scholarly journals Development of multivalent nanobodies blocking SARS-CoV-2 infection by targeting RBD of spike protein

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qizhong Lu ◽  
Zongliang Zhang ◽  
Hexian Li ◽  
Kunhong Zhong ◽  
Qin Zhao ◽  
...  
Keyword(s):  
Therapeutic Agent ◽  
Economic Consequences ◽  
Spike Protein ◽  
Global Public Health ◽  
Single Chain ◽  
Rapid Acquisition ◽  
High Affinity ◽  
Neutralizing Capacity ◽  
Potential Resource

Abstract Background The outbreak and pandemic of coronavirus SARS-CoV-2 caused significant threaten to global public health and economic consequences. It is extremely urgent that global people must take actions to develop safe and effective preventions and therapeutics. Nanobodies, which are derived from single‑chain camelid antibodies, had shown antiviral properties in various challenge viruses. In this study, multivalent nanobodies with high affinity blocking SARS-CoV-2 spike interaction with ACE2 protein were developed. Results Totally, four specific nanobodies against spike protein and its RBD domain were screened from a naïve VHH library. Among them, Nb91-hFc and Nb3-hFc demonstrated antiviral activity by neutralizing spike pseudotyped viruses in vitro. Subsequently, multivalent nanobodies were constructed to improve the neutralizing capacity. As a result, heterodimer nanobody Nb91-Nb3-hFc exhibited the strongest RBD-binding affinity and neutralizing ability against SARS-CoV-2 pseudoviruses with an IC50 value at approximately 1.54 nM. Conclusions The present study indicated that naïve VHH library could be used as a potential resource for rapid acquisition and exploitation of antiviral nanobodies. Heterodimer nanobody Nb91-Nb3-hFc may serve as a potential therapeutic agent for the treatment of COVID-19.

scholarly journals Development of Multivalent Nanobodies Blocking SARS CoV 2 Infection by Targeting RBD of Spike Protein

2020 ◽  
Author(s):  
Qizhong Lu ◽  
Zongliang Zhang ◽  
Hexian Li ◽  
Kunhong Zhong ◽  
Qin Zhao ◽  
...  
Keyword(s):  
Therapeutic Agent ◽  
Economic Consequences ◽  
Spike Protein ◽  
Global Public Health ◽  
Single Chain ◽  
Rapid Acquisition ◽  
Neutralizing Capacity ◽  
Ic50 Value ◽  
Potential Resource

Abstract Background: The outbreak and pandemic of coronavirus SARS CoV 2 caused significant threaten to global public health and economic consequences. It is extremely urgent that global people must take actions to develop safe and effective preventions and therapeutics. Nanobodies, which are derived from single‑chain camelid antibodies, had shown antiviral properties in various challenge viruses. In this study, multivalent nanobodies with high affinity blocking SARS CoV 2 spike interaction with ACE2 protein were developed.Results: Totally, four specific nanobodies against spike protein and its RBD domain were screened from a naïve VHH library. Among them, Nb91 hFc and Nb3 hFc demonstrated antiviral activity by neutralizing spike pseudotyped viruses in vitro. Subsequently, multivalent nanobodies were constructed to improve the neutralizing capacity. As a result, heterodimer nanobody Nb91 Nb3 hFc exhibited the strongest RBD binding affinity and neutralizing ability against SARS CoV 2 pseudoviruses with an IC50 value at approximately 1.54 nM. Conclusions: The present study indicated that naïve VHH library could be used as a potential resource for rapid acquisition and exploitation of antiviral nanobodies. Heterodimer nanobody Nb91 Nb3 hFc may serve as a potential therapeutic agent for the treatment of COVID 19.

scholarly journals Antibodies that potently inhibit or enhance SARS-CoV-2 spike protein-ACE2 interaction isolated from synthetic single-chain antibody libraries

2020 ◽  
Author(s):  
Matthew D. Beasley ◽  
Sanja Aracic ◽  
Fiona M. Gracey ◽  
Ruban Kannan ◽  
Avisa Masarati ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Single Chain ◽  
Single Chain Antibody ◽  
High Affinity ◽  
Scfv Library ◽  
Antibody Libraries ◽  
Prophylactic Use

AbstractAntibodies with high affinity against the receptor binding domain (RBD) of the SARS-CoV-2 S1 ectodomain were identified from screens using the Retained Display™ (ReD) platform employing a 1 × 1011 clone single-chain antibody (scFv) library. Numerous unique scFv clones capable of inhibiting binding of the viral S1 ectodomain to the ACE2 receptor in vitro were characterized. To maximize avidity, selected clones were reformatted as bivalent diabodies and monoclonal antibodies (mAb). The highest affinity mAb completely neutralized live SARS-CoV-2 virus in cell culture for four days at a concentration of 6.7 nM, suggesting potential therapeutic and/or prophylactic use. Furthermore, scFvs were identified that greatly increased the interaction of the viral S1 trimer with the ACE2 receptor, with potential implications for vaccine development.

