Systemic and Mucosal HIV-Immunity by Hsp-Gp96 Vaccines

描述(申请人提供)：热休克蛋白gp96是一种重要的内质网(ER)多肽和蛋白质伴侣。部分gp96相关多肽被修剪并装载到MHC I上，用于将抗原呈递给CD8细胞。与抗原肽相关的游离gp96具有很强的免疫原性。免疫系统使用ARC和DC上的gp96受体作为一个复杂的系统来检测细胞损伤，并通过吞噬热休克蛋白并将其交叉呈递给CD8细胞来监测与释放的热休克蛋白相关的抗原肽(交叉启动)。与完整蛋白相比，多肽与gp96的结合可将CD8细胞的抗原交叉免疫增强10,000至1,000,000倍。通过用lgG1-Fc部分取代人gp96的KDEL保留信号，我们产生了从转基因细胞分泌的gp96-融合蛋白(gp96-LG)。我们的研究表明，gp96-LG能在体内介导强大的、抗原特异性的CD8-CTL扩增，引起肿瘤排斥反应，并产生长期的抗肿瘤免疫。Gp96-lg疫苗治疗癌症的临床试验正在进行中。我们的数据显示，细胞分泌gp96-LG会触发树突状细胞的募集和激活。DC募集和激活NK细胞，并诱导Th1环境，使同源CD8 CTL在交叉呈递的刺激下强烈扩张，最初的gp96相关多肽被DC吞噬。我们还发现，腹膜免疫后分泌gp96-Ig的细胞在包括上皮内CD8细胞(IEL)在内的粘膜部位诱导出强烈的抗原特异性CD8反应。结果表明，分泌gp96-LG的细胞可诱导基于黏膜和全身CD8-CTL的免疫。因此，由HIV抗原表达细胞分泌的gp96-LG有望在系统和粘膜部位提供强大的抗HIV免疫，并提供免受感染的保护。这些假设将在申请中得到检验。在具体目标1中，我们将研究gp96免疫途径与不同系统和粘膜部位的抗原特异性IgA和CD8反应的关系。此外，还将检查gp96疫苗的多特异性。在特定的目标2中，我们将研究gp96疫苗诱导的粘膜和系统部位的CD8记忆反应，并将CD8反应与对病毒攻击的抵抗力相关联。该模型系统将使用HLAA2转基因小鼠和表达牛痘病毒的HIV。

DESCRIPTION (provided by applicant): Heat shock protein gp96 is an important endoplasmic reticulum (ER) chaperone for peptides and proteins. A fraction of the gp96-associated peptides are trimmed and loaded onto MHC I for antigen presentation to CD8 cells. Cell free gp96 associated with antigenic peptides is highly immunogenic. The immune system uses gp96-receptors on ARC and DC as a sophisticated system to detect cell damage and monitor antigenic peptides that are associated with liberated heat shock proteins by engulfing them and cross presenting them to CD8 cells (cross priming). Compared to intact proteins, the association of peptides with gp96 enhances antigen cross priming of CD8 cells between 10,000 to 1 million fold. By replacing the KDEL retention signal of human gp96 with the lgG1-Fc portion we have generated a gp96-fusion protein (gp96-lg) that is secreted from transfected cells. We have shown that gp96-lg secreted from transfected tumor cells in vivo mediated strong, antigen specific CD8-CTL expansion, caused tumor rejection and generated long term anti-tumor immunity. Clinical trials with gp96-lg vaccines in cancer are ongoing. Our data shows that cell secreted gp96-lg triggers recruitment and activation of dendritic cells. DC recruit and activate NK cells and induce a Th1 environment for strong expansion of cognate CD8 CTL stimulated by cross presented, originally gp96-associated peptides engulfed by DC. We also show that cell secreted gp96-Ig upon intraperitoneal immunization induces strong antigen specific CD8 responses in mucosal sites including intraepithelial CD8 cells (IEL). The data indicates that cell secreted gp96-lg induces both, mucosal and systemic CD8-CTL based immunity. Gp96-lg secreted from HIV-antigen expressing cells therefore is expected to provide strong anti HIV immunity systemically and at mucosal sites and provide protection from infection. These hypotheses will be examined in the application. In specific Aim 1, we will examine the route of gp96-immunization in relation to antigen specific IgA and CD8 responses at various systemic and mucosal sites. In addition, polyspecificity of gp96 vaccines will be examined. In specific aim 2, we will study the gp96-vaccine induced CD8 memory response at mucosal and systemic sites and correlate CD8 responses with resistance to viral challenge. The model system will use HLA A2 transgenic mice and HIV expressing vaccinia virus.