
==== Front
bioRxiv
BIORXIV
bioRxiv
Cold Spring Harbor Laboratory

10.1101/2024.05.23.595634
preprint
1
Article
Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance
Dadonaite Bernadeta http://orcid.org/0000-0003-0908-6982

Ahn Jenny J http://orcid.org/0009-0000-3912-7162

Ort Jordan T
Yu Jin
Furey Colleen
Dosey Annie
Hannon William W
Baker Amy Vincent
Webby Richard J http://orcid.org/0000-0002-4397-7132

King Neil P
Liu Yan
Hensley Scott E
Peacock Thomas P http://orcid.org/0000-0001-7077-2928

Moncla Louise H http://orcid.org/0000-0001-5722-1988

Bloom Jesse D http://orcid.org/0000-0003-1267-3408

24 5 2024
2024.05.23.595634http://biorxiv.org/lookup/doi/10.1101/2024.05.23.595634
nihpp-2024.05.23.595634.pdf
H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple non-human mammalian species. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here we use pseudovirus deep mutational scanning to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids, and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine strains. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza.
==== Body
pmc
