Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus based assay

Nie, Jianhui; Li, Qianqian; Wu, Jiajing; Zhao, Chenyan; Hao, Huan; Liu, Huan; Zhang, Li; Nie, Lingling; Qin, Haiyang; Wang, Meng; Lu, Qiong; Li, Xiaoyü; Sun, Qiyu; Liu, Junkai; Fan, Changfa; Huang, Weijin; Xu, Miao; Wang, Youchun

doi:10.21203/rs.3.pex-941/v1

Cited by 55 publications

(80 citation statements)

References 15 publications

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“…SARS-CoV-2 pseudovirus neutralization assay for the original (Wuhan-Hu-1) strain and variant B.1.1.7, B.1.351 and P.1. strains (Beijing Tiantan Pharmaceutical Biotechnology Development Co. Ltd.) were conducted as previously described (40), with some modifications. To evaluate the SARS-CoV-2 pseudovirus neutralization activity of antisera, samples were first heat-inactivated for 30 min and serially diluted (3-fold), incubated with an equal volume of 650 TCID50 pseudovirus at 37°C for 1 h, along with virus-alone (positive control) and cell-alone (negative control).…”

Section: Pseudovirus Neutralization Assaysmentioning

confidence: 99%

Broad neutralization against SARS-CoV-2 variants induced by a modified B.1.351 protein-based COVID-19 vaccine candidate

et al. 2021

Preprint

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Beginning in late 2020, the emergence and spread of multiple variant SARS-CoV-2 strains harboring mutations which may enable immune escape necessitates the rapid evaluation of second generation COVID-19 vaccines, with the goal of inducing optimized immune responses that are broadly protective. Here we demonstrate in a mouse immunogenicity study that two doses of a modified B.1.351 spike (S)-Trimer vaccine (B.1.351 S-Trimer) candidate can induce strong humoral immune responses that can broadly neutralize both the original SARS-CoV-2 strain (Wuhan-Hu-1) and Variants of Concern (VOCs), including the UK variant (B.1.1.7), South African variant (B.1.351) and Brazil variant (P.1). Furthermore, while immunization with two doses (prime-boost) of Prototype S-Trimer vaccine (based on the original SARS-CoV-2 strain) induced lower levels of cross-reactive neutralization against the B.1.351 variant, a third dose (booster) administered with either Prototype S-Trimer or B.1.351 S-Trimer was able to increase neutralizing antibody titers against B.1.351 to levels comparable to neutralizing antibody titers against the original strain elicited by two doses of Prototype S-Trimer.

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Section: Pseudovirus Neutralization Assaysmentioning

confidence: 99%

Broad neutralization against SARS-CoV-2 variants induced by a modified B.1.351 protein-based COVID-19 vaccine candidate

et al. 2021

Preprint

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“…This assay can also be used to deconvolute mechanisms of action of anti-SARS-CoV-2 hits from live SARS-CoV-2 screens to identify compounds that act on viral entry versus viral replication. Viral pseudotyped assays have been commonly used to examine neutralization activity of antibodies and other biologics that block spike protein binding to the ACE2 receptor 13 . However, unlike testing neutralizing antibodies, which have a clear target and mechanism of action, there is less certainty about the molecular target and the mechanism of inhibition when screening cell penetrant small molecules, which introduces additional mechanisms for false positives, such as inhibition of luciferase reporter expression.…”

Section: Hit Confirmation In a Sars-cov-2 Cytopathic Effect (Cpe) Assaymentioning

confidence: 99%

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Pradhan

Gorshkov

et al. 2021

Preprint

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Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

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“…The virucidal activity of OSCN − was rstly investigated with a recombinant Vesicular Stomatitis Virus (rVSV), encoding the reported gene luciferase instead of the viral glycoprotein. The rVSV can be easily manipulated under biosafety level 2 conditions and pseudotyped by the S protein obtaining a virus (rVSV-S) with a tropism dictated by the heterologous S envelope which represents an excellent surrogate of SARS-CoV-2 to study the virus entry and the viral neutralization [8,10,11]. Indeed, rVSV-S was incubated with different OSCN − concentrations for 60 min at 37 °C (pre-treatment), and then Vero cells were infected at multiplicity of infection (MOI) 0.065 FFU/cell.…”

Section: Virucidal Activity Of Oscnand Oscn -/Lfmentioning

confidence: 99%

In-vitro Virucidal Activity of Hypothiocyanite and Hypothiocyanite/lactoferrin Mix Against SARS-CoV-2

Cegolon

Mirandola

Salaris

et al. 2020

Preprint

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SARS-CoV-2 replicates efficiently in the upper airways during the prodromal stage, with resulting viral shedding into the environment from patients with active COVID-19 as well as from asymptomatic individuals. There is a need to find pharmacological interventions to mitigate the spread of COVID-19. Hypothiocyanite and lactoferrin are molecules of the innate immune system with a large spectrum cidal activity and easy to administer by aerosol inhalation. The combination of the above two molecules was designated as orphan drug by the Food and Drug Administration and the European Medicines Agency. Here we found a dose-dependent as well as time-dependent virucidal activity of hypothiocyanite at micromolar concentrations, slightly improved by lactoferrin, against SARS-CoV-2. The two substances individually tested were devoid of any cytotoxicity.

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Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus based assay

Cited by 55 publications

References 15 publications

Broad neutralization against SARS-CoV-2 variants induced by a modified B.1.351 protein-based COVID-19 vaccine candidate

Broad neutralization against SARS-CoV-2 variants induced by a modified B.1.351 protein-based COVID-19 vaccine candidate

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

In-vitro Virucidal Activity of Hypothiocyanite and Hypothiocyanite/lactoferrin Mix Against SARS-CoV-2

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