Recombinant SARS-CoV-2 spike proteins for sero-surveillance and epitope mapping

The receptor binding domain (RBD) of the coronavirus spike protein (S) has been verified to be the main target for potent neutralizing antibodies (nAbs) in most coronaviruses, and the N-terminal domain (NTD) of some betacoronaviruses has also been indicated to induce nAbs. For alphacoronavirus HCoV-229E, its RBD has been shown to have neutralizing epitopes, and these epitopes could change over time. However, whether neutralizing epitopes exist on the NTD and whether these epitopes change like those of the RBD are still unknown. Here, we verified that neutralizing epitopes exist on the NTD of HCoV-229E. Furthermore, we characterized an NTD targeting nAb 5H10, which could neutralize both pseudotyped and authentic HCoV-229E VR740 in vitro. Epitope mapping indicated that 5H10 targeted motif E1 (147-167 aa) and identified F159 as critical for 5H10 binding. More importantly, our results revealed that motif E1 was highly conserved among clinical isolates except for F159. Further data proved that mutations at position 159 gradually appeared over time and could completely abolish the neutralizing ability of 5H10, supporting the notion that position 159 may be under selective pressure during the human epidemic. In addition, we also found that contemporary clinical serum has a stronger binding capacity for the NTD of contemporary strains than historic strains, proving that the epitope on the NTD could change over time. In summary, these findings define a novel neutralizing epitope on the NTD of HCoV-229E S and provide a theoretical basis for the design of vaccines against HCoV-229E or related coronaviruses. Importance Characterization of the neutralizing epitope of the spike (S) protein, the major invasion protein of coronaviruses, can help us better understand the evolutionary characteristics of these viruses and promote vaccine development. To date, the neutralizing epitope distribution of alphacoronaviruses is not well known. Here, we identified a neutralizing antibody that targeted the N-terminal domain (NTD) of the alphacoronavirus HCoV-229E S protein. Epitope mapping revealed a novel epitope that was not previously discovered in HCoV-229E. Further studies identified an important residue, F159. Mutations that gradually appeared over time at this site abolished the neutralizing ability of 5H10, indicating that selective pressure occurred at this position in the spread of HCoV-229E. Furthermore, we found that the epitopes within the NTD also changed over time. Taken together, our findings defined a novel neutralizing epitope and highlighted the role of the NTD in the future prevention and control of HCoV-229E or related coronaviruses.

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Vaccine Design against Coronavirus Spike (S) Glycoprotein in Chicken: Immunoinformatic and Computational Approaches

10.21203/rs.3.rs-25375/v2 ◽

2020 ◽

Author(s):

Eman Ali Awadelkareem ◽

Sumaia Awad Elkariem Ali

Keyword(s):

T Cell ◽

Rna Virus ◽

Protein S ◽

Economic Loss ◽

Protein Characterization ◽

Spike Protein ◽

T Cell Epitopes ◽

Resistance Response ◽

S Protein ◽

Infectious Bronchitis

Abstract Background: Infectious bronchitis (IB) is a highly contagious respiratory disease in chickens and produces economic loss within the poultry industry. This disease is caused by a single stranded RNA virus belonging to Cronaviridae family. This study aimed to design a potential multi-epitopes vaccine against Infectious bronchitis virus spike protein (S). Protein characterization was also performed for IBV spike protein.Methods: The present study used various tools in Immune Epitope Database (IEDB) to predict conserved B and T cell epitopes against IBV spike (S) protein that may perform a significant role in provoking the resistance response to IBV infection. Results: In B cell prediction methods, three epitopes (1139KKSSYY1144, 1140KSSYYT1145, 1141SSYYT1145) were selected as surface, linear and antigenic epitopes. Many MHCI and MHCII epitopes were predicted for IBV S protein. Among them 982YYITARDMY990 and 983YITARDMYM991 epitopes displayed high antigenicity, no allergenicity and no toxicity as well as great linkage with MHCI and MHCII alleles. Moreover, docking analysis of MHCI epitope produced strong binding affinity with BF2 alleles. Conclusion: Five conserved epitopes were expected from spike glycoprotein of IBV as the best B and T cell epitopes due to high antigenicity, no allergenicity and no toxicity. In addition, MHC epitopes showed great linkage with MHC alleles as well as strong interaction with BF2 alleles. These epitopes should be designed and incorporated and then tested as multi-epitope vaccine against IBV.

