Optimization of Surface Display of DENV2 E Protein on a Nanoparticle to Induce Virus Specific Neutralizing Antibody Responses. 
The dengue virus (DENV) causes over 350 million infections, resulting in ∼25,000 deaths per year globally.  An effective dengue vaccine requires generation of strong and balanced neutralizing antibodies against all four antigenically distinct serotypes of DENV.  The leading live-attenuated tetravalent dengue virus vaccine platform has shown partial efficacy, with an unbalanced response across the four serotypes in clinical trials.  DENV subunit vaccine platforms are being developed because they provide a strong safety profile and are expected to avoid the unbalanced immunization issues associated with live multivalent vaccines.  Subunit vaccines often lack immunogenicity, requiring either a particulate or adjuvanted formulation.  Particulate formulations adsorbing monomeric DENV-E antigen to the particle surface incite a strong immune response, but have no control of antigen presentation.  Highly neutralizing epitopes are displayed by DENV-E quaternary structures.  To control the display of DENV-E and produce quaternary structures, particulate formulations that covalently attach DENV-E to the particle surface are needed.  Here we develop a surface attached DENV2-E particulate formulation, as well as analysis tools, using PEG hydrogel nanoparticles created with particle replication in nonwetting templates (PRINT) technology.  We found that adding Tween-20 to the conjugation buffer controls DENV-E adsorption to the particle surface during conjugation, improving both protein stability and epitope display.  Immunizations with the anionic but not the cationic DENV2-E conjugated particles were able to produce DENV-specific and virus neutralizing antibody in mice.  This work optimized the display of DENV-E conjugated to the surface of a nanoparticle through EDC/NHS chemistry, establishing a platform that can be expanded upon in future work to fully control the display of DENV-E.