Viruses with Pseudolethal Mutations as Vaccines

描述（由申请人提供）：我们提出了一种新的病毒类别 由具有假致死性的复制能力病毒组成的疫苗 使它们不能形成传染性后代的突变。我们 这表明，由于这种病毒将保留细胞内的能力， 复制，它们可以诱导免疫反应，相当于由 感染这类疫苗不能按“常规”配制和使用 传染性疫苗然而，如果以感染性DNA形式制成， 可以联合收割机的效率，感染性病毒在诱导保护性 免疫应答、非感染性疫苗制剂的安全性以及 DNA疫苗生产和维护成本低。以下目标 将在拟议的研究中加以讨论。1)我们将设计黄病毒 感染性DNA构建体，其能够启动生产性感染， 将质粒DNA转染易感细胞并接种小鼠。 我们开发了一种革命性的方法， 黄病毒的稳定感染性DNA构建体。我们将准备传染性DNA 西尼罗河病毒（WN），最近出现在美国和疫苗 以及黄热病17 D疫苗株的 （YF 17 D）病毒，其作为疫苗载体非常有吸引力。2)我们将 将突变引入WN和YF 17 D感染性DNA构建体， 使这些病毒不能形成感染性后代。我们将介绍 衣壳基因中的缺失，其将消除病毒体组装，和/或修饰 C-prM连接处的位点在病毒蛋白酶裂解之前或期间 病毒体组装。这种修饰不会影响RNA的早期过程 病毒蛋白质的复制、合成和加工，从而使 病毒复制的细胞内阶段基本上完好无损。我们将描述 这种突变体的复制和感染特性。3)使用DNA 免疫方法，我们将评估突变的免疫原性潜力， WN和YF 17 D病毒将只能进行单轮病毒感染。 细胞内复制，并与“经典”非复制 DNA疫苗和传染性病毒。采用ELISA、Western blot和FAGS， 将表征所得免疫的体液、GIL和Th 1/Th 2谱 应答最后，我们将确定它们在病毒中的保护潜力， 挑战实验WN感染性克隆将促进 WN疫苗，并将在本研究过程中评价一种候选疫苗。

DESCRIPTION (provided by applicant): We propose a new category of viral vaccines consisting of replication competent viruses with pseudolethal mutations that render them incapable to form the infectious progeny. We suggest, that since such viruses will retain the capabilty for intracellular replication, they can induce immune responses comparable to that resulted from infection. This class of vaccines cannot be prepared and used as "regular" infectious vaccines. However, if made in the infectious DNA form, such vaccines could combine the efficiency of infectious viruses in induction of protective immune responses, the safety of non-infectious vaccine preparations, and the low cost of DNA vaccine production and maintenance. The following objectives will be addressed in the proposed study. 1) We will design flavivirus infectious DNA constructs capable of initiating productive infection upon transfection of plasmid DNA into susceptible cells and inoculation into mice. We have developed a revolutionary approach affording design of remarkably stable infectious DNA constructs of flaviruses. We will prepare infectious DNA of West Nile (WN) virus, which recently emerged in United States and vaccine for which is not available, and of the 17D vaccine strain of yellow fever (YF17D) virus, which is very attractive as a vaccine vector. 2) We will introduce mutations into WN and YF17D infectious DNA constructs that will render these viruses incapable of forming infectious progeny. We will introduce deletions in the capsid gene that will abolish virion assembly, and/or modify the site at C-prM junction cleaved by the viral protease prior to or during virion assembly. Such modifications will not affect earlier processes of RNA replication, synthesis and processing of viral proteins, thus leaving the intracellular phase of viral replication largely intact. We will characterize replication and infectious properties of such mutants. 3) Using DNA immunization methodology, we will evaluate the immunogenic potential of mutated WN and YF17D viruses that will be capable of only a single round ol intracellular replication, and compare with that of a "classic" non-replicating DNA vaccine and of an infectious virus. Using ELISA, Western blot, and FAGS, we will characterize humoral, GIL and Th1/Th2 profiles of the resulting immune responses. Finally, we will determine their protective potential in virus challenge experiments. The WN infectious clone will facilitate development of a WN vaccine, and one candidate will be evaluated in the course of this study.