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While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in\n                      <jats:italic toggle=\"yes\">Escherichia coli<\/jats:italic>\n                      , and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      . Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (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 these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      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 proteins made in bacteria. 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\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      . We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized 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.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.01804-24","type":"journal-article","created":{"date-parts":[[2024,7,22]],"date-time":"2024-07-22T09:01:20Z","timestamp":1721638880000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":4,"title":["Exploiting the Affimer platform against influenza A virus"],"prefix":"10.1128","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8239-2738","authenticated-orcid":false,"given":"Oliver","family":"Debski-Antoniak","sequence":"first","affiliation":[{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alex","family":"Flynn","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David P.","family":"Klebl","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mois\u00e9s H.","family":"Rojas Rechy","sequence":"additional","affiliation":[{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christian","family":"Tiede","sequence":"additional","affiliation":[{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ian A.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Integrative Structural and Computational Biology, The Scripps Research Institute","place":["La Jolla, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen P.","family":"Muench","sequence":"additional","affiliation":[{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Darren","family":"Tomlinson","sequence":"additional","affiliation":[{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9084-2927","authenticated-orcid":false,"given":"Juan","family":"Fontana","sequence":"additional","affiliation":[{"name":"School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds","place":["Leeds, United Kingdom"]},{"name":"Astbury Centre for Structural and Molecular Biology, University of Leeds","place":["Leeds, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.3390\/v13112250"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms18081650"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.2147\/IDR.S105473"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.15167\/2421-4248\/jpmh2019.60.1.1205"},{"key":"e_1_3_4_6_2","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.16777"},{"key":"e_1_3_4_7_2","doi-asserted-by":"publisher","DOI":"10.3201\/eid1906.121478"},{"key":"e_1_3_4_8_2","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1177\/00333549101250S305","article-title":"Influenza: the once and future pandemic","volume":"125","author":"Taubenberger JK","year":"2010","unstructured":"Taubenberger JK, Morens DM. 2010. 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