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.
Highlights
Zika virus (ZIKV) and the dengue viruses (DENVs) are mosquito-borne members of the flaviviridea family, which can cause severe neurological and hemorrhagic syndromes in humans[1,2,3]
The expression and purity of soluble recombinant antigen (sRecE) monomers from both DENV2 and ZIKV was analyzed by SDS-PAGE and western blot using anti-His mabs (Fig. 1B)
Both Coomassie Brilliant Blue staining and western blotting show the presence of pure proteins with the predicted molecular mass for DENV2 (~48 kDa) and ZIKV sRecE (~47 kDa)
Summary
Zika virus (ZIKV) and the dengue viruses (DENVs) are mosquito-borne members of the flaviviridea family, which can cause severe neurological and hemorrhagic syndromes in humans[1,2,3]. Recent studies have established that complex quaternary structure epitopes displayed by E oligomers on the viral surface but not E monomers are targets of strongly neutralizing and protective human antibodies[6, 7]. SRecE from flaviviruses are in a dynamic equilibrium that favors the monomer over the dimer, which likely explains the poor binding of strongly neutralizing quaternary epitope directed human antibodies and the overall poor immunogenicity in pre-clinical studies[6, 20, 21]. From a patient who had recovered from a primary DENV serotype 2 infection, we previously isolated and characterized a DENV2 serotype-specific strongly neutralizing mAb, 2D22, that binds to a E protein dimer-dependent quaternary epitope (Fig. 1A)[22,23,24]. The ability to assemble E homo dimers displaying quaternary structure antibody epitopes is an important advance with applications in flavivirus vaccine development and diagnostics
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