A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV

ABSTRACTRecently, highly transmissible SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 were identified in India with mutations within the spike proteins. The spike protein of Kappa contains four mutations E154K, L452R, E484Q and P681R, and Delta contains L452R, T478K and P681R, while B.1.618 spike harbors mutations Δ145-146 and E484K. However, it remains unknown whether these variants have altered in their entry efficiency, host tropism, and sensitivity to neutralizing antibodies as well as entry inhibitors. In this study, we found that Kappa, Delta or B.1.618 spike uses human ACE2 with no or slightly increased efficiency, while gains a significantly increased binding affinity with mouse, marmoset and koala ACE2 orthologs, which exhibits limited binding with WT spike. Furthermore, the P618R mutation leads to enhanced spike cleavage, which could facilitate viral entry. In addition, Kappa, Delta and B.1.618 exhibits a reduced sensitivity to neutralization by convalescent sera owning to the mutation of E484Q, T478K, Δ145-146 or E484K, but remains sensitive to entry inhibitors-ACE2-lg decoy receptor. Collectively, our study revealed that enhanced human and mouse ACE2 receptor engagement, increased spike cleavage and reduced sensitivity to neutralization antibodies of Kappa, Delta and B.1.618 may contribute to the rapid spread of these variants and expanded host range. Furthermore, our result also highlighted that ACE2-lg could be developed as broad-spectrum antiviral strategy against SARS-CoV-2 variants.

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Tiger team: a panel of human neutralizing mAbs targeting SARS-CoV-2 spike at multiple epitopes

10.1101/2020.05.20.106609 ◽

2020 ◽

Author(s):

Tal Noy-Porat ◽

Efi Makdasi ◽

Ron Alcalay ◽

Adva Mechaly ◽

Yinon Levy ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Neutralizing Antibodies ◽

Phage Display Library ◽

Therapeutic Drug ◽

The Novel ◽

Receptor Binding Domain ◽

Human Coronavirus ◽

Isolation And Characterization ◽

Emerging Virus

AbstractThe novel highly transmissible human coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Thus far, there is no approved therapeutic drug, specifically targeting this emerging virus. Here we report the isolation and characterization of a panel of human neutralizing monoclonal antibodies targeting the SARS-CoV-2 receptor binding domain (RBD). These antibodies were selected from a phage display library constructed using peripheral circulatory lymphocytes collected from patients at the acute phase of the disease. These neutralizing antibodies are shown to recognize distinct epitopes on the viral spike RBD, therefore they represent a promising basis for the design of efficient combined post-exposure therapy for SARS-CoV-2 infection.

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Virtual Screening of Curcumin and Its Analogs Against the Spike Surface Glycoprotein of SARS-CoV-2 and SARS-CoV

10.26434/chemrxiv.12142383.v2 ◽

2020 ◽

Author(s):

Ashish Patel ◽

Malathi Rajendran ◽

Suresh B Pakala ◽

Ashish Shah ◽

Harnisha Patel ◽

...

Keyword(s):

