ELISPOT Peptide Mapping Assay to Identify Novel Candidate CMV Vaccine Antigens

描述(申请人提供)：对人类巨细胞病毒(HCMV)的免疫是复杂的，既需要体液免疫反应(主要是针对包膜糖蛋白的抗体)，也需要细胞免疫(对多种结构/调节蛋白的CD4+和CD8+反应)。由于先天性巨细胞病毒感染导致终身残疾，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早孕疫苗的目标至关重要。一种佐剂糖蛋白B(GB)疫苗的临床试验显示，在育龄的年轻女性中有一些希望，但免疫力减弱和适度的疗效(~50%)需要考虑其他策略。预防HCMV病的一个关键是发展MHC-I类限制性的细胞毒性CD8+T淋巴细胞反应。量化这些细胞的效应器功能和细胞因子谱的能力是评估HCMV疫苗有效性的一个关键方面。特别是，测量干扰素-γ的精确度(IF-？)免疫后的T细胞是至关重要的，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)模型为评估疫苗介导的保护作用提供了一个有用的系统，但不幸的是，T细胞反应的评估一直存在问题，这主要是由于缺乏免疫学分析和试剂用于豚鼠研究。为了解决这一不足，本申请的目标1建议开发一种新颖的IF-？Elispot使用一组最近开发的单抗对豚鼠进行检测。在目标2中，这一检测将用于使用重叠的多肽库来检查和验证对已知GPCMV T细胞靶标GP83(HCMV pp65同源)的反应。在GPCMV感染的近交系2豚鼠的GP83特异性反应中关键的精确多肽表位(S)将被绘制出来。此外，我们将使用ELISPOT通过多肽库来询问T细胞对GPCMV ORF GP32、GP48、GP48a、GP55(GB同源物)、GP82、GP99、GP122(IE2)和GP123(IE1)的反应。这些ORF在豚鼠对GPCMV感染的细胞反应中可能是重要的，因为：1)在HCMV感染和其他CMV感染后，T细胞反应频繁；2)在HCMV感染的背景下，这些ORF同时激发CD4+和CD8+反应；以及3)ORF在GPCMV基因组中被很好地保守。这些实验利用R03机制，将支持新研究技术/方法的开发，允许对豚鼠T细胞反应进行中试和可行性研究，并能够识别在GPCMV感染中重要的特定多肽表位。这些研究将提供有关GPCMV的细胞免疫反应的新的、新的信息；将促进一种迄今为止尚不适用于豚鼠研究的重要分析方法的发展；并将对改进的HCMV疫苗的设计产生影响。最终，旨在增强基于抗体的糖蛋白疫苗产生的免疫力的多价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，这是一个主要的、尚未实现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.