Dengue virus (DV), a positive stranded RNA virus, is transmitted by the mosquitoes, and is the cause of a growing public health problem. Approximately 60-80 million persons are infected annually, and rates of infection are as high as 6% in some areas. The virus can be divided into 4 serotypes (DEN 1-4), and all 4 serotypes circulate in Caribbean, Asia and the Americas. There is no vaccine available against this virus, but there are concerted efforts being undertaken to develop a tetravalent live attenuated vaccine. One of the problems associated with the development of dengue vaccine is that pre-existing non-neutralizing antibodies are thought to be associated with enhanced infection. One way out would be to construct a designer vaccine that contains T-helper, CTL and neutralizing B-Cell epitopes, and does not contain any B cell-epitope those are targets of non-neutralizing antibodies. First step towards realizing the goal of constructing such a vaccine would be characterization of T and B cell epitopes from the 4 serotypes and/or cross reactive epitopes that can be recognized in association with variety of HLA for generalized immune responses. This application deals with identification of such epitopes that can be used in future designer vaccine. Our central hypothesis is that prM-E and NS-1 protein are targets of T cell mediated immune response during natural infection with DEN-3, and there are T-helper and CTL epitopes in these proteins. DEN-3 as well as these proteins have not been vigorously pursued for the definition of immune targets, and this will be the first organized attempt to identify immune targets of CD4 and CD8 cells in prM-E and NS-1 protein of DEN-3. We will use overlapping peptides to identify 20-mer Th/CTL targets. This will be followed by identification of minimal epitope by synthesizing deletion mutant of the 20-mer peptides and using them in ELISPOT assay. We will also determine functional properties of the CD4 and CD8 T cell clones like cytokine secretion pattern, ability to block/reduce replication of DEN-3 as well as other serotypes. ? ?
