Development and validation of a cell-based SEAP reporter assay for the detection of neutralizing antibodies against an anti-IL-13 therapeutic antibody

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to more than 4 million confirmed infections worldwide and over 300,000 deaths. While Remdesivir has recently received FDA emergency use authorization for treatment of SARS-CoV-2 infection, convalescent plasma (CP) with high titers of SARS-CoV-2 neutralizing antibodies (NAbs) from recovered donors remains a promising and widely accessible method to mitigate severe disease symptoms. Here, we describe the development and validation of a cell-free neutralization PCR assay using SARS-CoV-2 spike protein S1 and human ACE2 receptor-DNA conjugates. By comparing with samples collected prior to the outbreak, we confirmed that NAbs were specifically detected in COVID-19 cases. Using our unique assay, the NAb signals are detectable as early as 10 days after onset of symptoms and continue to rise, plateauing after 18 days. Notably, we showed that the use of licensed pathogen reduction technology to inactivate potentially contaminating infectious pathogens in CP did not alter NAb signals, paving a path to safely administer effective CP therapies. The described neutralization PCR assay can serve as a qualification tool to easily identify suitable CP donors of a potentially lifesaving therapy. In addition, this assay tool is readily deployable in standard laboratories with biosafety level 2 capability, and can yield results within 2-3 hr. This advancement can facilitate research on factors driving diverse COVID-19 disease manifestations, and to evaluate the impact of various CP processing protocols on CP therapeutic efficacy.

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Development and validation of a cell based assay for the detection of neutralizing antibodies against recombinant insulins

Journal of Immunological Methods ◽

10.1016/j.jim.2017.09.004 ◽

2018 ◽

Vol 452 ◽

pp. 53-62 ◽

Cited By ~ 5

Author(s):

Sanjukta Chatterjee ◽

Laxmikant Vashishta ◽

Vinit S. Waichale ◽

Vivek G. Nayak ◽

Ramakrishnan Melarkode ◽

...

Keyword(s):

Neutralizing Antibodies ◽

A Cell ◽

Development And Validation

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Development and validation of a cell-based bioassay for the detection of neutralizing antibodies against recombinant human erythropoietin in clinical studies

Journal of Immunological Methods ◽

10.1016/j.jim.2004.07.007 ◽

2004 ◽

Vol 293 (1-2) ◽

pp. 115-126 ◽

Cited By ~ 33

Author(s):

Xin Wei ◽

Steven J. Swanson ◽

Shalini Gupta

Keyword(s):

Clinical Studies ◽

Recombinant Human Erythropoietin ◽

Neutralizing Antibodies ◽

Human Erythropoietin ◽

A Cell ◽

Development And Validation

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SARS-CoV-2 Serology Status Detected by Commercialized Platforms Distinguishes Previous Infection and Vaccination Adaptive Immune Responses

10.1101/2021.03.10.21253299 ◽

2021 ◽

Author(s):

Raymond T. Suhandynata ◽

Nicholas J. Bevins ◽

Jenny T. Tran ◽

Deli Huang ◽

Melissa A. Hoffman ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Adaptive Immune Response ◽

