Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 angstrom resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera. Author summary Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the most lethal human pathogens in existence. No approved vaccine or therapies exist and rapid diagnosis of CCHFV is a critical aspect of disease management. We describe the selection and characterization of a high affinity non-antibody binding protein, Affimer-NP, that specifically recognises the CCHFV nucleocapsid protein (NP). Affimer-NP interferes with the RNA-binding function of CCHFV NP and inhibits CCHFV gene expression in mammalian cells. Solution of the crystallographic structure of the CCHFV NP/Affimer-NP complex at 2.84 angstrom resolution revealed the structural basis for this interference, and we validated the in vitro application of this novel molecule for the development of ELISA and lateral flow assays, presenting the first published prototype point-of-care test able to detect recombinant CCHFV NP in spiked human and animal sera. These findings present a possible starting point for the future development of anti-viral molecules targeted to CCHFV NP, and diagnostic assays for the detection of CCHFV NP, contributing to the preparedness for potential future outbreak scenarios.