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Establishment of zoonotic viruses, causing pandemics like the Spanish flu and Covid-19, requires adaptation to human receptors. Pandemic influenza A viruses (IAV) that crossed the avian-human species barrier switched from binding avian-type α2-3-linked sialic acid (2-3Sia) to human-type 2-6Sia receptors. Here, we show that this specificity switch is however less dichotomous as generally assumed. Binding and entry specificity were compared using mixed synthetic glycan gradients of 2-3Sia and 2-6Sia and by employing a genetically remodeled Sia repertoire on the surface of a Sia-free cell line and on a sialoglycoprotein secreted from these cells. Expression of a range of (mixed) 2-3Sia and 2-6Sia densities shows that non-binding human-type receptors efficiently enhanced avian IAV binding and entry provided the presence of a low density of high affinity avian-type receptors, and vice versa. Considering the heterogeneity of sialoglycan receptors encountered in vivo, hetero-multivalent binding is physiologically relevant and will impact evolutionary pathways leading to host adaptation.

Influenza A viruses pose a serious pandemic risk, while generation of efficient vaccines against seasonal variants remains challenging. There is thus a pressing need for new treatment options. We report here a set of macrocyclic peptides that inhibit influenza A virus infection at low nanomolar concentrations by binding to hemagglutinin, selected using ultrahigh-Throughput screening of a diverse peptide library. The peptides are active against both H1 and H5 variants, with no detectable cytotoxicity. Despite the high sequence diversity across hits, all tested peptides were found to bind to the same region in the hemagglutinin stem by HDX-MS epitope mapping. A mutation in this region identified in an escape variant confirmed the binding site. This stands in contrast to the immunodominance of the head region for antibody binding and suggests that macrocyclic peptides from in vitro display may be well suited for finding new druggable sites not revealed by antibodies. Functional analysis indicates that these peptides stabilize the prefusion conformation of the protein and thereby prevent virus-cell fusion. High-Throughput screening of macrocyclic peptides is thus shown here to be a powerful method for the discovery of novel broadly acting viral fusion inhibitors with therapeutic potential.