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Description, A stabilized dimer of the surface protein from dengue virus has been engineered to elicit antibodies that neutralize the virus., Dengue virus (DENV) is a worldwide health burden, and a safe vaccine is needed. Neutralizing antibodies bind to quaternary epitopes on DENV envelope (E) protein homodimers. However, recombinantly expressed soluble E proteins are monomers under vaccination conditions and do not present these quaternary epitopes, partly explaining their limited success as vaccine antigens. Using molecular modeling, we found DENV2 E protein mutations that induce dimerization at low concentrations (

Dengue viruses (DENV serotypes 1-4) and Zika virus (ZIKV) are related flaviviruses that continue to be a public health concern, infecting hundreds of millions of people annually. The traditional live-attenuated virus vaccine approach has been challenging for the four DENV serotypes because of the need to achieve balanced replication of four independent vaccine components. Subunit vaccines represent an alternative approach that may circumvent problems inherent with live-attenuated DENV vaccines. In mature virus particles, the envelope (E) protein forms a homodimer that covers the surface of the virus and is the major target of neutralizing antibodies. Many neutralizing antibodies bind to quaternary epitopes that span across both E proteins in the homodimer. For soluble E (sE) protein to be a viable subunit vaccine, the antigens should be easy to produce and retain quaternary epitopes recognized by neutralizing antibodies. However, WT sE proteins are primarily monomeric at conditions relevant for vaccination and exhibit low expression yields. Previously, we identified amino acid mutations that stabilize the sE homodimer from DENV2 and dramatically raise expression yields. Here, we tested whether these same mutations raise the stability of sE from other DENV serotypes and ZIKV. We show that the mutations raise thermostability for sE from all the viruses, increase production yields from 4-fold to 250-fold, stabilize the homodimer, and promote binding to dimer-specific neutralizing antibodies. Our findings suggest that these sE variants could be valuable resources in the efforts to develop effective subunit vaccines for DENV serotypes 1 to 4 and ZIKV.

Dengue virus (DENV) is responsible for the most prevalent and significant arthropod-borne viral infection of humans. The leading DENV vaccines are based on tetravalent live-attenuated virus platforms. In practice, it has been challenging to induce balanced and effective responses to each of the four DENV serotypes because of differences in the replication efficiency and immunogenicity of individual vaccine components. Unlike live vaccines, tetravalent DENV envelope (E) protein subunit vaccines are likely to stimulate balanced immune responses, because immunogenicity is replication independent. However, E protein subunit vaccines have historically performed poorly, in part because the antigens utilized were mainly monomers that did not display quaternary-structure epitopes found on E dimers and higher-order structures that form the viral envelope. In this study, we compared the immunogenicity of DENV2 E homodimers and DENV2 E monomers. The stabilized DENV2 homodimers, but not monomers, were efficiently recognized by virus-specific and flavivirus cross-reactive potently neutralizing antibodies that have been mapped to quaternary-structure epitopes displayed on the viral surface. In mice, the dimers stimulated 3-fold-higher levels of virus-specific neutralizing IgG that recognized epitopes different from those recognized by lower-level neutralizing antibodies induced by monomers. The dimer induced a stronger E domain I (EDI)- and EDII-targeted response, while the monomer antigens stimulated an EDIII epitope response and induced fusion loop epitope antibodies that are known to facilitate antibody-dependent enhancement (ADE). This study shows that DENV E subunit antigens that have been designed to mimic the structural organization of the viral surface are better vaccine antigens than E protein monomers. IMPORTANCE Dengue virus vaccine development is particularly challenging because vaccines have to provide protection against four different dengue virus stereotypes. The leading dengue virus vaccine candidates in clinical testing are all based on live-virus vaccine platforms and struggle to induce balanced immunity. Envelope subunit antigens have the potential to overcome these limitations but have historically performed poorly as vaccine antigens, because the versions tested previously were presented as monomers and not in their natural dimer configuration. This study shows that the authentic presentation of DENV2 E-based subunits has a strong impact on antibody responses, underscoring the importance of mimicking the complex protein structures that are found on DENV particle surfaces when designing subunit vaccines.