A Novel Method of Generating Hepatitis C Virus-Like Particles using Lentivirus

利用慢病毒产生丙型肝炎病毒样颗粒的新方法

• 批准号: 7748047
• 负责人: Boro Dropulic
• 金额: $ 28.29万
• 依托单位: LENTIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7748047
• 关键词: Hepatitis C virus; Methods; Subfamily lentivirinae; Virus-like particle; novel

DESCRIPTION (provided by applicant): Retrovirus derived VLPs (rVLPs) are excellent vaccine candidates, not only for retroviral diseases such as AIDS but also for a wide range of other viral diseases of clinical interest such as hepatitis C, yellow fever, West Nile fever, dengue fever, and influenza. Indeed, the retroviral core protein Gag can be modified to incorporate epitopes of interest and/or be pseudotyped with glycoproteins from a large host of families of enveloped viruses; E1E2 of hepatitis C virus, prM and E of yellow fever virus, prM and E of West Nile virus, E of Dengue virus and HA of influenza virus can all be efficiently pseudotyped onto rVLPs. Noteworthy, the pseudotyped glycoproteins retain their wild type conformation. We have engineered expression cassettes that produce rVLPs pseudotyped with one or both of the HCV envelope proteins E1 and E2 (HCV-E1-rVLP or HCV-E1E2-rVLP). We have shown that mice primed with a recombinant viral vector expressing the HCV envelope proteins E1 and E2 will mount unprecedented neutralizing humoral immune responses upon boosting with pseudotyped rVLPs, compared with a second immunization with the recombinant viral vector. However, while these pseudotyped rVLPs make highly promising candidates for a vaccine against Hepatitis C, they are cumbersome to produce and thus their production calls for a new efficient and safe system. We have previously compared HCV-rVLPs produced via baculovirus vectors in an insect cell line or via DNA vectors in a human cell line. The baculovirus vector system gives rise to considerably higher HCV-rVLP titers than the DNA vector system. However, HCV-rVLPS produced from the DNA vector system induce far better humoral immune responses, likely due to a correct glycosylation pattern provided by the human cell line. We believe that a lentivirus vector system could alleviate the production bottleneck. Indeed, lentiviral vectors can be stably incorporated at high copy numbers per cell, and they are readily expressed in human cell lines. Therefore, the aim of this grant application is the construction of a lentiviral vector expressing HCVrVLPs, and validation of its use for high titer production of HCV-rVLPs from human cells stably transduced with the lentiviral vector. Such a system would pave the way for continued development of the Hepatitis C vaccine candidate presented here, as well as for any vaccine candidate consisting of rVLPs. PUBLIC HEALTH RELEVANCE: The Heptaitis C virus is the most common blood-borne virus and HCV infection represents a major public health concern; approximately 3% of the world's population (200 million people) is chronically infected. Causing fibrosis and cirrhosis of the liver and eventually hepatocellular carcinoma, it is the leading cause of liver transplantation in the U.S. No vaccine is available and the current standard of care, a combination of Pegylated-Interferon alpha and the antiviral drug Ribavirin, is effective in only about 50% of patients completing therapy, is lengthy, and is often poorly tolerated. New anti-viral drugs are in the early clinical stage of development, but the available pre-clinical and clinical data indicate that drugresistant variants are likely to emerge during treatment. Thus, the patient population is still underserved and treatment needs remain unmet. Therefore, there is a strong need for new alternative therapies that act through different mechanisms, such as prophylactic and therapeutic vaccines. Lentigen has developed a novel LENTIMAXTM Lentiviral vector gene delivery system that can deliver payload with 100% efficiency in mammalian cells, resulting in very high levels of protein production. Lentiviral vectors (LVs) have recently been shown to achieve HIGH and STABLE gene delivery in a variety of cell lines used for the manufacture of proteins and vaccines. Therefore they are the ideal system for the rapid and efficient delivery of genes encoding proteins into cells for high yielding production of target proteins. Lentigen has exclusive intellectual property in the production of proteins using Lentiviral vector technology. Lentigen's LENTIMAX(tm) system is inherently flexible in that any cell line can be efficiently and stably engineered with any gene(s) of interest into any mammalian cell type. The technology can be used in multiple formats, and in particular related to this RFA it can be used for rapid production of virus-like particles from human cells.

