A novel immunogen comprising a bc loop and mutant fusion loop epitopes generates potent neutralization and protective abilities against flaviviruses without risk of disease enhancement

Flaviviruses, including Dengue virus (DENV), Zika virus (ZIKV) and Japanese encephalitis virus (JEV), remain major global health threats, and currently, there are no widely available vaccines for humans. The fusion loop region of the flavivirus envelope protein plays a crucial role in eliciting neutralizing antibodies and providing protection against secondary DENV infections. However, these antibodies often exhibit dual functionalities, with both neutralizing and enhancing activities, posing a challenge for vaccine development. In this study, we focused on optimizing the fusion loop epitope as the primary immunogen and incorporated the adjacent bc loop which we had reported previously as a complementary element, aiming to enhance the immunogen capable of robust neutralization and protection without inducing the risk of antibody-dependent enhancement (ADE). This newly designed immunogen was named as muBCFL which comprises sequences spanning from amino acid 69 to 116 primarily on DENV-2 envelope protein, along with four specific mutations (T76A, W101A, G106Q, and L107D). The synthesized muBCFL peptide elicited neutralizing antibodies against all four DENV serotypes, ZIKV, and JEV, with particularly strong neutralization activity against DENV-2, ZIKV, and JEV. Besides, compared to pre-immune sera, muBCFL-immune sera significantly reduced viremia levels in DENV- or ZIKV-infected AG129 mice and increased the survival rates of JEV-challenged ICR mice. Furthermore, in vitro and in vivo ADE assays validated that muBCFL-immune sera did not induce ADE compared with the control 4G2 monoclonal antibody. These findings indicated that the muBCFL sequence holds great potential as a safe and effective immunogen for developing a flavivirus vaccine in the future.