IMMUNOLOGIC STUDIES FROM BONE MARROW DONORS AND VOLUNTEERS FOR THE STUDY OF CMV

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。先前几个实验室的结果表明，巨细胞病毒的pp65片段蛋白是该病毒血清阳性个体识别的主要免疫原性蛋白。我们的实验室已经鉴定出在10种不同的HLA I类等位基因背景下识别pp65的CTL克隆。为这些克隆确定的表位库代表了大约95%的美国种族人口。评估这些CTL表位是否在一般人群中主要使用，需要分析刺激CMV血清阳性hla型个体外周血淋巴细胞方法的免疫原性，该技术将游离肽表位高浓度添加到微孔培养的PBMC中。我们对pp65特异性CTL表位和HLA A*0201、A*1101、A*2402、A*6901和B*0702进行了研究。在每种情况下，我们检查了少于5名血清阳性的健康志愿者，并显示对HLA等位基因特异性的CTL表位有反应，它们表达。CD8+CTL可以识别装载肽和巨细胞病毒感染的靶标，在两周内扩增100倍。使用体外刺激程序可以灵敏地确定个体是否对CMV产生CTL反应，以及pp65是否是该反应的组成部分。我们希望证明至少有5个，如果可能的话，10个随机选择的个体会对单个表位产生反应，这表明表达相同限制性I类等位基因的所有个体对该表位产生普遍反应的可能性。这为生产含有一个或多个这些抗原表位的疫苗分子的方法提供了基本原理，以使高危个体免疫CMV感染。获得外周血和活检的临床程序是临床方案的核心部分，最好在GCRC的主持下进行。我们已经开发了表达HLA等位基因的个体队列，我们从pp65和最近的pp150中获得了表位。我们希望开展这些研究，以表征CMV pp65和pp150的所有主要表位，它们在人类对病毒的免疫反应中是重要的。进行这些研究的目的是确定一种适用于所有高危人群的肽类疫苗。我们还将继续获得新的巨细胞病毒表位，以完成我们的表位库，以确保尽可能完整地代表构成美国人口的不同种族的个体。有几个同意表（A-D）是有效的协议。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Previous results from several laboratories have indicated that the pp65 segament protein from CMV is the major immunogenic protein recognized by individuals who are seropositive for the virus. CTL clones, which recognize pp65 in the context of ten different HLA Class I alleles, have been identified by our laboratory. The repertoire of epitopes that have been defined for these clones are representative of approximately 95% of the ethnic population of the United States. Evaluation of whether these CTL epitopes are predominantly used in the general population requires an analysis of the immunogenicity of the approach of stimulating peripheral blood lymphocytes from HLA-typed individuals who are seropositive for CMV, in a technique in which the free peptide epitope is added at a high concentration to PBMC in microwell cultures. We have used this approach with CTL epitopes specific for pp65 and restricted by HLA A*0201, A*1101, A*2402, A*6901 and B*0702. In each case, we have examined less than five healthy volunteers who are seropositive and have been shown to respond to the CTL epitopes that are specific for the HLA allele, which they express. CD8+CTL, which recognize both peptide loaded and CMV infected targets, are amplified 100-fold in a two-week period. The use of the in vitro stimulation procedure allows a sensitive determination of whether an individual is making a CTL response to CMV, and whether pp65 is a component of that response. We wish to demonstrate that at least five, and if possible, ten randomly chosen individuals will respond to a single epitope, suggesting the potential for a universal response to that epitope from ll individuals who express the same restricting Class I allele. This provides the rationale for an approach to producing vaccine molecules, containing one or more of these epitopes to immunize at-risk individuals against CMV infection. The clinical procedures of obtaining both peripheral blood and biopsy are the central part of the clinical protocol and could be best carried out under the auspices of the GCRC. We have developed cohorts of individuals who express the HLA alleles for which we have epitopes from pp65, and more recently, pp150. We wish to carry out these studies, to characterize all of the major epitopes of CMV pp65 and pp150, which are important in the human immune response to the virus. The purpose for carrying on these studies is to define a peptide-based vaccine that would be applicable to all at-risk individuals. We will also continue to derive new CMV epitopes, to complete our repertoire of epitopes that would ensure as complete a representation as possible of individuals of different ethnicities that make up the American population. There are several consent forms (A-D) which are active for the protocol.