ARTES Biotechnology GmbH, Elisabeth-Selbert-Straße 9, 40764, Langenfeld, Germany.
Laboratory of Plant and Process Design, Technical University of Dortmund, Emil-Figge-Straße 70, 44227, Dortmund, Germany.
Microb Cell Fact. 2018 Feb 5;17(1):17. doi: 10.1186/s12934-018-0868-0.
Chimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field.
In this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C.
The dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.
嵌合病毒样颗粒（VLP）允许在其表面展示外源抗原，并且已被证明在开发安全的亚单位疫苗或药物输送方面具有价值。然而，寻找一种廉价的生产系统和一种允许稳定掺入多种大型外源抗原的 VLP 支架仍然是该领域的主要挑战。
本研究建立了一种用于嵌合 VLP 开发的多功能且具有成本效益的平台。鸭乙型肝炎病毒的膜整合小表面蛋白（dS）被选为 VLP 支架，而工业上应用且安全的酵母汉逊德巴利酵母（同义词：Pichia angusta、Ogataea polymorpha）则作为异源表达宿主。来自四种感染动物的病毒的八种不同的、分子量高达 412 个氨基酸的大分子抗原被遗传融合到 dS 上，并分离出重组生产菌株。在所有情况下，融合蛋白都得到了很好的表达，并且在与 dS 共同生产时，可以生成包含这两种蛋白的嵌合 VLP。通过从重组乙型肝炎 VLP 疫苗的生产过程中改编的下游工艺进行纯化。嵌合 VLP 在蛋白质水平上纯度高达 95%，并且包含高达 33%的融合蛋白。免疫学数据支持外源抗原在天然 VLP 上的表面暴露。每 100g 干细胞重量可分离约 40mg 的嵌合 VLP。这与优化生产人类乙型肝炎 VLP 报告的值非常相似。纯化的嵌合 VLP在 4°C 下可稳定保存 6 个月。
基于 dS 的 VLP 支架可耐受多种大型分子量外源蛋白序列的掺入。它适用于展示来自多种病原体的高免疫原性抗原。基于酵母的生产系统允许进行具有成本效益的生产，不受小规模基础研究的限制。因此，基于 dS 的 VLP 平台在抗原呈递方面非常高效，应该在未来疫苗的开发中得到考虑。
