Helmholtz Centre for Infection Research (VAM), Braunschweig, Germany.
Microb Cell Fact. 2011 Jun 26;10:48. doi: 10.1186/1475-2859-10-48.
A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification.
High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell.
It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.
通过给予单一病毒多肽，即乙型肝炎表面抗原（HBsAg），可以获得针对乙型肝炎感染的保护性免疫应答。因此，乙型肝炎疫苗是通过利用重组 DNA 技术生成的，优选使用酵母基表达系统。然而，该多肽需要组装成球形颗粒，即所谓的病毒样颗粒（VLPs），以引发所需的保护性免疫应答。到目前为止，尚无明确的证据表明 HBsAg 是在酵母细胞内体内组装成 VLPs 的，还是在下游处理和纯化过程中体外组装的。
使用甲醇诱导的 AOX1 表达系统，用重组毕赤酵母进行了 HBsAg 的高水平生产。在去污剂处理的细胞裂解物的几个下游步骤后，将重组疫苗分离为 VLPs 形式。使用电子显微镜研究结合免疫金标记物对抗原的细胞内定位进行搜索，结果表明 HBsAg 存在于延伸的内质网中，在那里它被发现组装成定义明确的多层、层状结构。两层之间的距离确定为~6nm，表明这些层片代表排列整齐的 HBsAg 亚基的单层。我们没有发现内质网内或酵母细胞其他部位存在 VLPs 的任何证据。
得出的结论是，高水平的生产和内在的缓慢 HBsAg VLP 组装动力学导致抗原在内质网中的保留和积累，在那里它至少部分组装成定义明确的层状结构。HBsAg 向高尔基体的进一步运输受到损害，从而导致分泌途径功能障碍和内质网的延伸，该内质网向外膨出形成不规则的云状形成物。由于未在细胞内发现 VLPs，因此可以得出结论，VLP 组装过程必须在去污剂介导的 HBsAg 层片解组装和随后的 HBsAg 重新组装成球形 VLPs 的下游处理过程中进行。
