DEVELOPMENT OF A VIRUS-FREE DNA VACCINE AGAINST SMALLPOX

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 天花是生物恐怖袭击事件中的一种威胁，因此开发针对这种病毒的安全疫苗非常重要。有一个重要的未接种疫苗的免疫功能低下的人群，不能安全地接种目前的疫苗。其中包括移植患者、接受免疫抑制治疗的患者、孕妇和老年人。我们有兴趣开发一种更安全的疫苗，保护人们免受可能的攻击。我们推测，免疫增强剂介导的分子抗原免疫应答将控制感染小鼠的病毒负荷。然而，由于天花只是可能的威胁之一，该项目将提供一个很好的模型来测试分子佐剂的假设，这将有助于建立一个系统，可适用于其他A类，B和C类优先病原体。将用编码牛痘病毒蛋白C7 L和A55 R基因的质粒DNA免疫小鼠，所述牛痘病毒蛋白C7 L和A55 R基因是已知产生细胞免疫应答的非结构蛋白，以及最近发现的E6 R和L3 L核心蛋白。据我们所知，这些分子的免疫学尚未得到测试。动物将用每种构建体单独或与两种分子佐剂（趋化因子MIP-1-α和编码树突状细胞特异性生长因子Flt-3L的另一种构建体）组合接种，所述组合已知增加树突状细胞的募集和扩增。据我们所知，这种分子佐剂的组合从未在天花疫苗接种的背景下进行过测试。将分别通过ELISPOT和ELISA测定每种抗原的细胞和体液介导的免疫应答。将对每种抗原进行分子作图，以鉴定优势表位。将用致死剂量的牛痘病毒攻毒动物，并通过qPCR分析测定病毒载量。将测定记忆、活化和增殖，以检测每种抗原对诱导免疫应答的持久作用。预期针对牛痘病毒的强细胞和体液免疫应答使得可以在没有暴露于活病毒的危险的情况下开发保护性免疫记忆。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Smallpox is a threat in an event of a bioterrorist attack, therefore it is important to develop a safe vaccine against this virus. There is an important population of non-vaccinated immunocompromised people that could not be safely vaccinated with the current vaccine. Within these are patients of transplants, under immunosupressive therapy, pregnant women, and the elderly. We are interested in the development of a safer vaccine that protects the population against a possible attack. We hypothesize that the adjuvant-mediated enhancement of the molecular antigen immune response will control the viral burden on infected mice. However, as smallpox is only one of possible threats, this project will provide an excellent model to test the hypothesis of a molecular adjuvant that will help set up a system that could be applied to other Category A, B and C Priority Pathogens. Mice will be immunized with plasmid DNA encoding for the vaccinia virus proteins C7L and A55R genes that are non structural proteins known to generate a cellular immune response, and the recently discovered E6R and L3L core proteins. The immunology of those molecules has not been tested to the best of our knowledge. Animals will be vaccinated with each construct individually or combined with two molecular adjuvants, the chemokine MIP-1-alpha and another construct which encodes for the dendritic cell-specific growth factor Flt-3L, a combination that is known to increase the recruitment and expansion of dendritic cells. To the best of our knowledge, this combination of molecular adjuvants has never been tested in the context of smallpox vaccination. Cell- and humoral-mediated immune responses from each antigen will be determined by ELISPOT and ELISA, respectively. Molecular mapping of each antigen will be performed to identify dominant epitopes. Animals will be challenged with a lethal dose of vaccinia virus, and the viral load will be determined by qPCR analysis. Memory, activation and proliferation will be determined to detect the long-lasting effect of each antigen on the induction of an immune response. Strong cellular and humoral immune responses against vaccinia virus are expected so that protecting immune memory could be developed without the hazard of being exposed to live virus.