Characterization of two engineered dimeric Zika virus envelope proteins as immunogens for neutralizing antibody selection and vaccine design

The envelope protein of Zika virus (ZIKV) exists as a dimer on the mature viral surface and is an attractive antiviral target because it mediates viral entry. However, recombinant soluble wild-type ZIKV envelope (wtZE) might preferentially exist as monomer (monZE). Recently, it has been shown that the A264C substitution could promote formation of dimeric ZIKV envelope protein (ZE(A264C)), requiring further characterization of purified ZE(A264C) for its potential applications in vaccine development. We also noted that ZE(A264C), connected by disulfide bond, might be different from the noncovalent native envelope dimer on the virion surface. Because the antibody Fc fragment exists as dimer and is widely used for fusion protein construction, here we fused wtZE to human immunoglobulin G1 (IgG1) Fc fragment (ZE-Fc) for noncovalent wtZE dimerization. Using a multistep purification procedure, we separated dimeric ZE(A264C) and ZE-Fc, revealing that they both exhibit typical beta-sheet-rich secondary structures and stabilities similar to those of monZE. The binding activities of monZE, ZE(A264C), and ZE-Fc to neutralizing antibodies targeting different epitopes indicated that ZE(A264C) and ZE-Fc could better mimic the native dimeric status, especially in terms of the formation of tertiary and quaternary epitopes. Both ZE(A264C) and ZE-Fc recognize a ZIKV-sensitive cell line as does monZE, indicating that the two constructs are still functional. Furthermore, a murine immunization assay disclose that ZE(A264C) and ZE-Fc elicit more neutralizing antibody responses than monZE does. These results suggest that the two immunogen candidates ZE(A264C) and ZE-Fc have potential utility for neutralizing antibody selection and vaccine design against ZIKV.