Biopharmaceutical New Technologies (BioNTech) Protein Therapeutics Corporation, An der Goldgrube 12, 55131, Mainz, Germany.
Department of Internal Medicine III, Translational and Experimental Oncology, University Medical Center of Johannes Gutenberg University, Langenbeckstrasse 1, 55131, Mainz, Germany.
J Nanobiotechnology. 2018 Apr 13;16(1):39. doi: 10.1186/s12951-018-0363-0.
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.
病毒样颗粒（VLPs）是一种有吸引力的纳米颗粒支架，可广泛应用于材料/生物科学和医学领域。在对其进行功能化之前，必须进行特定的适应性改造。这些调整经常会导致颗粒无序，但颗粒的完整性是 VLP 适用性的一个重要因素。因此，存在对颗粒稳定化的主要需求。本研究的目的是评估新型稳定化元件，用于功能化嵌合乙型肝炎病毒核心抗原病毒样颗粒（HBcAg-VLP），这些元件具有疫苗开发、成像或递药的有益特性。
评估了羧基末端多组氨酸肽和二硫键内二聚体对临床前批准的嵌合 HBcAg-VLP 的稳定性的影响。我们纯化了带有不同修饰 C 末端的重组嵌合 HBcAg-VLP，并通过定量蛋白质生化和生物物理技术比较了它们的物理和化学颗粒稳定性。我们观察到 T=3-VLP（三角化数）衣壳的化学稳定性低于 T=4-VLP，并且组装的 VLPs 中六组氨酸肽的可及性受到严重损害。附着在 C 末端的组氨酸取决于组氨酸部分的数量与机械和/或化学颗粒稳定性相关。基于冷冻电镜和生物层干涉测量法的分子建模方法揭示了增强 VLPs 完整性的潜在结构机制。触发衣壳稳定的相互作用发生在 HBcAg 单体上以及相邻单体的六组氨酸肽上的高度保守残基上。这种新的稳定化机制似乎模拟了肝病毒核心蛋白的一种进化保守的稳定化概念。
这些发现确立了遗传上简单可转移的 C 末端多组氨酸肽作为嵌合 HBcAg-VLP 的通用稳定化元件，以提高其适用性。
