Dengue Epitope Vaccine,Tetravalent & MHCII-Targeted

描述(由申请人提供)：这项多项目提案描述了几项新技术，旨在开发一种新形式的基于表位的登革热基因疫苗，编码一组选定的抗原肽单位(表位)，包含适用于所有血清型(四价)和遗传多样性的人类群体的有效疫苗所需的最低抗体和细胞抗原序列，并且缺乏与出血热相关的细胞毒性T细胞表位。结合抗原表位选择，针对MHC II隔室，以及对免疫反应的体外分析与特征良好的患者队列样本，可以预测获得一种优于基于其他策略的疫苗，并适用于其他病原体。生物信息学计算分析(项目1)将被应用于识别表位序列，这些表位序列被选择用于与代表多个人类淋巴细胞抗原(人类淋巴细胞抗原)等位基因(混杂表位)的主要组织相容类(MHC)基序簇结合，并与四种登革热血清型(泛登革热表位)中的每一种都有效。这将包括MHC II结合基序，用于将表位呈递给在免疫反应系统中发挥关键作用的CD4+辅助T细胞。还将研究CD8+细胞毒性T细胞表位的MHC I结合基序，以确定它们在预防感染中的可能作用，以及在登革出血热病原学中的拟议负面作用。 人类体外T细胞激活试验将用于分析选定的候选表位的生物相关性(项目2)。分析人类对抗原表位的反应对于疫苗开发至关重要，特别是在对人类白细胞抗原限制性T细胞表位的反应中。外周血液样本将从具有良好特征的登革热受试者队列中获得(核心B)。体外淋巴细胞刺激试验将确定那些刺激自然诱导的T细胞和B细胞的表位，并与多种HLA等位基因混杂。这些结果将被用于改进生物信息学模型，用于疾病严重程度的相关性，以及疫苗的构建。选定的表位最初将作为多肽进行测试，随后将作为核酸编码的嵌合抗原进行测试(项目4)。 一种新的聚合酶链式反应分析将用于登革热血清型和其他疾病的鉴别诊断(项目3)。适当选择患者血液样本需要对具有类似症状的其他疾病(其他黄病毒、阿拉伯病毒和猎人病毒)进行快速和准确的鉴别诊断。聚合酶链式反应(PCR)分析中的一种新的多重形式将提供病毒的快速鉴定，并另外设计用于细胞复制病毒的特异性定量。这项测试将应用于核心B队列的患者选择。一种以MHC II为靶点的四价、泛HLA、MHC II靶向登革热DNA疫苗(项目4)将把这些表位整合到针对MHC II隔室的抗原嵌合体中，通过靶向MHC II共定位的溶酶体相关膜蛋白(LAMP)的信号来激活所需的CD4+辅助T细胞，并增强免疫反应的所有手臂的CD4+、CD8+和抗体。表位映射技术将应用于鼠和人的抗血清，以确定作为中和抗体表位有效的病毒包膜蛋白的最小序列。疫苗构建将通过人类体外检测系统(项目2)验证人类登革热病毒特异性T和B细胞反应，并通过小鼠免疫验证中和抗体反应(项目4)。核心A将提供该计划的中央行政和财务管理，核心B将提供登革热患者队列的外周血液样本。此外，核心将维护一个定制的关系数据库系统，旨在支持和简化疫苗开发过程的所有方面。因此，它将包括集成的数据库“模块”，用于输入、搜索、跟踪和分析从队列患者的早期血液样本收集的关键过程数据，通过初步分析，并最终在候选登革热表位定义的LAMP嵌合体的疫苗试验中。

DESCRIPTION (provided by applicant): This multi-project proposal describes several novel technologies directed at the development of a new form of epitope-based dengue genetic vaccine encoding an ensemble of selected antigen peptide units (epitopes) containing the minimum antibody and cellular antigen sequences required for an effective vaccine applicable to all serotypes (tetravalent) and to genetically diverse human populations, and lacking cytotoxic T-cell epitopes associated with hemorrhagic fever. The combination of epitope selection, targeting to the MHC II compartment, and ex vivo analysis of the immune responses with samples of a well-characterized patient cohort can be predicted to achieve a vaccine superior to those based on other strategies, and to be applicable to other pathogens. Bioinformatic computational analysis (Project 1) will be applied to identify epitope sequences selected for binding to clusters of major histocompatability class (MHC) motifs representing multiple human lymphocyte antigen (HLA) alleles (promiscuous epitopes) and effective with each of the four dengue serotypes (pan-dengue epitopes). This will include MHC II binding motifs for epitope presentation to CD4+ helper T-cells which play a critical role in the immune response system. MHC I binding motifs of CD8+ cytotoxic T-cell epitopes will also be examined both for their possible role in prevention of infection and proposed negative role in the etiology of dengue hemorrhagic fever. A human ex vivo T-cell activation assay will be used to analyze the biological correlates of the selected epitope candidates (Project 2). Analysis of human responses to epitopes is critical for vaccine development, particularly in the response to HLA-restricted T-cell epitopes. Peripheral blood samples will be obtained from a well-characterized cohort of dengue subjects (Core B). An ex vivo lymphocyte stimulation assay will identify those epitopes that stimulate the naturally induced T- and B-cells and are promiscuous to multiple HLA alleles. The results will be used to refine the bioinformatic models, for correlations to the severity of disease, and for vaccine construction. Selected epitopes will be tested initially as peptides and subsequently as nucleic acid encoded chimeric antigens (Project 4). A novel PCR assay will be used for differential diagnosis of dengue serotypes and other diseases (Project 3). The appropriate selection of patient blood samples requires rapid and accurate differential diagnosis of other diseases with similar symptoms (other flaviviruses, arenaviruses and huntaviruses). A novel multiplex format in a polymerase chain reaction (PCR) assay will provide rapid identification of viruses and additionally is designed for specific quantification of cellular replicating virus. This assay will be applied to patient selection for the Core B cohort. A tetravalent, pan-HLA, MHC II-targeted dengue DNA vaccine (Project 4) will incorporate the epitopes into an antigen chimera directed to the MHC II compartment for the required activation of CD4+ helper T-cells by targeting signals of the lysosome-associated membrane protein (LAMP) that is colocalized by MHC II and enhances all arms of the immune response CD4+, CD8+ and antibody. Epitope mapping technologies will be applied with both mouse and human antisera to identify the minimum sequences of the viral envelope proteins effective as neutralizing antibody epitopes. The vaccine constructs will be validated for human dengue virus specific T- and B-cell responses by the human ex vivo assay system (Project 2), and by mouse immunization for neutralizing antibody response (Project 4). Core A will provide central administration and financial management of the program and Core B, the peripheral blood samples of the dengue patient cohort. Additionally, the Cores will maintain a customized relational database system designed to support and streamline all aspects of the vaccine development process. As such, it will comprise integrated database "modules" for the entry, searching, tracking, and analysis of process-critical data from the early collection of blood samples from cohort patients, through the preliminary assays, and culminating in the vaccine trials of the candidate dengue epitope-defined LAMP chimera.