Citation
- Authors: Durie IA. et al.
- Year: 2022
- Journal: Nat Commun 13 7298
- Applications: in vitro / DNA / FectoPRO
- Cell type: Expi293F
Description: Human embryonic kidney Fibroblast
Known as: Expi 293-F, Expi, HEK-293 Expi
Method
Cchfv Gp38 Production:
- Expi293 cells (Thermo Fischer) were maintained in Expi293 Media (Thermo Fischer) based on their specifications. Expi293 were transfected with FectoPro reagent (Polyplus). CCHFV GP38 expression plasmids were co-transfected with pCDNA3.1-human furin plasmid, generously provided by N.G. Seidah (Clinical Research Institute of Montreal), at a ratio of 4:1 in Expi293 cells.
- Four hours after transfection, kifunesine was added to a final concentration of 5µM to inhibit the processing of complex glycans, which could interfere in the crystallization process.
- The cell supernatant was harvested between 5–6 days then the cells and debris were removed by centrifugation and filtered through a 0.2 µm filter.
- The sodium chloride concentration of media was adjusted to 500 mM final using 5 M NaCl solution. Supernatants were then run over a Histrap-Excel nickel column (Cytiva) and eluted using 500mM Imidazole. The eluted proteins were digested overnight with HRV3C protease. The cleaved GP38 was purified by size exclusion chromatography on a Superdex 200 column.
Abstract
Crimean-Congo Hemorrhagic Fever Virus (CCHFV) causes a life-threatening disease with up to a 40% mortality rate. With no approved medical countermeasures, CCHFV is considered a public health priority agent. The non-neutralizing mouse monoclonal antibody (mAb) 13G8 targets CCHFV glycoprotein GP38 and protects mice from lethal CCHFV challenge when administered prophylactically or therapeutically. Here, we reveal the structures of GP38 bound with a human chimeric 13G8 mAb and a newly isolated CC5-17 mAb from a human survivor. These mAbs bind overlapping epitopes with a shifted angle. The broad-spectrum potential of c13G8 and CC5-17 and the practicality of using them against Aigai virus, a closely related nairovirus were examined. Binding studies demonstrate that the presence of non-conserved amino acids in Aigai virus corresponding region prevent CCHFV mAbs from binding Aigai virus GP38. This information, coupled with in vivo efficacy, paves the way for future mAb therapeutics effective against a wide swath of CCHFV strains.