Systemic and Mucosal HIV-Immunity by Hsp-Gp96 Vaccines

描述（由申请人提供）：热休克蛋白gp96是肽和蛋白质的重要内质网（ER）伴侣。gp96相关肽的一部分被修剪并装载到MHC I上，用于抗原呈递到CD8细胞。与抗原肽相关的无细胞gp96具有高度的免疫原性。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和监测与释放的热休克蛋白相关的抗原肽，通过吞噬它们并将它们交叉呈递给CD8细胞（交叉启动）。与完整的蛋白相比，肽与gp96的结合使CD8细胞的抗原交叉引物增强了1万到100万倍。通过用lgG1-Fc部分取代人gp96的KDEL保留信号，我们产生了一个gp96融合蛋白（gp96-lg），该蛋白从转染的细胞中分泌。我们已经证明，从转染的肿瘤细胞分泌的gp96-lg在体内介导强烈的抗原特异性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疫苗在癌症中的临床试验正在进行中。我们的数据表明，细胞分泌的gp96-lg触发树突状细胞的招募和激活。DC招募和激活NK细胞，诱导Th1环境，同源CD8 CTL通过被DC吞噬的交叉呈现的gp96相关肽刺激强烈扩增。我们还发现，经腹腔免疫后分泌gp96-Ig的细胞在粘膜部位（包括上皮内CD8细胞（IEL））诱导了强烈的抗原特异性CD8反应。这些数据表明，细胞分泌的gp96-lg可诱导粘膜和全身CD8-CTL免疫。因此，从表达HIV抗原的细胞中分泌的Gp96-lg有望在全身和粘膜部位提供强大的抗HIV免疫，并提供免受感染的保护。这些假设将在应用程序中进行检验。在特定的Aim 1中，我们将研究gp96免疫途径与各种全身和粘膜部位抗原特异性IgA和CD8反应的关系。此外，还将检验gp96疫苗的多特异性。在特定目标2中，我们将研究gp96疫苗在粘膜和全身部位诱导的CD8记忆反应，并将CD8反应与病毒攻击的抗性联系起来。该模型系统将使用HLA A2转基因小鼠和表达痘苗病毒的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.