Dengue-2 virus-like particle (VLP) based vaccine elicits the highest titers of neutralizing antibodies when produced at reduced temperature.

A dengue vaccine capable of rapidly eliciting a robust and balanced immunity against the four virus serotypes after only a few immunizations is greatly needed. We describe a new strategy to develop dengue vaccines based on the assembly of virus-like particles (VLPs) utilizing the structural proteins CprME together with a modified complex of the NS2B/NS3 protease, which enhances particle formation and yield. These VLPs are produced in mammalian cells and resemble native dengue virus as demonstrated by negative staining and immunogold labelling electron microscopy (EM). We found that VLPs produced at lower temperature (31 °C) were recognized by conformational monoclonal antibodies (MAbs) 4G2, 3H5 and C10 whereas VLPs produced at higher temperature (37 °C) were not recognized by these MAbs. To investigate the significance of these conformational discrepancies in vaccine performance, we tested the immunogenicity of VLP vaccines produced at 31 °C or 37 °C. Mice immunized with the VLP vaccine produced at 31 °C (VLP-31 °C) elicited the highest titer of neutralizing antibodies when compared to those elicited by equivalent doses of the vaccine produced at 37 °C (VLP-37 °C), inactivated dengue virus vaccine or to the titer of a human anti-dengue-2 convalescence serum reference. Our results demonstrate that the conformation of the E protein displayed on the VLP vaccine plays a critical role in the induction of highly neutralizing antibodies. These findings will guide development of a tetravalent vaccine capable of eliciting a robust and balanced neutralizing response against the four-dengue serotypes regardless of background immunity.