LIPOSOMAL TARGETING OF ANTIGENS TO DENDRITIC CELLS

描述(改编自申请人的摘要)：抗体的诱导-和 细胞毒性T淋巴细胞介导的保护性粘膜免疫以及 系统免疫，在HIV疫苗的开发中非常重要。一项关键战略 将抗原定向到强效的专门化抗原呈递 细胞(APC)，树突状细胞(DC)，在共同的诱导部位 粘膜免疫。这项提案的主要目标是将抗原定位于 粘膜组织和皮肤中的DC。这将通过使用消毒药来完成 针对DC细胞表面蛋白DEC-205的稳定脂质体(SSL)。 用抗DEC-205单抗偶联的SSL向DC递送抗原 抗体NLDC-145将使用体外检测方法进行定量：方法 是设计一种最小限度地被巨噬细胞摄取的脂质体配方 通过空间稳定，它有能力特异性地结合到 通过抗体偶联获得DC。除了抗体介导的靶向 单产李斯特氏菌的溶血素DC，LLO，将 共包裹在DC免疫靶向脂质体中。您好 脂质体与抗原蛋白共包裹可介导 将蛋白质送入胞浆中的抗原提呈途径 在巨噬细胞中。因此，含有LLO的脂质体提供了一种潜在的方法 MHC I类和MHC II类限制性抗原的靶向抗原 演示文稿。利用小鼠骨髓来源的树突状细胞和荧光 技术，申请者计划测量两个具体的增强 树突状细胞通过NLDC-145连接的脂质体摄取和部分递送 脂质体内容物进入DC胞浆。另外，一种模型抗原， 卵清蛋白(OVA)，将被包裹在脂质体中以监测 NLDC-145-偶联、含LLO的脂质体和MHC向DC的传递 OVA的I类和MHC II类限制性抗原提呈将是 化验过了。在体外研究中开发的最佳配方将是 小鼠体内定位脂质体及其在肠道DC中的含量 还有皮肤。用DC靶向SSL配方免疫的小鼠将被 通过测量卵清蛋白特异性抗体来检测粘膜免疫诱导 分泌物进入阴道液。这里提出的脂质体制剂可以 导致安全而有效的艾滋病毒疫苗的开发，这种疫苗可以诱导 抗体和抗体介导的粘膜和系统保护性免疫 细胞毒性T淋巴细胞反应。

DESCRIPTION (adapted from applicant's abstract): Induction of antibody- and cytotoxic T lymphocyte-mediated protective mucosal immunity, as well as systemic immunity, is important in HIV vaccine development. A key strategy will be to direct antigens to the potent specialized antigen presenting cells (APC), the dendritic cells (DC), in the common inductive sites of mucosal immunity. The major goal of this proposal is to target antigens to DC in mucosal tissues and skin. This will be done using sterically stabilized liposomes (SSL) targeted to DEC-205, a DC cell surface protein. Delivery of antigen to DC, using SSL conjugated with anti-DEC-205 monoclonal antibody NLDC- 145, will be quantified using in vitro assays: the approach is to devise a liposome formulation that has minimal uptake by macrophages via steric stabilization, and that has the ability to specifically bind to DC via antibody conjugation. In addition to antibody-mediated targeting to DC, the hemolysin from Listeria monocvtogenes, listeriolysin O (LLO), will be co-encapsulated inside the DC-immunotargeted liposomes. LLO co-encapsulated with antigenic protein inside liposomes can mediate the delivery of the protein into the cytosolic pathway of antigen presentation in macrophages. Thus, the LLO-containing liposomes offer a potential way of targeting antigens for both MHC class I- and MHC class II-restricted antigen presentation. Using mouse bone marrow-derived DC and fluorescence techniques, the applicants plan to measure both the specific enhancement of liposome uptake by DC via NLDC-145 conjugation and the delivery of a portion of liposomal content into DC cytosol. Additionally, a model antigen, ovalbumin (OVA), will be encapsulated inside liposomes to monitor the delivery to DC by the NLDC-145-conjugated, LLO-containing liposomes, and MHC class I- and MHC class II-restricted antigen presentation of OVA will be assayed. The optimal formulation developed in the in vitro studies will be tested in mice, localizing liposomes and their contents in DC of intestine and skin. Mice immunized with the DC-targeted SSL formulations will be tested for induction of mucosal immunity, by measuring OVA-specific antibody secretion into vaginal fluid. The liposome formulations proposed here could lead to the development of a safe yet efficient HIV vaccine that can elicit both mucosal and systemic protective immunity mediated by antibody and cytotoxic T lymphocyte responses.