High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples

AbstractBackgroundThe SARS-CoV-2 virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than a million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection.Study Design and MethodsTo extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike specific antibodies in the plasma of convalescent donors.Results and ConclusionWe found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations, but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.

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Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike

10.1101/2020.06.08.140244 ◽

2020 ◽

Cited By ~ 6

Author(s):

Jérémie Prévost ◽

Romain Gasser ◽

Guillaume Beaudoin-Bussières ◽

Jonathan Richard ◽

Ralf Duerr ◽

...

Keyword(s):

Viral Entry ◽

Neutralizing Antibodies ◽

Cross Sectional Study ◽

Health Interventions ◽

Cross Sectional ◽

Neutralizing Activity ◽

Main Target ◽

Specific Igg ◽

Human Coronaviruses ◽

Humoral Responses

ABSTRACTThe SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we performed a cross-sectional study on 106 SARS-CoV-2-infected individuals to evaluate humoral responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased after symptoms resolution. Some of the elicited antibodies cross-reacted with other human coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing antibodies within the first two weeks of infection, the level of neutralizing activity was significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.

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High-Throughput Assays Using a Luciferase-Expressing Replicon, Virus-Like Particles, and Full-Length Virus for West Nile Virus Drug Discovery

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.12.4980-4988.2005 ◽

2005 ◽

Vol 49 (12) ◽

pp. 4980-4988 ◽

Cited By ~ 88

Author(s):

Francesc Puig-Basagoiti ◽

Tia S. Deas ◽

Ping Ren ◽

Mark Tilgner ◽

David M. Ferguson ◽

...

Keyword(s):

West Nile Virus ◽

Drug Discovery ◽

High Throughput ◽

Viral Entry ◽

High Throughput Screening ◽

Full Length ◽

West Nile ◽

Subgenomic Replicon ◽

Virion Assembly ◽

Virus Like Particles

ABSTRACT Many flaviviruses cause significant human disease worldwide. The development of flavivirus chemotherapy requires reliable high-throughput screening (HTS) assays. Although genetic systems have been developed for many flaviviruses, their usage in antiviral HTS assays has not been well explored. Here we compare three cell-based HTS assays for West Nile virus (WNV) drug discovery: (i) an assay that uses a cell line harboring a persistently replicating subgenomic replicon (containing a deletion of viral structural genes), (ii) an assay that uses packaged virus-like particles containing replicon RNA, and (iii) an assay that uses a full-length reporting virus. A Renilla luciferase gene was engineered into the replicon or into the full-length viral genome to monitor viral replication. Potential inhibitors could be identified through suppression of luciferase signals upon compound incubation. The antiviral assays were optimized in a 96-well format, validated with known WNV inhibitors, and proved useful in identifying a new inhibitor(s) through HTS of a compound library. In addition, because each assay encompasses multiple but discrete steps of the viral life cycle, the three systems could potentially be used to discriminate the mode of action of any inhibitor among viral entry (detected by assays ii and iii but not by assay i), replication (including viral translation and RNA synthesis; detected by assays i to iii), and virion assembly (detected by assay iii but not by assays i and ii). The approaches described in this study should be applicable to the development of cell-based assays for other flaviviruses.

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High-throughput Detection of Protein Kinase Activities in Cell Lysate Based on the Aggregation of Gold Nanoparticles with Peptides

10.1557/proc-1241-xx08-08 ◽

2009 ◽

Author(s):

Yoshiki Katayama ◽

Hirotaro Kitazaki ◽

Jeong-Hun Kang ◽

Xiaoming Han ◽

Takeshi Mori ◽

...

Keyword(s):

Gold Nanoparticles ◽

Protein Kinase ◽

High Throughput ◽

Cell Lysate ◽

High Throughput Detection

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Faculty Opinions recommendation of High throughput detection of antibody self-interaction by bio-layer interferometry.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725357018.793504037 ◽

2015 ◽

Author(s):

Michael Doyle

Keyword(s):

High Throughput ◽

High Throughput Detection

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Faculty Opinions recommendation of A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726184750.793540024 ◽

2017 ◽

Author(s):

Cheng-Hock Toh

Keyword(s):

High Throughput ◽

Immune Complexes ◽

Neutrophil Extracellular Traps ◽

Extracellular Traps ◽

High Throughput Detection ◽

Novel Method ◽

Net Release ◽

Detection And Quantification

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High-Throughput Detection of an Alkaloidal Plant Metabolome in Plant Extracts Using LC-ESI-QTOF-MS

Journal of Proteome Research ◽

10.1021/acs.jproteome.1c00111 ◽

2021 ◽

Author(s):

Bibi Zareena ◽

Adeeba Khadim ◽

Syed Usama Y. Jeelani ◽

Saddam Hussain ◽

Arslan Ali ◽

...

Keyword(s):

High Throughput ◽

Plant Extracts ◽

High Throughput Detection

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Functional Genomic Identification of Cadmium Resistance Genes from a High GC Clone Library by Coupling the Sanger and PacBio Sequencing Strategies

Genes ◽

10.3390/genes11010007 ◽

2019 ◽

Vol 11 (1) ◽

pp. 7

Author(s):

Jinghao Chen ◽

Chao Xing ◽

Xin Zheng ◽

Xiaofang Li

Keyword(s):

High Throughput ◽

Resistance Genes ◽

Sanger Sequencing ◽

Genomic Library ◽

Full Length ◽

Host Cells ◽

Full Genome Sequence ◽

Functional Screening ◽

Functional Genomic ◽

Pacbio Sequencing

Functional (meta) genomics allows the high-throughput identification of functional genes in a premise-free way. However, it is still difficult to perform Sanger sequencing for high GC DNA templates, which hinders the functional genomic exploration of a high GC genomic library. Here, we developed a procedure to resolve this problem by coupling the Sanger and PacBio sequencing strategies. Identification of cadmium (Cd) resistance genes from a small-insert high GC genomic library was performed to test the procedure. The library was generated from a high GC (75.35%) bacterial genome. Nineteen clones that conferred Cd resistance to Escherichia coli subject to Sanger sequencing directly. The positive clones were in parallel subject to in vivo amplification in host cells, from which recombinant plasmids were extracted and linearized by selected restriction endonucleases. PacBio sequencing was performed to obtain the full-length sequences. As the identities, partial sequences from Sanger sequencing were aligned to the full-length sequences from PacBio sequencing, which led to the identification of seven unique full-length sequences. The unique sequences were further aligned to the full genome sequence of the source strain. Functional screening showed that the identified positive clones were all able to improve Cd resistance of the host cells. The functional genomic procedure developed here couples the Sanger and PacBio sequencing methods and overcomes the difficulties in PCR approaches for high GC DNA. The procedure can be a promising option for the high-throughput sequencing of functional genomic libraries, and realize a cost-effective and time-efficient identification of the positive clones, particularly for high GC genetic materials.

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