Assessment of Immunity to Vaccinia and MVA

尽管它对天花感染非常有效，但由其产生的保护性免疫参数 对牛痘的了解仍然不完全。大量个体在以下情况下有严重中毒的高风险 接种牛痘疫苗或接触牛痘疫苗，以及开发更安全的天花疫苗的需要，突显了 了解这种保护的免疫学基础的重要性。该项目将承担一项详细的 严密控制条件下牛痘和MVA免疫后T、B细胞反应的检测 在正常受试者中，以及特应性皮炎患者或恶性血液病患者和 造血干细胞移植后的免疫缺陷。这些研究将测量中和和其他 结合抗体，31F胸腺嘧啶核苷掺入的淋巴增殖反应，51Cr的细胞毒性T细胞活性 释放试验和ELISPOT的T细胞γ干扰素反应。接种了其中一种疫苗的正常受试者 Vacinia或MVA，将用Vacinia挑战，以确定是否提供了针对挑战的保护。 免疫反应的测量将在多个时间点顺序进行，包括之前和 一次又一次挑战。将比较接种疫苗和MVA接受者之间的应答率和应答模式，以及 正常人和患者队列。 B和T细胞反应的特异性将使用生物信息学方法和分子检测 该中心研究资源技术开发部分的技术。这些研究将测量 生物信息学鉴定的一组重组牛痘病毒编码蛋白的抗体反应 重要的中和作用，包括DIL的EGF样结构域(SPGF)。因为我们最初研究的所有科目都将 如果人类白细胞抗原A*0201阳性，我们将能够利用重组痘苗病毒蛋白中的多肽来鉴定 ELISPOT、细胞因子流式细胞术和多肽-MHC四聚体的I类等位基因特异性T细胞反应。非I类 等位基因特异性T细胞反应将使用完整的重组蛋白进行检测。光谱型分析也将 用于识别主要的T细胞表位。这些研究将提供免疫的全面图景。 对接种牛痘疫苗和MVA疫苗的反应，并确定针对哪些重要反应的表位。 这些研究还将证明两组高危患者对MVA的反应是否与 法线。第t年和第2年的拟议研究侧重于MVA，但类似的方法将用于研究其他 针对天花的候选疫苗，以及针对生物防御中重要的其他病原体的疫苗。

Despite its extraordinary effectiveness against smallpox infection, the parameters of protective immunity generated by vaccinia remain incompletely understood. Large numbers of individuals are at high-risk for serious toxicities if vaccinated with or exposed to vaccinia, and the need to develop safer vaccines against smallpox underscores the importance of understanding the immunologic basis for such protection. This project will undertake a detailed examination of T and B cell responses to immunization with vaccinia and MVA under carefully controlled conditions in normal subjects, and to MVA in patients with atopic dermatitis or patients with hematologic malignancies and immune deficiency after hematopoietic stem cell transplantation. The studies will measure neutralizing and other binding antibodies, lymphoproliferative responses by 31f Thymidine incorporation, cytotoxic T-cell activity by a 51Cr release assay, and T-cell gamma interferon responses by ELISPOT. Normal subjects who were vaccinated with either vaccinia or MVA, will be challenged with vaccinia to determine whether protection against challenge was conferred. Measurements of immune responses will be conducted sequentially at multiple time points, including those before and after challenge. Rates and patterns of responses wilt be compared between vaccinia and MVA recipients, and between normals and the patient cohorts. The specificity of the B and T cell responses will be examined employing bioinformatics approaches and molecular techniques of the Research Resource Technical Development Component of the Center. These studies will measure antibody responses to a panel of recombinant vaccinia-encoded proteins identified by bioinformatics as potentially important in neutralizati on, including the EGF-like domain of DIL (SPGF). Since all subjects in our initial studies will be HLA-A*0201 positive, we will be able to utilize peptides from the recombinant vaccinia proteins to characterize class I allele specific T-cell responses by ELISPOT, cytokine flow cytometry, and peptide-MHC tetramers. Non-class I allele specific T-cell responses will be examined using whole recombinant proteins. Spectratype analysis will also be used to identify predominant T-cell epitopes. These studies wilt provide a comprehensive picture of the immune responses to vaccination with vaccinia and MVA, and identify epitopes against which important responses are directed. These studies will also demonstrate whether 2 groups of high-risk patients respond to MVA in a similar fashion to normals. The proposed studies in years t and 2 focus on MVA, but similar approaches would be utilized to study other candidate vaccines against smallpox, as well as vaccines against other agents important in biodefense.