Immune Targets During Natural Dengue Infection

描述(申请人提供)：登革病毒(DV)是一种阳性滞留的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6000-8000万人感染，一些地区的感染率高达6%。该病毒可分为4个血清型(DEN1-4)，所有4个血清型都在加勒比海、亚洲和美洲流行。感染一种毒株并不能提供对其他毒株的保护性免疫。缺乏有效的登革热疫苗有几个原因。众所周知，在二次感染过程中，预先存在的非中和抗体确实会增强病毒的复制。有鉴于此，理想的疫苗将只包括保护性表位，以避免有害影响。然而，构建多表位疫苗的想法目前还不能实现，因为还没有在所有4种血清型中鉴定出足够数量的免疫靶点。这项申请涉及识别这样的靶点，但我们的建议仍然局限于T细胞表位。我们计划在DEN-3的PRM-E和NS-1蛋白中确定CTL和/或T辅助表位。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点，并且这些蛋白中存在T辅助细胞和CTL表位。我们将开发DV3特异性T细胞株和克隆。这些克隆将被鉴定为CD4和CD8表型，并用于在ELISPOT试验中鉴定T辅助和CTL表位。这些克隆将首先针对包含全长PrM-E和NS1的重叠多肽进行测试，然后针对缺失多肽进行筛选。将确定新确定的表位的人类白细胞抗原限制性元件。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. Infection with one strain does not provide protective immunity against other strains. There are several reasons for the lack of an effective vaccine against DV. It is known that pre-existing non neutralizing antibodies indeed enhance the virus replication during secondary infection. In view of this an ideal vaccine will include only protective epitopes so that deleterious effect can be avoided. However idea of construction of a polyepitope vaccine cannot be realized at this time because adequate numbers of immune targets have not been identified in all 4 serotypes. This application deals with identification of such targets but our proposal remains limited to T cell epitopes. We plan to identify CTL and/or T-helper epitopes in prM-E and NS-1 protein of the DEN-3. 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. We will develop DV3-specific T cell lines and clones. These clones will be characterized for CD4 and CD8 phenotype and used for the identification of T-helper and CTL epitopes in ELISPOT assays. The clones will be first tested against overlapping peptides encompassing full length prM-E and NS1, followed by screening against deletion peptides. The HLA restriction element for the newly identified epitopes will be determined. 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.