Universal building blocks for antigen presentation to uncover the flavivirus-directed antibody response

Projektinformationen

FLAVIR

ID Finanzhilfevereinbarung: 101077640

DOI

10.3030/101077640

EK-Unterschriftsdatum 11 April 2023

Startdatum 1 August 2023

Enddatum 31 Juli 2028

Finanziert unter

• European Research Council (ERC)

Gesamtkosten

€ 1 687 500,00

EU-Beitrag

€ 1 687 500,00

Koordiniert durch

UNIVERSITEIT UTRECHT
Netherlands

Periodic Reporting for period 1 - FLAVIR (Universal building blocks for antigen presentation to uncover the flavivirus-directed antibody response)

Berichtszeitraum: 2023-08-01 bis 2026-01-31

Zusammenfassung vom Kontext und den Gesamtzielen des Projekts

Flaviviruses are a large group of viruses spread mainly by mosquitoes and ticks, causing major global health problems. Well-known examples include dengue, Zika, yellow fever, and West Nile virus. Dengue infects around 100 million people each year, and Zika’s 2015 outbreak was linked to birth defects. These viruses are mostly found in tropical and subtropical areas, but as the climate warms, their mosquito carriers are spreading northward, bringing local outbreaks to parts of the U.S. and Europe.

Other related viruses, such as Japanese encephalitis and tick-borne encephalitis, also threaten many regions. These viruses often infect the brain and can be deadly. While vaccines exist for yellow fever and Japanese encephalitis, safe and effective vaccines for dengue and other flaviviruses remain a major challenge. One reason is that antibodies from a previous infection or vaccination can sometimes worsen disease instead of preventing it—a phenomenon called antibody-dependent enhancement (ADE).

A key viral protein, called the Envelope (E) glycoprotein, is the main target for neutralizing antibodies. Some antibodies strongly block infection by recognizing specific parts of the E protein, while others bind more weakly and can actually promote infection through ADE. However, most current studies focus on isolated antibodies, not the complex mix of (polyclonal) antibodies found in blood. Understanding this broader response could greatly improve vaccine and antibody design.

The proposed project, FLAVIR, aims to develop new ways to display and study the E protein, allowing detailed analysis of how antibodies recognize and neutralize flaviviruses. Using advanced techniques in mass spectrometry, electron microscopy, and glycoproteomics, the project will reveal how different antibodies and sugar modifications affect immune recognition. These methods will also help design better vaccines and diagnostic tools by stabilizing and presenting E proteins in forms that mimic real viruses.

Ultimately, FLAVIR will build a flexible platform for studying many flaviviruses, helping to uncover how our immune system fights these infections—and how we can use that knowledge to prevent them.

Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse

We are now testing several new methods to stabilize the flavivirus Envelope glycoprotein E in its prefusion dimer form. Using databases of natural protein structures, we have identified 20 promising molecular “tags” that may help form stable dimers of the E protein. These designs are being screened using advanced mass spectrometry and electron microscopy to confirm proper assembly. Early results with dengue and Zika E-dimers look promising, and we are expanding the work to other flaviviruses such as yellow fever, Japanese encephalitis, and West Nile virus.

We are also building virus-like particles (VLPs) from these stabilized dimers. These particles mimic the virus surface but are completely non-infectious, allowing detailed study and safe vaccine development. By attaching E-dimers to self-assembling protein scaffolds, we aim to recreate the full icosahedral structure of the virus. This approach lets us control the exact sequence and presentation of antigens, including removing features that can trigger antibody-dependent enhancement (ADE). We are testing several nanoparticle systems and exploring “mosaic” particles that mix E proteins from different virus strains to promote broader immune protection.

In parallel, we are analyzing how sugar molecules (glycans) attached to the E protein influence antibody binding. Using state-of-the-art glycoproteomics and cryo-EM, we can map these glycans at each attachment site and see which forms help or hinder antibody recognition. We are currently comparing how different antibodies—both monoclonal and polyclonal—interact with specific glycoforms.

Finally, we are sequencing the complex mixtures of antibodies produced in response to flavivirus infection. Our new mass spectrometry–based antibody sequencing methods allow us to identify the exact sequences of antibodies directly from serum, without needing to isolate B cells. Combining this with structural mapping, we are now visualizing which regions of the E protein are targeted by antibodies and how this differs between protective and enhancing responses.

Together, these experiments are revealing—in unprecedented detail—how antibodies recognize flaviviruses. The insights gained will help design safer, more effective vaccines and guide next-generation diagnostics for dengue, Zika, and related infections.

Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts)

We are producing recombinant flavivirus Envelope (E) glycoproteins in mammalian cell culture to study how these proteins assemble and interact with antibodies. Our system allows us to rapidly test new designs and scale up promising candidates for detailed analysis. Using advanced mass spectrometry, electron microscopy, and antibody-binding assays, we are studying how different mutations and molecular linkers stabilize the E-dimer—the key structure recognized by neutralizing antibodies—while minimizing exposure of regions linked to unwanted immune responses. So far, we have created several stable E-dimer designs and continue refining them to optimize antigen presentation.

We are also building self-assembling nanocages that display multiple copies of the E-dimer, mimicking the natural virus surface. Early imaging shows these particles assemble as designed, and we are improving their stability and yield. We are now advancing toward the next goal: assembling complete virus-like particles that safely reproduce the full structure of the flavivirus shell.

In parallel, we have mapped the sugar modifications (glycans) on E-dimers from Zika and all four dengue serotypes, showing they match those on real viruses. Initial antibody-binding experiments suggest that bulkier glycans can shield key epitopes from immune recognition, an effect we are now studying in more detail.

Finally, we are combining mass spectrometry–based antibody sequencing with electron microscopy to identify which antibodies recognize which parts of the E protein. We have improved our data analysis tools, developed new software for sequence visualization, and obtained serum samples from dengue-infected individuals to apply these methods in real-world immune responses.

Continued work on this project will provide new molecular tools to study flavivirus immunity in unprecedented detail. These advances will help researchers design better vaccines, improve antibody discovery pipelines, and expand structural biology methods for complex viral systems.