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疫苗开发中很重要。 一项关键战略 是将抗原引导到有效的特化抗原呈递系统 细胞（APC），树突状细胞（DC），在共同的诱导位点， 粘膜免疫 该提案的主要目标是将抗原靶向于 粘膜组织和皮肤中的DC。 这将通过空间上使用 靶向DEC-205（DC细胞表面蛋白）的稳定脂质体（SSL）。 使用与抗DEC-205单克隆抗体缀合的SSL将抗原递送至DC 抗体NLDC- 145将使用体外测定进行定量：该方法 是设计一种脂质体制剂， 通过空间位阻稳定，并且具有特异性结合 DC通过抗体缀合。 除了抗体介导的靶向 DC，来自单核细胞增生李斯特菌的溶血素，溶血素O（LLO）， 将其共包封在DC免疫靶向脂质体内。 LLO 与脂质体内的抗原蛋白共包封可以介导 将蛋白质递送到抗原呈递的胞质途径中 在巨噬细胞中。 因此，含有LLO的脂质体提供了一种潜在的方式， 针对MHC I类和MHC II类限制性抗原的靶向抗原 演示文稿. 使用小鼠骨髓来源的DC和荧光 技术，申请人计划衡量的具体增强， DC通过NLDC-145缀合的脂质体摄取和部分脂质体的递送 脂质体内容物进入DC胞质溶胶。 另外，模型抗原， 卵清蛋白（OVA）将被封装在脂质体内，以监测 通过NLDC-145缀合的含LLO的脂质体和MHC递送至DC OVA的I类和MHCII类限制性抗原呈递将是 化验。 体外研究中开发的最佳制剂为 在小鼠中进行测试，定位脂质体及其在肠DC中的含量 和皮肤。 用靶向DC的SSL制剂免疫的小鼠将被免疫。 通过测量OVA特异性抗体检测粘膜免疫诱导 分泌物进入阴道液。 这里提出的脂质体制剂可以 从而开发出一种安全有效的艾滋病毒疫苗， 由抗体介导的粘膜和全身保护性免疫， 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.