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. 2018 Mar 26;12(3):e0006366.
doi: 10.1371/journal.pntd.0006366. eCollection 2018 Mar.

Sensitive and specific detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-Specific IgM and IgG antibodies in human sera using recombinant CCHFV nucleoprotein as antigen in μ-capture and IgG immune complex (IC) ELISA tests

Affiliations

Sensitive and specific detection of Crimean-Congo Hemorrhagic Fever Virus (CCHFV)-Specific IgM and IgG antibodies in human sera using recombinant CCHFV nucleoprotein as antigen in μ-capture and IgG immune complex (IC) ELISA tests

Petra Emmerich et al. PLoS Negl Trop Dis. .

Abstract

As the most widespread tick-borne arbovirus causing infections in numerous countries in Asia, Africa and Europe, Crimean-Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) was included in the WHO priority list of emerging pathogens needing urgent Research & Development attention. To ensure preparedness for potential future outbreak scenarios, reliable diagnostic tools for identification of acute cases as well as for performance of seroprevalence studies are necessary. Here, the CCHFV ortholog of the major bunyavirus antigen, the nucleoprotein (NP), was recombinantly expressed in E.coli, purified and directly labeled with horseradish peroxidase (HRP). Employing this antigen, two serological tests, a μ-capture ELISA for the detection of CCHFV-specific IgM antibodies (BLACKBOX CCHFV IgM) and an IgG immune complex (IC) ELISA for the detection of CCHFV-specific IgG antibodies (BLACKBOX CCHFV IgG), were developed. Test performance was evaluated and compared with both in-house gold standard testing by IgM/IgG indirect immunofluorescence (IIF) and commercially available ELISA tests (VectoCrimean-CHF-IgM/IgG, Vector-Best, Russia) using a serum panel comprising paired samples collected in Kosovo during the years 2013-2016 from 15 patients with an acute, RT-PCR-confirmed CCHFV infection, and 12 follow-up sera of the same patients collected approximately one year after having overcome the infection. Reliably detecting IgM antibodies in all acute phase sera collected later than day 4 after onset of symptoms, both IgM ELISAs displayed excellent diagnostic and analytical sensitivity (100%, 95% confidence interval (CI): 85.2%-100.0%). While both IgG ELISAs readily detected the high IgG titers present in convalescent patients approximately one year after having overcome the infection (sensitivity 100%, 95% CI: 73.5%-100.0%), the newly developed BLACKBOX CCHFV IgG ELISA was superior to the commercial IgG ELISA in detecting the rising IgG titers during the acute phase of the disease. While all samples collected between day 11 and 19 after onset of symptoms tested positive in both the in-house gold standard IIFT and the BLACKBOX CCHFV IgG ELISA (sensitivity 100%, 95% CI: 71.5%-100.0%), only 27% (95% CI: 6.0%-61.0%) of those samples were tested positive in the commercial IgG ELISA. No false positive signals were observed in either IgM/IgG ELISA when analyzing a priori CCHFV IgM/IgG negative serum samples from healthy blood donors, malaria patients and flavivirus infected patients as well as CCHFV IgM/IgG IIFT negative serum samples from healthy Kosovar blood donors (for BLACKBOX CCHFV IgM/IgG: n = 218, 100% specificity, 95% CI: 98.3%-100.0%, for VectoCrimean-CHF-IgM/IgG: n = 113, 100% specificity, 95% CI: 96.8%-100.0%).

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Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests: PE and HS are inventors of the IgG immune complex technology employing recombinant CD32 which is protected by European (EP2492689) and international (CN103460048, HK1192320, CA2823107, US2014080120) patents owned by the Bernhard Nocht Institute for Tropical Medicine (BNITM).

