Orthopox immunization in patients with cancer or eczema

描述（由申请人提供）：虽然天花在世界范围内的根除代表了20世纪世纪医学的一项重大成就，但将这种病毒用作针对我们大多数易受疾病影响的平民人口的生物恐怖主义制剂可能导致前所未有的死亡率。有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，因此必须反对大规模的疫苗接种。免疫学的最新发展，无论是关于适应性和先天性免疫反应的机制的理解，现在允许对正痘病毒和其他类型的病毒的保护性免疫的细胞和体液基础的评价。这些进展包括结构水平上的免疫识别、抗原呈递、细胞迁移和T细胞记忆的细节。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鉴定提供保护性人类免疫的关键正痘抗原表位。项目1将根据项目2中确定的参数，在病毒疫苗接种试验期间检测正常和高危患者对牛痘病毒的保护性免疫。项目2将使用生物信息学和位置特异性评分矩阵通过全基因组比较来识别牛痘、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法进行确认。通过疫苗接种引发的抗原特异性T记忆细胞将通过pMHC四聚体、T细胞记忆的常规和新生物标志物以及在疫苗接种后不同时间通过单细胞PCR检查的分子详细的T细胞记忆库进行评估。 同样，将使用重组正痘蛋白、BIAcore、ELISA和中和研究来鉴定针对牛痘和天花的人中和抗体的靶标和生物物理参数，后者与CDC结合。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过正痘疫苗的正常人或特应性皮炎患者的人类皮肤成分的生产性病毒感染，并比较和对比其中的中枢记忆和皮肤归巢效应T细胞的性质。将利用使用生物反应调节剂和转基因小鼠的小鼠模型来检查皮肤环境的操纵如何改变疫苗接种效力。项目4将利用现代分子遗传学技术，通过删除免疫逃逸功能，但保持宿主范围、复制和免疫原性，使痘苗病毒-惠氏株突变，以降低毒力。 致病性和免疫原性评估将在C57 BL/6、转基因或突变小鼠中使用系统性、粘膜和皮肤划痕感染途径进行。建议设立教育部分、试点项目部分和研究资源技术开发部分，以迅速传播该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.