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Please see the preprint for additional details: doi.org/10.64898/202... Thanks to @bdadonaite.bsky.social for leading study, and Annie Dosey, @jahn0.bsky.social, @timyu.bsky.social, Sara Sunshine, Ariana Farrell, and @kinglabipd.bsky.social for valuable contributions.

Defining the molecular interaction between influenza hemagglutinin and MHC-IIThe hemagglutinin (HA) of some influenza viruses can interact with major histocompatibility complex class II (MHC-II), but how these proteins interact is unclear. Here we demonstrate that diverse H5 HAs can use MHC-II to enter cells, with avian MHC-II enabling more efficient entry than human MHC-II for most H5 HAs. To define the molecular interface, we use pseudovirus deep mutational scanning to measure how mutations to H5 HA affect its interaction with tufted duck MHC-II, and identify mutations that restrict HA to exclusively MHC-II or sialic acid receptors. We leverage identification of H5 HA mutations that increase binding to tufted duck MHC-II to determine a 4.8 Å cryo-EM model of the complex. To support the structural model, we measure how all mutations to tufted duck MHC-II affect its interaction with H5 HA, and find the alpha chain is the dominant determinant but beta chain sites near the peptide-binding groove also contribute. To generalize these findings, we use deep mutational scanning to show that a H7 HA interacts with MHC-II similarly to H5 HA. Finally, we show that H1, H2, H3, and H9 HAs interact with avian or human MHC-II, although interactions vary among strains that evolved in different hosts. ### Competing Interest Statement J.D.B. and B.D. are inventors on Fred Hutch licensed patents related to the pseudovirus deep mutational scanning and a provisional patent on MHC-II binding deficient HA vaccine antigens. J.D.B consults for Apriori Bio, GSK, Merck, and Pfizer. J.D.B. holds stock options in the Vaccine Company. N.P.K. is a paid consultant of AstraZeneca. National Institute of Allergy and Infectious Diseases, 75N93021C00015, U19AI181881 National Cancer Institute, P30CA015704 Washington Research Foundation, https://ror.org/00hasdx88, postdoctoral fellowship to Sara Sunshine Howard Hughes Medical Institute, https://ror.org/006w34k90doi.org

Note our study used pseudoviruses and conditionally replicative virions to ensure biosafety, and reports deep mutational scanning only for HA usage of tufted duck MHC-II to limit any information hazard concerns.

All our data is available along with rich set of interactive plots: dms-vep.org/Flu-H5N1-Ame...

Although breadth of MHC-II usage among HAs suggests evolutionary selection in some strains/hosts, further work needed to understand biological relevance. Hypotheses include that it could impact cell entry or immunogenicity in actual infection of some hosts.

We also showed H7 HA binds tufted duck MHC-II similarly to H5 HA, & some H1, H2, H3, & H9 HAs also can use avian or human MHC-II. But patterns vary among strains. For instance, an avian influenza HA and the 1918 HA can use tufted duck MHC-II, but later human strains cannot.

Structure & deep mutational scanning suggest identity of peptide bound to MHC-II could influence interaction of HA & MHC-II. Also, HA binding would likely block ability of MHC-II to interact with T-cell (perhaps analogous to how EBV gp42 can bind to MHC-II to block T cell activation).

So although structure only 4.8 A, it is corroborated by deep mutational scanning of both HA and MHC-II showing that sites in both proteins that affect binding are at structural interface. (Sites where mutations decrease binding are red in structure below)

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To validate structure, we used inverted pseudotyping deep mutational scanning to measure how mutations to tufted duck MHC-II affect binding to H5 HA. Most mutations with big impact in alpha chain, but beta-chain mutations near peptide-binding groove also have effect.

To determine structure, we produced H5 HA protein w mutations that increased binding to tufted duck MHC-II. This HA increased fraction of particles bound to MHC-II in ns-EM, and we were able to use it solve cryo-EM structure of H5 HA bound to tufted duck MHC-II.

The mutations that reduced MHC-II entry clustered in a region on HA head defining the MHC-II binding surface. We also directly measured how HA mutations affect binding to tufted duck MHC-II, and identified same binding surface.

To understand how HA interacts w MHC-II, we measured how all H5 HA mutations affect pseudovirus entry via sialic acid or tufted duck MHC-II. Identified loss-of-function mutants that could only use MHC-II or sialic acid.

We tested 80 H5 HAs: most but not all could enter cells via tufted duck & to lesser extent human MHC-II. Note MHC-II highly variable within and between species. See dms-vep.org/Flu-H5N1-Ame... for interactive version of below plot.

We first measured ability of two H5 HAs to enter cells via sialic acid or MHC-II. As shown below, both HAs could use tufted duck & to lesser extent human MHC-II. [Note: experiments used pseudoviruses, which can only undergo single round of cell entry, providing safe way to study HA]

As background, influenza long known to use sialic acid as entry receptor. ~7 yrs ago was shown bat flu can enter cells via MHC-II (www.nature.com/articles/s41...); later shown for some H2 & H3 strains. We set out to assess how common MHC-II usage is & define how HA interacts w MHC-II.

MHC class II proteins mediate cross-species entry of bat influenza viruses - NatureThe DR isotype of the human leukocyte antigen of the MHC class II—or its homologues in bats, pigs, mice and chickens—is an essential cell entry determinant for bat influenza A viruses.www.nature.com

See the full paper for additional details and analyses: www.pnas.org/doi/10.1073/... Thanks to Brendan Larsen for leading study & our collaborators in @veeslerlab.bsky.social lab.

We also defined how F mutations affect neutralization by a panel of monoclonal antibodies. This allowed us to quantify the resilience of different antibodies to escape, and predict which antibodies also neutralize the related Hendra virus.

A strategy for vaccines is to stabilize F in pre-fusion conformation. We identified sites where mutations to proline (which blocks helix formation) are disfavored. This identifies new candidate mutations for stabilizing F vaccine immunogens.

For this study, we used pseudoviruses that can only undergo a single round of cell entry (& so are not human pathogens) to measure how mutations to F affect its fusion function. We found F is more functionally constrained than the other Nipah surface protein, RBP.

The final version of this article has been published in @natecoevo.nature.com alongside a nice News and Views by @seth-zost.bsky.social: www.nature.com/articles/s41...

Learning a viral protein’s vocabulary - Nature Ecology & EvolutionA powerful technique for probing the effects of amino acid substitutions on protein function sheds light on the evolutionary constraints of a rapidly evolving influenza virus glycoprotein.www.nature.com

The final version of record of this study has been published in @elife.bsky.social alongside a nice insight perspective: elifesciences.org/articles/110...

Virus Evolution: Scaling up efforts to target evolving virusesHigh-throughput neutralisation tests could lead to a better understanding of the evolution of human influenza.elifesciences.org