Published in Antiviral Res: A new image-based and high-throughput platform to identify broad-spectrum coronavirus antivirals

The CARE member, Johnson & Johnson Innovative Medicine, developed a high-content imaging platform compatible with high-throughput screening, with the aim of identifying compounds with antiviral activity and to deprioritize those that induce undesirable phenotypes in host cells.

The immunofluorescence-based assay to assess the antiviral compounds against coronaviruses was adapted for high-throughput screening by shortening assay run times, miniaturizing well size and volumes, combining permeabilization and staining steps, and by using automated image analysis. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled identification of compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity.

This platform was easily adapted to different SARS-CoV-2 variants (B1, Omicron BA.5 and Omicron XBB.1.5), SARS-CoV and human coronavirus 229E using different antibodies and cell types. With the platform, ~900K compounds were screened and hits were triaged in just four weeks, thereby allowing the identification of potential anti-coronaviral compounds carrying broad-spectrum activity with limited off-target effects.

This new platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. By developing and running the antiviral assay against different coronavirus strains, it was demonstrated that the assay can most likely be efficiently adapted to test against new SARS-CoV-2 variants or new coronavirus species as soon as they emerge. Given the high risk for future coronavirus outbreaks or other pandemics from new zoonotic reservoirs, the flexibility and broad applicability of this assay are of particular interest.

To learn more, click here: A flexible, image-based, high-throughput platform encompassing in-depth cell profiling to identify broad-spectrum coronavirus antivirals with limited off-target effects

Introducing Utrecht University – a CARE academic organisation

Established in 1636, Utrecht University has a long history and is rich in tradition. Utrecht University was formed by the provincial government in 1636. Recognized as the leading Dutch university according to the Shanghai Ranking, it offers 45 undergraduate and 167 graduate programs, has some 30,000 students served by 7,500 academic staff. Today the university has seven faculties in Veterinary Medicine, Medicine, Science, Geosciences, Social and Behavioural Sciences, Humanities and Law, Economics and Governance.

The primary contributor to CARE from the university is the Virology section at the Faculty of Veterinary Medicine. The Virology section is studying various viruses (coronavirus, picornavirus, influenza, paramyxovirus) with the aim of developing innovative antiviral drugs, therapeutic antibodies and vaccine approaches.

The Virology section was started in 1971 by Prof. Marian Horzinek, who is considered a “Founding Father” of (veterinary) virology in The Netherlands. He started virology research at the university studying Feline Infectious Peritonitis (FIP), a deadly disease in cats due to infection with a feline coronavirus (FCoV). The section has longstanding and extensive experience in coronavirus research, specifically in

1. Receptor identification and virus-receptor interactions
2. Structure-function studies of the viral Spike protein, which is the protein involved in binding receptor(s) and mediating cellular entry and the main target for humoral immune responses, and as such a key viral protein for development of intervention strategies
3. Determinants of coronavirus inter-species transmissibility
4. Viral genome replication and virus-host interactions
5. Development of intervention strategies (antibodies, vaccines, antiviral drugs) and diagnostics for (emerging) coronaviruses

The Virology section comprises approximately 45 members, including researchers at all seniority levels and support staff, rising to up to 60 researchers when housing students performing their MSc internships.

Why did Utrecht University choose to get involved in CARE?

The Virology Section has 50+ years of experience in coronavirus biology and possesses extensive expertise in both human and animal coronaviruses. They have particular expertise in virus structure, virus-receptor interactions, virus entry mechanisms, genome replication and virus-host interactions. The CARE project enabled them to quickly contribute their knowledge with many other research groups, while also benefitting from enhanced collaboration with the CARE partners.

What has Utrecht University delivered for CARE?

UU’s contributions span across various innovations and findings in Work Packages (WPs) 1, 2, 4 and 5. Overall, their efforts aim to identify potential antiviral drugs, peptides, nanobodies and antibodies, to understand their mechanisms of action, and to explore host factors involved in coronavirus infection, which could inform the development of effective therapies against SARS-CoV-2 and other related coronaviruses.

