells ◽  
Neutralization Activity ◽  
Rapid Responses ◽  
Therapeutic Development ◽  
Single Domain Antibodies

Abstract SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

scholarly journals A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

2020 ◽  
Author(s):  
Tingting Li ◽  
Hongmin Cai ◽  
Hebang Yao ◽  
Bingjie Zhou ◽  
Ning Zhang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Binding Domain ◽  
Host Cells ◽  
Neutralization Activity ◽  
Rapid Responses ◽  
Therapeutic Development ◽  
Single Domain Antibodies ◽  
Direct Delivery

ABSTRACTSARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22–7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8–18. Llama-derived single-domain antibodies (nanobodies, ∼15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL−1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL−1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

scholarly journals Therapeutic antibodies, targeting the SARS-CoV-2 spike N-terminal domain, protect lethally infected K18-hACE2 mice

2021 ◽  
Author(s):  
Tal Noy-Porat ◽  
Adva Mechaly ◽  
Yinon Levy ◽  
Efi Makdasi ◽  
Ron Alcalay ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Phage Display Library ◽  
Host Cells ◽  
Terminal Domain ◽  
Isolation And Characterization ◽  
Recent Emergence ◽  
Therapeutic Monoclonal Antibodies

AbstractSince the onset of the current COVID-19 pandemic, high priority is given to the development of neutralizing antibodies, as a key approach for the design of therapeutic strategies to countermeasure and eradicate the disease. Previously, we reported the development of human therapeutic monoclonal antibodies (mAbs) exhibiting very high protective ability. These mAbs recognize epitopes on the spike receptor binding domain (RBD) of SARS-CoV-2 that is considered to represent the main rout of receptor engagement by the SARS-CoV-2 virus. The recent emergence of viral variants emphasizes the notion that efficient antibody treatments need to rely on mAbs against several distinct key epitopes in order to circumvent the occurrence of therapy escape-mutants. Here we report the isolation and characterization of 12 neutralizing mAbs, identified by screening a phage-display library constructed from lymphatic cells collected from severe COVID-19 patients. The antibodies target three distinct epitopes on the spike N-terminal domain (NTD) of SARS-CoV-2, one of them defining a major site of vulnerability of the virus. Extensive characterization of these mAbs suggests a neutralization mechanism which relies both on amino-acid and N-glycan recognition on the virus, and involvement of receptors other than the hACE2 on the target cell. Two of the selected mAbs, which demonstrated superior neutralization potency in vitro, were further evaluated in vivo, demonstrating their ability to fully protect K18-hACE2 transgenic mice even when administered at low doses and late after infection. The study demonstrates the high potential of the mAbs for therapy of SARS-CoV-2 infection and underlines the possible role of the NTD in mediating infection of host cells via alternative cellular portals other than the canonical ACE2 receptor.

scholarly journals Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naïve human antibody library

2021 ◽  
Author(s):  
Benjamin Nikola Bell ◽  
Abigail E. Powell ◽  
Carlos Rodriguez ◽  
Jennifer R Cochran ◽  
Peter S. Kim
Keyword(s):  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
In Vitro Selection ◽  
Surface Display ◽  
Binding Domain ◽  
Yeast Surface Display ◽  
Surface Glycoprotein ◽  
Human Antibody ◽  
Receptor Binding Domain

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naive antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naive antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60 ng/mL in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naive antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.

scholarly journals A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tingting Li ◽  
Hongmin Cai ◽  
Hebang Yao ◽  
Bingjie Zhou ◽  
Ning Zhang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
In Vitro Selection ◽  
Rapid Development ◽  
Clinical Signs ◽  
Live Virus ◽  
Fusion Construct ◽  
Virus Challenge ◽  
Medical Interventions ◽  
Single Domain Antibodies

AbstractSARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1–6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7–17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 μg mL−1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

PENGEMBANGAN TEKNOLOGI SELEKSI IN VITRO BERULANG (REPEAT CYCLING–IN VITRO SELECTION) TOLERAN STRES KEKERINGAN UNTUK PERBAIKAN MUTU GENETIK DAN PRODUKSI TANAMAN KEDELAI BERMIKORIZA

