PDF(Pubmed)
Abstract：
Influenza A virus (IAV) is well known for its pandemic potential. 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 Escherichia coli, 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 in vitro. 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.
OBJECTIVE: 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 in vitro. 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.
OBJECTIVE: 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 in vitro. 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.
摘要：
甲型流感病毒(IAV)以其大流行潜力而闻名。虽然目前的监测和疫苗接种策略非常有效，由于IAV的高突变率,治疗方法通常是短暂的.最近,单克隆抗体（mAb）已经成为一种有前途的治疗方法，既针对当前的毒株，也针对未来的IAV大流行。除了MABS，存在几种抗体样替代品，旨在提高单克隆抗体水平。其中,资助者因其短暂的发展时间而脱颖而出，在大肠杆菌中的高表达水平，和无动物生产。在这项研究中,我们利用Affimer平台来分离和生产特异性和有效的IAV抑制剂。使用IAV三聚体血凝素（HA）融合蛋白的单体版本，我们分离了12种体外抑制IAV感染的Affimer。这些Affimers中的两个进行了详细的表征，并表现出对靶H3HA蛋白的纳摩尔结合亲和力，特异性结合到HA1头部结构域。低温电子显微镜（cryo-EM），采用一种新颖的喷雾方法来制备低温网格，允许我们对HA-Affimer复合物进行成像。结合功能测定，我们确定这些Affimer通过阻断HA与宿主细胞受体的相互作用来抑制IAV，唾液酸。此外，这些Affimer抑制与用于分离的IAV菌株密切相关。总的来说,我们的结果支持使用Affimmer作为现有IAV靶向治疗的可行替代方案,并突出了其作为诊断试剂的潜力.
目的：甲型流感病毒是少数能引起毁灭性大流行的病毒之一。由于这种病毒的高突变率,每年需要接种疫苗，抗病毒药物是短暂的。单克隆抗体提供了解决流感病毒感染的有希望的方法，但存在一些局限性。为了改进这一战略，我们探索了Affimer平台，是细菌中产生的抗体样蛋白。通过对流感病毒融合蛋白的单体版本进行噬菌体展示，一个既定的病毒靶标，我们能够分离体外抑制流感病毒感染的Affimers.我们通过使用针对病毒复制周期的不同阶段的测定来表征Affimers的抑制机制。我们还对HA-Affimer复合物结构进行了表征，使用一种新颖的方法来制备低温电子显微镜样品。总的来说,这些结果表明Affimers是一种有前途的抗流感病毒感染的工具。
目的：甲型流感病毒是少数能引起毁灭性大流行的病毒之一。由于这种病毒的高突变率,每年需要接种疫苗，抗病毒药物是短暂的。单克隆抗体提供了解决流感病毒感染的有希望的方法，但存在一些局限性。为了改进这一战略，我们探索了Affimer平台，是细菌中产生的抗体样蛋白。通过对流感病毒融合蛋白的单体版本进行噬菌体展示，一个既定的病毒靶标，我们能够分离体外抑制流感病毒感染的Affimers.我们通过使用针对病毒复制周期的不同阶段的测定来表征Affimers的抑制机制。我们还对HA-Affimer复合物结构进行了表征，使用一种新颖的方法来制备低温电子显微镜样品。总的来说,这些结果表明Affimers是一种有前途的抗流感病毒感染的工具。
