ELISPOT Peptide Mapping Assay to Identify Novel Candidate CMV Vaccine Antigens

描述（由申请人提供）：对人巨细胞病毒（HCMV）的免疫是复杂的，需要体液免疫反应（主要是包膜糖蛋白抗体）和细胞免疫反应（对多种结构/调节蛋白的CD4+和CD8+反应）。由于先天性HCMV感染可导致终身残疾，因此了解保护发育中的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。一种糖蛋白B （gB）佐剂疫苗的临床试验在年轻育龄妇女中显示出一些希望，但免疫力下降和疗效不高（约50%）需要考虑其他策略。预防HCMV疾病的关键是MHC I类受限的细胞毒性CD8+ t淋巴细胞反应的发展。量化这些细胞的效应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，在接种疫苗后，测量T细胞对干扰素γ (IF- γ)的分泌是至关重要的，因为这种细胞因子在保护中起着关键作用。豚鼠巨细胞病毒（GPCMV）先天性感染模型为评估疫苗介导的保护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，主要是由于缺乏用于豚鼠研究的免疫学分析和试剂。为了解决这一缺陷，本申请的目标1建议开发一种新的IF-？ELISPOT检测豚鼠，使用一组最近开发的单克隆抗体。在目标2中，该试验将使用重叠肽库来检查和验证对已知GPCMV t细胞靶点GP83 （HCMV pp65同源物）的反应。在gpcmv感染的近交系2豚鼠的GP83特异性应答中，精确的肽表位将被绘制出来。此外，我们将使用ELISPOT和肽库来询问T细胞对GPCMV ORFs GP32、GP48、GP48a、GP55 （gB同源物）、GP82、GP99、GP122 （IE2）和GP123 （IE1）的反应。假设这些orf在豚鼠对GPCMV感染的细胞反应中很重要，因为：1)t细胞反应在HCMV感染以及其他cmv感染后频繁发生；2)在HCMV感染的情况下，这些orf同时引起CD4+和CD8+反应；3) orf在GPCMV基因组中具有较好的保守性。这些利用R03机制的实验将支持新的研究技术/方法的开发，允许对豚鼠T细胞反应进行试点和可行性研究，并使鉴定在GPCMV感染中重要的特异性肽表位成为可能。这些研究将为GPCMV的细胞免疫反应提供新的信息；将促进开发一种迄今无法用于豚鼠研究的重要测定方法；并将对改进HCMV疫苗的设计产生影响。最终，多价t细胞疫苗旨在增强基于抗体的糖蛋白疫苗所赋予的免疫力，这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.