SARS-COV-2 Delta variant displays moderate resistance to neutralizing antibodies and spike protein properties of higher soluble ACE2 sensitivity, enhanced cleavage and fusogenic activity

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. The spike proteins of B.1.617.1, B.1.617.2, and AY.1 variants have several substitutions in the receptor binding domain (RBD), including L452R+E484Q, L452R+T478K, and K417N+L452R+T478K, respectively, that could potentially reduce effectiveness of therapeutic antibodies and current vaccines. Here we compared B.1.617 variants, and their single and double RBD substitutions for resistance to neutralization by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. Pseudoviruses with the B.1.617.1, B.1.617.2, and AY.1 spike showed a modest 1.5 to 4.4-fold reduction in neutralization titer by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for pseudoviruses with C.37, P.1, R.1, and B.1.526 spikes, but seven- and sixteen-fold reduction for vaccine-elicited and convalescent sera, respectively, was seen for pseudoviruses with the B.1.351 spike. Four of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses due to the L452R substitution, whereas six of twenty-three therapeutic neutralizing antibodies showed either complete or partial loss of neutralization against B.1.617.1 pseudoviruses due to either the E484Q or L452R substitution. Against AY.1 pseudoviruses, the L452R and K417N substitutions accounted for the loss of neutralization by four antibodies and one antibody, respectively, whereas one antibody lost potency that could not be fully accounted for by a single RBD substitution. The modest resistance of B.1.617 variants to vaccine-elicited sera suggest that current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants, but the therapeutic antibodies need to be carefully selected based on their resistance profiles. Finally, the spike proteins of B.1.617 variants are more efficiently cleaved due to the P681R substitution, and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

Download Full-text

SARS-CoV-2 Delta Variant Displays Moderate Resistance to Neutralizing Antibodies and Spike Protein Properties of Higher Soluble ACE2 Sensitivity, Enhanced Cleavage and Fusogenic Activity

Viruses ◽

10.3390/v13122485 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2485

Author(s):

Sabari Nath Neerukonda ◽

Russell Vassell ◽

Sabrina Lusvarghi ◽

Richard Wang ◽

Fernando Echegaray ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Neutralization Assay ◽

Spike Protein ◽

Therapeutic Antibodies ◽

Partial Loss ◽

Fusogenic Activity ◽

Fold Reduction ◽

Protein Properties ◽

Moderate Resistance ◽

Second Wave

The SARS-CoV-2 B.1.617 lineage variants, Kappa (B.1.617.1) and Delta (B.1.617.2, AY) emerged during the second wave of infections in India, but the Delta variants have become dominant worldwide and continue to evolve. Here, we compared B.1.617 variants for neutralization resistance by convalescent sera, mRNA vaccine-elicited sera, and therapeutic neutralizing antibodies using a pseudovirus neutralization assay. B.1.617.1, B.1.617.2, and AY.1 pseudoviruses showed a modest 1.5- to 4.4-fold reduction in neutralization by convalescent sera and vaccine-elicited sera. In comparison, similar modest reductions were also observed for C.37, P.1, R.1, and B.1.526 pseudoviruses, but 7- and 16-fold reductions for vaccine-elicited and convalescent sera, respectively, were seen for B.1.351 pseudoviruses. Among twenty-three therapeutic antibodies tested, four antibodies showed either complete or partial loss of neutralization against B.1.617.2 pseudoviruses and six antibodies showed either complete or partial loss of neutralization against B.1.617.1 and AY.1 pseudoviruses. Our results indicate that the current mRNA-based vaccines will likely remain effective in protecting against B.1.617 variants. Finally, the P681R substitution confers efficient cleavage of B.1.617 variants’ spike proteins and the spike of Delta variants exhibited greater sensitivity to soluble ACE2 neutralization, as well as fusogenic activity, which may contribute to enhanced spread of Delta variants.

