Plant-produced Crimean-Congo haemorrhagic fever virus nucleoprotein for use in indirect ELISA

• Published in: Journal of virological methods 2016-10, Vol.236, p.170-177
• Main Authors: Atkinson, Richard, Burt, Felicity, Rybicki, Edward P., Meyers, Ann E.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Viral - blood; Antigens, Viral - genetics; Antigens, Viral - immunology; Antigens, Viral - isolation & purification; Antigens, Viral - metabolism; Crimean-Congo haemorrhagic fever; Enzyme-Linked Immunosorbent Assay - methods; Hemorrhagic Fever, Crimean - diagnosis; Humans; Immunoglobulin G - blood; Mice; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana benthamiana; Nucleoprotein; Nucleoproteins - genetics; Nucleoproteins - immunology; Nucleoproteins - isolation & purification; Nucleoproteins - metabolism; Plants, Genetically Modified; Recombinant antigen; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Serologic Tests - methods
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2016.07.025

•CCHFV NP protein was successfully expressed in plants.•Plant-produced CCHFV NP production was successfully scaled up and purified.•CCHFV NP detected anti-CCHFV IgG in sera from convalescent patients by ELISA.•CCHFV NP distinguished between sera from infected and uninfected individuals. Crimean-Congo haemorrhagic fever (CCHF) is a disease of serious public concern caused by the CCHF virus (CCHFV). Anti-CCHFV IgG in humans can be detected using ELISA with native antigen prepared from cell cultures which have been infected with virus or from brain tissue of suckling mice which have been inoculated with virus. However, the preparation of these reagents requires high biosafety levels and is expensive. A safer, more cost-effective recombinantly-produced NP reagent is desirable. Recently, plants have been shown to be a cost-effective and safe system for expression of recombinant proteins. This work describes cloning of the CCHFV NP gene into three different plant expression systems and comparison of expression in Nicotiana benthamiana. The highest expressing construct was selected. Expressed NP was purified by ammonium sulphate fractionation prior to histidine affinity chromatography. Purified NP was tested in an indirect ELISA to determine if the recombinant antigen was able to detect anti-CCHFV IgG in sera from convalescent patients. Plant-produced NP detected IgG antibodies against CCHFV in 13/13 serum samples from convalescent patients and 0/13 samples collected from volunteers with no history of CCHFV infection. Results were compared with commercially available immunofluorescent assays and 100% concordance was obtained between the two assays. This suggests that a full evaluation of the plant produced NP for application as a safe recombinant is warranted.