In WP1, they evaluated a large number of small molecules for antiviral activity against SARS-CoV-2 and other coronaviruses, characterized antiviral effects and performed Mechanism of Action studies to reveal their molecular mechanisms.

In WP2, they developed macrocyclic peptide inhibitors and nanobodies targeting the Spike protein of SARS-CoV-2. The team studied the site in Spike that is targeted by the macrocyclic peptide using cryo-Electron Microscopy (cryo-EM). This revealed that the peptide binds to a conserved region of the SARS-CoV-2 spike protein that has hitherto not been exploited by antibodies or small molecules and has not been mutated in any of the variants of concern.

In WP4, they established assays, reagents and structural biology workflows for the assessment and characterization of human neutralizing antibodies (nAbs) against SARS-CoV-2 and variants. The team generated bispecific antibodies to enhance potency and breadth of nAbs against SARS-CoV-2 and related coronaviruses. They also developed broad-spectrum nAbs against porcine deltacoronavirus (PDCoV), an emerging coronavirus with pandemic potential. Finally, they isolated and characterized human antibodies that target host receptor aminopeptidase N (APN). These antibodies are highly resistant to viral escape and can be employed for coronaviruses that share the same receptor for entry, increasing our pandemic preparedness against newly emerging coronaviruses.

In WP5, they determined changes in the metabolome in coronavirus-infected cells and identifying essential host factors for coronavirus infection via genetic CRISPR-cas9 screens. In follow-up experiments, they are investigating the physiological importance of the virus-induced changes in metabolism and the role of the identified host factors for efficient virus replication and/or evasion of infection-limiting host antiviral responses.

For more information about the different work packages, please click here

Who is working in the CARE team at Utrecht University?

The Utrecht University team is led by two Principal Investigators: Professor Dr Frank van Kuppeveld who studies the interaction between viruses and their host; and Dr Berend Jan Bosch (Associate Professor) who studies virus-receptor interactions and cell entry mechanism of membrane-enveloped viruses, particularly coronaviruses.

During the CARE project, Dr Daniel Hurdiss, a structural virologist, was promoted from a post-doctoral researcher to Assistant Professor. He is a structural virologist who, among others, uses cryo-EM to study the 3D structure of viral proteins and understand their functional implications.

Many of UU’s virologists have been involved in CARE from the beginning, all aiming to translate their knowledge into intervention strategies targeting either viral entry and/or replication: Frank Buitenwerf, Marianthi Chatziandreou, Oliver Debski Antoniak, Tim Donselaar, Wenjuan Du, Preeti Hooda, Ruben Hulswit, Yifei Lang, Joline van der Lee, Rutger Luteijn, Wendy Meijer, Vera Nijman, Lonneke Nouwen, Collins Owino, Judith Oymans, Itziar Serna Martin, Jill Ver Eecke, Marleen Zwaagstra. In the first year of CARE, the group of Alexandre Bonvin (Computational Structural Biology) at the university’s Faculty of Science also contributed to CARE.

What benefits have Utrecht University experienced through being part of CARE?

The Utrecht University Virology team has enjoyed the following benefits:

• Access to a broader range of expertise, technologies, and facilities.
• Opportunities to work alongside industry partners, other academic institutions, and governmental agencies which fosters knowledge exchange, interdisciplinary research, and the pooling of diverse perspectives and skills.
• Academic researchers’ opportunities for networking, professional development, and establishing collaborations with leading experts and institutions in the field.
• Support to conduct cutting-edge research, accelerate progress towards the development of effective therapies, and make meaningful contributions to the global effort to combat infectious diseases.

Want to know more about Utrecht University?

https://www.uu.nl/en/organisation/faculty-of-veterinary-medicine/veterinary-research/one-health/infection-immunity/virology

CARE Partner perspectives – Frank van Kuppeveld

The Covid-19 pandemic is over so what is the value of CARE? Principal investigators comment on CARE’s importance for future pandemic preparedness. Today, see the response from Prof. Dr. Frank van Kuppeveld, head of the Virology Division of the Faculty of Veterinary Medicine at Utrecht University. 

