scholarly journals Evaluation of cell-based and surrogate SARS-CoV-2 neutralization assays

2021 ◽  
Author(s):  
Anton M. Sholukh ◽  
Andrew Fiore-Gartland ◽  
Emily S. Ford ◽  
Maurine D. Miner ◽  
Yixuan J. Hou ◽  
...  
Keyword(s):  
Geometric Mean ◽  
Live Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Protein Receptor ◽  
Virus Assay ◽  
Viral Particles ◽  
Antibody Assays ◽  
High Concordance ◽  
Highly Correlated ◽  
Transmembrane Serine Protease

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using forty plasma samples from convalescent individuals with mild-to-moderate COVID-19: four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate ELISA-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor, human angiotensin converting enzyme 2 (hACE2). Vero, Vero E6, HEK293T expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND50) geometric mean titers (GMTs) that were highly correlated (Pearson r = 0.81–0.89) and ranged within 3.4-fold. The live-virus assay and LV-pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers: 141 and 178, respectively. ND50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike and RBD ( r = 0.63–0.89), but moderately correlated with nucleoprotein IgG ( r = 0.46–0.73). ND80 GMTs mirrored ND50 data and showed similar correlation between assays and with IgG concentrations. The VSV-pseudovirus assay and LV-pseudovirus assay with HEK293T/hACE2 cells in low and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms. 249

scholarly journals Evaluation of SARS-CoV-2 neutralization assays for antibody monitoring in natural infection and vaccine trials

2020 ◽  
Author(s):  
Anton M Sholukh ◽  
Andrew Fiore-Gartland ◽  
Emily S Ford ◽  
Yixuan Hou ◽  
Longping Victor Tse ◽  
...  
Keyword(s):  
Natural Infection ◽  
Geometric Mean ◽  
Neutralization Assay ◽  
Elisa Test ◽  
Live Virus ◽  
Moderate Disease ◽  
Single Region ◽  
Antibody Assays ◽  
Highly Correlated ◽  
Transmembrane Serine Protease

Determinants of protective immunity against SARS-CoV-2 infection require the development of well-standardized, reproducible antibody assays to be utilized in concert with clinical trials to establish correlates of risk and protection. This need has led to the appearance of a variety of neutralization assays used by different laboratories and companies. Using plasma samples from COVID-19 convalescent individuals with mild-to-moderate disease from a localized outbreak in a single region of the western US, we compared three platforms for SARS-CoV-2 neutralization: assay with live SARS-CoV-2, pseudovirus assay utilizing lentiviral (LV) and vesicular stomatitis virus (VSV) packaging, and a surrogate ELISA test. Vero, Vero E6, HEK293T cells expressing human angiotensin converting enzyme 2 (hACE2), and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 (TMPRSS2) were evaluated. Live-virus and LV-pseudovirus assay with HEK293T cells showed similar geometric mean titers (GMTs) ranging 141–178, but VSV-pseudovirus assay yielded significantly higher GMT (310 95%CI 211-454; p < 0.001). Fifty percent neutralizing dilution (ND50) titers from live-virus and all pseudovirus assay readouts were highly correlated (Pearson r = 0.81–0.89). ND50 titers positively correlated with plasma concentration of IgG against SARS-CoV-2 spike and receptor binding domain (RBD) (r = 0.63–0.89), but moderately correlated with nucleoprotein IgG (r = 0.46-0.73). There was a moderate positive correlation between age and spike (Spearman's rho=0.37, p=0.02), RBD (rho=0.39, p=0.013) and nucleoprotein IgG (rho=0.45, p=0.003). ND80 showed stronger correlation with age than ND50 (ND80 rho=0.51 (p=0.001), ND50 rho=0.28 (p=0.075)). Our data demonstrate high concordance between cell-based assays with live and pseudotyped virions.

scholarly journals Adjuvanting a subunit SARS-CoV-2 nanoparticle vaccine to induce protective immunity in non-human primates

2021 ◽  
Author(s):  
Prabhu S. Arunachalam ◽  
Alexandra C. Walls ◽  
Nadia Golden ◽  
Caroline Atyeo ◽  
Stephanie Fischinger ◽  
...  
Keyword(s):  
Neutralizing Antibody ◽  
Alpha Tocopherol ◽  
Water Emulsion ◽  
Live Virus ◽  
Protein Receptor ◽  
Oil In Water ◽  
A Subunit ◽  
Highly Correlated ◽  
Oil In Water Emulsion ◽  
Protection Against Infection

AbstractThe development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

scholarly journals Diosmectite inhibits the interaction between SARS-CoV-2 and human enterocytes by trapping viral particles, thereby preventing NF-kappaB activation and CXCL10 secretion

