New approach to T cell study for Vaccine in Uganda

描述（由申请人提供）：艾滋病已达到世界范围的流行比例，疫苗的开发似乎是最有效的方法来遏制艾滋病毒1感染世界各地。目前用于引发T细胞应答的HIV特异性抗原包括重组牛痘病毒、重组全蛋白或合成肽，其中许多在非B亚型中不可用。这些问题对在资源贫乏的国家开展疫苗和艾滋病毒免疫学研究造成了重大障碍。该基金旨在评估一种新型HIV抗原表达载体，该载体可能会取代重组牛痘病毒或大量重叠合成肽的使用。我们评估了含有HIV-1抗原的炭疽杆菌的遗传脱毒致死毒素（LFn-HIV），发现CTL应答是可重复检测的。含有多达500个氨基酸的HIV抗原的LFn-HIV的生产在技术上是简单的，相对便宜的，并且因此是非B进化枝抗原以及多表位构建体的有吸引力的替代来源。因此，我们建议在功能和定量测定中评估LFn-HIV，以研究细胞免疫应答。将探索该技术以解决抗原表达的功效以及抗原加工和呈递的潜在途径，并最终在乌干达引入该概念以开始评估交叉进化枝免疫应答。 这项建议的具体目标是： 1)利用基因脱毒的炭疽致死因子开发并测试一种新的MHC I类抗原递送系统。我们将开始使用来自乌干达的明确定义的CTL细胞系或克隆来开发和测试亚型A和D构建体。 2)探讨LFn-HIV的抗原加工和呈递功能。我们将确定进入胞质所需的最佳LFn序列。我们还将确定不同的APC是否更有效地表达LFn-HIV。最后，我们将探讨是否LFn-HIV eliminating辅助性T细胞反应，以及这种抗原处理途径是否与MHC I类途径部分共享。 3)在乌干达等资源有限的环境中测试LFn-HIV。我们将介绍多个进化枝（A、B和D）LFn-HIV的使用，并使用ELIspot和流式细胞术评估该群体中跨进化枝活性的水平。

DESCRIPTION (provided by applicant): AIDS has reached epidemic proportions worldwide and the development of a vaccine appears the most effective approach to curbing HIV-1 infection worldwide. Current HIV-specific antigens used to elicit T cell responses included recombinant vaccinia viruses, recombinant whole proteins or synthetic peptides, many of which are not available in non-B subtypes. These issues raise significant obstacles to conducting vaccine and HIV immunology studies in resource-poor countries. This grant intends to evaluate a novel HIV antigen expression vector that might obviate the use of recombinant vaccinia viruses or large pools of overlapping synthetic peptides. We evaluated genetically detoxified lethal toxin of Bacillus anthracis containing HIV-1 antigen (LFn-HIV) and found that CTL responses are reproducibly detectable. Production of LFn-HIV containing HIV antigens up to 500 amino acids is technically simple, relatively inexpensive, and is, therefore, an attractive alternative source of non-B clade antigens as well as multiple-epitopes construct. We therefore propose to evaluate LFn-HIV in both functional and quantitative assays to study the cellular immune response. This technology will be explored to address the efficacy of antigen expression as well as potential pathway of the antigen processing and presentation, and finally introduce the concept in Uganda to begin evaluating cross-clade immune response. The specific aims of this proposal are: 1) To develop and test a new MHC class I antigen delivery system using genetically detoxified anthrax lethal factor. We will begin to develop and test subtype A and D constructs using well defined CTL cell lines or clones from Uganda. 2) To evaluate the antigen processing and presentation of LFn-HIV. We will identify the optimal LFn sequence required for cytosolic entry. We will also determine whether different APCs express LFn-HIV more efficiently. Finally we will explore whether LFn-HIV elicits T helper response and whether this antigen processing pathway is partially shared with the MHC class I pathway. 3) To test the LFn-HIV in a resource-limited setting such as Uganda. We will introduce the use of LFn-HIV of multiple clades (A, B and D) and assess the level of cross-clade activity in this population using both ELIspot and flow based cytometry.