Virofight has completed its first year and the first Periodic Report has been approved by our EIC-Project Office. Please find here the publishable summary of the first 12 months of our exciting project.

Summary of the context and overall objectives of the project 
Viral infections affect millions of people every year and cause tremendous human suffering and costs to society. For approximately 70% of all WHO-listed viruses, no treatment is available, and the antiviral drugs that do exist typically must be applied very early after infection to be effective. The VIROFIGHT consortium proposes a radically new approach to fight viral infections, help reduce the scale and sheer impact of viral infections, address the problem of a lack of broadly applicable antiviral treatments, and create means for combating emerging pathogens. Instead of targeting virusspecific proteins or enzymes by small molecules as current antivirals do, we construct synthetic nano-shells that can specifically recognize and engulf entire viruses to efficiently neutralize the pathogen by occlusion. To achieve this technological target, our interdisciplinary project integrates supramolecular chemistry, molecular nanoengineering, and virology with the mission to develop and test prototypes of engulfing nano-shells that have the principal capacity to neutralize any given virus. We fabricate biocompatible nano-shells through DNA origami and protein design and attach virusspecific molecules modularly to them in a multivalent fashion, to exploit avidity effects for strong virus binding. The virus binder is identified through rapid in vitro selection processes designed to be applicable to any target virus. Neutralization assays are used to test our concept on a variety of viruses.

Work performed within the first year 
The VIROFIGHT consortium combines two complementary approaches to combat human viruses. On the one hand, we use prefabricated nano-shells to trap entire viral particles. On the other hand, we polymerize molecular building blocks into virus-neutralizing shells dynamically on the surface of viruses. Both approaches will inhibit the interaction of viral particles with host cells, with the goal to reduce the viral load in acute infections. For this purpose, macromolecular shells are needed that accommodate entire virus particles. To create such shells, the consortium decided to use to complementary bottom-up fabrication techniques: DNA origami and de novo protein design. In WP1, discrete shells were created using DNA origami methods that feature cavity diameters ranging from 40 nm to 280 nm. These shells are thus large enough to accommodate most human spherical viruses. The shells were further modified to feature apertures to allow the trapping of viral pathogens. The structures of the artificial DNA-origami subunits and assembled shells were validated using negative stain TEM and cryogenic electron microscopy (cryo- EM). In WP2, DNA origami shell concepts were developed where the subunits can polymerize on the surface of viruses forming a thick engulfing DNA layer around the viral pathogen. In addition to these systems, protein engineering and de-novo design was employed in WP2 to create prototypes of viral particle engulfing net (inspired by the natural protein). Also, a nucleic-acid based “sticky-tape” is being developed that can wrap around viruses. To trap viral particles inside the nano-shells, or to allow dynamic assembly of shell-forming building blocks around viruses, specific virus-binding molecules are needed in addition to the shell-forming building blocks. One type of virus-binder that is being considered by the VIROFIGHT consortium are aptamers. Nucleic acid-based aptamers have gained attention as alternatives to antibodies due to their ease of production, low immunogenicity, high thermal and chemical stability and small size, while they retain comparable target binding and specificity. Moreover, they can easily be conjugated to the DNAbased virus-engulfing carrier structures. Therefore, in WP3 we successfully established a workflow that allows the development of RNA aptamers against a viral protein of choice. Exemplarily, we deployed this procedure onto SARS-Cov2 spike proteins. The best candidate to come out of this screen was tested for binding to viral proteins and virus-like particles (VLP). We successfully developed an RNA aptamer that is binding with high affinity to the spike protein of SARS-CoV-2 and have started to investigate the capacity of this aptamer to neutralize SARS-CoV-2 by itself, and as a “glue” in the DNA origami shells for trapping SARS-Cov-2 pseudotyped VLP. For selecting aptamers and testing the neutralization of the antiviral nano-shells, the VIROFIGHT

consortium produces various enveloped and non-enveloped viruses, virus-like-particles (VLPs), or lentiviral vectors (LV) in WP4. In particular, we fabricated HIV-Gag-derived VLPs presenting SARSCoV- 2 spike proteins on the surface used for the aptamer selection process. In conclusion, we successfully fabricated nano-shells that are large enough to engulf a variety of viruses by either trapping the viral particles in pre-assembled shells or polymerizing the shells on the surface of viruses. Moreover, we developed an aptamer selection pipeline to produce specific aptamers for any virus protein. Finally, viruses and virus-like particles were produced for aptamer selection and neutralization experiments. Therefore, we successfully completed all tasks that were envisioned for this first reporting period.

