BackgroundVirus-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.ResultsThe 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.ConclusionsThese findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.Electronic supplementary materialThe online version of this article (10.1186/s12951-018-0363-0) contains supplementary material, which is available to authorized users.

We introduce aqueous phases comprising single, highly compacted self-amplifying messenger RNA (saRNA) molecules, that are suitable for prophylactic and therapeutic application, specifically for vaccination against infectious diseases. The formulations are formed in the presence of the positively charged polymer polyethylenimine (PEI), which leads to condensation of the single saRNA molecules into a globular organization with high packing density, low mass fraction of polymer, and, consequently, very small size. In this format, they display improved biological activity in comparison to previously described saRNA/PEI nanoparticlulate formulations, both in vitro and in vivo. Application of the ultra-compacted single saRNA formulation for vaccination, via intramuscular route, results in relevant titers for practical use at lower doses compared to the nanoparticle formulations. These novel saRNA vaccine products can be obtained by straight-forward manufacturing routes, and they can be readily frozen or lyophilized. With these characteristics they can be particularly of interest for future vaccine products even for application under challenging conditions, where requirements such as high activity, good thermostability, low cost of goods, and facilitated logistics need to be fulfilled at the same time. One-Sentence Summary: single saRNA molecules, condensed into an unusually compact globular organization with very small size by an oppositely charged polyelectrolyte (polyethylenimine), are applicable as vaccines, which induce very strong immunological responses at very low doses.

We present a generically applicable approach to determine an extensive set of size-dependent critical quality attributes inside nanoparticulate pharmaceutical products. By coupling asymmetrical-flow field-flow fractionation (AF4) measurements directly in-line with solution small angle X-ray scattering (SAXS), vital information such as (i) quantitative, absolute size distribution profiles, (ii) drug loading, (iii) size-dependent internal structures, and (iv) quantitative information on free drug is obtained. Here the validity of the method was demonstrated by characterizing complex mRNA-based lipid nanoparticle products. The approach is particularly applicable to particles in the size range of 100 nm and below, which is highly relevant for pharmaceutical products – both biologics and nanoparticles. The method can be applied as well in other fields, including structural biology and environmental sciences.