Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

Abstract

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

pH-responsive Virus-like Nanoparticles with Enhanced Tumour-targeting Ligands for Cancer Drug Delivery

“…Hepatitis B core antigen (HBcAg) self-assembles into VLNPs, which have been shown to be some of the most powerful protein engineering tools employed to display immunogens and cell-targeting peptides, as well as for the packaging of genetic materials10. An HBcAg mutant, namely truncated HBcAg (tHBcAg), also self-assembles into icosahedral nanoparticles of approximately 35 nm, which can be used to package green fluorescent protein (GFP)111213.…”

Virus‐like particles: Next‐generation nanoparticles for targeted therapeutic delivery

“…This sequence has been easily extended to incorporate mRNAs, micro RNAs, and small interfering RNAs . HBVc, P22, and CCMV VLPs have all been shown to load RNA through electrostatic interactions between the nucleic acid and the coat proteins . HBVc and Qβ VLPs have also used similar principles to load DNA .…”

“…HBVc, P22, and CCMV VLPs have all been shown to load RNA through electrostatic interactions between the nucleic acid and the coat proteins . HBVc and Qβ VLPs have also used similar principles to load DNA . These nucleic acid‐loaded VLPs have been developed for various uses including vaccines and vaccine adjuvants, gene delivery systems, micro RNA delivery systems, gene knockdown systems, and gene replacement by delivering guide RNA for the CRISPR system .…”

Designs of Antigen Structure and Composition for Improved Protein-Based Vaccine Efficacy

“…PRR agonists can also be encapsulated by antigen when working with proteins that self-assemble into organized matrixes, as has been demonstrated by the "packaging" of nucleic acids and proteins within various VLPs (88)(89)(90)(91)(92)(93). Post-expression encapsulation can be achieved via simple diffusion through vaccine matrix pores when working with nucleic acids, though assembly and disassembly cycling may be necessary to encapsulate larger particles, such as proteins.…”