Publication Spotlight: Applying Nextstrain and iCn3D to modify and expand an existing activity for undergraduate students characterizing potential binding of antibodies to mutations in the pathogens: Influenza, RSV, or Enterovirus D68

Submitted by EA Stewart on 2 April, 2025.

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A crucial part of building activities for students is choosing a topic that will be engaging and foster the kind of interest that will make students want to take the activity to new heights. The Antibody Hackathon team did just that in choosing to build activities and teaching protocols around the ability of antibodies to bind to SARS-CoV-2, given the global significance of that virus. Viruses have played a very prominent part in our recent human history, with losses due to Sars-CoV-2 still very much in our minds, and viral illnesses still claiming lives every year. One of the defenses we have in our battle against viruses is neutralization, during which an antibody protein binds to a viral protein and blocks its ability to cause illness. This makes interactions between viruses and antibodies, as well as the potential for immune escape [mutations in a pathogen's proteins which keep an antibody from binding], a critical part of research in infectious diseases, and an important topic in a biotechnology curriculum.

As the Antibody Hackathon's protocols were put to use, feedback from students and faculty made it clear that the activities were so successful that participants wanted to branch out and apply the protocols to other pathogens. A recent Journal of Advanced Technological Education paper titled "Applying Nextstrain and iCn3D to modify and expand an existing activity for undergraduate students characterizing potential binding of antibodies to mutations in the pathogens Influenza, Respiratory Syncytial Virus (RSV), or Enterovirus D68" describes the authors' steps as they worked to build new guidelines for applying the original protocols to new pathogens.

The authors chose four pathogens (Hookworms, Influenza, RSV, and Enterovirus) and searched for information in sequence and protein structure databases that would support bioinformatics experimentation. As they did this, they built a series of guidelines that can help instructors as they work to build their own data sets for other pathogens. Their first step was similar to the original SARS-CoV-2 protocol, in which they needed to find structure files that contain antibodies bound to pathogen proteins of interest. After this step they then needed to align protein sequence data from antigenic variants to the identified pathogen protein sequence in the structure. In order to do these two steps they searched SAbDab for structural data and Nextstrain and the Influenza Virus database* for strain sequences. Unfortunately, none of the hookworms of interest, although a significant global public health issue, were found in the Nexstrain database, nor were any hookworm protein-antibody structures found in SAbDab. Thus, as a first guideline for possible new pathogens, it is important to make sure that the sequence of the reference strain is available.

Further exploration with the other three pathogens yielded additional guidelines--available in the Supplemental Materials of the JATE manuscript. The authors found that it is important that there are a large number of protein variant sequences for the pathogen, available with an NCBI accession number format, or a FASTA sequence. As a crucial part of the experiment is examining structures, PDB files containing pathogen protein/antibody complexes are also needed. And, to avoid unnecessary and possibly confounding complexities, the authors recommend focusing on structures that contain a single antibody-antigen interaction.

Expansion of the original protocols was shown to be possible, although there is a critical need for additional sequence and structural data The new guidelines will make it easier for an important biotechnology resource to expand and continue to capture the interest and enthusiasm of new students.

* The Influenza Virus database was redirected, as of Fall 2024, to NCBI Virus. For the most up-to-date information, use NCBI Virus.

E. Lannan, A. Sterling, Y. Hu, and S. Porter. Applying Nextstrain and iCn3D to modify and expand an existing activity for undergraduate students characterizing potential binding of antibodies to mutations in the pathogens Influenza, Respiratory Syncytial Virus (RSV), or Enterovirus D68, Journal of Advanced Technological Education. Vol. 3, Issue 2, 2024. DOI: 10.5281/zenodo.13357348