New approach to T cell study for Vaccine in Uganda

说明（由申请人提供）：艾滋病在世界范围内已达到流行病的程度，研制疫苗似乎是在世界范围内遏制艾滋病毒-1感染的最有效办法。目前用于引起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，其中许多抗原在非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。该资助旨在评估一种新的HIV抗原表达载体，该载体可能避免使用重组痘苗病毒或大量重叠的合成肽。我们评估了含有HIV-1抗原的炭疽芽孢杆菌的遗传解毒致死毒素（LFn-HIV），发现CTL反应是可重复检测的。生产含有多达500个氨基酸的HIV抗原的LFn-HIV在技术上简单，相对便宜，因此是非b进化支抗原和多表位结构的有吸引力的替代来源。因此，我们建议在功能和定量分析中评估LFn-HIV，以研究细胞免疫反应。这项技术将被探索以解决抗原表达的功效以及抗原加工和递呈的潜在途径，并最终在乌干达引入这一概念，开始评估跨进化的免疫反应。

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.