Antibody Assay ◽

Adaptive Immune Responses ◽

Adaptive Immune ◽

Previous Infection ◽

A Cell

AbstractBackgroundThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected over 110 million individuals and led to 2.5 million deaths worldwide. As more individuals are vaccinated, the clinical performance and utility of SARS-CoV-2 serology platforms needs to be evaluated.MethodsThe ability of four commercial SARS-CoV-2 serology platforms to detect previous infection or vaccination were evaluated using a cohort of 53 SARS-CoV-2 PCR-positive patients, 89 SARS-CoV-2-vaccinated healthcare workers (Pfizer or Moderna), and 127 SARS-CoV-2 negative patients. Serology results were compared to a cell based SARS-CoV-2 pseudovirus (PSV) neutralizing antibodies assay.ResultsThe Roche S-(spike) antibody and Diazyme neutralizing antibodies (NAbs) assays detected adaptive immune response in 100.0% and 90.1% of vaccinated individuals who received two-doses of vaccine (initial and booster), respectively. The Roche N-(nucleocapsid) antibody assay and Diazyme IgG assay did not detect adaptive immune response in vaccinated individuals. The Diazyme Nabs assay correlated with the PSV SARS-CoV-2 ID50 neutralization titers (R2= 0.70), while correlation of the Roche S-antibody assay was weaker (R2= 0.39). Median PSV SARS-CoV-2 ID50 titers more than doubled in vaccinated individuals who received two-doses of the Moderna vaccine (ID50: 597) compared to individuals that received a single dose (ID50: 284).ConclusionsThe Roche S-antibody and Diazyme NAbs assays robustly detected adaptive immune responses in SARS-CoV-2 vaccinated individuals and SARS-CoV-2 infected individuals. The Diazyme NAbs assay strongly correlates with the PSV SARS-CoV-2 NAbs in vaccinated individuals. Understanding the reactivity of commercially available serology platforms is important when distinguishing vaccination response versus natural infection.SummaryThe Roche S (spike protein)-antibody and Diazyme neutralizing-antibodies (NAbs) assays were evaluated for their clinical utility in the detection of SARS-CoV-2 related adaptive immune responses by testing SARS-CoV-2 PCR-confirmed patients, SARS-CoV-2-vaccinated individuals, and SARS-CoV-2-negative individuals. Commercial serology results were compared to results generated using a cell-based SARS-CoV-2 pseudovirus (PSV) NAbs assay and previously validated SARS-CoV-2 commercial serology assays (Roche N (nucleocapsid protein) antibody and Diazyme IgG). We demonstrate that the Roche S-antibody and Diazyme NAbs assays detected adaptive immune response in SARS-CoV-2 vaccinated individuals and the presence of SARS-CoV-2 PSV NAbs. The Roche S-antibody assay had an observed positive percent agreement (PPA) of 100% for individuals who received two doses of the Pfizer or Moderna vaccine. By contrast, the Roche N assay and Diazyme IgG assay did not detect vaccine adaptive immune responses. Our findings also indicate that the Diazyme NAbs assay correlates strongly with the levels of SARS-CoV-2 ID50 neutralization titers using the PSV Nab assay in vaccinated individuals.

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Development of a Cell-Based Assay for the Detection of Neutralizing Antibodies to PF-06730512 Using Homogenous Time-Resolved Fluorescence

The AAPS Journal ◽

10.1208/s12248-020-0431-x ◽

2020 ◽

Vol 22 (2) ◽

Author(s):

Michael Luong ◽

Ying Wang ◽

Stephen P. Berasi ◽

Janet E. Buhlmann ◽

Hongying Yang ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Time Resolved Fluorescence ◽

Time Resolved ◽

A Cell

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Thermostable ricin vaccine protects rhesus macaques against aerosolized ricin: Epitope-specific neutralizing antibodies correlate with protection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502585112 ◽

2015 ◽

Vol 112 (12) ◽

pp. 3782-3787 ◽

Cited By ~ 40

Author(s):

Chad J. Roy ◽

Robert N. Brey ◽

Nicholas J. Mantis ◽

Kelly Mapes ◽

Iliodora V. Pop ◽

...

Keyword(s):

Castor Oil ◽

Mammalian Cells ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Lethal Dose ◽

