Specific detection of dengue and Zika virus antibodies using envelope proteins with mutations in the conserved fusion loop

Abstract

Detection of antibodies is widely used for the diagnosis of infections with arthropod-borne flaviviruses including dengue (DENV) and Zika virus (ZIKV). Due to the emergence of ZIKV in areas endemic for DENV, massive co-circulation is observed and methods to specifically diagnose these infections and differentiate them from each other are mandatory. However, serological assays for flaviviruses in general, and for DENV and ZIKV in particular, are compromised by the high degree of similarities in their proteins which can lead to cross-reacting antibodies and false-positive test results. Cross-reacting flavivirus antibodies mainly target the highly conserved fusion loop (FL) domain in the viral envelope (E-) protein, and we and others have shown previously that recombinant E-proteins bearing FL-mutations strongly reduce cross-reactivity. Here we investigate whether such mutant E-proteins can be used to specifically detect antibodies against DENV and ZIKV in an ELISA-format. IgM antibodies against DENV and ZIKV virus were detected with 100% and 94.2% specificity and 90.7% and 87.5% sensitivity, respectively. For IgG the mutant E-proteins showed cross-reactivity, which was overcome by pre-incubation of the sera with the heterologous antigen. This resulted in specificities of 97.1% and 97.9% and in sensitivities of 100% and 100% for the DENV and ZIKV antigens, respectively. Our results suggest that E-proteins bearing mutations in the FL-domain have a high potential for the development of serological DENV and ZIKV tests with high specificity.

Specific detection and differentiation of tick‐borne encephalitis and West Nile virus induced IgG antibodies in humans and horses

“…For the serological differentiation of WNV and TBEV IgG positive sera we conducted indirect ELISA measurements with cross‐reactivity reduced recombinant envelope (Equad) proteins bearing four amino acid mutations in and near the conserved fusion‐loop domain that have been previously characterized for WNV, DENV and ZIKV (Chabierski et al, ; Rockstroh et al, , ).…”

“…The assay described here uses mutant forms of insect‐cell derived flavivirus E‐proteins, a technique that has been shown to enable specific diagnosis of DENV and ZIKV infections (Rockstroh et al, , ). In addition, a bacterially expressed Equad protein was shown to reduce cross‐reactivities in human WNV diagnosis, but the wild‐type protein had to be taken along in order to yield specific results by determining the ratios between values obtained with the wild‐type and the Equad version (Chabierski et al, ).…”

“…Most cross‐reacting antibodies target the highly conserved fusion‐loop (FL‐) domain within the flaviviral envelope (E‐) protein, a major structural protein. We and others have previously shown that the insertion of mutations in the FL‐domain leads to a strong reduction in cross‐reactive antibody binding (Chabierski et al, ; Crill & Chang, ; Roberson, Crill, & Chang, ; Rockstroh et al, , ). A bacterially expressed quadruple mutant E‐protein (Equad) of WNV may be used to discriminate human WNV infections from infections by other flaviviruses when comparing the antibody signals with those obtained by using the wild‐type protein (Chabierski et al, ).…”

Comprehensive Evaluation of Differential Serodiagnosis between Zika and Dengue Viral Infections

“…Use of the ZIKV/DENV ratio to differentiate between ZIKV and DENV infections. Previous studies suggested that a significant proportion of anti-E antibodies were GR antibodies that recognized the highly conserved fusion peptide during flavivirus infection (26,33). To avoid the binding of such GR antibodies on the ZIKV VLP, a fusion peptide mutant (GL106/107KD) ZIKV VLP (ZIKV FP-VLP) was generated for MAC-ELISA, and the proper cutoff value was determined similarly (see Fig.…”

Comprehensive evaluation of differential serodiagnosis between Zika and dengue viral infection

“…Previous studies suggested that a significant proportion of anti-E antibodies were GR antibodies that recognized the highly conserved fusion peptide during flavi virus infection. 24, 28 To avoid the binding of such GR antibodies on the ZIKV VLP, a fusion peptide mutant ZIKV VLP (ZIKV FP-VLP) was generated for MAC-ELISA and the proper cutoff value was determined (Fig S1). A significant decrease of the cross-reactivity to ZIKV FP-VLP among the DENV serum panel was noticed; in addition, a similar positive proportion among the ZIKV serum panel was detected (Table S1).…”