Optimization of Surface Display of DENV2 E Protein on a Nanoparticle to Induce Virus Specific Neutralizing Antibody Responses

Abstract

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.

Considerations for Size, Surface Charge, Polymer Degradation, Co‐Delivery, and Manufacturability in the Development of Polymeric Particle Vaccines for Infectious Diseases

“…Neutral nm-μm Elevated Dengue [166] Influenza [167,168] Malaria [167] Ovalbumin [169] Streptococcus [167] Salting out Similar to an coacervation, except a salt is used to impede dissolving of organic phase into the aqueous phase. [170] Semicontinuous Aqueous and organic solvent…”

Nanoparticles as Vaccines to Prevent Arbovirus Infection: A Long Road Ahead

“…Several types of materials were used to develop vaccine-based nanoparticles. For dengue vaccines, it seemed that several types of organic (bovine serum albumin, lipid) and inorganic materials (calcium chloride, carbon, and calcium phosphate) were used [33,[53][54][55][56][57][58][59][60][61]. For ZIKV vaccines, only lipid and poly(amidoamine) nanoparticles were used [62][63][64][65][66][67], and for JEV vaccines, protein-VLPs, chitosan, and poly(gamma-glutamic acid) nanoparticles were tested [68][69][70][71][72][73][74][75].…”

“…DNA, RNA, VLPs, inactivated viruses, recombinant viral vectors, peptides, and subunit vaccines are used as experimental vaccines against arboviruses. Besides these several approaches, subunits-based vaccines are the most used [33,54,55,[58][59][60][76][77][78][79]83,87]. Subunit vaccines are developed from selected fragments of the pathogen, such as proteins or polysaccharides.…”

“…However, only studies evaluated the nanoparticles' vaccine potential to induce an effective immune response to DENV [33,[54][55][56]59,83] and WNV [76,77] used subunit antigens. In general, the subunit antigens used on these nanoparticles were structural proteins (membrane and envelope proteins) produced and purified in a heterologous expression system (E. coli, mammalian, and insect cells) [54,55,[58][59][60][76][77][78][79]83,87]. As shown in Figure 3B, nanoparticles carrying RNA were tested on ZIKV [62][63][64]66,67], CHIKV [81], and DENV [61] in preclinical immunization assays.…”

Vaccines Against Dengue and West Nile Viruses in India: The Need of the Hour

“…The enormity of DEN infection, severity, and four serotypes, along with other flaviviruses circulating in the same region largely complicate matters. Based on information seen in other virus vaccines, it has become clear that monomeric E protein does not work on its own and needs an adjuvant (24). Preclinical studies have explored DNA plasmid expressing different proteins singly or in combination (25,122).…”