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. 2017 Jul 3;7(1):4524.
doi: 10.1038/s41598-017-04767-6.

In Vitro Assembly and Stabilization of Dengue and Zika Virus Envelope Protein Homo-Dimers

Affiliations

In Vitro Assembly and Stabilization of Dengue and Zika Virus Envelope Protein Homo-Dimers

Stefan W Metz et al. Sci Rep. .

Abstract

Zika virus (ZIKV) and the 4 dengue virus (DENV) serotypes are mosquito-borne Flaviviruses that are associated with severe neuronal and hemorrhagic syndromes. The mature flavivirus infectious virion has 90 envelope (E) protein homo-dimers that pack tightly to form a smooth protein coat with icosahedral symmetry. Human antibodies that strongly neutralize ZIKV and DENVs recognize complex quaternary structure epitopes displayed on E-homo-dimers and higher order structures. The ZIKV and DENV E protein expressed as a soluble protein is mainly a monomer that does not display quaternary epitopes, which may explain the modest success with soluble recombinant E (sRecE) as a vaccine and diagnostic antigen. New strategies are needed to design recombinant immunogens that display these critical immune targets. Here we present two novel methods for building or stabilizing in vitro E-protein homo-dimers that display quaternary epitopes. In the first approach we immobilize sRecE to enable subsequent dimer generation. As an alternate method, we describe the use of human mAbs to stabilize homo-dimers in solution. The ability to produce recombinant E protein dimers displaying quaternary structure epitopes is an important advance with applications in flavivirus diagnostics and vaccine development.

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Conflict of interest statement

Aravinda de Silva has consulted on dengue vaccines for Takeda Vaccines, GSK and Merck Pharmaceuticals. He is also an inventor in patents related to dengue vaccines.

Figures

Figure 1
Figure 1
DENV2 and ZIKV sRecE expression and characterization. (A) The flavivirus E protein consists of three beta-barrel domains designated domains I (red), II (yellow) and III (blue), with the native protein forming a head-to-tail homo-dimer. The quaternary epitopes recognized by human dimer-dependent Mabs 2D22 (cyan), A11 (green) and C8 (magenta) are indicated, . (B) DENV and ZIKV sRecE expression was analyzed by Western Blot (anti-His mAbs), CBB (C) and by ELISA using 4G2, 1M7, 2D22, A11, B7 (EDE2), C8 and C10 (EDE1) mAbs.
Figure 2
Figure 2
DENV2 sRecE dimer assembly on Ni2+-coated surfaces. (A) Indicated amounts of DENV2 sRecE were loaded and reloaded on Ni2+-coated plates and analyzed for monomeric (4G2) or dimeric protein structures (2D22). Increased dimer formation is displayed as the 2D22/4G2 signal ratio. (B) The same assay was performed with EDE1 (A11, B7), EDE2 (C8, C10) and C) 3F9 and 1M7 mAbs. No antibody signals were detected when no protein was loaded or reloaded and groups 500 + 0 and 500 + 500 were statistically compared by student T-test (p < 0.05).
Figure 3
Figure 3
Temperature effects on sRecE dimer formation. Indicated amounts of DENV2 sRecE were loaded and reloaded at different temperatures, resulting in the following temperature regiments: (A) Load at 37 °C and reload at 37 °C, (B) load at 37 °C and reload at 21 °C, (C) load at 21 °C and reload at 37 °C and (D) load at 21 °C and reload at 21 °C. Dimer formation was analyzed by 4G2 and 2D22 binding. Groups 500 + 0 and 500 + 500 were statistically compared by student T-test (p < 0.05).
Figure 4
Figure 4
Antibody mediated dimer assembly. Indicated amounts of DENV and ZIKV sRecE were incubated with 500 ng of (A) 2D22, 4G2, (B) A11, B7, C8 and C10 mAbs in solution. Protein/mAb complexes were loaded on Ni2+-coated plates and captured antibodies were detected. Statistical analysis was performed using the student T-test.
Figure 5
Figure 5
Antibody mediated dimer assembly is a temperature dependent process. The effect of temperature on mAb mediated dimer assembly was analyzed by incubating 500 ng mAb with 500 ng DENV2 or ZIKV sRecE. sRecE-mAb incubations were performed at 21 °C or 37 °C and loaded on Ni2+-coated plates and captured antibodies were detected.
Figure 6
Figure 6
Schematic representation of the postulated model for sRecE-dimer assembly from immobilized monomers. (1) sRecE is chelated to Ni2+-coated plates. The immobilization of sRecE at its C-terminal end presumably locks the protein in a specific conformation. (2) The plates are subsequently blocked and (3) reloaded with sRecE at high protein concentrations. (4) This enables interaction of the immobilized sRecE with the reloaded proteins and generates quaternary epitopes that can be recognized by E-dimer epitope dependent mAbs.

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