Progress beyond the state of the art, expected results until the end of the project and potential impacts
Viral infections represent a huge socio-economic burden to society, as we are currently experiencing it with the COVID-19 pandemic. The VIROFIGHT project aiming at a radical new line of antiviral technology for eradicating multiple viruses therefore has an enormous potential for decreasing the burden for patients and saving costs to society. Our system may also be the foundation for a new ecosystem of antiviral drugs, with potential to enable routine treatment of many types of viral infection with a drastic impact for European citizens and healthcare systems. Our scientific advancements and their future use will greatly impact the European technology sector by boosting the use of nanotechnology and molecular medicine. Our technology has a high translational potential for the treatment of major disease threats. Furthermore, VIROFIGHT can impact seemingly distant fields, such as the purification of food or drinking water from viral pathogens by trapping the  pathogens in filters equipped with our virus-binding systems. The mission of this project is to develop and test prototypes of engulfing nano-shells that have the

principal capacity to neutralize any given virus. A new therapeutic solution that could fight different viruses using the same platform would represent a real breakthrough for antiviral medicine and could save lives and cost to society. Our radical new technology builds on two recent scientific insights: (1) identification of molecular binders against user-defined targets through in vitro selection processes enables producing molecules that confer specificity to any given target virus; (2) fabrication of fully addressable synthetic virus-sized nanoparticles enables building “shell-forming” nanoparticles and attaching the virus-binders to them. Such shells should be able to recognize viruses specifically and engulf them (see picture below). In this first reporting period, we successfully established the technological foundation for our
therapeutic concept, which allows us to move on to testing the neutralization capacity of these shells in human cell cultures and potentially mouse models in the next funding period.

Critical milestone reached in radical new antiviral treatment approach

The VIROFIGHT team has succeeded with a first proof-of-concept for its virus engulfing and neutralizing nano-shells

Instead of targeting virus-specific proteins or enzymes by small molecules as done by current antivirals, researchers of the EU-funded VIROFIGHT project develop nano-shells that engulf and neutralize entire viruses. This novel approach may one day help fight multiple viruses such as Sars-CoV-2, HIV, influenza and hepatitis B with one and the same platform. A year after the project has started, the team led by project coordinator Hendrik Dietz of the Technical University of Munich (TUM) has now managed to demonstrate the feasibility of the virus engulfing and neutralizing nano-shells.

 Need for a broadly applicable antiviral treatment

 Viral infections affect millions of people every year and cause tremendous human suffering and costs to society. For approximately 70% of all WHO listed viruses, no treatment is available and the antiviral drugs that do exist must be applied very early after infection to be effective. The current COVID-19 pandemic is only one such example. The vision of the VIROFIGHT consortium is therefore to develop and test prototypes of nano-shells that may have the principal capacity to neutralize any given virus by engulfing them.

Experiments reveal virus engulfing and -neutralizing nature of the nano-shells

The team around Hendrik Dietz now demonstrated the virus-trapping concept of this approach. The researchers designed nano-shells using the concept of DNA-origami and by mimicking symmetry principles found in natural virus capsids. The artificial shells feature  internal cavity diameters up to 280 nm – big enough to fit most human viruses. When applying the nano-shells to human cell cultures infected with Adeno-associated virus in a first series of tests, the number of infected cells is significantly decreased. Cryo-EM images revealed that indeed the nano-shells neutralized the virus by engulfing viral particles inside their cavities. The results were recently published in the  journal Nature Materials.

DNA nano-shells trapping Hepatitis B virus particles

“These promising results mark an important milestone. But more work is still needed to validate this new avenue for antiviral technology” says Hendrik Dietz, professor of Biomolecular Nanotechnology at the Technical University of Munich (TUM) and Principal Investigator at TUM’s Munich School of BioEngineering. “The next steps for the VIROFIGHT team include how to trap and neutralize many different viruses without the need for antibodies, and testing the system in organisms.”

Ulrike Protzer, professor of Virology at TUM and co-author of the publication adds: „If the idea can be realized to simply eliminate viruses mechanically, this would be broadly applicable and thus an important breakthrough, especially for newly emerging viruses.”

Multidisciplinary consortium to further validate the approach

The interdisciplinary VIROFIGHT consortium integrates experts on supramolecular chemistry, molecular nanoengineering, and virology across Europe. Project partners are Aarhus University (Denmark), ARTTIC S.A.S. (France), ARTTIC Innovation GmbH (Germany), National Institute of Chemistry (Slovenia), University of Regensburg (Germany), Technical University of Munich (Germany) (coordinator). VIROFIGHT is supported by a 3.88 million Euro grant from the European Union’s Horizon 2020 funding programme. The project kicked-off on June 1st 2020 and will run until 31 May 2024.

* featured image shows Christian Sigl preparing nano-shells   for virus trapping. 
  photo by Daniel Delang / TUM