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Repurposing Therapeutics for COVID-19: Supercomputer-Based Docking to the SARS-CoV-2 Viral Spike Protein and Viral Spike Protein-Human ACE2 Interface

10.26434/chemrxiv.11871402.v3 ◽

2020 ◽

Cited By ~ 13

Author(s):

Micholas Smith ◽

Jeremy C. Smith

Keyword(s):

Small Molecules ◽

High Throughput Screening ◽

Protein S ◽

Host Cells ◽

Spike Protein ◽

The Novel ◽

S Protein ◽

Host Virus Interactions ◽

Virus Interactions ◽

Ranked List

The novel Wuhan coronavirus (SARS-CoV-2) has been sequenced, and the virus shares substantial similarity with SARS-CoV. Here, using a computational model of the spike protein (S-protein) of SARS-CoV-2 interacting with the human ACE2 receptor, we make use of the world's most powerful supercomputer, SUMMIT, to enact an ensemble docking virtual high-throughput screening campaign and identify small-molecules which bind to either the isolated Viral S-protein at its host receptor region or to the S protein-human ACE2 interface. We hypothesize the identified small-molecules may be repurposed to limit viral recognition of host cells and/or disrupt host-virus interactions. A ranked list of compounds is given that can be tested experimentally.

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SARS-CoV-2 spike protein arrested in the closed state induces potent neutralizing responses

10.1101/2021.01.14.426695 ◽

2021 ◽

Author(s):

George W. Carnell ◽

Katarzyna A. Ciazynska ◽

David A. Wells ◽

Xiaoli Xiong ◽

Ernest T. Aguinam ◽

...

Keyword(s):

Receptor Binding ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Protein S ◽

Spike Protein ◽

Receptor Binding Site ◽

S Protein ◽

Closed State ◽

S Proteins ◽

Protein Constructs

AbstractThe majority of SARS-CoV-2 vaccines in use or in advanced clinical development are based on the viral spike protein (S) as their immunogen. S is present on virions as pre-fusion trimers in which the receptor binding domain (RBD) is stochastically open or closed. Neutralizing antibodies have been described that act against both open and closed conformations. The long-term success of vaccination strategies will depend upon inducing antibodies that provide long-lasting broad immunity against evolving, circulating SARS-CoV-2 strains, while avoiding the risk of antibody dependent enhancement as observed with other Coronavirus vaccines. Here we have assessed the results of immunization in a mouse model using an S protein trimer that is arrested in the closed state to prevent exposure of the receptor binding site and therefore interaction with the receptor. We compared this with a range of other modified S protein constructs, including representatives used in current vaccines. We found that all trimeric S proteins induce a long-lived, strongly neutralizing antibody response as well as T-cell responses. Notably, the protein binding properties of sera induced by the closed spike differed from those induced by standard S protein constructs. Closed S proteins induced more potent neutralising responses than expected based on the degree to which they inhibit interactions between the RBD and ACE2. These observations suggest that closed spikes recruit different, but equally potent, virus-inhibiting immune responses than open spikes, and that this is likely to include neutralizing antibodies against conformational epitopes present in the closed conformation. Together with their improved stability and storage properties we suggest that closed spikes may be a valuable component of refined, next-generation vaccines.

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Extracellular vesicles containing ACE2 efficiently prevent infection by SARS-CoV-2 Spike protein-containing virus

10.1101/2020.07.08.193672 ◽

2020 ◽

Cited By ~ 1

Author(s):

Federico Cocozza ◽

Ester Piovesana ◽

Nathalie Névo ◽

Xavier Lahaye ◽

Julian Buchrieser ◽

...

Keyword(s):

Membrane Fusion ◽

Extracellular Vesicles ◽

Protein S ◽

Spike Protein ◽

S Protein ◽

Surface Receptor

ABSTRACTSARS-CoV-2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by TMPRRS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2-EVs are efficient decoys for SARS-CoV-2 S protein-containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is 500 to 1500 times more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2.

