Viruses with Pseudolethal Mutations as Vaccines

描述(由申请人提供)：我们提出了一个新的病毒类别 由具有复制能力的伪致死性病毒组成的疫苗 突变使他们不能形成有感染力的后代。我们 表明，由于这种病毒将保留对细胞内的能力 复制，它们可以诱导免疫反应，其结果与 感染。这类疫苗不能作为“常规”疫苗制备和使用 传染性疫苗。然而，如果以传染性DNA的形式制造，这种疫苗 可以结合感染性病毒在诱导保护作用方面的效率 免疫反应，非传染性疫苗制剂的安全性，以及 低成本的DNA疫苗生产和维护。以下目标 将在拟议的研究中予以解决。1)我们将设计黄病毒 能够引发生产性感染的感染性DNA构建体 将质粒DNA导入敏感细胞并接种于小鼠体内。 我们开发了一种革命性的方法，为设计提供非凡的 稳定的具有感染性的黄病毒DNA结构。我们将准备具有传染性的DNA 最近在美国出现的西尼罗河(WN)病毒和疫苗 以及黄热病的17D疫苗株 (YF17D)病毒，作为疫苗载体非常有吸引力。2)我们会 将突变引入WN和YF17D感染性DNA结构，将 使这些病毒不能形成感染性后代。我们将介绍 衣壳基因中的缺失将取消病毒粒子组装，和/或修改 C-PrM连接处被病毒蛋白水解酶切割的部位 病毒粒子组装。这样的修饰不会影响RNA的早期过程 病毒蛋白的复制、合成和加工，从而使 细胞内阶段的病毒复制基本完好无损。我们将描述 这种突变体的复制和感染特性。3)使用DNA 免疫方法学，我们将评估突变的免疫原性 WN和YF17D病毒将只能感染单轮病毒 细胞内复制，并与经典的非复制 DNA疫苗和一种传染性病毒。使用ELISA法、Western印迹法和Fags法，我们 将表征产生的免疫的体液、GIL和Th1/Th2谱 回应。最后，我们将确定它们在病毒中的保护潜力。 挑战实验。WN感染性克隆将促进一种 WN疫苗，在这项研究过程中将对一种候选疫苗进行评估。

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.