New approach to T cell study for Vaccine in Uganda

描述(申请人提供)：艾滋病在全世界已经达到流行的程度，疫苗的开发似乎是在全球范围内遏制艾滋病毒-1感染的最有效的方法。目前用于激发T细胞反应的HIV特异性抗原包括重组痘苗病毒、重组全蛋白或合成肽，其中许多在非B亚型中是不存在的。这些问题对在资源贫乏的国家进行疫苗和艾滋病毒免疫学研究造成了重大障碍。这笔赠款旨在评估一种新的艾滋病毒抗原表达载体，该载体可能会避免使用重组牛痘病毒或大量重叠的合成多肽。我们评估了含有HIV-1抗原的炭疽芽孢杆菌(LFN-HIV)的基因解毒致死毒素(LFN-HIV)，发现CTL反应是可重复检测的。生产含有高达500个氨基酸的HIV抗原的LFN-HIV技术简单，相对便宜，因此是一种有吸引力的非B分支抗原和多表位构建的替代来源。因此，我们建议从功能和定量两个方面评估LFN-HIV，以研究细胞免疫反应。将探索这项技术，以解决抗原表达的有效性以及抗原处理和呈递的潜在途径，并最终将这一概念引入乌干达，开始评估跨分支免疫反应。 这项建议的具体目标是： 1)利用基因解毒的炭疽致死因子，开发和测试新型MHC-I类抗原递送系统。我们将开始使用乌干达定义良好的CTL细胞系或克隆来开发和测试A和D亚型结构。 2)评价LFN-HIV的抗原加工和提呈。我们将确定胞浆进入所需的最佳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，我们将探索LFN-HIV是否引起T辅助反应，以及这一抗原处理途径是否与MHC-I类途径部分共享。 3)在资源有限的地方，如乌干达，测试LFN-HIV。我们将介绍LFN-HIV在多个分支(A、B和D)中的使用，并使用ELISPOT和基于流式的细胞术来评估该人群中的跨分支活动水平。

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.