Binding Energy ◽

Viral Entry ◽

Curcuma Longa ◽

Binding Energies ◽

Surface Glycoprotein ◽

Spike Protein ◽

Pharmacokinetic Parameters ◽

The Novel ◽

Angiotensin Converting Enzyme 2 ◽

Entry Inhibitors

COVID-19, a new pandemic caused by SARS-CoV-2, was first identified in 2019 in Wuhan, China. The novel corona virus SARS-CoV-2 and the 2002 SARS-CoV have 74 % identity and use similar mechanisms to gain entry into the cell. Both the viruses enter the host cell by binding of the viral spike glycoprotein to the host receptor, angiotensin converting enzyme 2 (ACE2). Targeting entry of the virus has a better advantage than inhibiting the later stages of the viral life cycle. The crystal structure of the SARS-CoV (6CRV: full length S protein) and SARS-CoV-2 Spike proteins (6M0J: Receptor binding domain, RBD) was used to determine potential small molecule inhibitors. Curcumin, a naturally occurring phytochemical in Curcuma longa, is known to have broad pharmacological properties. In the present study, curcumin and its derivatives were docked, using Autodock 4.2, onto the 6CRV and 6M0J to study their capability to act as inhibitors of the spike protein and thereby, viral entry. The curcumin and its derivatives displayed binding energies, ΔG, ranging from -10.98 to -5.12 kcal/mol (6CRV) and -10.01 to -5.33 kcal/mol (6M0J). The least binding energy was seen in bis-demethoxycurcumin with: ΔG = -10.98 kcal/mol (6CRV) and -10.01 kcal/mol (6M0J). A good binding energy, drug likeness and efficient pharmacokinetic parameters suggest the potential of curcumin and few of its derivatives as SARS-CoV-2 spike protein inhibitors. However, further research is necessary to investigate the ability of these compounds as viral entry inhibitors.<br>

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Keep out! SARS-CoV-2 entry inhibitors: their role and utility as COVID-19 therapeutics

Virology Journal ◽

10.1186/s12985-021-01624-x ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Lennox Chitsike ◽

Penelope Duerksen-Hughes

Keyword(s):

Viral Entry ◽

Neutralizing Antibodies ◽

Herd Immunity ◽

Economic Life ◽

Antigenic Drift ◽

Early Management ◽

Entry Inhibitors ◽

Current Availability ◽

Exposure Prophylaxis ◽

The Impact

AbstractThe COVID-19 pandemic has put healthcare infrastructures and our social and economic lives under unprecedented strain. Effective solutions are needed to end the pandemic while significantly lessening its further impact on mortality and social and economic life. Effective and widely-available vaccines have appropriately long been seen as the best way to end the pandemic. Indeed, the current availability of several effective vaccines are already making a significant progress towards achieving that goal. Nevertheless, concerns have risen due to new SARS-CoV-2 variants that harbor mutations against which current vaccines are less effective. Furthermore, some individuals are unwilling or unable to take the vaccine. As health officials across the globe scramble to vaccinate their populations to reach herd immunity, the challenges noted above indicate that COVID-19 therapeutics are still needed to work alongside the vaccines. Here we describe the impact that neutralizing antibodies have had on those with early or mild COVID-19, and what their approval for early management of COVID-19 means for other viral entry inhibitors that have a similar mechanism of action. Importantly, we also highlight studies that show that therapeutic strategies involving various viral entry inhibitors such as multivalent antibodies, recombinant ACE2 and miniproteins can be effective not only for pre-exposure prophylaxis, but also in protecting against SARS-CoV-2 antigenic drift and future zoonotic sarbecoviruses.

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Humanized Single Domain Antibodies Neutralize SARS-CoV-2 by Targeting Spike Receptor Binding Domain

10.1101/2020.04.14.042010 ◽

2020 ◽

Cited By ~ 9

Author(s):

Xiaojing Chi ◽

Xiuying Liu ◽

Conghui Wang ◽

Xinhui Zhang ◽

Lili Ren ◽

...

Keyword(s):

Viral Entry ◽

Neutralizing Antibodies ◽

Neutralization Assay ◽

Single Domain ◽

The Novel ◽

Binding Data ◽

Single Domain Antibodies ◽

Human Igg1 ◽

Equilibrium Dissociation ◽

Medical Burden

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread across more than 200 countries and regions, leading to an unprecedented medical burden and live lost. SARS-CoV-2 specific antivirals or prophylactic vaccines are not available. Neutralizing antibodies provide efficient blockade for viral infection and are a promising category of biological therapies. Using SARS-CoV-2 spike RBD as a bait, we have discovered a panel of humanized single domain antibodies (sdAbs). These sdAbs revealed binding kinetics with the equilibrium dissociation constant (KD) of 0.7~33 nM. The monomeric sdAbs showed half maximal inhibitory concentration (IC50) of 0.003~0.3 μg/mL in pseudotyped particle neutralization assay, and 0.23~0.50 μg/mL in authentic SARS-CoV-2 neutralization assay. Competitive ligand-binding data suggested that the sdAbs either completely blocked or significantly inhibited the association between SARS-CoV-2 RBD and viral entry receptor ACE2. Finally, we showed that fusion of the human IgG1 Fc to sdAbs improved their neutralization activity by tens of times. These results reveal the novel SARS-CoV-2 RBD targeting sdAbs and pave a road for antibody drug development.

