Preparation of recombinant antigens for demonstrating antibody responses in patients with Crimean-Congo haemorrhagic fever virus infections
Samudzi, Rudo Ruth
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Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne viral zoonosis widely distributed in Africa, Asia, Russia and the Balkans. The causative agent, CCHF virus (CCHFV) has the propensity to cause nosocomial infections with a high fatality rate. Cases of CCHF are diagnosed annually in southern Africa. Increasing numbers of cases are seen in regions of Asia and in the past ten years CCHFV has emerged in several countries in the Balkans and re-emergence in south-western regions of the Russian Federation. Diagnosis of CCHFV infections during the acute phase is based on isolation of the virus or amplification of viral RNA. Patients that survive the infection have a demonstrable IgG and IgM antibody response, usually from day 5 to 7 after onset of illness. Current serological diagnostic assays based on ELISA or IF use inactivated virus which requires biosafety level 4 facilities for culturing the virus and therefore limits the number of laboratories that can prepare suitable reagents. Preparation of recombinant antigens would enable laboratories to perform serological diagnosis of CCHFV infections and surveillance studies. The purpose of this study was to prepare a recombinant CCHFV nucleoprotein using a bacterial expression system, to determine if the protein was immunogenic and to determine if the protein was able to detect IgG antibodies in survivors of CCHFV infection. The complete open reading frame of the gene encoding the NP of CCHFV was amplified by RT-PCR using primers specifically designed with restriction sites engineered to the primers to facilitate cloning. The amplicon was cloned into pGEM® T Easy vector using T/A cloning and the gene sequenced to confirm that the correct gene had been amplified and cloned into the vector for downstream cloning and expression applications. Initially we aimed to express the native gene using a bacterial expression system and the NP gene was rescued from the recombinant plasmid and cloned into pQE-80L vector using the BamH1 and Pst1 restriction sites present in the multiple cloning site on the vector. Various attempts were made to express the CCHFV NP protein however no protein was detectable using SDS PAGE methods or Western blot. The nucleotide sequence that we had determined for the open reading frame of our gene encoding the NP was analysed using the Rare Codon Analysis Tool software and we elected to codon optimize the gene for expression in E. coli. The optimized gene was synthesized by GenScript and supplied cloned in the multiple cloning site of pUC57. The optimized gene was excised from pUC57 and cloned into pColdTF bacterial expression vector. A 106 kDa protein was expressed from the construct likely representing the HIS tagged TF chaperone protein fused to the CCHFV NP protein and confirmed by Western blot analysis. A higher yield of the protein was present in the insoluble phase and as optimization of the growth and induction conditions did not significantly alter the insoluble to soluble ratio of the expressed protein, the protein was harvested from the insoluble phase by denaturing, purification and refolding of the protein. The biological activity of the recombinant protein was confirmed using immunoassays and by immunizing mice to determine if the antibodies induced by the recombinant protein could be detected using an antigen prepared from the whole virus. Four of five mice immunized with the recombinant NP had a detectable antibody response using an immunofluorescent assay. Serum samples from acute and convalescent patients collected at varying stages after onset of illness were reacted in a Western blot with the recombinant CCHFV NP protein. The recombinant antigen was able to detect IgG antibody in all the convalescent patient sera except two sera collected on days 14 and 15 during the acute phase. In contrast all the samples were detected using the recombinant antigen in an ELISA. Due to the potential biohazardous nature of samples only samples collected two weeks after onset of illness were tested. The results showed 100% concordance with the results obtained in an ELISA using mouse brain derived antigen. The assay was shown to be reproducible and stability studies showed that four months after preparation the protein was still active. A full validation of the protein using a large panel of serum samples from confirmed CCHF patients is now required. The results suggest that bacterially expressed proteins lacking post translational modifications and folding that occur with mammalian and baculovirus expression can be used in ELISA to detect IgG antibody against CCHFV in human sera which finds application in diagnostics, epidemiologic and surveillance studies.