Optimization of Zika virus envelope protein production for ELISA and correlation of antibody titers with virus neutralization in Mexican patients from an arbovirus endemic region

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.Electronic supplementary materialThe online version of this article (10.1186/s12985-018-1104-6) contains supplementary material, which is available to authorized users.

Assessment of Immunogenicity and Efficacy of a Zika Vaccine Using Modified Vaccinia Ankara Virus as Carriers

“…To test the expression of ZIKV antigens, BHK-21 cells were transfected with rMVA. At 24 h after transfection, BHK-21 cell extracts were tested by Western blot as previously described [19]. Briefly, samples added to Laemmli sample buffer containing 20 mM β-mercaptoethanol, boiled for 5 min, and loaded on a Mini-PROTEAN TGX protein gel with Bio-Rad, Precision Plus Protein TM WesternC TM standards.…”

“…Enzyme-linked immunosorbent assay (ELISA)-recombinant ZIKV Envelope protein was used to measure anti-ZIKV envelope antibody concentrations in vaccinated mice by ELISA as previously described [19]. Briefly, Nunc Maxisorp Immuno ELISA plates were coated with Zika virus envelope antigen (Env-CD4) diluted in PBS to a final concentration of 2 µg/mL and left at room temperature (RT )overnight.…”

“…Serum samples were collected at 4 weeks and 12 weeks post immunisation. (a) Antibody responses elicited by MVA-ZIKV vaccine candidates at 4 weeks and (b) 12 weeks post immunisation were quantified by ELISA in plates coated with a ZIKV E protein[19]. (c) Cellular immune responses to MVA-ZIKV vaccine candidates.…”

Immunogenicity and Efficacy of Zika Virus Envelope Domain III in DNA, Protein, and ChAdOx1 Adenoviral-Vectored Vaccines

“…For ELISA in BALB/c mice before challenge, antibody endpoint titres are the highest reciprocal serum dilution that resulted in an absorbance >2-fold over the background values, as calculate elsewhere [36]. Anti-Zika envelope antibody concentrations in A129 mice sera before challenge was measured by IgG ELISA using Zika Env antigen as previously described [48].…”

A Zika vaccine based on chimpanzee adenovirus ChAdOx1 elicits lineage-transcending sterile immunity and prevents colonisation of brain and ovaries

“…Interestingly, three of the vaccine candidates provided 100% protection: the Asian-lineage based vaccines ChAdOx1 prME and ChAdOx1 prME ∆ TM, as well as the African-lineage based vaccine ChAdOx1 CprME/NS. We sought to analyse the levels of anti-ZIKV envelope antibodies at 1 dbc by using ELISA plates coated ZIKV AS envelope 23 (Figure 3b). Sera from control groups were negative and Log10 reciprocal titers showed that all ChAdOx1 ZIKV vaccines elicited anti-envelope responses.…”