What is your role in the LWNVIVAT project?

We lead WP1, which is dedicated to the computational design of a vaccine that can provide robust protection against WNV. Our focus is on identifying and stabilizing conserved viral epitopes while minimizing cross-reactivity with other flaviviruses, using state-of-the-art bioinformatic tools.

We work in close collaboration with the project coordinator, Jorge Carrillo (IrsiCaixa), to ensure alignment across all stages of the project. This includes coordinating with WP2, led by Carlo Carolis (CRG), to assess protein production and antigenicity, and WP4 and WP5, led by Julia García Prado (IrsiCaixa) and Jorge Carrillo, respectively, for comprehensive immunogenicity testing of our vaccine candidates. These collaborative efforts are essential for integrating computational design with the validation and immune response assessments performed at experimental labs.

What is the primary goal of your research?

Our group works in the area of structural bioinformatics, developing methods and tools for the prediction of protein properties, in this case applied to the design of vaccines. In more detail, we contribute with protein analysis and design tools to the general aim of developing a structurally stabilized WNV vaccine able to promote potent, long-lasting immune responses, preventing cross-reactive immune reactions with other flaviviruses.

What tools do you use to predict which proteins or genes trigger the strongest response against WNV?

More than identifying new proteins we focus on predicting T-cell epitopes for all the proteome – all potential immunogenic regions. In the case of antibody recognition sites triggered by B-cell epitopes, we focus on a well-known target, i.e. the E protein, which is crucial for viral entry into host cells.

For this work we use a large set of openly accessible tools, some developed in-house at BSC’s Electronic and Atomic Protein Modeling group and the Computational Biology lab, along with other publicly available resources. These include:

• NOAH, a structural-based major histocompatibility complex (MHC)-I binding affinity
• NetCleave, a neural network predictor of C-terminal antigen processing for MHC-I and – II
• PredIG, an interpretable predictor of T-cell epitope
• Brewpitopes, a pipeline to refine computational B-cell epitope predictions based on epitope surface exposition.

What factors do you consider determining whether a protein is immunogenic and capable of activating the immune system?

When determining whether a protein is immunogenic, the factors we consider include its ability to induce neutralizing antibodies and T-cell responses, conservation of the epitopes across WNV strains, and its potential structural stability. We use evolutionary analysis to exclude regions that might induce cross-reactivity with other flaviviruses.

Can you explain what cross-reactivity is?

Cross-reactivity occurs when an immune response generated against one pathogen (e.g., WNV) also recognizes and interacts with related pathogens (e.g., dengue or Zika viruses) due to shared antigenic regions. This can lead to unintended immune effects, such as ADE, where non-neutralizing antibodies enhance viral entry into host cells.

What are the latest results you have achieved?

We have conducted an analysis to identify B-cell epitopes on the WNV E protein with high neutralization potential, conserved across WNV variants, and minimal cross-reactivity potential with other flaviviruses.

Following this, we have designed stabilized recombinant E proteins to serve as immunogens. By modelling these proteins with AI techniques and performing protein-wide mutation screenings, we have identified mutations that stabilize key conformations, enhance epitope exposure and disrupt regions with cross-reactivity potential. These optimized designs are now ready for experimental validation.

What are the next steps in your research?

We are working on two different tasks:

• Exploring the creation of smaller, epitope-enriched domains to serve as independent immunogens with potentially reduced cross-reactivity.
• Predicting and selecting a set of immunogenic T-cell epitopes to develop a T-cell- based vaccine that addresses the worldwide genetic diversity of HLA genes. Accounting for this diversity is crucial to ensure effective antigen presentation and robust cellular immune responses on a global scale