scholarly journals A Neutralization Assay Based on Pseudo-Typed Lentivirus with SARS CoV-2 Spike Protein in ACE2-Expressing CRFK Cells

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 153
Author(s):  
Yalçın Pısıl ◽  
Hisatoshi Shida ◽  
Tomoyuki Miura
Keyword(s):  
Monoclonal Antibodies ◽  
Inhibitory Concentration ◽  
Immunoglobulin A ◽  
Hek293t Cell ◽  
Neutralization Assay ◽  
Cell System ◽  
Immunoglobulin M ◽  
Spike Protein ◽  
Highly Pathogenic ◽  
Angiotensin Converting Enzyme 2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic zoonotic virus that spreads rapidly. In this work, we improve the hitherto existing neutralization assay system to assess SARS-CoV-2 inhibitors using a pseudo-typed lentivirus coated with the SARS-CoV-2 spike protein (LpVspike +) and angiotensin-converting enzyme 2 (ACE2)-transfected cat Crandell–Rees feline kidney (CRFK) cells as the host cell line. Our method was 10-fold more sensitive compared to the typical human embryonic kidney 293T (HEK293T) cell system, and it was successfully applied to quantify the titers of convalescent antisera and monoclonal anti-spike antibodies required for pseudo virus neutralization. The 50% inhibition dilution (ID50) of two human convalescent sera, SARS-CoV-2 immunoglobulin G (IgG) and SARS-CoV-2 immunoglobulin M (IgM), which were 1:350 (±1:20) and 1:1250 (±1:350), respectively. The 50% inhibitory concentration (IC50) of the IgG, IgM and immunoglobulin A (IgA) anti-SARS-CoV-2 monoclonal antibodies (mAbs) against LpVspike(+) were 0.45 (±0.1), 0.002 (±0.001) and 0.004 (±0.001) µg mL−1, respectively. We also found that reagents typically used to enhance infection were not effective in the CFRK system. This methodology is both efficient and safe; it can be employed by researchers to evaluate neutralizing monoclonal antibodies and contribute to the discovery of new antiviral inhibitors against SARS-CoV-2.

scholarly journals Monoclonal antibodies targeting the influenza virus N6 neuraminidase

2021 ◽  
Author(s):  
Shirin Strohmeier ◽  
Fatima Amanat ◽  
Juan Manuel Carreño ◽  
Florian Krammer
Keyword(s):  
Monoclonal Antibodies ◽  
Influenza A ◽  
Neutralization Assay ◽  
Escape Mutant ◽  
Influenza A Viruses ◽  
Highly Pathogenic ◽  
Lateral Ridge

AbstractInfluenza A viruses are a diverse species that include 16 hemagglutinin (HA) subtypes and 9 neuraminidase (NA) subtypes. While the antigenicity of many HA subtypes is reasonably well studied, less is known about NA antigenicity, especially when it comes to non-human subtypes that only circulate in animal reservoirs. The N6 NA subtypes are mostly found in viruses infecting birds. However, they have also been identified in viruses that infect mammals, such as swine and seals. More recently, highly pathogenic H5N6 subtype viruses have caused rare infections and mortality in humans. Here, we generated murine mAbs to the N6 NA, characterized their breadth and antiviral properties in vitro and in vivo and mapped their epitopes by generating escape mutant viruses. We found that the antibodies had broad reactivity across the American and Eurasian N6 lineages, but relatively little binding and inhibition of the H5N6 NA. Several of the antibodies exhibited strong NA inhibition activity and some also showed activity in the antibody dependent cellular cytotoxicity reporter assay and neutralization assay. In addition, we generated escape mutant viruses for six monoclonal antibodies and found mutations on the lateral ridge of the NA. Lastly, we observed variable protection in H4N6 and H5N6 mouse challenge models when the antibodies were given prophylactically.ImportanceThe N6 NA has recently gained prominence due to the emergence of highly pathogenic H5N6 viruses. Currently, there is limited characterization of the antigenicity of avian N6 neuraminidase. Our data is an important first step towards a better understanding of the N6 NA antigenicity.

scholarly journals Cross-Neutralizing Activity of Monoclonal Antibodies Against N501Y Mutant Strain of SARS-CoV-2

