Immune Targets During Natural Dengue Infection

描述（由申请人提供）：登革热病毒（DV）是一种正链RNA病毒，由蚊子传播，是日益严重的公共卫生问题的原因。每年约有6000万至8000万人受到感染，某些地区的感染率高达6%。该病毒可分为4种血清型（DEN 1-4），所有4种血清型均在加勒比、亚洲和美洲流行。目前还没有针对这种病毒的疫苗，但正在作出协调一致的努力，开发一种四价减毒活疫苗。与开发登革热疫苗相关的一个问题是，人们认为预先存在的非中和抗体与感染增强有关。一种方法是构建一种含有t辅助细胞、CTL和中和性B细胞表位的设计疫苗，而不含任何作为非中和性抗体靶点的B细胞表位。实现构建这种疫苗的目标的第一步将是鉴定4种血清型的T和B细胞表位和/或交叉反应性表位，这些表位可以识别与各种HLA相关的广泛免疫反应。这一申请涉及这些抗原表位的鉴定，这些抗原表位可用于未来的设计疫苗。我们的中心假设是，在DEN-3自然感染过程中，prM-E和NS-1蛋白是T细胞介导的免疫反应的靶标，并且这些蛋白中存在T辅助蛋白和CTL表位。DEN-3以及这些蛋白尚未被大力用于免疫靶点的定义，这将是首次有组织地尝试确定DEN-3的prM-E和NS-1蛋白中CD4和CD8细胞的免疫靶点。我们将使用重叠肽来鉴定20-mer Th/CTL靶点。随后将通过合成20-mer肽的缺失突变体并将其用于ELISPOT检测来鉴定最小表位。我们还将确定CD4和CD8 T细胞克隆的功能特性，如细胞因子分泌模式，阻断/减少DEN-3复制的能力以及其他血清型。

DESCRIPTION (provided by applicant): 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.