Recombinant Vaccinia Virus with Reduced Virulence

牛痘病毒（VV）-用于预防天花的惠氏基础疫苗比其他疫苗具有安全边际。 可靠的疫苗株。相比之下，专门衰减的VV不能提供强大的保护。这一目标 建议使用当代分子遗传学来特异性地突变VV-惠氏菌株，以便：（i）降低 通过特异性删除免疫调节和抗炎功能，同时保持宿主范围， 复制和免疫原性，如通过在原代人成纤维细胞和角质形成细胞中的复制测量的，MRC 5。 Vero，RK13。BHK和NIH 3 t3细胞，以及IP、鼻内或 皮肤划痕VV感染。Kieff实验室已经制造了用于表达EB病毒基因的重组VV 并且与威尔士实验室合作表征v-IL 10对VV病毒感染的作用， 免疫反应。威尔士实验室研究小鼠对VV感染的反应已有20多年， NK细胞和NK细胞亚群的活化和抗病毒作用，7_ T细胞的活化和抗病毒作用，异源 免疫反应，从而记忆a[_ T细胞特异性其他病毒改变VV发病机制，和（在合作 与LiisaSelin博士）定义了I类Kb限制的免疫显性VV表位。VV合成的动力学， C57 BL/6小鼠全身和粘膜感染后的传播、免疫应答和发病率， 已经很明确了。具有改变的免疫系统组分的遗传操作的C57 BL/6小鼠，例如 抗病毒细胞因子和淋巴细胞亚群将有助于评估免疫受损的hosl如何应答。 一种更弱但仍具有免疫原性的疫苗。表达人HLA-A2的小鼠也是可获得的，并且将在 用于评估人类T细胞表位的重要性。我们的目标是对VV毒力的新见解，并期望 定义对人类疫苗有用的突变。(ii)增加VMV免疫效力， VMV T细胞表位是VV缺失的。这些实验将与威尔士教授合作完成 和Reinherz，由于人类HLA-A2小鼠的实用性，最初将集中于A2表位。及（iii） 通过分子遗传学和免疫学的研究，增加VV毒力和致病机理的知识 特异性VV开放阅读框的影响，其是基于毒力的免疫调节的主要候选者。

Vaccinia Virus (VV)-Wyeth base vaccines for the prevention of small pox have a margin of safety over other dependable vaccine strains. In contrast, specifically attenuated VV do not offer robust protection. The objectives of this proposal are use contemporary molecular genetics to specifically mutate the VV-Wyeth strain so as to: (i) Lower virulence by specifically deleting immune modulatory and anti-inflammatory functions, while maintaining host range, replication, and immunogenicity as measured by replication in primary human fibroblasts and keratinocytes, MRC5. Vero, RK13. BHK and NIH3t3 cells and by replication and immune potency in murine models of IP, intranasal, or dermal scarification VV infection. The Kieff lab has made recombinant VV for expression of Epstein-Barr Virus genes in the past and has collaborated with the Welsh lab in characterizing the effect of v-ILl0 on VV virus infection and immune responses. The Welsh lab has studied mouse responses to VV infection for over 20 years, discovering the activation and antiviral role of NK cells and NK cell subsets, the activation and antiviral role of 7_ T cells, heterologous immune response, whereby memory a[_ T cells specific to other viruses alter VV pathogenesis, and (in collaboration with Dr. Liisa Selin) defined a class I Kb - restricted, immunodominant, VV epitope. The kinetics of VV synthesis, dissemination, immune responses, and morbidity following systemic and mucosal infection of C57BL/6 mice are already well defined. Genetically manipulated C57BL/6 mice with altered immune system components, such as antiviral cytokines and lymphocyte subsets, will assist in evaluating how an immune- compromised hosl may respond to a more attenuated, but still immunogenic vaccine. Mice expressing human HLA-A2 are also available and will be used to assess the significance of human T cell epitopes. We aim for novel insights into VV virulence and expect to define mutations that will be useful for human vaccines. (ii) Increase VMV immune potency by including potential VMV T cell epitopes that are missing from VV. These experiments will be done in collaboration with Professors Welsh and Reinherz and will initially focus on A2 epitopes because of the utility of the human HLA-A2 mice. And (iii) Increase knowledge of VV virulence and pathogenesis, by investigation of the molecular genetic and immunological effects of specific VV open reading frames that are prime candidates for immune modulation based virulence.