2017 ◽  
Vol 1 (1) ◽  
pp. 74-84
Author(s):  
Ahmad Riduan ◽  
Rainiyati Rainiyati ◽  
Yulia Alia
Keyword(s):  
Arbuscular Mycorrhizae ◽  
In Vitro Selection ◽  
Soil Samples ◽  
Single Spore ◽  
Isolation And Characterization ◽  
Single Spore Culture ◽  
Spore Culture ◽  
Glomus Sp

Every plant rhizospheres in any ecosystem there are various living microorganisms including Arbuscular Mycorrhizae Fungi (AMF).  An isolation and characterization is required to investigate the species or type of the AMF. This research was aimed at studying the isolation and characterization of AMF sporulation in soybean rhizospheres in Jambi Province. The results of evaluation on soil samples before trapping showed that there are spores from three genus of AMF twelve types Glomus , two types Acaulospora and one type of Enthrophospora.  Following single spore culture in soybean rhizosphere, 5 spore types were obtained:  Glomus sp-1, Glomus sp-4, Glomus sp-7, Glomus sp-8 Glomus sp-10.

scholarly journals Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

2021 ◽  
Author(s):  
Jimmy D Gollihar ◽  
Jason S McLellan ◽  
Daniel R Boutz ◽  
Jule Goike ◽  
Andrew Horton ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Surface Display ◽  
Functional Characterization ◽  
Yeast Surface Display ◽  
Spike Protein ◽  
Emerging Pathogens ◽  
Effective Interventions ◽  
Yeast Surface

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

In Vitro Selection and Characterization of DNA Aptamers to a Small Molecule Target

2017 ◽  
Vol 9 (4) ◽  
pp. 233-268 ◽  
Author(s):  
Annamaria Ruscito ◽  
Erin M. McConnell ◽  
Anna Koudrina ◽  
Ranganathan Velu ◽  
Christopher Mattice ◽  
...  
Keyword(s):  
Small Molecule ◽  
In Vitro Selection ◽  
Dna Aptamers

Impact of the B.1.1.7 variant on neutralizing monoclonal antibodies recognizing diverse epitopes on SARS–CoV–2 Spike

2021 ◽  
Author(s):  
Carl Graham ◽  
Jeffrey Seow ◽  
Isabella Huettner ◽  
Hataf Khan ◽  
Neophytos Kouphou ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Infectious Virus ◽  
Antigenic Drift ◽  
Host Cells ◽  
Receptor Binding Domain ◽  
Neutralization Activity ◽  
Neutralizing Activity ◽  
Neutralizing Epitopes

The interaction of the SARS–CoV–2 Spike receptor binding domain (RBD) with the ACE2 receptor on host cells is essential for viral entry. RBD is the dominant target for neutralizing antibodies and several neutralizing epitopes on RBD have been molecularly characterized. Analysis of circulating SARS–CoV–2 variants has revealed mutations arising in the RBD, the N–terminal domain (NTD) and S2 subunits of Spike. To fully understand how these mutations affect the antigenicity of Spike, we have isolated and characterized neutralizing antibodies targeting epitopes beyond the already identified RBD epitopes. Using recombinant Spike as a sorting bait, we isolated >100 Spike–reactive monoclonal antibodies from SARS–CoV–2 infected individuals. ≈45% showed neutralizing activity of which ≈20% were NTD–specific. None of the S2–specific antibodies showed neutralizing activity. Competition ELISA revealed that NTD–specific mAbs formed two distinct groups: the first group was highly potent against infectious virus, whereas the second was less potent and displayed glycan–dependant neutralization activity. Importantly, mutations present in B.1.1.7 Spike frequently conferred resistance to neutralization by the NTD–specific neutralizing antibodies. This work demonstrates that neutralizing antibodies targeting subdominant epitopes need to be considered when investigating antigenic drift in emerging variants.

Sign in / Sign up

Export Citation Format

Share Document