scholarly journals SARS-CoV-2 RBD in vitro evolution parrots and predicts contagious mutation spread

2021 ◽  
Author(s):  
Gideon Schreiber ◽  
Jiri Zahradník ◽  
Shir Marciano ◽  
Maya Shemesh ◽  
Eyal Zoler ◽  
...  
Keyword(s):  
South African ◽  
Convergent Evolution ◽  
Vaccine Development ◽  
Spike Protein ◽  
Structural Basis ◽  
Receptor Binding Domain ◽  
In Vitro Evolution ◽  
High Affinity ◽  
Future Drug

Abstract SARS-CoV-2 is continually evolving, with more contagious mutations spreading rapidly. Using in vitro evolution to affinity maturate the receptor-binding domain (RBD) of the spike protein towards ACE2 resulted in the more contagious mutations, S477N, E484K, and N501Y, to be among the first selected, explaining the convergent evolution of the “European” (20E-EU1), “British” (501.V1),”South African” (501.V2), and Brazilian variants (501.V3). Plotting the binding affinity to ACE2 of all RBD mutations against their incidence in the population shows a strong correlation between the two. Further in vitro evolution enhancing binding by 600-fold provides guidelines towards potentially new evolving mutations with even higher infectivity. For example, Q498R epistatic to N501Y. Nevertheless, the high-affinity RBD is also an efficient drug, inhibiting SARS-CoV-2 infection. The 2.9Å Cryo-EM structure of the high-affinity complex, including all rapidly spreading mutations, provides a structural basis for future drug and vaccine development and for in silico evaluation of known antibodies.

scholarly journals High affinity binding of SARS-CoV-2 spike protein enhances ACE2 carboxypeptidase activity

2020 ◽  
Vol 295 (52) ◽  
pp. 18579-18588 ◽  
Author(s):  
Jinghua Lu ◽  
Peter D. Sun
Keyword(s):  
Spike Protein ◽  
The Novel ◽  
Global Public Health ◽  
Health Crisis ◽  
Peptide Substrates ◽  
Angiotensin Converting Enzyme 2 ◽  
High Affinity ◽  
Enzymatic Function ◽  
Biological Substrates ◽  
Entry Receptor

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) has emerged to a pandemic and caused global public health crisis. Human angiotensin-converting enzyme 2(ACE2) was identified as the entry receptor for SARS-CoV-2. As a carboxypeptidase, ACE2 cleaves many biological substrates besides angiotensin II to control vasodilatation and vascular permeability. Given the nanomolar high affinity between ACE2 and SARS-CoV-2 spike protein, we investigated how this interaction would affect the enzymatic activity of ACE2. Surprisingly, SARS-CoV-2 trimeric spike protein increased ACE2 proteolytic activity ∼3-10 fold against model peptide substrates, such as caspase-1 substrate and Bradykinin-analog. The enhancement in ACE2 enzymatic function was mediated by the binding of SARS-CoV-2 spike RBD domain. These results highlighted the potential for SARS-CoV-2 infection to enhance ACE2 activity, which may be relevant to the cardiovascular symptoms associated with COVID-19.

scholarly journals Dalbavancin binds ACE2 to block its interaction with SARS-CoV-2 spike protein and is effective in inhibiting SARS-CoV-2 infection in animal models

Cell Research ◽  
2020 ◽  
Vol 31 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Gan Wang ◽  
Meng-Li Yang ◽  
Zi-Lei Duan ◽  
Feng-Liang Liu ◽  
Lin Jin ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Spike Protein ◽  
Drug Candidate ◽  
Peptide Drug ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
High Affinity ◽  
Plasma Half Life

AbstractInfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic worldwide. Currently, however, no effective drug or vaccine is available to treat or prevent the resulting coronavirus disease 2019 (COVID-19). Here, we report our discovery of a promising anti-COVID-19 drug candidate, the lipoglycopeptide antibiotic dalbavancin, based on virtual screening of the FDA-approved peptide drug library combined with in vitro and in vivo functional antiviral assays. Our results showed that dalbavancin directly binds to human angiotensin-converting enzyme 2 (ACE2) with high affinity, thereby blocking its interaction with the SARS-CoV-2 spike protein. Furthermore, dalbavancin effectively prevents SARS-CoV-2 replication in Vero E6 cells with an EC50 of ~12 nM. In both mouse and rhesus macaque models, viral replication and histopathological injuries caused by SARS-CoV-2 infection are significantly inhibited by dalbavancin administration. Given its high safety and long plasma half-life (8–10 days) shown in previous clinical trials, our data indicate that dalbavancin is a promising anti-COVID-19 drug candidate.