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The use of pseudotyped coronaviruses for the screening of entry inhibitors: Green tea extract inhibits the entry of SARS-CoV-1, MERS-CoV, and SARS-CoV-2 by blocking receptor-spike interaction

Current Pharmaceutical Biotechnology ◽

10.2174/1389201022666210810111716 ◽

2021 ◽

Vol 22 ◽

Author(s):

Jeswin Joseph ◽

Thankamani Karthika ◽

V.R. Akshay Das ◽

V. Stalin Raj

Keyword(s):

Green Tea ◽

Viral Entry ◽

Neutralizing Antibodies ◽

High Titer ◽

Antiviral Agents ◽

Natural Compounds ◽

Cell Surface Receptor ◽

Soluble Form ◽

Entry Inhibitors ◽

Wide Range

Background: Coronaviruses (CoVs) infect a wide range of animals and birds. Their tropism is primarily determined by the ability of the spike protein to bind to a host cell surface receptor. The ongoing outbreak of SARS-CoV-2 inculcates the need for the development of effective intervention strategies. Objectives: In this study, we aim to produce pseudotyped coronaviruses of SARS-CoV-1, MERS-CoV, and SARS-CoV-2 and show its applications, including virus entry, neutralization, and screening of entry inhibitors from natural products. Methods: Here, we generated VSV-based pseudotyped coronaviruses (CoV-PVs) for SARS-CoV-1, MERS-CoV, and SARS-CoV-2. Recombinant spike proteins of SARS-CoV-1, MERS-CoV, and SARS-CoV-2 were transiently expressed in HEK293T cells followed by infection with recombinant VSV. High titer pseudoviruses were harvested and subjected to distinct validation assays, which confirms the proper spike pseudotyping. Further, specific receptor-mediated entry was confirmed by antibody neutralization and soluble form of receptor inhibition assay on Vero E6 cells. Next, these CoV-PVs were used for screening of antiviral activity of natural compounds such as green tea and Spirulina extract. Results: Medicinal plants and natural compounds have been traditionally used as antiviral agents. In the first series of experiments, we demonstrated that pseudotyped viruses specifically bind to their receptors for cellular entry. SARS-CoV-1 and MERS-CoV anti-sera neutralize SARS-CoV-1-PV and SARS-CoV-2-PV, and MERS-CoV-PV, respectively. Incubation of soluble ACE2 with CoV-PVs inhibited entry of SARS-CoV-1 and SARS-CoV-2 PVs but not MERS-CoV-PV. Also, transient expression of ACE2 and DPP4 in non-permissive BHK21 cells enabled infection by SARS-CoV-1-PV, SARS-CoV-2-PV, and MERS-CoV-PV, respectively. Next, we showed the antiviral properties of known entry inhibitors of enveloped viruses, Spirulina, and green tea extracts against CoV-PVs. SARS-CoV-1-PV, MERS-CoV-PV, and SARS-CoV-2-PV entry was blocked with higher efficiency when preincubated with either green tea or Spirulina extracts. Green tea provided a better inhibitory effect by binding to the S1 domain of the spike and blocking the spike interaction with its receptor. Conclusion: In summary, we demonstrated that pseudotyped viruses are an ideal tool for studying viral entry, quantification of neutralizing antibodies, and screening of entry inhibitors in a BSL-2 facility. Moreover, green tea might be a promising natural remedy against emerging coronaviruses.

