Published in MCP: Assessment of Kinome-Wide Activity Remodeling upon Picornavirus Infection

The CARE partner Utrecht University (UU) conducted an overview of the (de)regulation of kinase activities (kinome) by two picornaviruses during infection using a targeted mass spectrometry-based system-wide kinase activation assay (covering ~40% of the human kinome). The family Picornaviridae, a large family of small, non-enveloped viruses with a single stranded positive sense RNA genome, includes many well-known human and animal pathogens and are known to deregulate key host-cellular processes.

UU observed a considerable overlap in the kinome activity remodeling of the host cells infected by either coxsackievirus B3 (CVB3), an enterovirus, and encephalomyocarditis (EMCV), a cardiovirus. Both activate the MAPK pathways and the DNA damage response (DDR), although the dynamics of activation of specific kinases within these pathways were different for CVB3 or EMCV.

Moreover, during both CVB3 and EMCV infection, several kinases involved in regulating the cell cycle were inhibited – most of them having a role in mitosis – suggesting that both viruses regulate cell cycle, especially mitosis.

As the observed activation of several kinases upon CVB3 or EMCV infection suggests a role of these kinases in viral reproduction, UU administered 16 kinase inhibitors including MAPK pathway inhibitors, DDR inhibitors, an inhibitor of CDK9, and a pan-PAK kinase inhibitor. Nearly all 16 inhibitors could decrease viral reproduction, although most of them often only at relatively high concentrations. However, almost all inhibitors also reduced cell viability at these higher concentrations, illustrating a likely narrow therapeutic window balancing effective treatment and cellular toxicity.

Altogether, the data provide a quantitative understanding of the regulation of kinome activity induced by picornavirus infection, providing a resource important for developing novel antiviral therapeutic interventions. This work was important to establish assays and methodology to evaluate the impact of coronaviruses on host cells in CARE.

To learn more, click here:  Assessment of Kinome-Wide Activity Remodeling upon Picornavirus infection

CARE’s Young Researchers – Introducing Oliver Debski-Antoniak PhD, Utrecht University

Read about how Oliver’s work in the discovery and advancement of potential antibody treatments for both existing and emerging coronavirus infections is making a significant contribution towards efforts in prevention and alleviation of economic and health strains caused by endemics and pandemics in the future.

CARE (Corona Accelerated R&D in Europe) is the largest European research initiative addressing the challenges of COVID-19. It comprises 38 partners, from both industry and academia, in a set-up of eight multidisciplinary work-packages (WPs). In this series, we highlight the work of some of the young researchers involved in CARE as part of their PhD or postdoctoral work. Here, we learn how this opportunity has benefited Oliver, while simultaneously benefiting CARE and its ambition to help society defeat COVID-19 and future pandemics.

Why did you decide to get involved in CARE?

My decision to join the CARE consortium was driven by a profound desire to contribute to pandemic prevention and preparedness on a global scale. This aspiration led me to the virology department in Utrecht University, where I could actively participate in the groundbreaking efforts through fantastic collaborations via the CARE consortium. I was particularly inspired by the innovative work emerging from this collaboration, such as the discovery of P5C3, a highly potent antibody against SARS-CoV-2. This work, spearheaded by Craig Fenwick et al, highlighted work coming from the CARE consortium is at the leading edge of research.

Tell us about the work you have been doing in the CARE consortium

My work is centered around two primary objectives:

1. The discovery and advancement of potential treatments for both existing and emerging coronavirus infections.
2. Enhancing our understanding of the initial stages of both novel and known coronavirus infections, primarily through studying the structure, function, and mechanism of action of the viral spike protein, which facilitates recognition and entry into the host cell.

I apply my expertise in cryo-EM, a technique that allows me to visualize the virus directly. This method offers detailed insights into the molecular interactions between the virus and the host cell, as well as between the virus and potential therapeutic candidates, thereby revealing the virus’s vulnerabilities.

Through my contributions to the CARE consortium, several pieces of work have been published. These include the characterization of a promising cyclic peptide with potential as a pan-Sarbecovirus therapeutic. This peptide binds to a highly conserved region of the spike protein, enabling it to remain active against all SARS-CoV-2 variants of concern and neutralize other members of the Sarbecoviruses, a group of coronaviruses with the potential to cross over to humans, which could have catastrophic consequences.

