ELISPOT Peptide Mapping Assay to Identify Novel Candidate CMV Vaccine Antigens

描述（由申请方提供）：对人巨细胞病毒（HCMV）的免疫是复杂的，需要体液免疫应答（主要是针对包膜糖蛋白的抗体）和细胞免疫应答（针对多种结构/调节蛋白的CD 4+和CD 8+应答）。由于先天性HCMV感染引起的终身残疾，了解保护发育中胎儿的宿主防御决定因素对于开发有效的孕前疫苗至关重要。含佐剂的糖蛋白B（gB）疫苗的临床试验显示，在育龄期的年轻女性中有一定的前景，但免疫力下降和适度的疗效（~50%）需要考虑其他策略。保护免受HCMV疾病的关键是MHC I类限制性细胞毒性CD 8 + T淋巴细胞应答的发展。量化这些细胞的效应子功能和细胞因子谱的能力是评估HCMV疫苗有效性的关键方面。特别是，测量干扰素γ（IF-？）疫苗接种后T细胞的免疫应答是至关重要的，因为这种细胞因子在保护中起着关键作用。先天性感染的豚鼠巨细胞病毒（GPCMV）模型提供了一个有用的系统，用于评估疫苗介导的保护，但不幸的是，T细胞反应的评价一直存在问题，主要是由于缺乏免疫学检测和试剂的豚鼠研究。为了解决这一缺陷，本申请的目的1提出开发一种新的IF-？使用一组最近开发的单克隆抗体对豚鼠进行ELISPOT测定。在目的2中，该测定将用于使用重叠肽文库检查和验证对已知GPCMV T细胞靶标GP 83（HCMV pp 65同源物）的应答。将绘制GPCV感染的近交系2豚鼠的GP 83特异性应答中关键的精确肽表位。此外，我们将使用ELISPOT与肽库一起询问T细胞对GPCV ORF GP 32、GP 48、GP 48 a、GP 55（gB同源物）、GP 82、GP 99、GP 122（IE 2）和GP 123（IE 1）的应答。假设这些ORF在豚鼠对GPCV感染的细胞应答中是重要的，因为：1）T细胞应答在HCMV感染以及其他CMV感染后是频繁的; 2）这些ORF在HCMV感染的情况下引发CD 4+和CD 8+应答;以及3）ORF在GPCV基因组中是高度保守的。利用R 03机制的这些实验将支持新研究技术/方法的开发，允许豚鼠T细胞应答的试点和可行性研究，并能够鉴定在GPCMV感染中重要的特异性肽表位。这些研究将提供关于对GPCMV的细胞免疫应答的新颖的、新的信息;将促进迄今为止豚鼠研究不可用的重要测定的开发;并且将对改进的HCMV疫苗的设计具有影响。最终，旨在增强基于抗体的糖蛋白疫苗所赋予的免疫力的多价T细胞疫苗可能会改善保护婴儿免受先天性HCMV感染的前景，这是一个主要且未得到满足的公共卫生优先事项。

DESCRIPTION (provided by applicant): Immunity to human cytomegalovirus (HCMV) is complex, and requires both humoral immune responses (predominately antibody to envelope glycoproteins) and cellular immunity (CD4+ and CD8+ responses to multiple structural/regulatory proteins). Because of lifelong disabilities caused by congenital HCMV infection, understanding the host defense determinants that protect the developing fetus is critical, toward the goal of developing an effective preconception vaccine. Clinical trials of an adjuvanted glycoprotein B (gB) vaccine showed some promise in young women of childbearing age, but waning immunity and modest efficacy (~50%) necessitate consideration of other strategies. A key to protection against HCMV disease is the development of MHC class I restricted, cytotoxic CD8+ T-lymphocyte responses. The ability to quantify both the effector functions and cytokine profiles of these cells is a critical aspect of the evaluation of the effectiveness of HCMV vaccines. In particular, measuring the elaboration of interferon gamma (IF-?) by T cells following vaccination is vital, since this cytokine plays a key role in protectio. The guinea pig cytomegalovirus (GPCMV) model of congenital infection provides a useful system for evaluating vaccine-mediated protection, but, unfortunately, evaluation of T cell responses has been problematic, largely due to a lack of immunological assays and reagents for guinea pig research. To address this deficiency, aim 1 of this application proposes to develop a novel IF-? ELISPOT assays for the guinea pig, using a panel of recently developed monoclonal antibodies. In aim 2, this assay will be used to examine and validate, using overlapping peptide libraries, the response to a known GPCMV T-cell target, GP83 (HCMV pp65 homolog). The precise peptide epitope(s) critical in the GP83- specific response of GPCMV-infected inbred strain 2 guinea pigs will be mapped. In addition, we will use ELISPOT to interrogate, with peptide libraries, the T cell response to GPCMV ORFs GP32, GP48, GP48a, GP55 (gB homolog), GP82, GP99, GP122 (IE2), and GP123 (IE1). These ORFs are hypothesized to be important in the guinea pig cellular response to GPCMV infection, since: 1) T-cell responses are frequent following HCMV infection, as well as other CMVs; 2) these ORFs elicit both CD4+ and CD8+ responses in the setting of HCMV infection; and 3) the ORFs are well-conserved in the GPCMV genome. These experiments, utilizing the R03 mechanism, will support development of new research technologies/methodologies, allow pilot and feasibility studies of the guinea pig T cell response, and enable identification of specific peptide epitopes important in GPCMV infection. These studies will provide novel, new information about the cellular immune response to GPCMV; will facilitate development of an important assay heretofore unavailable for guinea pig research; and will have implications for design of improved HCMV vaccines. Eventually, polyvalent T-cell vaccines aimed at augmenting immunity conferred by antibody-based glycoprotein vaccines may improve prospects for protecting infants against congenital HCMV infection, a major and unmet public health priority.