Assessment of Immunity to Vaccinia and MVA

尽管它对天花感染有非凡的效果，但它产生的保护性免疫的参数 对牛痘的认识还不完全。大量个体处于严重毒性的高风险中， 接种或接触过牛痘，需要开发更安全的天花疫苗， 了解这种保护的免疫学基础的重要性。该项目将进行详细的 在严格控制的条件下，检测T和B细胞对牛痘和MVA免疫的应答 在正常受试者中，以及在特应性皮炎患者或恶性血液病患者中， 造血干细胞移植后免疫缺陷这些研究将测量中和和其他 结合抗体，通过31 f胸苷掺入的淋巴增殖反应，通过51 Cr 释放测定和通过ELISPOT的T细胞γ干扰素应答。接种了以下任一疫苗的正常受试者 牛痘或MVA，将用牛痘攻击以确定是否赋予了针对攻击的保护。 免疫应答的测量将在多个时间点依次进行，包括之前和之后的时间点。 挑战之后。反应率和模式将比较牛痘和MVA接受者之间， 正常人和患者队列。 B和T细胞应答的特异性将采用生物信息学方法和分子生物学方法进行检查。 该中心的研究资源技术开发组件的技术。这些研究将衡量 对一组重组牛痘编码蛋白的抗体应答， 在中和中起重要作用，包括DIL的EGF样结构域（SPGF）。由于我们初步研究中的所有受试者都将 HLA-A*0201阳性，我们将能够利用来自重组牛痘蛋白的肽来表征 通过ELISPOT、细胞因子流式细胞术和肽-MHC四聚体测定I类等位基因特异性T细胞应答。非I类 等位基因特异性T细胞应答将使用完整的重组蛋白进行检查。光谱分析也将是 用于鉴定主要的T细胞表位。这些研究将提供一个全面的图片免疫 本发明的目的在于提供针对牛痘和MVA疫苗接种的免疫应答的表位，并鉴定针对其的重要应答的表位。 这些研究还将证明2组高危患者对MVA的反应是否与 正常人t年和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，以及针对在生物防御中重要的其他因子的疫苗。

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.