2020 ◽  
Vol 9 (4) ◽  
pp. 41-45
Author(s):  
Ruxia Ding ◽  
Haixin Wang ◽  
Yi Yang ◽  
Liangshu Xie ◽  
Li Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Mutant Strain ◽  
Wild Type Strain ◽  
Neutralization Assay ◽  
Spike Protein ◽  
Wild Type ◽  
Neutralizing Activity ◽  
Theory Result ◽  
Global Concern ◽  
The Uk

The dominant N501Y mutation in the spike protein that SARS-CoV-2 virus uses to bind to the human ACE2 receptor were found in the UK, which has aroused global concern and worried. Mutations in spike protein may, in theory, result in more infectious and spreading more easily. In order to evaluate the broad-spectrum protective effect of the monoclonal antibodies(mAbs), we compared the neutralization activities of six prepared mAbs against SARS-CoV-2 with pseudovirus neutralization assay. Only one of them showed a decrease of 6 folds in neutralizing activity to N501Y mutant strain, compared with the wild type strain. We should continue to monitor emergence of new variants in different regions to study their infectivity and neutralization effect.

scholarly journals Synthetic antibodies neutralize SARS-CoV-2 infection of mammalian cells

2020 ◽  
Author(s):  
Shane Miersch ◽  
Mart Ustav ◽  
Zhijie Li ◽  
James B. Case ◽  
Safder Ganaie ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Receptor Binding ◽  
Mammalian Cells ◽  
Rna Viruses ◽  
Large Family ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Highly Pathogenic ◽  
Zoonotic Infections

ABSTRACTCoronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.

scholarly journals Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

2020 ◽  
Author(s):  
Sabari Nath Neerukonda ◽  
Russell Vassell ◽  
Rachel Herrup ◽  
Shufeng Liu ◽  
Tony Wang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
293T Cell ◽  
Neutralization Assay ◽  
Target Cells ◽  
Highly Pathogenic ◽  
Angiotensin Converting Enzyme 2 ◽  
Pathogenic Viruses ◽  
Screening Assays ◽  
Assay Precision ◽  
Transmembrane Serine Protease

AbstractPseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

scholarly journals Sequence variation of SARS-CoV-2 spike protein may facilitate stronger interaction with ACE2 promoting high infectivity

2020 ◽  
Author(s):  
Masaud Shah ◽  
Bilal Ahmad ◽  
Sangdun Choi ◽  
Hyun Goo Woo
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Sequence Variation ◽  
Spike Protein ◽  
Cell Recognition ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Binding Domains ◽  
Conformational Epitopes ◽  
High Infectivity

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19), is a novel beta coronavirus emerged in China in 2019. Coronavirus uses spike glycoprotein to interact with host angiotensin-converting enzyme 2 (ACE2) and ensure cell recognition. High infectivity of SARS-CoV-2 raises questions on spike-ACE2 binding affinity and its neutralization by anti-SARS-CoV monoclonal antibodies (mAbs). Here, we observed Val-to-Lys417 mutation in the receptor-binding domains (RBD) of SARS-CoV-2, which established a Lys-Asp electrostatic interaction enhancing its ACE2-binding. Pro-to-Ala475 substitution and Gly482 insertion in the AGSTPCNGV-loop of RBD hindered neutralization of SARS-CoV-2 by anti-SARS-CoV mAbs. In addition, we identified unique and structurally conserved conformational-epitopes on RBDs, which can be potential therapeutic targets. Collectively, we provide new insights into the mechanisms underlying the high infectivity of SARS-CoV-2 and development of new effective neutralizing agents.

scholarly journals Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248348
Author(s):  
Sabari Nath Neerukonda ◽  
Russell Vassell ◽  
Rachel Herrup ◽  
Shufeng Liu ◽  
Tony Wang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
293T Cell ◽  
Neutralization Assay ◽  
Target Cells ◽  
Highly Pathogenic ◽  
Angiotensin Converting Enzyme 2 ◽  
Pathogenic Viruses ◽  
Screening Assays ◽  
Assay Precision ◽  
Transmembrane Serine Protease

Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.

scholarly journals Spherical Neutralizing Aptamer Inhibits SARS-CoV-2 Infection

2021 ◽  
Author(s):  
Miao Sun ◽  
Siwen Liu ◽  
Ting Song ◽  
Fude Chen ◽  
Jialu Zhang ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Conformational Transition ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Mutant Strains ◽  
Infection Mechanisms ◽  
Binding Interfaces ◽  
Potent Neutralization

