What is your role in the LWNVIVAT project?

Our team specializes in the expression, purification, and characterization of immunogens. We use advanced scientific tools to produce and carefully study these proteins.

What is the primary goal of your research?

Our research focuses on the production of immunogenic proteins that induce a strong immune response while minimizing cross-reactivity with other flaviviruses. This work is highly collaborative and benefits greatly from the expertise of our partners at the Barcelona Supercomputing Center. Led by Alfonso Valencia, their research group plays a key role in identifying and designing optimized protein variants.

How do you express a specific DNA sequence and produce proteins, particularly the LWNVIVAT immunogens for WNV?

Our team has extensive experience in protein production and characterization. In this project, we employ advanced tools and techniques to express proteins using both mammalian and insect cell systems.

This process involves:

• Making and inserting the genes: we design the DNA for each protein version (called an ORF variant) and insert it into a vector. These vectors are made to work well in both mammal and insect cells.
• Making the proteins: the vectors are put into cells, and those cells start producing the proteins. The proteins are then released into medium.
• Cleaning and checking the proteins: The proteins are collected and purified using special filters. Then, their quality is checked using advanced tools like:
  • Mass photometry – measures the size and weight of single protein molecules.
  • SEC-MALS – checks the size and shape of proteins.
  • nanoDSF – looks at how stable the protein is when heated.

These methods assess the oligomerization state, aggregation levels, and stability of the protein variants.

How do you ensure the proteins produced are the intended immunogens?

To confirm the identity and quality of the produced proteins, we implement rigorous validation steps:

• Sequencing: All immunogens undergo DNA sequencing to verify their identity.
• Expression and production screening: We measure expression levels and assess protein detection using Nano-Glo® HiBiT Lytic Detection System.
• Mass spectrometry (MS): This technique is employed to precisely identify and validate the produced protein variants.

What are the latest results you have achieved?

In recent months, we have successfully produced over 80 protein variants designed within the project. Our findings include:

• Identification of mutations that enhance protein expression and solubility, improving performance compared to original strains.
• Large-scale production of selected candidate proteins, which are now undergoing immunogenicity testing to evaluate their potential as effective immunogens.

What are the next steps in your research?

Our next goal is to further enhance our high-throughput (HTP) protein production pipeline.

This will allow us to screen a larger number of clones more efficiently and also to identify additional promising protein candidates that may induce a stronger immune response.

By continuously improving our production and screening processes, we aim to contribute to the development of effective immunogens for combating West Nile Virus (WNV) and related flaviviruses.