Abstract
BackgroundZika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Several ZIKV envelope (Env)-based vaccines have been developed recently. However, many commercially available ZIKV Env are based on the African lineage and produced in insect cells. Here, we sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications.MethodsWe designed various gene expression constructs to optimize the production of ZIKV using prM-Env and full or C-terminal truncations of Env; with or without a rat CD4 fusion partner to allow large-scale production of soluble protein in mammalian HEK293 cells. Protein expression was verified by mass spectrometry and western-blot with a pan-flavivirus antibody, a ZIKV Env monoclonal antibody and with immune sera from adenoviral (ChAdOx1) ZIKV Env-vaccinated mice. The resulting Env-CD4 was used as a coating reagent for immunoassay (ELISA) using both mouse and human seropositive sera.ResultsReplacement of the C-terminus transmembrane Env domain by a rat CD4 and addition of prM supported optimal expression and secretion of Env. Binding between the antigens and the antibodies was similar to binding when using commercially available ZIKV Env reagents. Furthermore, antibodies from ZIKV patients bound ZIKV Env-CD4 in ELISA assays, whereas sera from healthy blood donors yielded minimal OD background. The serological outcomes of this assay correlated also with ZIKV neutralisation capacity in vitro.ConclusionsResults obtained from this study indicate the potential of the Asian-lineage Zika Env-CD4 and Env proteins in ELISA assays to monitor humoral immune responses in upcoming clinical trials as well as a sero-diagnostic tool in ZIKV infection.
Highlights
Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics
ZIKV is classified into two lineages: African and Asian strains [2] but they share > 95% amino acid identity with a single serotype unlike the closely related flavivirus, dengue virus (DENV) that is composed of 4 different serotypes [3]
CD4 fusion tag increases the secretion of ZIKV Env proteins in mammalian cells We designed several expression plasmids for production of soluble Asian-lineage ZIKV Env (Fig. 1)
Summary
Zika virus (ZIKV) has become a global threat with immediate need for accurate diagnostics, efficacious vaccines and therapeutics. Many commercially available ZIKV Env are based on the African lineage and produced in insect cells. We sought to produce Asian-lineage ZIKV Env in mammalian cells for research and clinical applications. Zika virus (ZIKV) is an emerging arthropod-borne virus that belongs to the family Flaviviridae and the genus Flavivirus. It was first discovered in a sentinel rhesus monkey in the Zika forest of Uganda in 1947 [1]. ZIKV is classified into two lineages: African and Asian strains [2] but they share > 95% amino acid identity with a single serotype unlike the closely related flavivirus, dengue virus (DENV) that is composed of 4 different serotypes [3]. Most of ZIKV infections are asymptomatic or mild [10], but in some cases can lead to neurological complications including Guillain-Barré syndrome (GBS) in adults and microcephaly in foetuses [8, 9, 11]
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