<p>New neutralizing agents against SARS-CoV-2 and the associated mutant strains are urgently needed for the treatment and prophylaxis of COVID-19. Herein, we develop a <u>s</u>pherical cocktail <u>n</u>eutralizing <u>a</u>ptamer-gold nano<u>p</u>article (SNAP) to synergistically block the interaction of SARS-CoV-2 receptor-binding domain (RBD) and angiotensin-converting enzyme-2 (ACE2). Taking advantage of the simultaneous recognition of multi-homologous and multi-heterogenous neutralizing aptamers and dimensionally matched nano-scaffolds, the SNAP exhibits increased affinity to the RBD with a dissociation constant value of 5.46 pM and potent neutralization against authentic SARS-CoV-2 with a half-maximal inhibitory concentration of 142.80 aM. Additional benefits include the multi-epitope blocking capability of the aptamer cocktail and the steric hindrance of the nano-scaffold, which further covers the ACE2 binding interfaces and affects the conformational transition of the spike protein. As a result, the SNAP strategy exhibits broad neutralizing activity, almost completely blocking the infection of<a> N501Y</a> and D614G mutant strains. Overall, the SNAP strategy provides a new direction for development of anti-virus infection mechanisms, both to fight the COVID-19 pandemic and serve as a powerful technical reserve for future unknown pandemics.</p>

scholarly journals Spherical Neutralizing Aptamer Inhibits SARS-CoV-2 Infection

2021 ◽  
Author(s):  
Miao Sun ◽  
Siwen Liu ◽  
Ting Song ◽  
Fude Chen ◽  
Jialu Zhang ◽  
...  
Keyword(s):  
Inhibitory Concentration ◽  
Conformational Transition ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme 2 ◽  
Mutant Strains ◽  
Infection Mechanisms ◽  
Binding Interfaces ◽  
Potent Neutralization

<p>New neutralizing agents against SARS-CoV-2 and the associated mutant strains are urgently needed for the treatment and prophylaxis of COVID-19. Herein, we develop a <u>s</u>pherical cocktail <u>n</u>eutralizing <u>a</u>ptamer-gold nano<u>p</u>article (SNAP) to synergistically block the interaction of SARS-CoV-2 receptor-binding domain (RBD) and angiotensin-converting enzyme-2 (ACE2). Taking advantage of the simultaneous recognition of multi-homologous and multi-heterogenous neutralizing aptamers and dimensionally matched nano-scaffolds, the SNAP exhibits increased affinity to the RBD with a dissociation constant value of 5.46 pM and potent neutralization against authentic SARS-CoV-2 with a half-maximal inhibitory concentration of 142.80 aM. Additional benefits include the multi-epitope blocking capability of the aptamer cocktail and the steric hindrance of the nano-scaffold, which further covers the ACE2 binding interfaces and affects the conformational transition of the spike protein. As a result, the SNAP strategy exhibits broad neutralizing activity, almost completely blocking the infection of<a> N501Y</a> and D614G mutant strains. Overall, the SNAP strategy provides a new direction for development of anti-virus infection mechanisms, both to fight the COVID-19 pandemic and serve as a powerful technical reserve for future unknown pandemics.</p>

scholarly journals Potent Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by photosensitizers

2021 ◽  
Author(s):  
Shujuan Yu ◽  
Gaohui Sun ◽  
Yaqun Sui ◽  
Hanlin Li ◽  
Ning Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Antiviral Treatment ◽  
Spike Protein ◽  
Converting Enzyme ◽  
Viral Inactivation ◽  
Angiotensin Converting Enzyme 2 ◽  
Potent Inhibition

Abstract The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has exploded since December 2019, and causes more than 2 million death with more than 95 million people infected as of Jan. 21th, 2021 globally1,2. Angiotensin-converting enzyme 2 (ACE2), expressed in the lungs, arteries, heart, kidney, intestines, and nasal epithelium3, has been shown to be the primary entry point targeted by the surface spike protein of SARS-CoV-2. Currently, no proven antiviral treatment for SARS-CoV-2 infection is available. In this study, we screened a number of photosensitizers for photodynamic viral inactivation, and found compounds pentalysine β-carbonylphthalocyanine zinc (ZnPc5K) and chlorin e6 (ce6) potently inhibited the viral infection and replication in vitro with half-maximal effective concentrations (EC50) values at nanomolar level. Such viral inactivation strategy is implementable, and has unique advantages, including resistance to virus mutations, affordability compared to the monoclonal antibodies, and lack of long-term toxicity.

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