Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance

Authors: DADONAITE, BERNADETA - Fred Hutchinson Cancer Research Center; AHN, JENNY - University Of Washington; ORT, JORDAN - University Of Pennsylvania; YU, JIN - Imperial College; FUREY, COLLEEN - University Of Pennsylvania; DOSEY, ANNIE - University Of Washington; HANNON, WILLIAM - Fred Hutchinson Cancer Research Center; Baker, Amy; WEBBY, RICHARD - St Jude Children’s Research Hospital; KING, NEIL - University Of Washington; LIU, YAN - Imperial College; HENSLEY, SCOTT - University Of Pennsylvania; PEACOCK, THOMAS - Imperial College; MONCLA, LOUISE - University Of Pennsylvania; BLOOM, JESSE - Howard Hughes Medical Institute

Submitted to: PLoS Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/29/2024
Publication Date: 11/12/2024
Citation: Dadonaite, B., Ahn, J.J., Ort, J.T., Yu, J., Furey, C., Dosey, A., Hannon, W.W., Baker, A.L., Webby, R.J., King, N.P., Liu, Y., Hensley, S.E., Peacock, T.P., Moncla, L.H., Bloom, J.D. 2024. Deep mutational scanning of H5 hemagglutinin to inform influenza virus surveillance. PLoS Biology. 22(11). Article 3002916. https://doi.org/10.1371/journal.pbio.3002916.
DOI: https://doi.org/10.1371/journal.pbio.3002916

Interpretive Summary: Highly pathogenic avian influenza (HPAI) H5N1 causes major losses for poultry and dairy farmers and is considered a potential pandemic threat. H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple nonhuman mammalian species in the United States. Changes in the viral H5 hemagglutinin (HA) protein are detected by sequencing viruses from animal hosts, which may also inform pandemic potential in humans. However, laboratory experiments to test all viruses with sequence changes is unachievable. Here, we used a technique called pseudovirus deep mutational scanning to measure how changes to a 2.3.4.4b H5 HA protein affected virus properties. We identified mutations that allow HA to better bind mammalian host receptors and showed that some viruses detected in nature already carry mutations that stabilize HA. We also measured how HA mutations affected binding of antibodies from animals with immunity against 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired changes in the H5 protein that may create mismatches with candidate vaccine viruses, and we showed that a change present in some recent H5 HAs from field cases caused protective antibodies to no longer bind. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enabled comprehensive measurements of virus properties due to changes in the H5 protein. This information can inform real-time interpretation of sequence changes observed during surveillance of H5 influenza for animal and human health preparedness measures.

Technical Abstract: 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 nonhuman 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 a2-6-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 virus, and we show that a mutation present in some recent H5 HAs causes a large antigenic change. 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.