Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery.
The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins.
These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.
Virus-like-particles (VLPs) are self-assembled, proteinaceous nanoparticles derived from naturally occurring viruses. In last years, VLPs attracted broad scientific interest due to their properties as versatile scaffold in nanotechnology. These beneficial characteristics are mainly originated from their particle integrity. Since VLPs are not infectious and do not replicate, they are considered as a safe format in biomedicine.
One of the best characterized model VLP is based on the wildtype hepatitis B virus core antigen (HBcAg WT). The HBcAg WT monomer contains an assembly domain (1–149 aa) and a C-terminal domain (CTD) for binding of the nucleic acids. The assembly domain consists of five α-helices and the major immunodominant region (MIR), which is located between α-helix three and four and forms the spikes on the particles. In the assembly domain, cys61 forms an intradimer disulfide-bridge, which is not essential for capsid formation. HBcAg monomers associated into dimers and spontaneously assemble via interdimer contacts into small and large VLP capsids composed of 180 (T = 3-symmetry) or 240 (T = 4-symmetry) subunits. It has been hypothesized that T = 3 particles may be more stable as they better tolerate stress-inducing epitope insertions. HBcAg WT lead predominantly (> 90%) to T = 4 capsids.
Virus-like-particles per se are robust structures, and are highly immunogenic as the extremely repetitive and dense presentation of epitopes on the VLP surface efficiently stimulates B cells. When foreign epitopes are introduced into the MIR at the tips of the VLP spikes to obtain so called “chimeric VLP” for immunization, the T = 3/T = 4 capsid ratio can be altered, and the ability to build stable particles can be dramatically decreased. As the integrity of VLPs is critical for their immunogenicity, strategies to prevent destabilization of chimeric HBcAg-VLPs have been developed. The majorities of these alterations implicate disadvantages in vaccine development as they directly affect the foreign epitope sequence and may negatively change the epitope conformation. In addition they are located on a prominent area (e.g. MIR or spike) of the VLP surface, where they tend to display an unwanted, own immunogenicity. Therefore we have focused on novel capsid stabilizing sites/elements located proximal of the VLP to address these problems. An attractive element is a naturally occurring disulfide-bridge (cys61) which is situated inside the HBcAg-structure. Another interesting modification site of the HBcAg is the C-terminus, since it may be located in the particle lumen, has little interaction with the spike (MIR)-inserted epitope and is an essential determinant of VLP geometry and assembly.