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Antibodies and Vaccines Target RBD of SARS-CoV-2

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.671633 ◽

2021 ◽

Vol 8 ◽

Author(s):

Long Min ◽

Qiu Sun

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Development ◽

Neutralizing Antibody ◽

Protein S ◽

The Novel ◽

Global Public Health ◽

Human Coronavirus ◽

S Protein ◽

Important Target ◽

A Subunit

The novel human coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which gives rise to the coronavirus disease 2019 (COVID-19), has caused a serious threat to global public health. On March 11, 2020, the WHO had officially announced COVID-19 as a pandemic. Therefore, it is vital to find effective and safe neutralizing antibodies and vaccines for COVID-19. The critical neutralizing domain (CND) that is contained in the receptor-binding domain (RBD) of the spike protein (S protein) could lead to a highly potent neutralizing antibody response as well as the cross-protection of other strains of SARS. By using RBD as an antigen, many neutralizing antibodies are isolated that are essential to the therapeutics of COVID-19. Furthermore, a subunit vaccine, which is based on the RBD, is expected to be safer than others, thus the RBD in the S protein is a more important target for vaccine development. In this review, we focus on neutralizing antibodies that are targeting RBD as well as the vaccine based on RBD under current development.

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Virtual Screening of Curcumin and Its Analogs Against the Spike Surface Glycoprotein of SARS-CoV-2 and SARS-CoV

10.26434/chemrxiv.12142383 ◽

2020 ◽

Author(s):

Ashish Patel ◽

Malathi Rajendran ◽

Suresh B Pakala ◽

Ashish Shah ◽

Harnisha Patel ◽

...

Keyword(s):

Binding Energy ◽

Viral Entry ◽

Curcuma Longa ◽

Binding Energies ◽

Surface Glycoprotein ◽

Spike Protein ◽

Pharmacokinetic Parameters ◽

The Novel ◽

Angiotensin Converting Enzyme 2 ◽

Entry Inhibitors

COVID-19, a new pandemic caused by SARS-CoV-2, was first identified in 2019 in Wuhan, China. The novel corona virus SARS-CoV-2 and the 2002 SARS-CoV have 74 % identity and use similar mechanisms to gain entry into the cell. Both the viruses enter the host cell by binding of the viral spike glycoprotein to the host receptor, angiotensin converting enzyme 2 (ACE2). Targeting entry of the virus has a better advantage than inhibiting the later stages of the viral life cycle. The crystal structure of the SARS-CoV (6CRV: full length S protein) and SARS-CoV-2 Spike proteins (6M0J: Receptor binding domain, RBD) was used to determine potential small molecule inhibitors. Curcumin, a naturally occurring phytochemical in Curcuma longa, is known to have broad pharmacological properties. In the present study, curcumin and its derivatives were docked, using Autodock 4.2, onto the 6CRV and 6M0J to study their capability to act as inhibitors of the spike protein and thereby, viral entry. The curcumin and its derivatives displayed binding energies, ΔG, ranging from -10.98 to -5.12 kcal/mol (6CRV) and -10.01 to -5.33 kcal/mol (6M0J). The least binding energy was seen in bis-demethoxycurcumin with: ΔG = -10.98 kcal/mol (6CRV) and -10.01 kcal/mol (6M0J). A good binding energy, drug likeness and efficient pharmacokinetic parameters suggest the potential of curcumin and few of its derivatives as SARS-CoV-2 spike protein inhibitors. However, further research is necessary to investigate the ability of these compounds as viral entry inhibitors.<br>

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Virus-free and live-cell visualizing SARS-CoV-2 cell entry for studies of neutralizing antibodies and compound inhibitors

10.1101/2020.07.22.215236 ◽

2020 ◽

Author(s):

Yali Zhang ◽

Shaojuan Wang ◽

Yangtao Wu ◽

Wangheng Hou ◽

Lunzhi Yuan ◽

...

Keyword(s):

Viral Entry ◽

High Throughput Screening ◽

Neutralizing Antibodies ◽

Fluorescent Protein ◽

Expression System ◽

Live Cell ◽

Phenotypic Characterization ◽

Visualization System ◽

Entry Inhibitors ◽

Versatile Tool

AbstractThe ongoing COVID-19 pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and host ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system, we generated a recombinant fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process. In ACE2-expressing cells, we found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.

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