Nucleocapsid protein-specific monoclonal antibodies protect mice against Crimean-Congo hemorrhagic fever virus. Nature communications Crimean-Congo hemorrhagic fever virus (CCHFV) is a WHO priority pathogen. Antibody-based medical countermeasures offer an important strategy to mitigate severe disease caused by CCHFV. Most efforts have focused on targeting the viral glycoproteins. However, glycoproteins are poorly conserved among viral strains. The CCHFV nucleocapsid protein (NP) is highly conserved between CCHFV strains. Here, we investigate the protective efficacy of a CCHFV monoclonal antibody targeting the NP. We find that an anti-NP monoclonal antibody (mAb-9D5) protected female mice against lethal CCHFV infection or resulted in a significant delay in mean time-to-death in mice that succumbed to disease compared to isotype control animals. Antibody protection is independent of Fc-receptor functionality and complement activity. The antibody bound NP from several CCHFV strains and exhibited robust cross-protection against the heterologous CCHFV strain Afg09-2990. Our work demonstrates that the NP is a viable target for antibody-based therapeutics, providing another direction for developing immunotherapeutics against CCHFV. 10.1038/s41467-024-46110-4

Purification of Crimean Congo Hemorrhagic Fever Virus (CCHFV) Nucleocapsid Protein Using Detergent Gradient and Free Thawing. Bio-protocol Protein aggregation remains a major challenge in the purification of recombinant proteins in both eukaryotic and prokaryotic expression systems. One such protein is the nucleocapsid protein of Crimean Congo Hemorrhagic fever virus (CCHFV), which has high aggregation tendency and rapidly precipitates upon purification by NiNTA chromatography. Using the detergent gradient purification approach reported here, the freshly purified protein by NiNTA chromatography was mixed with the dilution buffer containing a high detergent concentration, followed by overnight freezing at -80 °C. Thawing the resulting mixture at room temperature triggered the formation of a detergent concentration gradient containing the active protein in the low detergent concentration zone towards the top of the gradient. The inactive aggregates migrated to the high detergent concentration zone towards the bottom of the gradient. The method prevented further aggregation and retained the activity of the native protein despite numerous freeze-thaw cycles. This simple approach creates an appropriate microenvironment towards the top of the gradient for correctly folded proteins, and it prevents aggregation by rapidly separating the preformed early aggregates from the correctly folded protein in the mixture. This unique approach will be of potential importance for the biotechnological industry, as well as other fields of protein biochemistry that routinely purify recombinant proteins and face the challenges of protein aggregation. Graphical abstract. 10.21769/BioProtoc.4483