Download Full-text

578. INO-4800 DNA Vaccine Induces Neutralizing Antibodies and T cell Activity Against Global SARS-CoV-2 Variants

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab466.776 ◽

2021 ◽

Vol 8 (Supplement_1) ◽

pp. S390-S391

Author(s):

Viviane M Andrade ◽

Aaron Christensen-Quick ◽

Joseph Agnes ◽

Jared Tur ◽

Charles C Reed ◽

...

Keyword(s):

T Cell ◽

Immune Responses ◽

Dna Vaccine ◽

Stock Options ◽

Neutralizing Antibodies ◽

Cell Activity ◽

Cellular Responses ◽

Neutralization Assay ◽

Material Support ◽

Fold Reduction

Abstract Background Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with increased transmissibility, disease severity, and resistance to neutralization by current vaccines under emergency use authorization (EUA). Here we assessed cross-immune responses of INO-4800 vaccinated subjects against SARS-CoV-2 VOCs. Methods We used a SARS-CoV-2 IgG ELISA and a pseudo neutralization assay to assess humoral responses, and an IFNγ ELISpot to measure cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. Results IgG binding titers were not impacted between wild-type (WT) and B.1.1.7 or B.1.351 variants. An average 1.9-fold reduction was observed for the P.1 variant in subjects tested at week 8 after receiving two doses of INO-4800 (Figure 1a). We performed a SARS-CoV-2 pseudovirus neutralization assay using sera collected from 13 subjects two weeks after administration of a third dose of either 0.5 mg, 1 mg, or 2 mg of INO-4800. Neutralization was detected against WT and the emerging variants in all samples tested. The mean ID50 titers for the WT, B.1.1.7, B.1.351 and P.1. were 643 (range: 70-729), 295 (range: 46-886), 105 (range: 25-309), and 664 (range: 25-2087), respectively. Compared to WT, there was a 2.1 and 6.9-fold reduction for B.1.1.7 and B.1.351, respectively, while there was no difference between WT and the P.1 variant (Figure 1b). Next, we compared cellular immune responses to WT and SARS-CoV-2 Spike variants elicited by INO-4800 vaccination. We observed similar cellular responses to WT (median = 82.2 IQR = 58.9-205.3), B.1.1.7 (79.4, IQR = 38.9- 179.7), B.1.351 (80, IQR = 40.0-208.6) and P.1 (78.3, IQR = 53.1-177.8) Spike peptides (Figure 2). Conclusion INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFNγ T cell responses were fully maintained against all variants tested. Disclosures Viviane M. Andrade, PhD, Inovio Pharmaceuticals Inc. (Employee) Aaron Christensen-Quick, PhD, Inovio Pharmaceuticals, Inc (Employee) Joseph Agnes, PhD, Inovio (Employee, Shareholder) Jared Tur, PhD, Inovio (Employee) Charles C. Reed, PhD, Inovio Pharmaceuticals (Employee, Shareholder) Richa Kalia, MS, Inovio Pharmaceuticals (Employee, Other Financial or Material Support, I have stock options with Inovio Pharmaceuticals as an employee.) Idania Marrero, MD, PhD, Inovio Pharmaceuticals (Employee, Shareholder) Dustin Elwood, PhD, Inovio Pharmaceuticals (Employee) Katherine Schultheis, MSc, Inovio Pharmaceuticals (Employee) Emma Reuschel, PhD, Inovio Pharmaceuticals (Employee) Trevor McMullan, MSc, Inovio (Shareholder) Patrick Pezzoli, BS, Inovio (Employee) Kimberly A. Kraynyak, PhD, Inovio Pharmaceuticals (Employee, Other Financial or Material Support, Stock options) Albert Sylvester, MS, Inovio (Employee, Shareholder) Mammen P. Mammen Jr., MD, Inovio Pharmaceuticals (Employee) J Joseph Kim, PhD, Inovio (Employee) David Weiner, PhD, Inovio (Board Member, Grant/Research Support, Shareholder, I serve on the SAB in addition to the above activities) Trevor R. F. Smith, PhD, Inovio (Employee, Shareholder) Stephanie Ramos, PhD, Inovio Pharmaceuticals (Employee) Laurent Humeau, PhD, Inovio Pharmaceuticals (Employee) Jean Boyer, PhD, Inovio (Employee) Kate Broderick, PhD, Inovio (Employee)

