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

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。先前几个实验室的结果表明，CMV的pp65节段蛋白是该病毒血清阳性个体识别的主要免疫原性蛋白。本实验室已鉴定出在10个不同的HLAI类等位基因背景下识别pp65的CTL克隆。已经为这些克隆定义的表位谱系代表了大约95%的美国种族人口。要评估这些CTL表位是否主要用于普通人群，需要分析刺激CMV血清阳性的HLA型个体的外周血淋巴细胞的免疫原性，在微孔培养中，将游离肽表位以高浓度添加到PBMC中。我们已经对pp65的CTL表位使用了这种方法，并受到HLAA*0201、A*1101、A*2402、A*6901和B*0702的限制。在每个病例中，我们检查了不到五名血清阳性的健康志愿者，他们被证明对他们表达的HLA等位基因的CTL表位有反应。识别多肽和CMV感染靶点的CD8+CTL在两周内被放大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是否是该反应的一个组成部分。我们希望证明，至少5个，如果可能，10个随机选择的个体将对单个表位做出反应，这表明表达相同限制性I类等位基因的11个个体对该表位做出普遍反应的可能性。这为生产疫苗分子的方法提供了理论基础，该分子包含一个或多个这些表位，以免疫高危个体免受CMV感染。获取外周血和活检的临床程序是临床方案的核心部分，最好是在GCRC的主持下进行。我们已经开发了一组表达HLA等位基因的个体，我们从pp65和最近的pp150获得了这些等位基因的表位。我们希望进行这些研究，以确定CMV pp65和pp150的所有主要表位，这两个表位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定义一种适用于所有高危个体的多肽疫苗。我们还将继续推出新的CMV表位，以完善我们的表位曲目，确保尽可能完整地代表组成美国人口的不同种族的个人。对于该议定书，有几种有效的同意书(A-D)。

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.