title: Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery.
abstract: Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, CA 94305;
authors.forename: Yuan
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, CA 94305;
authors.forename: Wei
authors.initials: W
authors.lastname: Chan
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, CA 94305;
authors.forename: Benjamin Y
authors.initials: BY
authors.lastname: Ko
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, CA 94305;
authors.forename: Christopher C
authors.initials: CC
authors.lastname: VanLang
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, CA 94305; Department of Bioengineering, Stanford University, Stanford, CA 94305 jswartz@stanford.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Swartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004220:Disulfides / Q000737:chemistry; D016503:Drug Delivery Systems / Q000379:methods*; D006509:Hepatitis B / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D017325:Hepatitis B Vaccines / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology; D006515:Hepatitis B virus / Q000235:genetics / Q000276:immunology / Q000502:physiology; D054884:Host-Pathogen Interactions / Q000276:immunology; D006801:Humans; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D053758:Nanoparticles / Q000008:administration & dosage / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D058425:Vaccines, Virus-Like Particle / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D004220:Disulfides; D017325:Hepatitis B Vaccines; D058425:Vaccines, Virus-Like Particle; D014758:Viral Core Proteins
keywords: cell-free protein synthesis; disulfide stabilization; engineered nanoparticles; hepatitis core protein; virus-like particle