| dc.description.abstract | Viral proteins are a potential candidate for subunit vaccine development as they are able to self-assemble into an authentic structure without the existence of an infectious genome. However, its poor immunogenicity limits its applications for vaccine delivery. Therefore, this work aims to develop an antigen-adjuvant complex to increase the immune response and cell viability with viral proteins, as well as investigate the mechanism of inflammasome signaling with the antigen-adjuvant complex in vitro.
Simian Virus 40 viral protein 1 (SV40 VP1) viral protein with a size of ~20 nm in diameter was produced as a recombinant protein using a baculovirus expression vector system, and verified using SDS-Page and Western Blot analysis. SV40 VP1 was then successfully encapsulated onto sacrificial CaCO3 microparticles using layer-by-layer deposition of biodegradable dextran/poly-L-arginine polyelectrolyte pairs. Zeta potential measurements, energy-dispersive spectroscopy, scanning electron and transmission electron microscopy confirmed the successful fabrication of the viral protein based polymeric multilayer capsule (VP-PMLC). Two approaches of viral protein encapsulation and different molecular weights (MW) of polyelectrolytes were investigated, and the highest encapsulation efficiency of 63% was observed using high MW polyelectrolyte pairs with protein deposited in the first layer.
In vitro release profiles were investigated with DC2.4 dendritic cells using fluorescent labeled dextran and 20nm fluorescent labeled silica beads to mimic the size of SV40 VP1. Confocal microscopy demonstrated that all particles were engulfed within 4h, and while leakage of silica beads was observed within 24h by encapsulation of low MW polyelectrolytes,
fabrication with high MW of polyelectrolytes did not result in leakage of silica beads until 48h to 72h.
The overall cell viability was ~ 80% with a particle/cell ratio of ~20. The expression of co-stimulatory molecules, CD40 and CD86, was used to evaluate immune response with the antigen and adjuvant. It was observed that VP-PMLCs stimulate higher immune response in bone marrow-derived dendritic cells (BMDCs). An examination of the secreted cytokine profile from exposed BMDC cultures showed significant secretion of the pro-inflammatory cytokine IL-1β but no detectable increase in the levels of other pro-inflammatory cytokines such as IL-12 and TNF-α. We then verified that cathepsin B was required for IL-1β secretion, but the actin polymerization was not necessary.
Together, our results demonstrate that SV40 VP1-based PMLCs are able to elicit stronger immune responses in dendritic cells while requiring lower doses of viral protein. | en |
