Protective orthopox immunization in normals and patients with cancer or eczema

描述(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，但使用这种病毒作为生物恐怖主义剂来对付我们主要易患疾病的平民人口可能会导致前所未有的死亡。面临活病毒疫苗并发症风险的个人，包括癌症和湿疹患者，占美国人口的很大比例，因此要求不要大规模接种疫苗。免疫学的最新发展，包括对适应性免疫反应和先天免疫反应的机制理解，现在允许评估针对正足病毒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华来确定提供保护性人类免疫的关键正位表位。项目1将根据项目2中确定的参数，检查正常和高危患者在病毒接种试验中对痘苗病毒的保护性免疫。项目2将通过生物信息学和特定位置评分矩阵的全基因组比较，并通过T细胞功能分析和质谱仪确认，确定牛痘、MVA和天花共有的T细胞表位。免疫后不同时间用pMHC四聚体、传统的和新的T细胞记忆生物标记物以及分子详细的T细胞记忆谱对免疫诱导产生的抗原特异性T记忆细胞进行鉴定。同样，针对牛痘和天花的人中和抗体的靶标和生物物理参数将通过重组正基因蛋白、Biaccore、酶联免疫吸附试验和中和研究来确定，后者与疾控中心一起使用。在项目3中，哈佛皮肤病研究中心的研究人员将检查接种正畸疫苗的正常人或特应性皮炎患者的皮肤成分是否具有生产性病毒感染，并比较和对比其中的中央记忆和皮肤归巢效应T细胞的性质。使用生物反应调节剂的小鼠模型和转基因小鼠将被用来检验皮肤环境的操纵如何改变疫苗接种效果。项目4将使用当代分子遗传学对痘苗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。将在C57BL/6、转基因或突变小鼠中进行致病性和免疫原性评估，采用系统、粘膜和皮肤划痕感染途径。提出了一个教育部分、试点项目部分和研究资源技术开发部分，以快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.