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Repurposing Therapeutics for COVID-19: Supercomputer-Based Docking to the SARS-CoV-2 Viral Spike Protein and Viral Spike Protein-Human ACE2 Interface

10.26434/chemrxiv.11871402.v4 ◽

2020 ◽

Cited By ~ 26

Author(s):

Micholas Smith ◽

Jeremy C. Smith

Keyword(s):

Small Molecules ◽

High Throughput Screening ◽

Protein S ◽

Host Cells ◽

Spike Protein ◽

The Novel ◽

S Protein ◽

Host Virus Interactions ◽

Virus Interactions ◽

Ranked List

The novel Wuhan coronavirus (SARS-CoV-2) has been sequenced, and the virus shares substantial similarity with SARS-CoV. Here, using a computational model of the spike protein (S-protein) of SARS-CoV-2 interacting with the human ACE2 receptor, we make use of the world's most powerful supercomputer, SUMMIT, to enact an ensemble docking virtual high-throughput screening campaign and identify small-molecules which bind to either the isolated Viral S-protein at its host receptor region or to the S protein-human ACE2 interface. We hypothesize the identified small-molecules may be repurposed to limit viral recognition of host cells and/or disrupt host-virus interactions. A ranked list of compounds is given that can be tested experimentally.

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Identification of a Potent Inhibitor Targeting the Spike Protein of Pandemic Human Coronavirus, SARS-CoV-2 by Computational Methods

10.26434/chemrxiv.12197934.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

SRUTHI UNNI ◽

Snehal Aouti ◽

Padmanabhan Balasundaram

Keyword(s):

Vaccine Development ◽

Protein S ◽

Dynamics Simulation ◽

Spike Protein ◽

Simulation Studies ◽

Viral Pathogen ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Chemical Libraries ◽

Pi Interactions

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is an emerging new viral pathogen that causes severe respiratory disease. SARS-CoV-2 is responsible for an outbreak of COVID-19 pandemic worldwide. As there are no confirmed antiviral drugs or vaccines currently available for the treatment of COVID-19, discovering potent inhibitors or vaccines are urgently required for the benefit of humanity. The glycosylated Spike protein (S-protein) directly interacts with human angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) of S-protein. As the S-protein is exposed to the surface and is essential for entry into the host, the S-protein can be considered as a first-line therapeutic target for antiviral therapy and vaccine development. In-silico screening, docking and molecular dynamics simulation studies were performed to identify repurposing drugs using DrugBank and PubChem library against the RBD of S-protein. The study identified a laxative drug, Bisoxatin (DB09219), which is used for the treatment of constipation and preparation of the colon for surgical procedures. It binds nicely at the S-protein – ACE2 interface by making substantial pi-pi interactions with Tyr505 in the ‘Site 1’ hook region of RBD and hydrophilic interactions with Glu406, Ser494 and Thr500. Bisoxatin consistently binds to the protein throughout the 100 ns simulation. Taken together, we propose that the discovered molecule, Bisoxatin may be a potent repurpose drug to develop new chemical libraries for inhibiting SARS-CoV-2 entry into the host.

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Identification of a Potent Inhibitor Targeting the Spike Protein of Pandemic Human Coronavirus, SARS-CoV-2 by Computational Methods

10.26434/chemrxiv.12197934 ◽

2020 ◽

Author(s):

SRUTHI UNNI ◽

Snehal Aouti ◽

Padmanabhan Balasundaram

Keyword(s):

