Dengue Epitope Vaccine,Tetravalent & MHCII-Targeted

描述（由申请人提供）：该多项目提案描述了几种新技术，旨在开发一种基于表位的新型登革热基因疫苗，该疫苗编码一组选定的抗原肽单元（表位），其中包含适用于所有血清型（四价）和遗传多样化人群所需的最少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。结合表位选择，靶向MHC II隔室，以及对具有良好特征的患者队列样本的免疫应答进行体外分析，可以预测获得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）将用于鉴定选择的表位序列，这些表位序列可与代表多个人类淋巴细胞抗原（HLA）等位基因的主要组织相容性类（MHC）基序结合（混杂表位），并对四种登革热血清型（泛登革热表位）中的每一种有效。这将包括MHC II结合基序，用于向CD4+辅助性t细胞呈递表位，这在免疫反应系统中起关键作用。CD8+细胞毒性t细胞表位的MHC I结合基序也将被检查其在预防感染中的可能作用以及在登革出血热病因学中的负面作用。

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.