Download Full-text

SARS-CoV-2 Omicron neutralization by therapeutic antibodies, convalescent sera, and post-mRNA vaccine booster

10.1101/2021.12.22.473880 ◽

2021 ◽

Author(s):

Sabrina Lusvarghi ◽

Simon D. Pollett ◽

Sabari Nath Neerukonda ◽

WEI WANG ◽

Richard Wang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Clinical Stage ◽

Booster Vaccination ◽

Neutralization Assay ◽

Therapeutic Antibodies ◽

Post Vaccination ◽

Spike Gene ◽

Rapid Spread ◽

Vaccine Booster ◽

High Level

The rapid spread of the highly contagious Omicron variant of SARS-CoV-2 along with its high number of mutations in the spike gene has raised alarm about the effectiveness of current medical countermeasures. To address this concern, we measured neutralizing antibodies against Omicron in three important settings: (1) post-vaccination sera after two and three immunizations with the Pfizer/BNT162b2 vaccine, (2) convalescent sera from unvaccinated individuals infected by different variants, and (3) clinical-stage therapeutic antibodies. Using a pseudovirus neutralization assay, we found that titers against Omicron were low or undetectable after two immunizations and in most convalescent sera. A booster vaccination significantly increased titers against Omicron to levels comparable to those seen against the ancestral (D614G) variant after two immunizations. Neither age nor sex were associated with differences in post-vaccination antibody responses. Only three of 24 therapeutic antibodies tested retained their full potency against Omicron and high-level resistance was seen against fifteen. These findings underscore the potential benefit of booster mRNA vaccines for protection against Omicron and the need for additional therapeutic antibodies that are more robust to highly mutated variants.

Download Full-text

Robust Neutralizing Antibody Levels Detected after Either SARS-CoV-2 Vaccination or One Year after Infection

Viruses ◽

10.3390/v13102003 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2003

Author(s):

Stefan Glöckner ◽

Franziska Hornung ◽

Michael Baier ◽

Sebastian Weis ◽

Mathias W. Pletz ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Booster Vaccination ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Neutralization Titer ◽

Neutralization Capacity ◽

A Cell ◽

One Year ◽

Study Participants ◽

Antibody Levels

Humoral immunity after infection or after vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been attributed a key part in mitigating the further transmission of the virus. In this study, we used a commercial anti-Spike immunoglobulin G (S-IgG) assay and developed a cell culture-based neutralization assay to understand the longitudinal course of neutralizing antibodies in both SARS-CoV2 infected or vaccinated individuals. We show that even more than one year after infection, about 78% of observed study participants remained seropositive concerning S-IgG antibodies. In addition, the serum of the individuals had stable neutralization capacity in a neutralization assay against a SARS-CoV-2 patient isolate from March 2020. We also examined volunteers after either homologous BNT162b2 prime-boost vaccination or heterologous AZD1222 prime/mRNA-based booster vaccination. Both the heterologous and the homologous vaccination regimens induced higher levels of neutralizing antibodies in healthy subjects when compared to subjects after a mild infection, showing the high effectiveness of available vaccines. In addition, we could demonstrate the reliability of S-IgG levels in predicting neutralization capacity, with 94.8% of seropositive samples showing a neutralization titer of ≥10, making it a viable yet cheap and easy-to-determine surrogate parameter for neutralization capacity.

