High-throughput screening of stabilizers for Respiratory Syncytial Virus: Identification of stabilizers and their effects on the conformational thermostability of viral particles

Respiratory syncytial virus (RSV) infects 99% of children by age 2 years and is a leading cause of serious lower respiratory tract infection (LRTI) and infant hospitalization in the United Kingdom. Identification of efficacious RSV therapeutics has been hindered by the lack of a robust and appropriate primary assay for high-throughput screening (HTS). Here we report an HTS cascade that identified inhibitors of RSV replication using a robust RSV replicon luminescence-reporter assay for the primary campaign. The performance of the assay was consistent and reliable at scale, with Z′ of 0.55 ± 0.08 across 150 assay plates and signal-to-background ratios >40. The HTS assay was used to screen the AstraZeneca compound library of 1 million compounds at a single concentration of 10 µM. Hits specifically targeting the RSV replicon were determined using a series of hit generation assays. Compounds nonspecifically causing cell toxicity were removed, and hits were confirmed in live viral inhibition assays exhibiting greater physiological relevance than the primary assay. In summary, we developed a robust screening cascade that identified hit molecules that specifically targeted RSV replication.

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Characterization of novel respiratory syncytial virus inhibitors identified by high throughput screen

Antiviral Research ◽

10.1016/j.antiviral.2014.12.012 ◽

2015 ◽

Vol 115 ◽

pp. 71-74 ◽

Cited By ~ 11

Author(s):

Valerie A. Laganas ◽

Ewan F. Dunn ◽

Robert E. McLaughlin ◽

Choi Lai Tiong-Yip ◽

Olga Yuzhakov ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

High Throughput ◽

High Throughput Screen ◽

Syncytial Virus

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Development and Validation of a High Throughput Screen for Inhibitors of Respiratory Syncytial Virus

Antiviral Research ◽

10.1016/j.antiviral.2007.01.085 ◽

2007 ◽

Vol 74 (3) ◽

pp. A59-A59

Author(s):

M HEIL ◽

M MCDOWELL ◽

C JONSSON ◽

W SEVERSON

Keyword(s):

Respiratory Syncytial Virus ◽

High Throughput ◽

High Throughput Screen ◽

Syncytial Virus ◽

Development And Validation

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Antigenic analysis of the F protein of the bovine respiratory syncytial virus: identification of two distinct antigenic sites involved in fusion inhibition

Archives of Virology ◽

10.1007/bf01315410 ◽

1995 ◽

Vol 140 (6) ◽

pp. 993-1005 ◽

Cited By ~ 13

Author(s):

J. P. Matheise ◽

K. Walravens ◽

A. Collard ◽

P. Coppe ◽

J. J. Letesson

Keyword(s):

Respiratory Syncytial Virus ◽

Bovine Respiratory Syncytial Virus ◽

Antigenic Analysis ◽

F Protein ◽

Virus Identification ◽

Antigenic Sites ◽

Fusion Inhibition ◽

Syncytial Virus

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Development and Standardization of a High-Throughput Multiplex Immunoassay for the Simultaneous Quantification of Specific Antibodies to Five Respiratory Syncytial Virus Proteins

mSphere ◽

10.1128/msphere.00236-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 7

Author(s):

Rutger M. Schepp ◽

Cornelis A. M. de Haan ◽

Deidre Wilkins ◽

Hans Layman ◽

Barney S. Graham ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

High Throughput ◽

Large Scale ◽

Dynamic Range ◽

Valuable Insight ◽

Conformational Structure ◽

Multiplex Immunoassay ◽

Rsv Infection ◽

Syncytial Virus ◽

Antibody Levels

ABSTRACT Human respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in (premature) newborns and causes respiratory illness in the elderly. Different monoclonal antibody (MAb) and vaccine candidates are in development worldwide and will hopefully become available within the near future. To implement such RSV vaccines, adequate decisions about immunization schedules and the different target group(s) need to be made, for which the assessment of antibody levels against RSV is essential. To survey RSV antigen-specific antibody levels, we developed a serological multiplex immunoassay (MIA) that determines and distinguishes antibodies against the five RSV glycoproteins postfusion F, prefusion F, Ga, Gb, and N simultaneously. The standardized RSV pentaplex MIA is sensitive, highly reproducible, and specific for the five RSV proteins. The preservation of the conformational structure of the immunodominant site Ø of prefusion F after conjugation to the beads has been confirmed. Importantly, good correlation is obtained between the microneutralization test and the MIA for all five proteins, resulting in an arbitrarily chosen cutoff value of prefusion F antibody levels for seropositivity in the microneutralization assay. The wide dynamic range requiring only two serum sample dilutions makes the RSV-MIA a high-throughput assay very suitable for (large-scale) serosurveillance and vaccine clinical studies. IMPORTANCE In view of vaccine and monoclonal development to reduce hospitalization and death due to lower respiratory tract infection caused by RSV, assessment of antibody levels against RSV is essential. This newly developed multiplex immunoassay is able to measure antibody levels against five RSV proteins simultaneously. This can provide valuable insight into the dynamics of (maternal) antibody levels and RSV infection in infants and toddlers during the first few years of life, when primary RSV infection occurs.

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A Fluorescence-Based High-Throughput Antiviral Compound Screening Assay Against Respiratory Syncytial Virus

Antiviral Methods and Protocols - Methods in Molecular Biology ◽

10.1007/978-1-62703-484-5_26 ◽

2013 ◽

pp. 337-344 ◽

Cited By ~ 9

Author(s):

Leen Kwanten ◽

Ben De Clerck ◽

Dirk Roymans

Keyword(s):

Respiratory Syncytial Virus ◽

High Throughput ◽

Screening Assay ◽

Antiviral Compound ◽

Compound Screening ◽

Syncytial Virus

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Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057104271956 ◽

2005 ◽

Vol 10 (2) ◽

pp. 108-117 ◽

Cited By ~ 4

Author(s):

Thomas Vanhercke ◽

Christophe Ampe ◽

Luc Tirry ◽

Peter Denolf

Keyword(s):

Phage Display ◽

High Throughput ◽

High Throughput Screening ◽

Enzyme Linked Immunosorbent Assay ◽

Selective Enrichment ◽

Phage Display Libraries ◽

Viral Particles ◽

Phage Libraries ◽

Rapid Characterization

Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phage-displayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 104-fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries.

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