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marco Poeta ◽  
Valentina Cioffi ◽  
Vittoria Buccigrossi ◽  
Merlin Nanayakkara ◽  
Melissa Baggieri ◽  
...  
Keyword(s):  
Nuclear Factor Kappa B ◽  
Direct Evidence ◽  
Spike Protein ◽  
Gi Tract ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Spread ◽  
Protein Receptor ◽  
Viral Particles ◽  
Nf Kappab ◽  
Apical Membranes

AbstractSARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.

scholarly journals Correlation between Commercial Anti-RBD IgG Titer and Neutralization Titer against SARS-CoV-2 Beta Variant

Diagnostics ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 2216
Author(s):  
Rosana Wing-Shan Poon ◽  
Lu Lu ◽  
Carol Ho-Yan Fong ◽  
Tak-Chuen Ip ◽  
Lin-Lei Chen ◽  
...  
Keyword(s):  
Antibody Titer ◽  
Neutralizing Antibodies ◽  
Geometric Mean ◽  
Neutralizing Antibody ◽  
Antibody Concentration ◽  
Wild Type ◽  
Neutralization Titer ◽  
Receptor Binding Site ◽  
Live Virus ◽  
Antibody Assays

Objectives: The emergence of SARS-CoV-2 variants of concern (VOCs) have diminished the effectiveness of vaccines and are associated with a rebound in the number of COVID-19 cases globally. These variants contain mutations at the spike (S) protein receptor binding site (RBD), which affect antibody binding. Current commercially available antibody assays were developed before the VOCs emerged. It is unclear whether the levels of these commercially available antibody assays can predict the neutralizing antibody titers against the VOCs. In this study, we sought to determine the correlation between the binding antibody concentration and microneutralization antibody titer against the beta variant. Methods: This study included 58 COVID-19 patients. The concentrations of IgG against the SARS-CoV-2 spike protein RBD and nucleocapsid (N) protein were measured using the Abbott SARS-CoV-2 IgG II Quant assay and the SARS-CoV-2 IgG assay, respectively. The neutralization antibody titer against the wild type lineage A SARS-CoV-2 and against the beta variant (B.1.351) was determined using a conventional live virus neutralization test. Results: The geometric mean MN titer (GMT) against the beta variant was significantly lower than that against the wild type lineage A virus (5.6 vs. 47.3, P<0.0001). The anti-RBD IgG had a better correlation with the neutralizing antibody titer than that of the anti-N IgG assay against the wild type lineage A virus (Spearman rho, 0.5901 vs. 0.3827). However, the correlation between the anti-RBD or the anti-N IgG and the MN titer against the beta variant was poor. Conclusions: Currently available commercial antibody assays may not predict the level of neutralizing antibodies against the variants. A new generation of antibody tests specific for variants are required.

scholarly journals An ultrapotent neutralizing bispecific antibody with broad spectrum against SARS-CoV-2 variants

2021 ◽  
Author(s):  
Hui Zhang ◽  
Haohui Huang ◽  
Rong Li ◽  
Lu Zhang ◽  
Zhiwei Wang ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Bispecific Antibody ◽  
Therapeutic Effects ◽  
Bodyweight Loss ◽  
Single Domain Antibody ◽  
Live Virus ◽  
Protection Efficiency ◽  
Angiotensin Converting Enzyme 2 ◽  
Virus Challenge ◽  
Virus Assay

In spite of the successful development of effective countermeasures against Covid-19, variants have and will continue to emerge that could compromise the efficacy of currently approved neutralizing antibodies and vaccines. Consequently, novel and more efficacious agents are urgently needed. We have developed a bispecific antibody, 2022, consisting of two antibodies, 2F8 and VHH18. 2F8 was isolated from our proprietary fully synthetic human IDEAL (Intelligently Designed and Engineered Antibody Library)-VH/VL library and VHH18 is a single domain antibody isolated from IDEAL-nanobody library. 2022 was constructed by attaching VHH18 to the C-terminal of Fc of 2F8. 2022 binds two non-overlapping epitopes simultaneously on the RBD of the SARS-CoV-2 spike protein and blocks the binding of RBD to human angiotensin-converting enzyme 2 (ACE2). 2022 potently neutralizes SARS-CoV-2 and all of the variants tested in both pseudovirus and live virus assays, including variants carrying mutations known to resist neutralizing antibodies approved under EUA and that reduce the protection efficiency of current effective vaccines. The half-maximum inhibitory concentration (IC50) of 2022 is 270 pM, 30 pM, 20 pM, and 1 pM, for wildtype, alpha, beta, and delta pseudovirus, respectively. In the live virus assay, 2022 has an IC50 of 30 pM, 13 pM, and 90 pM, for wildtype, beta, and delta live virus, respectively. In a mouse model of SARS-CoV-2, 2022 showed strong prophylactic and therapeutic effects. A single administration of 2022 intranasal (i.n.) or intraperitoneal (i.p.) 24 hours before virus challenge completely protected all mice from bodyweight loss, as compared with up to 20% loss of bodyweight in placebo treated mice. In addition, the lung viral titers were undetectable (FRNT assay) in all mice treated with 2022 either prophylactically or therapeutically, as compared with around 1X105 pfu/g lung tissue in placebo treated mice. In summary, bispecific antibody 2022 showed potent binding and neutralizing activity across a variety of SARS-CoV-2 variants and could be an attractive weapon to combat the ongoing waves of the COVID-19 pandemic propagated mainly by variants, especially, the much more contagious delta variant.