Download Full-text

mRNA-1273 and BNT162b2 mRNA vaccines have reduced neutralizing activity against the SARS-CoV-2 Omicron variant

10.1101/2021.12.20.473557 ◽

2021 ◽

Author(s):

Venkata-Viswanadh Edara ◽

Kelly E Manning ◽

Madison Ellis ◽

Lilin Lai ◽

Kathryn M Moore ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Antibody Responses ◽

Spike Protein ◽

Live Virus ◽

Neutralizing Activity ◽

Virus Neutralization Assay ◽

Fold Reduction

The BNT162b2 (Pfizer-BioNTech) and mRNA-1273 (Moderna) vaccines generate potent neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the global emergence of SARS-CoV-2 variants with mutations in the spike protein, the principal antigenic target of these vaccines, has raised concerns over the neutralizing activity of vaccine-induced antibody responses. The Omicron variant, which emerged in November 2021, consists of over 30 mutations within the spike protein. Here, we used an authentic live virus neutralization assay to examine the neutralizing activity of the SARS-CoV-2 Omicron variant against mRNA vaccine-induced antibody responses. Following the 2nd dose, we observed a 30-fold reduction in neutralizing activity against the omicron variant. Through six months after the 2nd dose, none of the sera from naive vaccinated subjects showed neutralizing activity against the Omicron variant. In contrast, recovered vaccinated individuals showed a 22-fold reduction with more than half of the subjects retaining neutralizing antibody responses. Following a booster shot (3rd dose), we observed a 14-fold reduction in neutralizing activity against the omicron variant and over 90% of boosted subjects showed neutralizing activity against the omicron variant. These findings show that a 3rd dose is required to provide robust neutralizing antibody responses against the Omicron variant.

Download Full-text

Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies

10.1101/2021.02.12.430472 ◽

2021 ◽

Author(s):

Delphine Planas ◽

Timothée Bruel ◽

Ludivine Grzelak ◽

Florence Guivel-Benhassine ◽

Isabelle Staropoli ◽

...

Keyword(s):

South Africa ◽

Humoral Immunity ◽

Partial Resistance ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Neutralization Assay ◽

Fold Reduction ◽

Nasal Swabs ◽

Reference Virus ◽

Antibody Levels

AbstractSARS-CoV-2 B.1.1.7 and B.1.351 variants emerged respectively in United Kingdom and South Africa and spread in many countries. Here, we isolated infectious B.1.1.7 and B.1.351 strains and examined their sensitivity to anti-SARS-CoV-2 antibodies present in sera and nasal swabs, in comparison with a D614G reference virus. We established a novel rapid neutralization assay, based on reporter cells that become GFP+ after overnight infection. B.1.1.7 was neutralized by 79/83 sera from convalescent patients collected up to 9 months post symptoms, almost similar to D614G. There was a mean 6-fold reduction in titers and even loss of activity against B.1.351 in 40% of convalescent sera after 9 months. Early sera from 19 vaccinated individuals were almost as potent against B.1.1.7 but less efficacious against B.1.351, when compared to D614G. Nasal swabs from vaccine recipients were not neutralizing, except in individuals who were diagnosed COVID-19+ before vaccination. Thus, faster-spreading variants acquired a partial resistance to humoral immunity generated by natural infection or vaccination, mostly visible in individuals with low antibody levels.

Download Full-text

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

Diagnostics ◽

10.3390/diagnostics11060994 ◽

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.

Download Full-text

Titers of Neutralizing Antibodies against SARS-CoV-2 Are Independent of Symptoms of Non-Severe COVID-19 in Young Adults

Viruses ◽

10.3390/v13020284 ◽

2021 ◽

Vol 13 (2) ◽

pp. 284

Author(s):

Hulda R. Jonsdottir ◽

Michel Bielecki ◽

Denise Siegrist ◽

Thomas W. Buehrer ◽

Roland Züst ◽

...