Vaccine Development ◽

Protein S ◽

Dynamics Simulation ◽

Spike Protein ◽

Simulation Studies ◽

Viral Pathogen ◽

S Protein ◽

Angiotensin Converting Enzyme 2 ◽

Chemical Libraries ◽

Pi Interactions

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) is an emerging new viral pathogen that causes severe respiratory disease. SARS-CoV-2 is responsible for an outbreak of COVID-19 pandemic worldwide. As there are no confirmed antiviral drugs or vaccines currently available for the treatment of COVID-19, discovering potent inhibitors or vaccines are urgently required for the benefit of humanity. The glycosylated Spike protein (S-protein) directly interacts with human angiotensin-converting enzyme 2 (ACE2) receptor through the receptor-binding domain (RBD) of S-protein. As the S-protein is exposed to the surface and is essential for entry into the host, the S-protein can be considered as a first-line therapeutic target for antiviral therapy and vaccine development. In-silico screening, docking and molecular dynamics simulation studies were performed to identify repurposing drugs using DrugBank and PubChem library against the RBD of S-protein. The study identified a laxative drug, Bisoxatin (DB09219), which is used for the treatment of constipation and preparation of the colon for surgical procedures. It binds nicely at the S-protein – ACE2 interface by making substantial pi-pi interactions with Tyr505 in the ‘Site 1’ hook region of RBD and hydrophilic interactions with Glu406, Ser494 and Thr500. Bisoxatin consistently binds to the protein throughout the 100 ns simulation. Taken together, we propose that the discovered molecule, Bisoxatin may be a potent repurpose drug to develop new chemical libraries for inhibiting SARS-CoV-2 entry into the host.

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An Impedimetric Approach for COVID-19 Detection

The Analyst ◽

10.1039/d1an01718g ◽

2021 ◽

Author(s):

Yudum Tepeli ◽

Burak Ekrem Citil ◽

U. Anik

Keyword(s):

Protein S ◽

Spike Protein ◽

S Protein ◽

Infection Mechanism ◽

Biosensor System ◽

Electrochemical Approach

In this study, an electrochemical approach for the determination of coronavirus disease (COVID-19) was developed. The biosensor system relied on the spike protein (S-protein) based infection mechanism of the virus...

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Abstract P508: Hyperglycemia Exacerbated Sars-cov-2 Spike Protein-induced Electrophysiological Dysfunctions In Hipsc-derived Cardiomyocytes

Circulation Research ◽

10.1161/res.129.suppl_1.p508 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Chloe Wong ◽

Thong B Nguyen ◽

Michael Tong ◽

Takashi Matsui ◽

Yiqiang Zhang

Keyword(s):

Microelectrode Array ◽

Field Potential ◽

Induced Pluripotent Stem Cell ◽

Protein S ◽

Spike Protein ◽

Cardiac Complications ◽

Diabetic Patients ◽

Structural Protein ◽

Cellular Mechanisms ◽

S Protein

Background: Cardiac arrhythmias, heart failure, and myocarditis are common symptoms associated with coronavirus 2 (SARS-CoV-2) infection. The cytopathic effect of SARS-CoV-2 is initiated by spike protein (S-protein) binding to the ACE2 receptor. However, cellular mechanisms of SARS-CoV-2 causing cardiac complications, particularly in a subset of the population such as diabetic patients, remain unclear. Aims: To determine electrophysiological cytopathic effects of SARS-CoV-2 S-protein in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and Results: CMs were differentiated from WTC-11, then re-plated on 48-well microelectrode array (MEA) plates and cultured under normal or hyperglycemic conditions (Glucose 5 mM (NG), and Glucose 20 mM (HG)) for three days. These HiPSC-CMs were treated with recombinant S-protein with or without insulin supplementation. Cardiac modules in AxIS MEA program were used to acquire and analyze electric and contractile functions. Beat period and field potential duration were significantly prolonged, while conductance velocity and spike slope were accelerated in HG without insulin compared to that in HG with insulin (p<0.05). S-protein-induced perturbations in local extracellular action potential (LEAP) matrices (e.g., LPD30, 50, 90) and Contractility matrices in HG were mitigated by insulin treatment. Higher concentration S-protein induced perturbations in normal hiPSC-CMs (NG+insulin). One day after S-protein treatment, CM functions appeared comparable to the pretreatment state, although changes in some electro-contractile matrices persisted. Conclusion: These results demonstrate SARS-CoV-2 viral structural protein can induce acute electrophysiological dysfunctions in hiPSC-CMs, which is influenced by glucose level. This establishes a new model for dissecting the mechanism of cardiac complications in viral infection.

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