scholarly journals Evaluation of a Pseudovirus Neutralization Assay for SARS-CoV-2 and Correlation with Live Virus-Based Micro Neutralization Assay

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 994
Author(s):  
Ahmed Majdi K. Tolah ◽  
Sayed S. Sohrab ◽  
Khaled Majdi K. Tolah ◽  
Ahmed M. Hassan ◽  
Sherif A. El-Kafrawy ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Epidemiological Studies ◽  
Neutralization Assay ◽  
Serum Samples ◽  
Live Virus ◽  
Spike Gene ◽  
Microneutralization Assay ◽  
Viral Particles ◽  
Reliable Performance ◽  
Prior Infection

The unusual cases of pneumonia outbreak were reported from Wuhan city in late December 2019. Serological testing provides a powerful tool for the identification of prior infection and for epidemiological studies. Pseudotype virus neutralization assays are widely used for many viruses and applications in the fields of serology. The accuracy of pseudotype neutralizing assay allows for its use in low biosafety lab and provides a safe and effective alternative to the use of wild-type viruses. In this study, we evaluated the performance of this assay compared to the standard microneutralization assay as a reference. The lentiviral pseudotype particles were generated harboring the Spike gene of SARS-CoV-2. The generated pseudotype particles assay was used to evaluate the activity of neutralizing antibodies in 300 human serum samples from a COVID-19 sero-epidemiological study. Testing of these samples resulted in 55 positive samples and 245 negative samples by pseudotype viral particles assay while microneutralization assay resulted in 64 positive and 236 negative by MN assay. Compared to the MN, the pseudotyped viral particles assay showed a sensitivity of 85.94% and a specificity of 100%. Based on the data generated from this study, the pseudotype-based neutralization assay showed a reliable performance for the detection of neutralizing antibodies against SARS-CoV-2 and can be used safely and efficiently as a diagnostic tool in a biosafety level 2 laboratory.

scholarly journals Caspofungin and LTX-315 inhibit SARS-CoV-2 replication by targeting the nsp12 polymerase

2020 ◽  
Author(s):  
Min Wang ◽  
Fei Ye ◽  
Jiaqi Su ◽  
Jingru Zhao ◽  
Bin Yuan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Vero Cells ◽  
Polymerase Activity ◽  
Structural Protein ◽  
Live Virus ◽  
Drug Candidates ◽  
Virtual Screen ◽  
Virus Assay ◽  
Promising Target

Abstract The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously designated as 2019-nCoV) outbreak has caused global concern1. Currently, there are no clinically approved specific drugs or vaccines available for this virus. The viral polymerase is a promising target for developing broad- spectrum antiviral drugs. Here, based on the highly similar structure of SARS- CoV non-structural protein 12 (nsp12) polymerase subunit2, we applied virtual screen for the available compounds, including both the FDA-approved and under- clinic drugs, to identify potential antiviral molecules against SARS-CoV-2. We found two drugs, the clinically approved anti-fungi drug Caspofungin Acetate (Cancidas) and the oncolytic peptide LTX-315, can bind SARS-CoV-2 nsp12 protein to block the polymerase activity in vitro. Further live virus assay revealed that both Caspofungin Acetate and LTX-315 can effectively inhibit SARS-CoV-2 replication in vero cells. These findings present promising drug candidates for treatment of related diseases and would also stimulate the development of pan- coronavirus antiviral agents.Authors Min Wang, Fei Ye, Jiaqi Su, Jingru Zhao, and Bin Yuan contributed equally to this work.

scholarly journals Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2

2020 ◽  
Author(s):  
Huihui Mou ◽  
Brian D. Quinlan ◽  
Haiyong Peng ◽  
Yan Guo ◽  
Shoujiao Peng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Angiotensin Converting Enzyme ◽  
Converting Enzyme ◽  
Viral Receptor ◽  
S Protein ◽  
Angiotensin Converting Enzyme 2 ◽  
Binding Domains ◽  
Protein Receptor ◽  
Horseshoe Bat ◽  
Horseshoe Bats

SUMMARYThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat (R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.

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