Keyword(s):

Young Adults ◽

Neutralizing Antibodies ◽

Severe Disease ◽

Humoral Response ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Asymptomatic Infection ◽

Homogeneous Population ◽

Humoral Immune ◽

Antibody Titers

Neutralizing antibodies are an important part of the humoral immune response to SARS-CoV-2. It is currently unclear to what extent such antibodies are produced after non-severe disease or asymptomatic infection. We studied a cluster of SARS-CoV-2 infections among a homogeneous population of 332 predominantly male Swiss soldiers and determined the neutralizing antibody response with a serum neutralization assay using a recombinant SARS-CoV-2-GFP. All patients with non-severe COVID-19 showed a swift humoral response within two weeks after the onset of symptoms, which remained stable for the duration of the study. One month after the outbreak, titers in COVID-19 convalescents did not differ from the titers of asymptomatically infected individuals. Furthermore, symptoms of COVID-19 did not correlate with neutralizing antibody titers. Therefore, we conclude that asymptomatic infection can induce the same humoral immunity as non-severe COVID-19 in young adults.

Download Full-text

Development of a high-throughput β-Gal-based neutralization assay for quantitation of herpes simplex virus-neutralizing antibodies in human samples

Vaccine ◽

10.1016/j.vaccine.2016.05.033 ◽

2016 ◽

Vol 34 (33) ◽

pp. 3901-3906 ◽

Cited By ~ 4

Author(s):

Amy Baccari ◽

Michael Cooney ◽

Tamara P. Blevins ◽

Lynda A. Morrison ◽

Shane Larson ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

High Throughput ◽

Neutralizing Antibodies ◽

Neutralization Assay ◽

Human Samples ◽

Simplex Virus

Download Full-text

Functional in vitro assessment of modified antibodies: Impact of label on protein properties

PLoS ONE ◽

10.1371/journal.pone.0257342 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0257342

Author(s):

Martin R. Edelmann ◽

Simon Hauri

Keyword(s):

Animal Experiments ◽

Size Exclusion ◽

Life Extension ◽

Spectrometric Analysis ◽

Pharmacokinetic Studies ◽

Therapeutic Antibodies ◽

Surface Binding ◽

Analytical Strategy ◽

Protein Properties

Labelling of therapeutic antibodies with radionuclides or fluorophores is routinely used to study their pharmacokinetic properties. A critical assumption in utilizing labelled therapeutic antibodies is that the label has no unfavourable effects on antibody charge, hydrophobicity, or receptor affinity. Ideally, the labelled protein should not have any significant deviations from the physiological properties of the original molecule. This article describes an established quality in vitro assessment workflow for labelled antibodies that ensures better prediction of changes in antibody pharmacokinetic (PK) properties after modifications. This analysis package considers degradation and aggregation analysis by size-exclusion chromatography, changes in neonatal-Fc-receptor (FcRn) affinity, and heparin interaction. FcRn binding is important for antibody recycling and half-life extension, whereas heparin affinity provides estimates on the rate of endocytosis through unspecific cell surface binding. Additionally, mass spectrometric analysis to determine the degree of labelling (DoL) completes the package and the combined analysis data allow to predict the label contribution to the PK properties of the modified antibody. This analytical strategy for labelling 11 IgGs has been investigated using 2 different IgG1 constructs and applying 7 different types of labels. Each labelling resulted in a change in the physicochemical properties of the protein. Not only can the DoL of modified IgGs lead to a change in protein properties, but the type of label also can. Furthermore, it was demonstrated that the labelling process can also influence the behaviour of labelled mAbs. An identical label on different constructs of IgG1 can cause different affinities for FcRn and heparin. Considering the assessment data, only 6 of the 11 modified antibodies from this study can be recommended for subsequent experiments. In conclusion, a suitability assessment of labelled antibodies prior to any pharmacokinetic studies is essential to reduce cost, allocate resources and reduce the number of animal experiments during pre-clinical drug development.

Download Full-text