Maria Rios Carrasco

Influenza A virus continues to pose a significant threat to global public health. Annually, the virus is estimated to cause approximately one billion infections, millions of which lead to severe illness and hundreds of thousands to death from respiratory complications. In addition to seasonal H1N1 and H3N2 viruses, highly pathogenic avian influenza viruses, such as H5Nx viruses, have spread widely in recent years. Specifically, clade 2.3.4.4b H5N1 viruses are increasingly detected in various mammalian species. This development has renewed concerns about further adaptation to mammalian hosts and the pandemic potential of these viruses.

This thesis focuses on the interactions between the influenza virus and sugar molecules (glycans) on the surface of host cells. These interactions are mediated by the viral hemagglutinin (HA) protein, which is responsible for the first step in the infection process: binding to cell receptors. Traditionally, avian influenza viruses are thought to primarily bind to α2,3-linked sialic acids, while human viruses prefer α2,6-linked sialic acids. However, increasingly more research shows that this receptor recognition is much more complex and is also determined by factors such as glycan branching and length, chemical modifications, multivalent presentation of HA, and receptor density on the cell surface.

In Chapter 2, the glycan distribution in the bovine mammary gland was investigated in relation to the binding of circulating H5N1 viruses from clade 2.3.4.4b. Using a panel of specific glycan-binding probes, the presence of various sialic acid variants was mapped. It was confirmed that the mammary gland contains both α2,3- and α2,6-linked sialic acids, but these do not necessarily correspond to human-type receptors. This supports the observation that efficient transmission to humans is unlikely. Furthermore, no relevant binding to the bovine respiratory epithelium was demonstrated, suggesting that the mammary gland may represent an alternative route for virus transmission.

In Chapter 3, the effect of the Q226L mutation, a known change in hemagglutinin associated with a shift from avian to human receptor binding, was investigated across three different H5 antigenic backgrounds. Depending on the context, this mutation led to three different phenotypes: loss of binding, dual binding to both α2,3- and α2,6-sialic acids, or a complete switch to α2,6-binding. In one of the backgrounds studied, the mutation even resulted in a complete switch from binding to chicken tissue to human respiratory tracts. These results underscore that the impact of adaptive mutations is highly dependent on the viral background.

In Chapter 4, the same Q226L mutation was studied in a bovine-origin H5 virus within clade 2.3.4.4b (H5TX). Although a shift towards α2,6-binding was also observed here, this was limited to complex and highly branched glycans that are only sparsely present in human respiratory tracts. The mutation alone was therefore insufficient to achieve efficient binding to human respiratory epithelium. Additionally, it was shown that the presence of a multibasic cleavage site in hemagglutinin can influence receptor binding, in a manner dependent on the viral background.

In Chapter 5, the role of multivalent presentation of hemagglutinin was investigated using the self-assembling nanoparticle platform I53-50. Here, HA trimers were compared in monomeric form, clustered via antibodies, and presented on nanoparticles. The results show that binding strength increases with higher HA presentation valency. By systematically varying the composition of functional and non-functional trimers on the nanoparticles, different binding mechanisms for linear and branched glycans could also be distinguished.

Taken together, the results of this thesis demonstrate that receptor recognition by influenza viruses is significantly more complex than the classical dichotomy into α2,3- and α2,6-linked sialic acids. Not only the type of receptor is critical, but also the spatial organization and presentation of glycans play a crucial role in virus binding. These insights contribute to a better understanding of the molecular determinants of host specificity and viral adaptation, and thus provide relevant knowledge for assessing the pandemic potential of emerging influenza viruses.