描述（由申请人提供）：逆转录病毒衍生的 VLP（rVLP）是极好的候选疫苗，不仅适用于艾滋病等逆转录病毒疾病，而且适用于临床感兴趣的各种其他病毒性疾病，如丙型肝炎、黄热病、西尼罗热、登革热和流感。事实上，逆转录病毒核心蛋白 Gag 可以被修饰以掺入感兴趣的表位和/或用来自大量包膜病毒家族的糖蛋白进行假型化；丙型肝炎病毒的E1E2、黄热病病毒的prM和E、西尼罗河病毒的prM和E、登革热病毒的E和流感病毒的HA都可以有效地假型化到rVLP上。值得注意的是，假型糖蛋白保留了其野生型构象。我们设计了表达盒，可产生用 HCV 包膜蛋白 E1 和 E2 之一或两者（HCV-E1-rVLP 或 HCV-E1E2-rVLP）假型化的 rVLP。我们已经证明，与用重组病毒载体进行第二次免疫相比，用表达 HCV 包膜蛋白 E1 和 E2 的重组病毒载体引发的小鼠在用假型 rVLP 加强后将产生前所未有的中和体液免疫反应。然而，虽然这些假型 rVLP 是丙型肝炎疫苗非常有希望的候选者，但它们的生产很麻烦，因此它们的生产需要新的高效且安全的系统。我们之前已经比较了通过昆虫细胞系中的杆状病毒载体或通过人类细胞系中的DNA载体产生的HCV-rVLP。杆状病毒载体系统产生比DNA载体系统高得多的HCV-rVLP滴度。然而，DNA载体系统产生的HCV-rVLPS可诱导更好的体液免疫反应，这可能是由于人类细胞系提供了正确的糖基化模式。我们相信慢病毒载体系统可以缓解生产瓶颈。事实上，慢病毒载体可以以高拷贝数稳定地掺入每个细胞，并且它们很容易在人类细胞系中表达。因此，本次资助申请的目的是构建表达HCVrVLP的慢病毒载体，并验证其用于从慢病毒载体稳定转导的人细胞中高效生产HCV-rVLP。这样的系统将为本文介绍的丙型肝炎候选疫苗以及由 rVLP 组成的任何候选疫苗的持续开发铺平道路。 公共卫生相关性：丙型肝炎病毒是最常见的血液传播病毒，HCV 感染是一个主要的公共卫生问题；世界人口中约 3%（2 亿人）患有慢性感染。它会导致肝脏纤维化和肝硬化，并最终导致肝细胞癌，是美国肝移植的主要原因。目前还没有可用的疫苗，目前的护理标准（聚乙二醇化干扰素 α 和抗病毒药物利巴韦林的组合）仅对大约 50% 的完成治疗的患者有效，且治疗时间长且耐受性往往较差。新的抗病毒药物正处于早期临床开发阶段，但现有的临床前和临床数据表明，治疗过程中很可能会出现耐药变异。因此，患者群体仍然得不到充分服务，治疗需求仍未得到满足。因此，迫切需要通过不同机制发挥作用的新替代疗法，例如预防性和治疗性疫苗。 Lentigen 开发了一种新型 LENTIMAXTM 慢病毒载体基因传递系统，可以在哺乳动物细胞中以 100% 的效率传递有效负载，从而产生非常高水平的蛋白质。最近，慢病毒载体 (LV) 已被证明可以在用于制造蛋白质和疫苗的多种细胞系中实现高且稳定的基因递送。因此，它们是快速有效地将编码蛋白质的基因递送到细胞中以高产地生产目标蛋白质的理想系统。 Lentigen 在使用慢病毒载体技术生产蛋白质方面拥有独家知识产权。 Lentigen 的 LENTIMAX(tm) 系统本质上是灵活的，因为任何细胞系都可以用任何感兴趣的基因有效且稳定地工程化到任何哺乳动物细胞类型中。该技术可以多种形式使用，特别是与 RFA 相关的技术，可以用于从人体细胞快速生产病毒样颗粒。