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

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.

scholarly journals Characterization of SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta) and B.1.618 on cell entry, host range, and sensitivity to convalescent plasma and ACE2 decoy receptor

2021 ◽  
Author(s):  
Wenlin Ren ◽  
Xiaohui Ju ◽  
Mingli Gong ◽  
Jun Lan ◽  
Yanying Yu ◽  
...  
Keyword(s):  
Host Range ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Cell Entry ◽  
Decoy Receptor ◽  
Host Tropism ◽  
Entry Inhibitors ◽  
Rapid Spread ◽  
Entry Efficiency

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.

scholarly journals Targeted destabilization of the HIV-1 gp120-gp41 interface leads to convergent evolution with mutations in the V1V2, HR1 and HR2 domains

2021 ◽  
Author(s):  
Alba Torrents de la Peña ◽  
Iván del Moral Sánchez ◽  
Judith A. Burger ◽  
Ilja Bontjer ◽  
Gözde Isik ◽  
...  
Keyword(s):  
Viral Entry ◽  
Envelope Glycoprotein ◽  
Convergent Evolution ◽  
High Throughput Screening ◽  
Neutralizing Antibodies ◽  
Dominant Role ◽  
Virus Evolution ◽  
Target Cells ◽  
Compensatory Mutations ◽  
Hiv 1

The HIV-1 envelope glycoprotein (Env) trimer is responsible for viral entry into target cells and is the sole target of neutralizing antibodies. The Env protein is therefore the focus of HIV-1 vaccine design. Env consists of two non-covalently linked subunits (gp120 and gp41) that form a trimer of heterodimers and this 6-subunit complex is metastable and conformationally flexible. Several approaches have been pursued to stabilize the Env trimer for vaccine purposes, which include structure-based design, high-throughput screening and selection by mammalian cell display. Here, we employed directed virus evolution to improve Env trimer stability. Accordingly, we deliberately destabilized the Env gp120-gp41 interface by mutagenesis in the context of replicating HIV-1 LAI virus and virus evolution over time. We identified compensatory changes that pointed at convergent evolution as they were largely restricted to specific Env regions, namely the V1V2-domain of gp120, and the the HR1 and HR2 domain of gp41. Specifically, S614G in V1V2 and Q567R in HR1 were frequently identified. Interestingly, the majority of the compensatory mutations were at distant locations from the original mutations and most likely strengthen inter-subunit interactions. These results show how the virus can overcome Env instability and illuminate the regions that play a dominant role in Env stability. Importance A successful HIV-1 vaccine most likely requires an envelope glycoprotein (Env) component, as the Env is the only viral protein on the surface of the virus and the target for neutralizing antibodies. However, HIV Env is metastable and flexible because of the weak interactions between the Env subunits, complicating the generation of recombinant mimics of native Env. Here, we used directed viral evolution to study Env stability. We deliberately destabilized the interface between Env subunits and explored the capacity of the virus to repair trimer instability by evolution. We identified compensatory mutations that converged in specific Env locations: the apex and the trimer interface. Selected mutations enhanced the stability of recombinant soluble Env trimer proteins. These results provided clues on understanding the structural mechanisms involved in Env trimer stability, which can guide future immunogen design.

scholarly journals A Comparative High-Throughput Screening Protocol to Identify Entry Inhibitors of Enveloped Viruses

2013 ◽  
Vol 19 (1) ◽  
pp. 100-107 ◽  
Author(s):  
Juan Wang ◽  
Han Cheng ◽  
Kiira Ratia ◽  
Elizabeth Varhegyi ◽  
William G. Hendrickson ◽  
...  
Keyword(s):  
High Throughput ◽  
Viral Entry ◽  
High Throughput Screening ◽  
Marburg Virus ◽  
Lassa Virus ◽  
Viral Pathogens ◽  
Enveloped Viruses ◽  
Entry Inhibitors ◽  
Screening Protocol ◽  
Therapeutic Prevention

Emerging and reemerging human viral pathogens pose great public health concerns since therapeutics against these viruses are limited. Thus, there is an urgent need to develop novel drugs that can block infection of either a specific virus or a number of viruses. Viral entry is thought to be an ideal target for potential therapeutic prevention. One of the challenges of developing antivirals is that most of these viruses are highly pathogenic and therefore require high biosafety-level containment. In this study, we have adopted a comparative high-throughput screening protocol to identify entry inhibitors for three enveloped viruses (Marburg virus, influenza virus H5N1, and Lassa virus) using a human immunodeficiency virus–based pseudotyping platform. We demonstrate the utility of this approach by screening a small compound library and identifying putative entry inhibitors for these viruses. One major advantage of this protocol is to reduce the number of false positives in hit selection, and we believe that the protocol is useful for inhibitor screening for many enveloped viruses.

scholarly journals Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera

2020 ◽  
Author(s):  
Kasopefoluwa Y. Oguntuyo ◽  
Christian S Stevens ◽  
Chuan-Tien Hung ◽  
Satoshi Ikegame ◽  
Joshua A. Acklin ◽  
...  
Keyword(s):  
Viral Entry ◽  
High Throughput Screening ◽  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Virus Neutralization ◽  
Strong Positive Correlation ◽  
Plasma Therapy ◽  
Live Virus ◽  
Virus Neutralization Assay ◽  
Marked Variability

