School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):3099-104. doi: 10.1073/pnas.0805532107. Epub 2010 Jan 27.
As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.
随着新的疾病威胁的出现以及现有病原体对传统干预措施产生耐药性，人们越来越关注开发疫苗以诱导保护性免疫反应。鉴于其令人钦佩的安全记录，蛋白质亚单位疫苗非常适合广泛的免疫接种，但它们的缺点包括免疫原性差和昂贵的制造。我们在这里表明，工程化的大肠杆菌外膜囊泡（OMV）是一种易于纯化的疫苗递送系统，能够在不添加佐剂的情况下大大增强低免疫原性蛋白质抗原的免疫原性。使用绿色荧光蛋白（GFP）作为模型亚单位抗原，将 GFP 与细菌溶血素 ClyA 进行基因融合，产生了一种嵌合蛋白，该蛋白在免疫小鼠中引发了强烈的抗 GFP 抗体滴度，而单独免疫 GFP 则没有引发这种滴度。利用 ClyA 的特异性分泌到 OMV 中，发现 ClyA-GFP 融合蛋白定位于 OMV 中，从而产生了工程重组 OMV。用工程 OMV 制剂免疫的小鼠中的抗 GFP 体液反应与单独纯化的 ClyA-GFP 融合蛋白的反应无法区分，与吸附到氢氧化铝上的纯化蛋白相当，氢氧化铝是一种标准佐剂。与当前实践相比，一个重大的改进是，含有 ClyA-GFP 的工程 OMV 可以通过超速离心轻松分离，有效地消除了从细胞培养表达系统中费力地纯化抗原的需要。通过与 ClyA 融合时可以将各种异源蛋白质功能性定位到 OMV 中，这项工作预示着 OMV 作为新一代预防性和治疗性疫苗的强大且可调节的技术平台的可能性。