Lung Damage ◽

Ricin Toxin ◽

A Cell ◽

A Chain ◽

Antibody Competition

Ricin toxin (RT) is the second most lethal toxin known; it has been designated by the CDC as a select agent. RT is made by the castor bean plant; an estimated 50,000 tons of RT are produced annually as a by-product of castor oil. RT has two subunits, a ribotoxic A chain (RTA) and galactose-binding B chain (RTB). RT binds to all mammalian cells and once internalized, a single RTA catalytically inactivates all of the ribosomes in a cell. Administered as an aerosol, RT causes rapid lung damage and fibrosis followed by death. There are no Food and Drug Administration-approved vaccines and treatments are only effective in the first few hours after exposure. We have developed a recombinant RTA vaccine that has two mutations V76M/Y80A (RiVax). The protein is expressed in Escherichia coli and is nontoxic and immunogenic in mice, rabbits, and humans. When vaccinated mice are challenged with injected, aerosolized, or orally administered (gavaged) RT, they are completely protected. We have now developed a thermostable, aluminum-adjuvant–containing formulation of RiVax and tested it in rhesus macaques. After three injections, the animals developed antibodies that completely protected them from a lethal dose of aerosolized RT. These antibodies neutralized RT and competed to varying degrees with a panel of neutralizing and nonneutralizing mouse monoclonal antibodies known to recognize specific epitopes on native RTA. The resulting antibody competition profile could represent an immunologic signature of protection. Importantly, the same signature was observed using sera from RiVax-immunized humans.

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Abstract 2555: A cell based bioluminescent reporter assay for rapid measuring function of PD-1 or PD-L1 therapeutic antibodies

10.1158/1538-7445.am2014-2555 ◽

2014 ◽

Author(s):

Mei Cong ◽

Natasha Karassina ◽

Jey Cheng ◽

Frank Fan

Keyword(s):

Therapeutic Antibodies ◽

Reporter Assay ◽

A Cell

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Neutralization Mechanism of a Monoclonal Antibody Targeting a Porcine Circovirus Type 2 Cap Protein Conformational Epitope

Journal of Virology ◽

10.1128/jvi.01836-19 ◽

2020 ◽

Vol 94 (9) ◽

Cited By ~ 1

Author(s):

Liping Huang ◽

Zhenzhao Sun ◽

Deli Xia ◽

Yanwu Wei ◽

Encheng Sun ◽

...

Keyword(s):

Amino Acids ◽

Monoclonal Antibody ◽

Neutralizing Antibodies ◽

Vaccine Design ◽

Binding Mode ◽

Porcine Circovirus Type ◽

Therapeutic Antibody ◽

Capsid Proteins ◽

Porcine Circovirus

ABSTRACT Porcine circovirus type 2 (PCV2) is an important pathogen in swine herds, and its infection of pigs has caused severe economic losses to the pig industry worldwide. The capsid protein of PCV2 is the only structural protein that is associated with PCV2 infection and immunity. Here, we report a neutralizing monoclonal antibody (MAb), MAb 3A5, that binds to intact PCV2 virions of the PCV2a, PCV2b, and PCV2d genotypes. MAb 3A5 neutralized PCV2 by blocking viral attachment to PK15 cells. To further explore the neutralization mechanism, we resolved the structure of the PCV2 virion in complex with MAb 3A5 Fab fragments by using cryo-electron microscopy single-particle analysis. The binding sites were located at the topmost edges around 5-fold icosahedral symmetry axes, with each footprint covering amino acids from two adjacent capsid proteins. Most of the epitope residues (15/18 residues) were conserved among 2,273 PCV2 strains. Mutations of some amino acids within the epitope had significant effects on the neutralizing activity of MAb 3A5. This study reveals the molecular and structural bases of this PCV2-neutralizing antibody and provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections. IMPORTANCE PCV2 is associated with several clinical manifestations collectively known as PCV2-associated diseases (PCVADs). Neutralizing antibodies play a crucial role in the prevention of PCVADs. We demonstrated previously that a MAb, MAb 3A5, neutralizes the PCV2a, PCV2b, and PCV2d genotypes with different degrees of efficiency, but the underlying mechanism remains elusive. Here, we report the neutralization mechanism of this MAb and the structure of the PCV2 virion in complex with MAb 3A5 Fabs, showing a binding mode in which one Fab interacted with more than two loops from two adjacent capsid proteins. This binding mode has not been observed previously for PCV2-neutralizing antibodies. Our work provides new and important information for vaccine design and therapeutic antibody development against PCV2 infections.

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