Periodic Reporting for period 2 - VIROFIGHT (General-purpose virus-neutralizing engulfing shells with modular target-specificity)
Reporting period: 2021-06-01 to 2022-11-30
Viruses cause a significant impact on human health and society, with millions of people affected by viral infections annually. Many of these infections result in suffering and large financial costs, as the entire human mankind has experienced over the last 3 years. Unfortunately, the majority of viruses listed by the World Health Organization (WHO) do not have any available treatment options, and the antiviral drugs that do exist are often only effective if administered at the early stages of infection. The VIROFIGHT consortium aims to combat viral infections through a revolutionary approach by creating synthetic nano-shells that can specifically recognize and engulf entire viruses in order to neutralize them efficiently. This approach is different from current antivirals that target specific virus proteins or enzymes with small molecules. The interdisciplinary project combines supramolecular chemistry, molecular nanoengineering, and virology to develop and test prototypes of these nano-shells that have the potential to neutralize any given virus. The biocompatible nano-shells are fabricated through DNA origami and protein design, and virus-specific molecules are attached to them in a multivalent fashion to enhance virus binding. Rapid in vitro selection processes are used to identify virus binders, and neutralization assays are used on various viruses. The goal is to help reduce the scale and impact of viral infections, address the lack of broadly applicable antiviral treatments, and create means for combating emerging pathogens.
The VIROFIGHT consortium is using two different methods to combat human viruses. One method involves using pre-made nano-shells to capture entire viral particles, while the other method involves creating virus-neutralizing shells on the surface of viruses. Both methods aim to inhibit the interaction of viral particles with host cells and reduce the viral load in acute infections. To create the shells, the consortium is using two techniques: DNA origami and de novo protein design. In the first method, discrete shells were made using DNA origami with cavity sizes ranging from 40 nm to 280 nm, large enough to hold most spherical human viruses. These shells were also modified to include openings to trap viral pathogens. The structures of the artificial DNA-origami subunits and assembled shells were confirmed using negative stain TEM and cryogenic electron microscopy (cryo-EM). Additionally, in the work package responsible for designing and producing the nano-shells we have succeeded in exploiting the broad-binding properties of heparan sulfates (HS) as virus binders to create a universal virus trapping platform. With the same HS-decorated shells, we trapped up to 10 different viruses and virus like particles (VLPs), all belonging to different families and with different surface complexities.
In the second work package the VIROFIGHT consortium developed concepts for DNA origami shells that can polymerize on the surface of viruses, forming a thick layer of DNA around the viral pathogen. We also used protein engineering and de novo design to create prototypes of viral particle engulfing nets, inspired by natural proteins. In addition to specific viral protein-binding protein domains, we have demonstrated efficient neutralization of oligomerized proteins that recognize glycosylation pattern of viral proteins that might be suitable for the neutralization of a wider range of viruses. Best neutralizers are efficient in the nanomolar range against life viruses. We are building upon recent advances in protein design to test several protein scaffolds to enhance viral neutralization. To effectively trap viral particles in the nano-shells or to allow dynamic assembly of shell-forming building blocks around viruses, the consortium is considering using specific virus-binding molecules such as aptamers. These aptamers are nucleic acid-based and have gained attention as alternatives to antibodies due to their ease of production, low immunogenicity, high thermal and chemical stability, and small size, while still retaining comparable target binding and specificity. They can also be easily conjugated to the DNA-based virus-engulfing carrier structures.
In the work package responsible for molecular binders we successfully established a workflow for developing RNA aptamers against a viral protein of choice. We applied this procedure to SARS-CoV-2 spike proteins and successfully developed an RNA aptamer that binds with high affinity to the spike protein of SARS-CoV-2. We have shown the capacity of this aptamer to neutralize SARS-CoV-2 by itself and have started to investigate its application as a "glue" in the DNA origami shells for trapping SARS-CoV-2 pseudo-typed VLP
In summary, the consortium has successfully fabricated nano-shells that are large enough to capture a variety of viruses by trapping the viral particles in pre-assembled shells or polymerizing the shells on the surface of viruses. We also developed an aptamer selection pipeline to produce specific aptamers for any virus protein. Viruses and virus-like particles were produced for aptamer selection and neutralization experiments, which are conducted in work package 4. All tasks that were planned for this reporting period have been completed successfully.
During the next funding period we will focus on the stability of the different shells and the coupling technology of binding moieties such as the aptamers and antibodies. Further, in vivo neutralization capacity of the nano-shells will be tested as well as a potential immune activity in animal models.
In the second work package the VIROFIGHT consortium developed concepts for DNA origami shells that can polymerize on the surface of viruses, forming a thick layer of DNA around the viral pathogen. We also used protein engineering and de novo design to create prototypes of viral particle engulfing nets, inspired by natural proteins. In addition to specific viral protein-binding protein domains, we have demonstrated efficient neutralization of oligomerized proteins that recognize glycosylation pattern of viral proteins that might be suitable for the neutralization of a wider range of viruses. Best neutralizers are efficient in the nanomolar range against life viruses. We are building upon recent advances in protein design to test several protein scaffolds to enhance viral neutralization. To effectively trap viral particles in the nano-shells or to allow dynamic assembly of shell-forming building blocks around viruses, the consortium is considering using specific virus-binding molecules such as aptamers. These aptamers are nucleic acid-based and have gained attention as alternatives to antibodies due to their ease of production, low immunogenicity, high thermal and chemical stability, and small size, while still retaining comparable target binding and specificity. They can also be easily conjugated to the DNA-based virus-engulfing carrier structures.
In the work package responsible for molecular binders we successfully established a workflow for developing RNA aptamers against a viral protein of choice. We applied this procedure to SARS-CoV-2 spike proteins and successfully developed an RNA aptamer that binds with high affinity to the spike protein of SARS-CoV-2. We have shown the capacity of this aptamer to neutralize SARS-CoV-2 by itself and have started to investigate its application as a "glue" in the DNA origami shells for trapping SARS-CoV-2 pseudo-typed VLP
In summary, the consortium has successfully fabricated nano-shells that are large enough to capture a variety of viruses by trapping the viral particles in pre-assembled shells or polymerizing the shells on the surface of viruses. We also developed an aptamer selection pipeline to produce specific aptamers for any virus protein. Viruses and virus-like particles were produced for aptamer selection and neutralization experiments, which are conducted in work package 4. All tasks that were planned for this reporting period have been completed successfully.
During the next funding period we will focus on the stability of the different shells and the coupling technology of binding moieties such as the aptamers and antibodies. Further, in vivo neutralization capacity of the nano-shells will be tested as well as a potential immune activity in animal models.
The VIROFIGHT project aims to develop a revolutionary new antiviral technology that can eradicate multiple viruses, with the potential to greatly benefit patients and reduce costs for society. The technology could also enable the routine treatment for various viral infections, greatly supporting thereby the European strategy and partnership on pandemic preparedness. The project's advancements in nanotechnology and molecular medicine could greatly impact the European technology industry, as new innovations have been developed and patents have been filed, but also the European labour market, as a first spin-off company has been already founded Additionally, such technology may have applications in fields like purifying food and water from viral pathogens. The project's goal is to create prototypes of nano-shells that can neutralize any virus based on two scientific breakthroughs; the ability to produce specific virus-binding molecules through in vitro selection, and the ability to create synthetic virus-sized nanoparticles that can engulf viruses specifically.