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

描述（由申请人提供）：逆转录病毒衍生的VLPs （rVLPs）是优秀的候选疫苗，不仅适用于艾滋病等逆转录病毒疾病，也适用于其他广泛的临床感兴趣的病毒性疾病，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，以纳入感兴趣的表位和/或与来自大量包膜病毒家族的糖蛋白假型；丙型肝炎病毒的E1E2、黄热病病毒的prM和E、西尼罗病毒的prM和E、登革热病毒的E和流感病毒的HA都能有效地假型到rvlp上。值得注意的是，假型糖蛋白保持其野生型构象。我们已经设计了表达盒，产生带有一种或两种HCV包膜蛋白E1和E2 （HCV-E1- rvlp或HCV- e1e2 - rvlp）的假型rvlp。我们已经证明，与用重组病毒载体第二次免疫相比，用表达HCV包膜蛋白E1和E2的重组病毒载体转染小鼠，在伪型rvlp增强后，会产生前所未有的中和性体液免疫反应。然而，虽然这些伪型rvlp是丙型肝炎疫苗的极有希望的候选物，但它们的生产很麻烦，因此需要一种新的高效和安全的系统。我们之前比较了在昆虫细胞系中通过杆状病毒载体或在人类细胞系中通过DNA载体产生的hcv - rvlp。杆状病毒载体系统比DNA载体系统产生更高的HCV-rVLP滴度。然而，由DNA载体系统产生的HCV-rVLPS诱导了更好的体液免疫反应，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，慢病毒载体可以稳定地以每个细胞的高拷贝数结合，并且它们很容易在人类细胞系中表达。因此，本基金申请的目的是构建表达HCVrVLPs的慢病毒载体，并验证其在用慢病毒载体稳定转导的人类细胞中高滴度生产HCV-rVLPs的用途。这样的系统将为本文介绍的丙型肝炎候选疫苗以及任何由rvlp组成的候选疫苗的持续开发铺平道路。

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.