We have also isolated and characterized potent monoclonal antibodies against the spike protein of Porcine Delta Coronavirus (PDCoV). PDCoV is a prevalent coronavirus in pig populations, causing significant economic impact on the porcine industry. However, PDCoV has also been found in humans in several isolated cases. This virus has considerable potential to cause future pandemics, necessitating extensive research to better understand this virus and develop therapeutics to mitigate the effects of an outbreak, should it achieve human-to-human transmission. Our antibodies, particularly antibody 67B12, which directly inhibits the spike’s ability to interact with the human cellular receptor required for entry, not only demonstrated excellent therapeutic potential but also contributed to our understanding of the mechanisms the spike uses to recognize host cells and facilitate cell entry.

What highlights can you share from your time in the CARE consortium so far?

Being a part of the CARE consortium has provided me with the unique opportunity to immerse myself in cutting-edge virology research. This experience has brought me into contact with the insights and expertise of some of the most distinguished and knowledgeable scientists in the field, globally. I’ve had the privilege of showcasing my work to international audiences and engaging in enriching discussions on pertinent topics. However, the most rewarding aspect is the knowledge that my contributions are advancing our understanding of a group of viruses that pose significant threats to global health and economy.

Why does this work matter?

The significance of this work lies in its potential to prevent and alleviate the economic and health strains caused by endemics and pandemics. The occurrence of the next pandemic is not a matter of ‘IF’, but rather ‘WHEN’. It is my hope that my contributions will play a part in ensuring that we never have to experience another pandemic-induced lockdown. The prospect of enduring another period of confinement, with virtual quizzes as the only form of entertainment, is something I would prefer not to contemplate.

How have you benefited from your involvement in CARE?

Participation in the CARE consortium has enriched me with an abundance of knowledge and experience in my area of passion. It has offered me exposure to the pinnacle of excellence in the field, sparking my imagination and shaping my approach to research inquiries and concepts.

What advice would you give to someone getting involved in a Public-Private Partnership?

Become a human sponge. Talk to people. Absorb all the knowledge and experience that is put in your path. Grow!!!!

Published in Nature: First insights into the Porcine Deltacoronavirus spike

Further to CARE’s SARS-CoV-2 research, CARE partner Utrecht University (UU) has studied the Porcine Deltacoronavirus (PDCoV) spike, providing crucial insights for developing antiviral strategies. PDCoV, an emerging enteric pathogen in pigs, has recently been detected in children with acute febrile illness. PDCoV enters cells by binding its spike (S) protein to the host receptor aminopeptidase N (APN) and can use the APN of different species, ranging from humans, felines to chickens, highlighting its capacity for interspecies transmission. Despite this zoonotic threat, the antigenic structure of PDCoV, i.e. the molecular structures on the surface of viruses that are recognized by the immune system and are capable of triggering immune responses, remains unknown.

UU and collaborators generated and characterized a set of human monoclonal antibodies (mAbs) targeting the S protein, which can effectively neutralize virus infection. Through an integrated approach involving functional and structural analyses, UU successfully pinpointed three vulnerable regions within the prefusion S trimer. The team showed that mAb binding to the spike S1A domain is important to shift the S1B domain towards an open conformation, which is necessary for binding to the APN receptor. Other antibodies targeting the S1B domain inhibited binding to the receptor, thereby preventing infection. The epitopes of these S1B-targeting antibodies were concealed in the prefusion S trimer conformation, suggestive of an intriguing immune evasion mechanism in PDCoV. One S1B mAb, broadly reactive against avian deltacoronaviruses, targeted a highly conserved epitope on S1B that entirely overlapped with APN-interacting residues.

The identification of neutralizing epitopes on the PDCoV S protein provides essential functional and structural insights for understanding humoral immune response against this zoonotic pathogen. These discoveries hold promise for developing potent tools for outbreak preparedness against prospective deltacoronaviruses in humans.

To learn more, click here: Neutralizing Antibodies Reveal Cryptic Vulnerabilities and Interdomain Crosstalk in the Porcine Deltacoronavirus Spike

CARE – Infographic – Work Package 5 – identifying immune markers contributing to the host immune responses to SARS-CoV-2 infection and their correlations with clinical and virological outcomes​

CARE has 8 Work Packages but do you know what each one does? Here, you can learn about the Work Package 5 team, their objectives, their partners, their breakthrough moments and more.