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVdeltaG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

A Homogeneous Fluorescent Live-Cell Assay for Measuring 7-Transmembrane Receptor Activity and Agonist Functional Selectivity Through Beta-Arrestin Recruitment

2009 ◽  
Vol 14 (7) ◽  
pp. 798-810 ◽  
Author(s):  
Bonnie J. Hanson ◽  
Justin Wetter ◽  
Mark R. Bercher ◽  
Leisha Kopp ◽  
Maya Fuerstenau-Sharp ◽  
...  
Keyword(s):  
G Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Second Messengers ◽  
Expression System ◽  
Live Cell ◽  
Assay Method ◽  
Protein Signaling ◽  
Functional Selectivity ◽  
7Tm Receptors

Seven-transmembrane (7TM) receptors play an essential role in the regulation of a wide variety of physiological processes, making them one of the top target classes for pharmaceuticals. 7TM receptor function is mediated and modulated through 2 primary processes: G-protein and beta-arrestin signaling. Classically, it has been recognized that these 2 processes can interact with one another during 7TM receptor desensitization, but it has more recently been recognized that these 2 processes can also act independently of one another and can activate parallel signaling pathways. As such, the methods used to interrogate 7TM receptor signaling, both from a biological and a pharmaceutical perspective, may need to be reevaluated and the question of whether functionally selective compounds (compounds that selectively activate one pathway over another) can be rationally developed must be raised. Although numerous high-throughput screening (HTS) compatible assays exist for studying second messengers arising from G-protein signaling, far fewer HTS compatible assays exist for studying beta-arrestin recruitment. The authors report on the Tango™ 7TM receptor assay technology, a high-throughput homogeneous assay method for monitoring beta-arrestin recruitment that uses a live-cell fluorescent readout. This assay format is broadly applicable to 7TM receptors, independent of G-protein coupling and, as such, has been used to produce assays for over 70 7TM receptor targets. The authors also show how flow cytometry can be used to select clones with desired pharmacological profiles and how an inducible expression system can increase the assay window for targets with high levels of constitutive activity. Finally, they demonstrate how the Tango™ system can be used in parallel with assays aimed at second-messenger signaling to enable functional selectivity studies. ( Journal of Biomolecular Screening 2009:798-810)

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

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.

scholarly journals Chimeric Filoviruses for Identification and Characterization of Monoclonal Antibodies

2016 ◽  
Vol 90 (8) ◽  
pp. 3890-3901 ◽  
Author(s):  
Philipp A. Ilinykh ◽  
Xiaoli Shen ◽  
Andrew I. Flyak ◽  
Natalia Kuzmina ◽  
Thomas G. Ksiazek ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
High Throughput Screening ◽  
Neutralizing Antibodies ◽  
Reverse Genetics ◽  
Ebola Virus ◽  
Fluorescent Protein ◽  
Marburg Virus ◽  
Antibody Neutralization ◽  
Surrogate Systems

ABSTRACTRecent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently use surrogate systems such as the rhabdovirus vesicular stomatitis Indiana virus (VSV), lentiviruses or gammaretroviruses with their envelope proteins replaced with EBOV GP or pseudotyped with EBOV GP. It is optimal for both screening and in-depth characterization of newly identified neutralizing MAbs to generate recombinant filoviruses that express a reporter fluorescent protein in order to more easily monitor and quantify the infection. Our study showed that unlike neutralization-sensitive chimeric VSV, authentic filoviruses are highly resistant to neutralization by MAbs. We used reverse genetics techniques to replace EBOV GP with its counterpart from the heterologous filoviruses Bundibugyo virus (BDBV), Sudan virus, and even Marburg virus and Lloviu virus, which belong to the heterologous genera in the filovirus family. This work resulted in generation of multiple chimeric filoviruses, demonstrating the ability of filoviruses to tolerate swapping of the envelope protein. The sensitivity of chimeric filoviruses to neutralizing MAbs was similar to that of authentic biologically derived filoviruses with the same GP. Moreover, disabling the expression of the secreted GP (sGP) resulted in an increased susceptibility of an engineered virus to the BDBV52 MAb isolated from a BDBV survivor, suggesting a role for sGP in evasion of antibody neutralization in the context of a human filovirus infection.IMPORTANCEThe study demonstrated that chimeric rhabdoviruses in which G protein is replaced with filovirus GP, widely used as surrogate targets for characterization of filovirus neutralizing antibodies, do not accurately predict the ability of antibodies to neutralize authentic filoviruses, which appeared to be resistant to neutralization. However, a recombinant EBOV expressing a fluorescent protein tolerated swapping of GP with counterparts from heterologous filoviruses, allowing high-throughput screening of B cell lines to isolate MAbs of any filovirus specificity. Human MAb BDBV52, which was isolated from a survivor of BDBV infection, was capable of partially neutralizing a chimeric EBOV carrying BDBV GP in which expression of sGP was disabled. In contrast, the parental virus expressing sGP was resistant to the MAb. Thus, the ability of filoviruses to tolerate swapping of GP can be used for identification of neutralizing MAbs specific to any filovirus and for the characterization of MAb specificity and mechanism of action.

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

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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