ELISPOT Peptide Mapping Assay to Identify Novel Candidate CMV Vaccine Antigens

ELISPOT 肽图分析法鉴定新型候选 CMV 疫苗抗原

• 批准号: 9016570
• 负责人: Mark R. Schleiss
• 金额: $ 7.52万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9016570
• 关键词: Address; Animal Model; Antibodies; Antibody Response; Antigens; Biological Assay; CD8-Positive T-Lymphocytes; CD8B1 gene; Cavia; Cells; Cellular Immunity; Child health care; Clinical Trials; Communicable Diseases; Complex; Congenital Abnormality; Contracts; Controlled Study; Coupled; Cytomegalovirus; Cytomegalovirus Infections; Cytomegalovirus Vaccines; Data; Development; Disease; Effectiveness; Ensure; Enzyme-Linked Immunosorbent Assay; Epitopes; Evaluation; Feasibility Studies; Female of child bearing age; Fetus; Frequencies; Future; Generations; Genes; Genome; Gifts; Glycoproteins; Goals; Guinea pig cytomegalovirus; Health; Homologous Gene; Host Defense; Human; Hybridomas; Immune; Immune Targeting; Immune response; Immunity; Inbred Strain; Infant; Infection; Interferon Type II; Interferons; Investigation; Knock-out; Knowledge; Laboratories; Libraries; MHC Class I Genes; Maps; Maternal-Fetal Transmission; Measures; Mediating; Mental Retardation; Methodology; Modeling; Molecular Conformation; Monoclonal Antibodies; Mus; N-terminal; Neurologic Symptoms; Newborn Infant; Open Reading Frames; Parents; Peptide Library; Peptide Mapping; Peptides; Phase II Clinical Trials; Pilot Projects; Placenta; Play; Preclinical Testing; Pregnant Women; Primary Infection; Process; Production; Public Health; Reagent; Recombinants; Research; Rodent; Specificity; Splenocyte; Subunit Vaccines; System; T cell response; T-Lymphocyte; Technology; Testing; United States; Vaccination; Vaccine Antigen; Vaccine Clinical Trial; Vaccine Design; Vaccine Research; Vaccines; Validation; Virus; Western Blotting; base; congenital cytomegalovirus; congenital infection; cytokine; cytotoxic; deafness; design; disability; efficacy testing; enzyme linked immunospot assay; gene product; genetic regulatory protein; improved; injured; insight; interest; mutant; novel; novel vaccines; pre-clinical; preclinical efficacy; prevent; public health priorities; recombinant peptide; research study; response; vaccine evaluation; vaccine trial; young woman

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.

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（主要是针对包膜糖蛋白的抗体）和细胞免疫（针对多种结构/调节蛋白的CD4+和CD8+反应）。由于先天性 HCMV 感染会导致终身残疾，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孕前疫苗的目标至关重要。含佐剂的糖蛋白 B (gB) 疫苗的临床试验显示出对年轻育龄女性的一些希望，但免疫力减弱且疗效有限（约 50%），因此需要考虑其他策略。预防 HCMV 疾病的关键是发展 I 类 MHC 限制性细胞毒性 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，测量疫苗接种后 T 细胞产生的干扰素 γ (IF-α) 至关重要，因为这种细胞因子在保护中发挥着关键作用。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，很大程度上是由于缺乏用于豚鼠研究的免疫测定和试剂。为了解决这一缺陷，本申请的目标 1 提出开发一种新颖的 IF-？使用一组最近开发的单克隆抗体对豚鼠进行 ELISPOT 检测。在目标 2 中，该测定将用于使用重叠肽库检查和验证对已知 GPCMV T 细胞靶标 GP83（HCMV pp65 同源物）的反应。将绘制在感染 GPCMV 的近交系 2 豚鼠的 GP83 特异性反应中至关重要的精确肽表位。此外，我们将使用 ELISPOT 和肽库来询问 T 细胞对 GPCMV ORF GP32、GP48、GP48a、GP55（gB 同源物）、GP82、GP99、GP122 (IE2) 和 GP123 (IE1) 的反应。假设这些 ORF 在豚鼠细胞对 GPCMV 感染的反应中很重要，因为： 1) HCMV 以及其他 CMV 感染后 T 细胞反应频繁； 2) 这些 ORF 在 HCMV 感染的情况下引发 CD4+ 和 CD8+ 反应； 3) ORF 在 GPCMV 基因组中保存完好。这些利用 R03 机制的实验将支持新研究技术/方法的开发，允许对豚鼠 T 细胞反应进行试点和可行性研究，并能够鉴定在 GPCMV 感染中重要的特定肽表位。这些研究将提供有关 GPCMV 细胞免疫反应的新颖、新信息；将促进迄今为止无法用于豚鼠研究的重要测定方法的开发；并将对改良 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，这是一个重大且尚未得到满足的公共卫生优先事项。