Figures

Fig 1
Fig 1. Prokaryotic expression and purification of CCHFV-NP.
(A) Primary structure of recombinant CCHFV-NP fusion protein encoded by the expression vector pOPINJ-CCHFV-NP (6xHis-GST-3C-CCHFV-NP). The full-length CCHFV-NP is N-terminally tagged with both a 6 x His-tag and GST. A 3C protease recognition site enables tag removal after purification. (B) Induction of protein expression by IPTG. Protein expression was induced by IPTG in E.coli pAPlacIQ cells transformed with pOPINJ-CCHFV-NP. Total bacterial lysates (pre: before induction, post: after induction) were analyzed by SDS-PAGE followed by Coomassie-staining. Arrow head: 6xHis-GST-3C-CCHFV-NP (calculated molecular weight: 82 kDa). (C) Ni-NTA affinity purification of 6xHis-GST-3C-CCHFV-NP under native conditions. E.coli pAPlacIQ harbouring 6xHis-GST-3C-CCHFV-NP were lyzed by lysozyme and sonification. After centrifugation, the soluble supernatant (SN) was applied to Ni-NTA agarose. Bound material was eluted by imidazole. FT: flow through, eluates: E1, E2, E3. Arrow head: 6xHis-GST-3C-CCHFV-NP (calculated molecular weight: 82 kDa). (D) Removal of 6xHis-GST tag by on-column 3C protease digest. Eluates from Ni-NTA affinity purification were pooled (In: Input) and applied to Glutathion HiCap matrix (FT: Flow-through). On-column cleavage was performed using recombinant, GST-tagged 3C-protease. E: Eluate from matrix (CCHFV-NP without tag, calculated molecular weight: 54 kDa), M: Proteins attached to the matrix after elution.
Fig 2
Fig 2. Detection range of BLACKBOX CCHFV IgM/IgG ELISAs.
(A) BLACKBOX CCHFV IgM ELISA. A CCHF patient serum (IIFT IgM titer 1: 640) was spiked into a negative human serum at different dilutions (1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512) and analyzed with the BLACKBOX CCHFV IgM ELISA. In parallel, 71 CCHFV IgM negative sera from healthy human blood donors (n = 49) and malaria infected (n = 22) patients were tested (mean optical density of signals obtained for CCHFV IgM negative sera: 0.032, standard deviation: 0.011). (B) BLACKBOX CCHFV IgG ELISA. A CCHF patient serum (IIFT IgG titer 1: 10240) was spiked into a negative human serum at different dilutions (1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400) and analyzed with the BLACKBOX CCHFV IgG ELISA. In parallel, 71 CCHFV IgG negative sera from healthy human blood donors (n = 49) and malaria infected (n = 22) patients were tested (mean optical density of signals obtained for CCHFV IgG negative sera: 0.072, standard deviation: 0.017).
Fig 3
Fig 3. Analysis of paired CCHF patient samples: Raw data (OD420 – OD620).
A serum panel consisting of 30 paired serum samples from 15 CCHF patients, serum samples from 12 CCHF patients collected approximately one year after recovery from CCHFV infection, a set of a priori CCHFV IgM/IgG negative serum samples (neg) ((A), (B): n = 120; (C), (D): n = 15, see S1 Fig), and 98 CCHFV IgM/IgG IIFT negative sera from healthy blood donors from Kosovo (HD K) were analyzed with the BLACKBOX CCHFV IgM ELISA (A), the BLACKBOX CCHFV IgG ELISA (B), the VectoCrimean-CHF-IgM ELISA (C) and the VectoCrimean-CHF-IgG ELISA (D). Cut-off values (represented by dotted lines) were determined by ROC analysis ((A): 0.129, (B): 0.161) or according to the manufacturer’s instructions ((C): 0.240, (D): 0.246), respectively (see S2 Fig). Filled circles indicate PCR positive serum samples.
Fig 4
Fig 4. Analysis of paired CCHF patient samples: IgG/IgM ratio.
Ratios of OD450 – OD620 values obtained with the BLACKBOX CCHFV IgG/IgM ELISA (A) and the VectoCrimean-CHF-IgG/IgM ELISA (B) were calculated for the analyzed serum samples. Filled circles indicate PCR positive serum samples. Statistical testing (One-way ANOVA/Tukey’s multiple comparison test) was performed with GraphPad Prism (ns: not significant (p > 0.05); **: p < 0.01; ***: p < 0.001; ****: p < 0.0001).
Fig 5
Fig 5. Analysis of CCHF patient samples: Raw data (OD450 – OD620), dependence from day after onset.
(A) BLACKBOX CCHFV IgM ELISA. (B) BLACKBOX CCHFV IgG ELISA. (C) VectoCrimean-CHF-IgM ELISA. (D) VectoCrimean-CHF-IgG ELISA. Paired serum samples from 15 CCHF patients were classified according to their time point of collection (days after onset of symptoms). Filled circles indicate PCR positive serum samples. Cut-off values (represented by dotted lines) were determined by ROC analysis ((A): 0.129, (B): 0.161) or according to the manufacturer’s instructions ((C): 0.240, (D): 0.246), respectively (see S2 Fig).
Fig 6
Fig 6. Analysis of CCHF patient samples: Raw data (OD450 – OD620), correlation with IIFT titer.
(A) BLACKBOX CCHFV IgM ELISA. (B) BLACKBOX CCHFV IgG ELISA. (C) VectoCrimean-CHF-IgM ELISA. (D) VectoCrimean-CHF-IgG ELISA. 42 serum samples from 15 CCHF patients were classified according to their CCHFV IgM (A, C) and IgG IIFT titer (B, D), respectively. IIFT was performed using acetone-fixed Vero cells infected with CCHFV. Cut-off values (represented by dotted lines) were determined by ROC analysis ((A): 0.129, (B): 0.161) or according to the manufacturer’s instructions ((C): 0.240, (D): 0.246), respectively (see S2 Fig). Time of sampling (days after onset of symptoms) is indicated by the assigned symbols.

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