Do you remember the egg shortage of 2024? It wasn’t just a supply chain issue; it was a consequence of the H5N1 avian influenza -or bird flu- sweeping through poultry populations worldwide. While primarily a bird disease, the virus occasionally infects humans, causing severe respiratory and neurological symptoms. Although human cases are rare and sustained human-to-human transmission has not occurred, the threat of mutation looms.
One of the barriers preventing bird flu from spreading efficiently in humans lies in the receptor specificity of hemagglutinin (HA)—a viral spike protein critical for entry into host cells and the antigenic protein. HA of avian flu binds to “avian-type” receptors with sialic acid in an α2-3 linkage, while human flu HA binds “human-type” receptors with sialic acid in an α2-6 linkage, abundant in the upper human airway. Acquisition of human-type receptor specificity is one of several features of pandemic influenza strains. Other pandemic HA features include cell entry, antibody evasion, and HA stability.
To stay ahead of the virus, the Bloom lab at Fred Hutch employed deep mutational scanning to assess the effects of single amino acid mutations in the HA protein of the bird flu. Their findings, recently published in PLOS Biology, provide critical insights into how these mutations influence viral entry, receptor specificity, stability, and antibody escape.
“H5 influenza poses a pandemic risk,” said Dr. Jesse Bloom, a professor in the Basic Sciences Division. “Our study makes it easier to identify mutations that might increase pandemic potential or affect vaccine efficacy.”
Deep mutational scanning involves creating libraries of pseudoviruses encoding every possible HA mutation, each linked to a unique genetic barcode for sequencing and identification. The team used this approach on the HA protein of a WHO-designated H5N1 candidate vaccine strain. This approach uses pseudoviruses that are not human pathogens and so can safely be studied at biosafety-level-2.These libraries were then tested for their ability to infect human 293T cells, which express both α2-3 and α2-6 sialic acid receptors. The results revealed significant variation in how HA mutations affect viral entry, with some HA regions tolerating mutations while others are highly constrained. Interestingly, mutations in the antigenic (surface-exposed) regions of HA that were intolerant to change may serve as promising targets for antibody-based therapies. The researchers also identified mutations that shifted receptor preference from avian-type to human-type receptors, validating their system by confirming mutations previously associated with increased transmissibility in past influenza pandemics.
“Mutations that increase HA stability are linked to higher transmissibility in influenza viruses,” the authors noted. By subjecting pseudoviruses to acidic conditions, mimicking the environment of human airways, the team identified stabilizing mutations that improve viral fitness. These findings could aid the design of more stable HA-based vaccines.
The team also explored HA mutations that allow escape from antibody neutralization, a critical concern for vaccine design. Using polyclonal sera from vaccinated or infected mice and ferrets, they pinpointed mutations in the HA head region that reduced neutralization. These escape mutations highlight the need to regularly update candidate vaccine strains to ensure effectiveness against evolving.
“We hope that this data can help in identifying avian influenza strains that may have phenotypic changes relevant to emergence of new pandemic variants,” explained Dr. Bernadeta Dadonaite, a staff scientist in the Bloom lab and leading author of the study. “We are now using H5 HA deep mutational scanning to better understand polyclonal responses to this virus infection which could be useful for better vaccine design,” Bernadeta added.
Using deep mutational scanning provides a rapid way to evaluate newly observed viral mutations and prioritized them for further study. This information could help identify avian flu strains with pandemic potential or guide the development of better vaccines. The Bloom lab’s work is helpful for pandemic preparedness, offering tools to monitor and respond to the ever-changing threat of influenza.
The spotlighted research was supported by grants from the National Institute of Health, Open Philanthropy, the Biotechnology and Biological Sciences Research, the UK Medical Research Council/Department for Environment, Food and Rural Affairs FluTrailMap-One Health consortium, the UK Medical Research Council and the Howard Hughes Medical Institute.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium members Dr. Jesse Bloom contributed to this work.
Dadonaite B, Ahn JJ, Ort JT, Yu J, Furey C, Dosey A, Hannon WW, Vincent Baker AL, Webby RJ, King NP, Liu Y, Hensley SE, Peacock TP, Moncla LH, Bloom JD. Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance. PLoS Biol. 2024
Joss Landazuri is a PhD candidate at the University of Washington in the Microbiology program working at the intersection of biomedical science, public policy, and science diplomacy. As a Latina scientist, communicator, and policy advocate, she is passionate about leveraging her academic training, personal background, and cultural heritage to engage underserved communities in both science and the policymaking process.
For the Media