The Role of Each Monomer in the Hemagglutinin Trimer of Influenza A Virus

Abstract

Influenza A virus (IAV) is able to enter the host cell trough receptor binding of hemagglutinin (HA) trimer to glycans with either α2,3- or α2,6-linked sialic acid (SA; avian-like or human-like receptor, respectively). IAV employs a multivalent system to enhance binding efficacy. By elucidating the multivalency of IAV, more efficient vaccines or high affinity inhibitors can be developed. This study aims to investigate the impact of each monomer on the binding of the HA trimer to its receptor, contributing to the understanding of the multivalent system employed by IAV. To accomplish this, we utilized the Triple Tandem Trimer design proposed by del Moral-Sánchez et al., 2024 , in which three HA proteins are linked and, after folding, form a trimer. We then introduced PR8e (avian-like HA), PR8d (human-like HA), or PR8Y98F (a nonbinding HA) into the trimer. This makes it possible to create a trimer with combinations of different HA’s (PR8EEE, PR8DEE, PR8DED, PR8DDD, PR8Y98FEE, PR8DY98FD). These proteins will be tested on their binding to its receptor through various assays, such as hemagglutination assay, ELISA, and FACS. Prior to this, the folding of these proteins is assessed using an ELISA with antibodies that target either the head or the stalk of the HA trimer. This revealed a structural similarity between the proteins and the controls PR8e and PR8d. The results of the binding assays suggest that incorporating either PR8e or PR8d into the HA trimer leads to a gradual shift in specificity. While each monomer contributes to the specificity and affinity of the trimer, modifying a monomer to a different specificity still enables it to contribute to binding, though to a reduced extent. Conversely, introducing a nonbinding HA into the trimer noticeably diminishes the binding affinity, though not completely.