No abstract

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 (<100 pM) and enhance production yield by more than 50-fold. Cross-dimer epitope antibodies bind to the stabilized dimers, and a crystal structure resembles the wild-type (WT) E protein bound to a dimer epitope antibody. Mice immunized with the stabilized dimers developed antibodies that bind to E dimers and not monomers and elicited higher levels of DENV2-neutralizing antibodies compared to mice immunized with WT E antigen. Our findings demonstrate the feasibility of using structure-based design to produce subunit vaccines for dengue and other flaviviruses.

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 potent 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 that were different from the epitopes recognized by lower level neutralizing antibodies induced by monomers. The dimer induced a stronger ED-I and ED-II targeted response, while the monomer antigens stimulated an ED-III epitope response and induced fusion loop epitope antibodies that are known to facilitate ADE. This study shows DENV E subunit antigens that have been designed to mimic the structural organization of 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 4 different dengue 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 previously tested versions 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 virus particle surfaces when designing subunit vaccines.