Exploiting the Affimer platform against influenza A virus

Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are short-lived due to the high mutation rates of IAV. Currently, monoclonal antibodies (mAbs) have emerged as a promising approach to tackle future IAV pandemics. Additionally, several antibody-like alternatives exist that aim to improve upon mAbs. Affimers, one such alternative, benefit from a short development time, high expression levels in E. coli , and complete animal-free production. Here we exploited the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Starting from a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV H3 subtype infection in vitro . Two of these Affimers were characterised in detail: they exhibited binding affinities to the target H3 HA protein in the nM range and bound specifically to the HA1 head domain. Cryo-EM employing a novel spray approach to prepare cryo-grids allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that the mode of inhibition of these Affimers is based on blocking the interaction of HA to the host-cell receptor - sialic acid. Additionally, these Affimers inhibited IAV strains closely related to the one employed for Affimer isolation. Overall, these results support the use of Affimers as an alternative to existing targeted therapies for IAV and pave the way for their use as diagnostic reagents. Importance Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections, but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like, bacterially made proteins. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro . We characterised the mechanism of inhibition of the Affimers by challenging with related influenza virus strains. We additionally characterised an HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.