Published in Antiviral Research: A new fully automated dual-reporter HTS antiviral assay to facilitate drug discovery against SARS-CoV-2.

CARE partner KU Leuven developed a dual-reporter high-throughput screening (HTS) assay to facilitate antiviral drug discovery against SARS-CoV-2 using a fully automated, high-containment robot system. 

For this purpose, by clonal selection, KU Leuven successfully obtained an A549 cell clone that expresses a strong fluorescent mCherry signal in the nucleus (for cytotoxicity evaluation) and that efficiently supports replication of a SARS-CoV-2 reporter virus that expresses the NeonGreen fluorescent reporter protein after infection (for antiviral potential), and this without being affected by virus-induced cytopathogenic effects. This dual-reporter system makes any staining steps redundant, and thus offers a convenient and robust strategy for phenotypic HTS. KU Leuven chose the A549 cell line, overexpressing the SARS-CoV-2 angiotensin converting enzyme 2 (ACE2) receptor and the spike priming protease transmembrane serine protease 2 (TMPRSS2) to develop this assay as this is a human airway epithelial cell line, a more relevant model than the Vero6 cell line, which is a monkey kidney-derived cell, often used for HTS campaigns against SARS-CoV-2.  

Image acquisition and analysis processes were automated using the Caps-It research infrastructure, a fully automated robotic system enclosed in an isolator, which facilitates HTS on high biosafety organisms, as developed by KU Leuven. An in-house algorithm was created for cell counting, segmentation, and validation for calculation of the percentage of infected cells. 

Two reference compounds with known antiviral activity against SARS-CoV-2, GS-441524 and PF-00835231, were tested in this assay and both resulted in antiviral activity against SARS-CoV-2 with similar EC50 and CC50 values reported in other publications, thus validating this novel assay.  

This newly developed high-content imaging-based HTS assay, which is automated, scalable, and robust, is suitable for screening of large compound libraries.   

To learn more, click here: Development of a robust and convenient dual-reporter high-throughput screening assay for SARS-CoV-2 antiviral drug discovery 

CARE External Newsletter – June 2024

The new issue of our biannual newsletter is out. In this edition we learn about cryo-EM and how this exciting new technology is benefiting our scientific endeavours across CARE, plus we introduce 3 CARE partners: Utrecht University and the Institute for Virology and Immunology (academic organisations) and Nuvisan (industry organisation).

Read the Newsletter here: CARE External Newsletter – June 2024

CARE’s Young Researchers – Introducing Thijs Steijaert, PhD student, Leiden University Medical Centre

Meet Thijs! Thijs Steijaert is a PhD student at Leiden University Medical Centre and in this short video, describes for us his role in CARE Work Package 5, where his work is contributing to growing scientific knowledge of our understanding of host pathways involved in replication and pathogenesis of the SARS-CoV-2 virus using proteomics and phosphoproteomics. Take a look at this 50 second video to find out why this work, and the opportunity to take part in the CARE consortium, matters to Thijs.

CARE – Infographic: Work Package 2 Target-based drug discovery and design.

CARE has 8 Work Packages but do you know what each one does? Here, you can learn about the Work Package 2 team, their objectives, their partners, their breakthrough moments and more.

The infographic is also available here

Published in Viruses: Lower infectivity of recent SARS-CoV-2 omicron sub–variants in Syrian Hamster.

The CARE partner, KU Leuven (KUL) evaluated the infectivity of two important sub-variants of omicron in Syrian hamsters. Indeed, since the emergence of the first omicron SARS-CoV-2 variant at the end of 2021, several sub-variants have evolved and become predominant in the human population, showing enhanced transmissibility and ability to (partly) escape the adaptive immune response. These include EG.5.1, an XBB sub-variant, and BA.2.86, a phylogenetically distinct variant, which were compared to the BA.5 sub-variant, a preceding BA.2 descendant. The BA.2 variant was not chosen as a comparator as it does not replicate efficiently in the KUL hamster model as compared to the BA.5 sub-variant.

Both EG.5.1 and BA.2.86 sub-variants are attenuated in Syrian hamsters as compared to the BA.5 sub-variant, as shown by lower infectious titers in the lung and lower lung pathology scores, while the viral RNA loads in throat swabs were comparable in the 3 strains. Interestingly, no virus titers in the lungs of the hamsters infected with the BA.2.86 sub-variant were observed although high viral RNA loads were detected. Therefore, viral RNA loads in the throat swabs and the lungs as well as lung histopathology scores could be a more suitable readout for vaccine and antiviral studies involving these two new sub-variants.

The continuous emergence of sub-variants will remain a challenge as it may require updating vaccines and therapeutic antibodies. Pre-clinical models with the evolving sub-variants are therefore crucial not only to study the virological characteristics of these new sub-variants, but more importantly to evaluate the efficacy of updated and also novel vaccines as well as therapeutic options for which the efficacy is virus variant dependent such as neutralizing antibodies.

To learn more, click here: Comparing the Infectivity of Recent SARS-CoV-2 Omicron Sub-Variants in Syrian Hamsters