Division of Viral Products, Center for Biologics Evaluations and Research, Food and Drug Administration, Bethesda, MD 20892, United States.
Vaccine. 2012 May 14;30(23):3413-22. doi: 10.1016/j.vaccine.2012.03.033. Epub 2012 Mar 29.
Several studies have described the production of influenza virus-like particles (VLP) using a variety of platform systems. These VLPs are non-replicating particles that spontaneously self-assemble from expressed influenza virus proteins and have been proposed as vaccine candidates for both seasonal and pandemic influenza. Although still in the early stages of development and evaluation as influenza vaccines, influenza VLPs have a variety of other valuable uses such as examining and understanding correlates of protection against influenza and investigating virus-cell interactions. The most common production system for influenza VLPs is the baculovirus-insect cell expression which has several attractive features including the ease in which new gene combinations can be constructed, the immunogenicity elicited and protection afforded by the produced VLPs, and the scalability offered by the system. However, there are differences between the influenza VLPs produced by baculovirus expression systems in insect cells and the influenza viruses produced for use as current vaccines or the virus produced during a productive clinical infection. We describe here the development of a modified vaccinia virus Ankara (MVA) system to generate mammalian influenza VLPs containing influenza H5N1 proteins. The MVA vector system is flexible for manipulating and generating various VLP constructs, expresses high level of influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M) proteins, and can be scaled up to produce VLPs in quantities sufficient for in vivo studies. We show that mammalian VLPs are generated from recombinant MVA vectors expressing H5N1 HA alone, but that increased VLP production can be achieved if NA is co-expressed. These mammalian H5N1 influenza VLPs have properties in common with live virus, as shown by electron microscopy analysis, their ability to hemagglutinate red blood cells, express neuraminidase activity, and to bind influenza specific antibodies. Importantly, these VLPs are able to elicit a protective immune response in a mouse challenge model, suggesting their utility in dissecting the correlates of immunity in such models. Mammalian derived VLPs may also provide a useful tool for studying virus-cell interactions and may have potential for development as pandemic vaccines.
已有多项研究描述了使用多种平台系统来生产流感病毒样颗粒（VLP）。这些 VLP 是无复制能力的颗粒，能够从表达的流感病毒蛋白中自发组装，被提议作为季节性和大流行性流感的候选疫苗。尽管作为流感疫苗仍处于早期开发和评估阶段，但流感 VLP 具有多种其他有价值的用途，例如检查和了解针对流感的保护相关性以及研究病毒-细胞相互作用。生产流感 VLP 的最常见系统是杆状病毒-昆虫细胞表达系统，该系统具有多种吸引人的特点，包括易于构建新的基因组合、所产生的 VLP 引发的免疫原性和提供的保护作用，以及系统提供的可扩展性。然而，杆状病毒表达系统在昆虫细胞中产生的流感 VLP 与目前用于疫苗的流感病毒以及在有产性临床感染期间产生的病毒之间存在差异。我们在这里描述了一种改良的禽痘病毒安卡拉（MVA）系统的开发，用于生成含有流感 H5N1 蛋白的哺乳动物流感 VLP。MVA 载体系统在操纵和生成各种 VLP 构建体方面具有灵活性，可高水平表达流感血凝素（HA）、神经氨酸酶（NA）和基质（M）蛋白，并可扩展规模以生产足够用于体内研究的 VLP。我们表明，仅表达 H5N1 HA 的重组 MVA 载体即可生成哺乳动物 VLP，但如果共同表达 NA，则可以提高 VLP 的产量。这些哺乳动物 H5N1 流感 VLP 具有与活病毒相似的特性，如电子显微镜分析所示，它们能够凝集红细胞、表达神经氨酸酶活性以及结合流感特异性抗体。重要的是，这些 VLP 能够在小鼠攻毒模型中引发保护性免疫反应，表明它们在这种模型中用于剖析免疫相关性的实用性。哺乳动物衍生的 VLP 也可能为研究病毒-细胞相互作用提供有用的工具，并可能有潜力开发为大流行性疫苗。