CARE – Infographic: Leadership and partnership

Do you whish to know who are particpating in this concortium and who are leading it? Check out this infographic for key details on CARE partnership.

The infographic is also available here

Introducing The Institute of Virology and Immunology (IVI) – a CARE academic organisation

In 1917, Swiss laws on epizootics were drawn up to combat foot-and-mouth disease effectively. In response, the Swiss Confederation built the Federal Vaccine Institution in 1942. In 1992, the institute was renamed to Institute of Virology and Immunoprophylaxis and moved to a new facility with a high containment laboratory in Mittelhäusern, approximately 15km from Bern. Since 2014, a merger with the Institute of Veterinary Virology at the Vetsuisse faculty led to the current Institute of Virology and Immunology (IVI). The connections to and involvement in the Vetsuisse Faculty are coordinated through a formal cooperation. Within the Vetsuisse Faculty, the IVI is associated with the Department of Infectious Diseases and Pathobiology.

The IVI is Switzerland’s reference laboratory for the diagnosis and research of highly infectious epizootic diseases, including viral zoonoses. The IVI reports to the Federal Food Safety and Veterinary Office (FSVO) within the Federal Deparment of Home Affairs (FDHA) of Switzerland. With its research in virology and immunology the IVI strives to develop solutions to combat and control viral diseases, for example African swine fever, Influenza viruses, bovine virus diarrhea virus, Japanese encephalitis and other flaviviruses, coronaviruses and emerging diseases in general.

The IVI employs approximately 120 staff at its sites in Bern and Mittelhäusern. These include scientists, students and support staff (engineers and technical staff, animal caretakers, administration and biosafety). Two divisions of IVI contribute to CARE. The team of Prof. Volker Thiel specialise in respiratory and enteric viral infections and zoonoses. The team of Prof. Charaf Benarafa are experts in molecular, cellular and in vivo pathways of innate immune responses in infectious and inflammatory disease

Why did IVI choose to get involved in CARE?

IVI decided to get involved with CARE primarily through the involvement in the SCORE project of which IVI were one of the core eight partners. All 8 partners from SCORE are also in the CARE consortium. Secondly, the Thiel team has more than 25 years of experience of working with coronaviruses, so the opportunity to combine efforts with the Benarafa team was a possibility that could not be missed due to their considerable expertise in dissecting the immune response using infectious disease models.

What has IVI delivered for CARE?

IVI’s achievements in CARE includes the production of fluorescent viruses in Work Package 1 (WP1) which were pivotal in the development of a high-throughput dual-reporter screening assay for antiviral drug discovery. This was possible due to the close collaboration with the team of Pieter Leyssen in KU Leuven (DOI: 10.1016/j.antiviral.2022.105506). Second, the IVI team also made a series of mutant viruses incorporating changes implicated in drug-resistance. This was done with the team of Dirk Jochmans from KU Leuven also within the context of WP1 (doi.org/10.1128/mbio.02815-22). In WP6, the IVI has contributed considerably by developing animal models for testing the efficacy of antivirals developed in the drug pipeline of the CARE project.

For more information about the different work packages, please click here

What benefits has IVI enjoyed through participating in CARE?

Both the Thiel and Benarafa teams have been able to broaden their networks, which have led to the instigation of a new EU-funded project to develop broad acting antivirals against RNA viruses with pandemic potential (PanViPrep), which began at the start of 2024. Other collaborations have also started with CARE colleagues working on other coronaviruses (including Feline Infectious Peritonitis). Undoubtedly, these opportunities have been facilitated through our participation in the CARE project.

In addition to Volker and Charaf, the IVI team includes

And Bettina Trüeb (Postdoctoral Research Scientist, WP1)

Want to know more about IVI?

https://www.ivi.admin.ch/ivi/en/home.html

https://www.ivi.unibe.ch/index_eng.html