scholarly journals Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2

Science ◽  
2020 ◽  
Vol 369 (6508) ◽  
pp. 1261-1265 ◽  
Author(s):  
Kui K. Chan ◽  
Danielle Dorosky ◽  
Preeti Sharma ◽  
Shawn A. Abbasi ◽  
John M. Dye ◽  
...  
Keyword(s):  
Host Cells ◽  
Viral Escape ◽  
Spike Protein ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
High Affinity ◽  
Decoy Receptors ◽  
Catalytically Active ◽  
Interaction Surface

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90–glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.

scholarly journals A broadly neutralizing biparatopic Nanobody protects mice from lethal challenge with SARS-CoV-2 variants of concern

2021 ◽  
Author(s):  
Teresa R. Wagner ◽  
Daniel Schnepf ◽  
Julius Beer ◽  
Karin Klingel ◽  
Natalia Ruetalo ◽  
...  
Keyword(s):  
Lethal Dose ◽  
Survival Rates ◽  
Drug Candidate ◽  
Lethal Challenge ◽  
Drug Candidates ◽  
High Affinity ◽  
Neutralizing Capacity ◽  
Future Drug

The ongoing COVID-19 pandemic and the frequent emergence of new SARS-CoV-2 variants of concern (VOCs), requires continued development of fast and effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nb) specific for the receptor-binding domain (RBD) of SARS-CoV-2, which are now being used as biparatopic Nbs (bipNbs) to investigate their potential as future drug candidates. Following detailed in vitro characterization, we chose NM1267 as the most promising candidate showing high affinity binding to several recently described SARS-CoV-2 VOCs and strong neutralizing capacity against a patient isolate of B.1.351 (Beta). To assess if bipNb NM1267 confers protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice were treated by intranasal route before infection with a lethal dose of SARS-CoV-2. NM1267-treated mice showed significantly reduced disease progression, increased survival rates and secreted less infectious virus via their nostrils. Histopathological analyses and in situ hybridization further revealed a drastically reduced viral load and inflammatory response in lungs of NM1267-treated mice. These data suggest, that bipNb NM1267 is a broadly active and easily applicable drug candidate against a variety of emerging SARS-CoV-2 VOCs.

scholarly journals Structural characterisation of a nanobody derived from a naïve library that neutralises SARS-CoV-2

2020 ◽  
Author(s):  
Jiangdong Huo ◽  
Audrey Le Bas ◽  
Reinis R. Ruza ◽  
Helen M.E. Duyvesteyn ◽  
Halina Mikolajek ◽  
...  
Keyword(s):  
Viral Entry ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Single Chain ◽  
Angiotensin Converting Enzyme 2 ◽  
Serious Illness ◽  
Structural Characterisation ◽  
High Affinity ◽  
Cryo Electron Microscopy ◽  
Seasonal Flu

Abstract The SARS-CoV-2 virus is more transmissible than previous coronaviruses and causes a more serious illness than seasonal flu. The SARS-CoV-2 receptor binding domain (RBD) of the Spike protein binds to the human angiotensin-converting enzyme 2 (ACE2) receptor as a prelude to viral entry into the cell. Using a naïve llama single chain nanobody library and PCR maturation we have produced a nanobody, H11-D4, with a KD 9 nM for RBD that blocks the binding of RBD to the ACE2. Single particle cryo-electron microscopy revealed that H11-D4 binds to each of the three RBDs in the Spike trimer. The 1.8 Å crystal structure of the H11-D4 – RBD complex has illuminated the molecular interactions that drive the high affinity. H11-D4 binds to an epitope on RBD that overlaps with the ACE2 binding, explaining the blocking of ACE2 binding. The nanobody showed potent neutralising activity against live SARS-CoV-2 virus.

scholarly journals Antibody-like proteins that capture and neutralize SARS-CoV-2

2020 ◽  
Vol 6 (42) ◽  
pp. eabd3916 ◽  
Author(s):  
T. Kondo ◽  
Y. Iwatani ◽  
K. Matsuoka ◽  
T. Fujino ◽  
S. Umemoto ◽  
...  
Keyword(s):  
High Speed ◽  
In Vitro Selection ◽  
Nasal Swab ◽  
Inhibitory Concentration ◽  
Rapid Development ◽  
Spike Protein ◽  
Emerging Pathogens ◽  
High Affinity ◽  
Selection Of

To combat severe acute respiratory syndrome–related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

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