Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol

Capcha, José Manuel Cóndor; Lambert, Guerline; Dykxhoorn, Derek M.; Salerno, Alessandro G.; Hare, Joshua M.; Whitt, Michael A.; Pahwa, Savita; Jayaweera, Dushyantha; Shehadeh, Lina A.

doi:10.3389/fcvm.2020.618651

Cited by 62 publications

(61 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Sample throughput may be increased by using microneutralization assays. Pseudovirus-based neutralization assays use replication deficient virus and do not require former safety measures [ 11 , 12 , 13 ]. They are also widely used and accepted, but they are also labor-intensive and not suitable for the analysis of a large cohort of subjects.…”

Section: Introductionmentioning

confidence: 99%

Utility of Different Surrogate Enzyme-Linked Immunosorbent Assays (sELISAs) for Detection of SARS-CoV-2 Neutralizing Antibodies

Kohmer

Rühl

Ciesek

et al. 2021

JCM

View full text Add to dashboard Cite

The plaque reduction neutralization test (PRNT) is a preferred method for the detection of functional, SARS-CoV-2 specific neutralizing antibodies from serum samples. Alternatively, surrogate enzyme-linked immunosorbent assays (ELISAs) using ACE2 as the target structure for the detection of neutralization-competent antibodies have been developed. They are capable of high throughput, have a short turnaround time, and can be performed under standard laboratory safety conditions. However, there are very limited data on their clinical performance and how they compare to the PRNT. We evaluated three surrogate immunoassays (GenScript SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript Biotech, Piscataway Township, NJ, USA), the TECO® SARS-CoV-2 Neutralization Antibody Assay (TECOmedical AG, Sissach, Switzerland), and the Leinco COVID-19 ImmunoRank™ Neutralization MICRO-ELISA (Leinco Technologies, Fenton, MO, USA)) and one automated quantitative SARS-CoV-2 Spike protein-based IgG antibody assay (Abbott GmbH, Wiesbaden, Germany) by testing 78 clinical samples, including several follow-up samples of six BNT162b2 (BioNTech/Pfizer, Mainz, Germany/New York, NY, USA) vaccinated individuals. Using the PRNT as a reference method, the overall sensitivity of the examined assays ranged from 93.8 to 100% and specificity ranged from 73.9 to 91.3%. Weighted kappa demonstrated a substantial to almost perfect agreement. The findings of our study allow these assays to be considered when a PRNT is not available. However, the latter still should be the preferred choice. For optimal clinical performance, the cut-off value of the TECO assay should be individually adapted.

show abstract

Section: Introductionmentioning

confidence: 99%

Utility of Different Surrogate Enzyme-Linked Immunosorbent Assays (sELISAs) for Detection of SARS-CoV-2 Neutralizing Antibodies

Kohmer

Rühl

Ciesek

et al. 2021

JCM

View full text Add to dashboard Cite

show abstract

“…A second confirmatory assay has been done with a different pseudovirus (SARS-CoV-2 spike plus GFP reporter bearing VSV-ΔG pseudovirus, i.e., vesicular stomatitis virus that lacks the VSV envelope glycoprotein) 89 and cell line (ACE2/Furin-overexpressing Vero-E6 cells). GFP fluorescence quantified using a live imaging system (Incucyte) was used as a measure of infection, and normalized values were fitted with regular concentration response curves as before.…”

Section: Resultsmentioning

confidence: 99%

“…For the VSV-ΔG based assay, the SARS-CoV-2 S bearing pseudovirus generated in-house was used as described before. 89 Vero-E6 cells (African Green Monkey renal epithelial cells; ATCC cat. no.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

et al. 2021

Self Cite

View full text Add to dashboard Cite

Inhibitors of the protein–protein interaction (PPI) between the SARS-CoV-2 spike protein and human ACE2 (hACE2), which acts as a ligand–receptor pair that initiates the viral attachment and cellular entry of this coronavirus causing the ongoing COVID-19 pandemic, are of considerable interest as potential antiviral agents. While blockade of such PPIs with small molecules is more challenging than that with antibodies, small-molecule inhibitors (SMIs) might offer alternatives that are less strain- and mutation-sensitive, suitable for oral or inhaled administration, and more controllable/less immunogenic. Here, we report the identification of SMIs of this PPI by screening our compound library focused around the chemical space of organic dyes. Among promising candidates identified, several dyes (Congo red, direct violet 1, Evans blue) and novel druglike compounds (DRI-C23041, DRI-C91005) inhibited the interaction of hACE2 with the spike proteins of SARS-CoV-2 as well as SARS-CoV with low micromolar activity in our cell-free ELISA-type assays (IC 50 ’s of 0.2–3.0 μM), whereas control compounds, such as sunset yellow FCF, chloroquine, and suramin, showed no activity. Protein thermal shift assays indicated that the SMIs of interest identified here bind SARS-CoV-2-S and not hACE2. While dyes seemed to be promiscuous inhibitors, DRI-C23041 showed some selectivity and inhibited the entry of two different SARS-CoV-2-S expressing pseudoviruses into hACE2-expressing cells in a concentration-dependent manner with low micromolar IC 50 ’s (6–7 μM). This provides proof-of-principle evidence for the feasibility of small-molecule inhibition of PPIs critical for SARS-CoV-2 attachment/entry and serves as a first guide in the search for SMI-based alternative antiviral therapies for the prevention and treatment of diseases caused by coronaviruses in general and COVID-19 in particular.

show abstract

“…The medium was replaced with 5%FCS-supplemented DMEM complemented with a mouse monoclonal anti-VSV-G antibody (CliniSciences, clone 8G5F11, final concentration 1 μg/mL) to neutralize residual viral input, as described (Condor Capcha et al, 2020). Cell supernatants containing pseudotyped VSV viruses were harvested 24h…”

Section: Spike Pseudotype Productionmentioning

confidence: 99%

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses

Rebendenne

Roy

Bonaventure

et al. 2021

Preprint

View full text Add to dashboard Cite

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

show abstract

Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol

Cited by 62 publications

References 36 publications

Utility of Different Surrogate Enzyme-Linked Immunosorbent Assays (sELISAs) for Detection of SARS-CoV-2 Neutralizing Antibodies

Utility of Different Surrogate Enzyme-Linked Immunosorbent Assays (sELISAs) for Detection of SARS-CoV-2 Neutralizing Antibodies

Small-Molecule Inhibitors of the Coronavirus Spike: ACE2 Protein–Protein Interaction as Blockers of Viral Attachment and Entry for SARS-CoV-2

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses

Contact Info

Product

Resources

About