The infographic is also available here

CARE’s Young Researchers – Introducing Manon Laporte, PhD, KU Leuven

Read about how Manon’s work in phenotypic antiviral screening enabled the discovery of a novel target in the replication cycle of coronaviruses and a small molecule inhibitor that can address that target, plus how this approach may benefit future pandemic preparedness across many virus types.

CARE (Corona Accelerated R&D in Europe) is the largest European research initiative addressing the challenges of COVID-19. It comprises 38 partners, from both industry and academia, in a set-up of eight multidisciplinary work-packages (WPs). In this series, we highlight the work of some of the young researchers involved in CARE as part of their PhD or postdoctoral work. Here, we learn how this opportunity has benefited Manon, while simultaneously benefiting CARE and its ambition to help society defeat COVID-19 and future pandemics.

What experience did you have working on a Public Private Partnership before joining CARE? 

I had no experience working on a PPP before but many of my current colleagues did.

How did your involvement in CARE come about?

I joined the group of Johan Neyts (the academic lead of Work Package 1) two years ago, approximately one year after the start of CARE. At that point, I had two years’ experience working with SARS-CoV-2, both at the Rega Institute in Leuven with my former Principal Investigator, Lieve Naesens and during my postdoc at the Garcia-Sastre lab in New York at the Icahn School of Medicine at Mount Sinai. Given my experience with SARS-CoV-2, I started working on the CARE antiviral projects together with Dirk Jochmans, research manager of Johan Neyts’ group.

Tell us about the work you have been doing in the CARE consortium

I’m part of the team working on Work Package 1: “Anti-coronavirus drug discovery in phenotypic virus-cell-based assays.” I plan and oversee the in vitro antiviral testing for different partners within CARE. I also helped to establish antiviral assays for high throughput screening using our unique lab-in-a box system CAPS-IT (https://rega.kuleuven.be/cmt/capsit) and I have set up several mode-of-action assays to elucidate the working mechanism of newly identified hits.

Our biggest project within CARE is the coronavirus M assembly inhibitor project together with CD3 (https://www.cd3.be/) and CISTIM (https://www.cistim.be/). When I joined two years ago, we had two interesting compound series. I have been very closely involved in the optimization process and in solving the molecular mechanism of action of ‘series 8’. These compounds inhibit the virus via an entirely novel and hitherto unknown way, namely by blocking the viral membrane (M) protein, the key regulator of coronavirus assembly.

What highlights can you share from your time in the CARE consortium so far?

Scientifically I feel very fortunate to be able to work on the coronavirus assembly inhibitor story. We discovered a novel druggable target in the replication cycle of coronaviruses and a small molecule inhibitor that can address that target.

In science, you sometimes have to be a little lucky and it is an amazing opportunity to work on such an exciting story with a great group of people, both within our team at KU Leuven, the medicinal chemists at CD3 and CISTIM, and our collaborators at other institutes. Within CARE, especially with the team of Daniel Hurdiss (Utrecht University) and the team of Eric Snijder (Leiden University Medical Centre).

Why does this work matter?

We prove again that phenotypic antiviral screening is a successful way of identifying novel antiviral targets. Until today, effective antiviral therapies for many viruses with epidemic and pandemic potential are still lacking (e.g., paramyxo-, pneumo-, bunya-, flavi-, toga-, filo-, enteroviruses). The mission of our lab (www.antivirals.be) is to develop small-molecule antiviral drugs (or combinations thereof) against these viruses using phenotypic high-throughput screening as a starting point. Hits from these screening campaigns are further optimized and we try to unravel their mode of action which often leads to the discovery of novel antiviral targets, as was the case for the coronavirus M targeting compounds. Besides my work on SARS-CoV-2, I am currently focusing on a small molecule discovery project for henipaviruses. Nipah virus is a highly lethal paramyxovirus (case fatality rate is estimated at 40% to 75%) that causes almost yearly outbreaks and can be transmitted from human to human. There is no treatment or vaccine available and Nipah virus is on the WHO R&D blueprint list of priority diseases. It is our goal to discover novel small-molecule inhibitors with pan-henipa coverage so that, in the event of a big outbreak we don’t lag behind with antiviral therapy as was the case during the COVID-19 outbreak.

How have you benefited from your involvement in CARE?

I have benefited by expanding my network significantly. Thanks to CARE it is possible to work with other experts in the field in a very efficient way. The amount of data we can collect by working together is truly unique.