Protective orthopox immunization in normals and patients with cancer or eczema

描述（由申请人提供）：虽然在世界范围内根除天花是20世纪医学的一项重大成就，但将这种病毒用作生物恐怖主义剂来对付我们大部分易受疾病影响的平民人口可能会导致前所未有的死亡率。面临活病毒疫苗并发症风险的个体，包括癌症和湿疹患者，占美国人口的很大比例，这就要求反对大规模接种活病毒疫苗。免疫学的最新发展，包括对适应性免疫和先天免疫反应的机制理解，现在允许评估针对正痘和其他类型病毒的保护性免疫的细胞和体液基础。这些进展包括结构水平上的免疫识别细节、抗原呈递、细胞迁移和T细胞记忆。在这里，四组研究人员将利用他们在疫苗学、病毒学、免疫学、皮肤生物学、结构和生物信息学方面的相当大的才能来确定提供保护性人类免疫的关键正痘表位。项目1将根据项目2确定的参数，检查在病毒疫苗接种试验中获得的正常和高危患者对牛痘病毒的保护性免疫。项目2将利用生物信息学和位置特异性评分矩阵进行全基因组比较，鉴定牛痘、MVA和天花共有的T细胞表位，并通过T细胞功能测定和质谱分析进行确认。通过疫苗接种诱导的抗原特异性T记忆细胞将通过pMHC四聚体、传统的和新的T细胞记忆生物标志物以及单细胞PCR在接种后不同时间检测的分子详细T细胞记忆谱来评估。同样，牛痘和天花（后者与CDC合作）的人中和抗体的靶点和生物物理参数将通过重组正痘蛋白、BIAcore、ELISA和中和研究来确定。在项目3中，来自哈佛皮肤病研究中心的研究人员将检查接种了正痘疫苗的正常人或特应性皮炎患者的人体皮肤成分，以检查其是否有繁殖性病毒感染，并比较和对比其中的中枢记忆和皮肤归家效应T细胞的性质。使用生物反应修饰剂和转基因小鼠的小鼠模型将被用来研究如何操纵皮肤环境改变疫苗接种效果。项目4将利用现代分子遗传学对牛痘病毒-惠氏株进行突变，通过删除免疫逃逸功能，但保持宿主范围、复制和免疫原性来降低毒力。致病性和免疫原性评估将在C57BL/6、转基因或突变小鼠中通过系统、粘膜和皮肤划伤感染途径进行。建议设立教育组成部分、试点项目组成部分和研究资源技术开发组成部分，以便迅速传播本中心努力产生的方法和试剂。

DESCRIPTION (provided by applicant): While worldwide eradication of smallpox represents a major accomplishment of medicine in the 20th century, use of this virus as a bioterrorism agent against our largely disease-susceptible civilian population could result in unprecedented mortality. Individuals at risk for live-virus vaccine complications, including those with cancer and eczema, comprise a large percentage of the US population, mandating against massive large-scale vaccination. Recent developments in immunology, both with regard to mechanistic understanding of adaptive and innate immune responses now allow for evaluation of the cellular and humoral bases of protective immunity against orthopox and other classes of viruses. These advances include details of immune recognition at a structural level, antigen presentation, cell migration and T cell memory. Here, four groups of investigators will utilize their considerable talents in vaccinology, virology, immunology, cutaneous biology, structure and bioinformatics to identify critical orthopox epitopes affording protective human immunity. Project 1 will examine protective immunity to vaccinia virus in normal and high-risk patients elicited during virus vaccination trials based on parameters identified in Project 2. Project 2 will identify T cell epitopes shared by vaccinia, MVA and smallpox by genome-wide comparison using bioinformatics and position-specific scoring matrices, and confirmed by T cell functional assays and mass spectrometry. Antigen-specific T memory cells elicited through vaccination will be assessed by pMHC tetramers, conventional and new biomarkers of T cell memory and molecularly detailed T cell memory repertoires as examined by single cell PCR at different times post-vaccination. Likewise, targets and biophysical parameters of human neutralizing antibodies to vaccinia and variola, the latter in conjunction with CDC, will be identified using recombinant orthopox proteins, BIAcore, ELISA and neutralization studies. In Project 3, investigators from the Harvard Skin Disease Research Center will examine human skin elements of orthopox vaccinated normals or atopic dermatitis patients for productive viral infection, and compare and contrast the nature of central memory and skin homing effector T cells therein. Murine models using biologic response modifiers and transgenic mice will be exploited to examine how manipulation of the cutaneous environment alters vaccination efficacy. Project 4 will use contemporary molecular genetics to mutate vaccinia virus-Wyeth strain to lower virulence by deleting immune escape functions but maintaining host range, replication and immunogenicity. Pathogenicity and immunogenicity assessment will be in C57BL/6, transgenic or mutant mice using systematic, mucosal and dermal scarification infectious routes. An Educational Component, Pilot Project Component and Research Resource Technical Development Component are proposed for rapid dissemination of methods and reagents resulting from this Center's effort.