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.
Showing the most recent 10 out of 38 publications
