(701e) Production of Robust Virus-Like Particles Via Disulfide-Bond Cross-Linking

Virus-like particles (VLPs) are protein macro-molecular complexes on the order of 10 to 100 nm in diameter and are typically icosahedral in shape and symmetry. VLPs have recently received considerable attention for vaccination, drug delivery, gene therapy and material science applications. VLP-based vaccines have repeatedly demonstrated high responses from low doses due to the strong antigenic nature of their repeated ordered surface. Examples of VLP-based vaccines include the Human Papillomavirus vaccine, Gardasil, approved by the FDA in June 2006 and the Hepatitis B vaccine. By genetically and/or chemically modifying VLPs, VLPs have also been used as efficient cell-specific drug/gene delivery devices and self-assembling nanostructure templates. For the above applications a robust capsid is desired which maintains its structural integrity during storage and while performing its function. One technique used to strengthen VLPs is cross-linking using innate or engineered disulfide bonds. We have assessed the ability to cross-link VLPs with disulfide bonds by either 1) modifying the redox potential during translation and VLP assembly or 2) during a post-assembly incubation for a number of VLPs. Hydrogen Peroxide has been shown to be an effective oxidizer for the formation of disulfide bonds and we also observed significant disulfide bond formation in VLPs by incubation with this oxidizer. However, due to hydrogen peroxide's high standard redox potential of 1.8 volts, non-specific reactions with protein residues which could compromise the integrity of the VLP are a concern. We report the ability to form disulfide-bond cross-linked VLPs by 1) changing the redox potential during protein translation and VLP assembly as well as 2) incubating the assembled VLP with more biologically compatible oxidizers. By optimizing the disulfide-bond cross-linking of VLPs we seek to demonstrate increased VLP stability which will in turn help increase the effectiveness of VLPs as vaccines, drug-delivery agents, and nanostructure templates.