RTB-mediated delivery: Orchestrating antigen trafficking to enhance cell immunity

描述（由申请人提供）：疫苗仍然是我们预防全球传染病的最有效工具。亚单位疫苗被认为比传统的全生物体灭活或减毒疫苗更安全，但通常缺乏提供长期保护的免疫原性。因此，迫切需要新的疫苗载体和佐剂来增强亚单位疫苗的效力，特别是那些能够指导强细胞介导免疫（CMI）的疫苗。这个探索性项目将评估基于植物凝集素的新型抗原载体RTB和RTBER的效用，以选择性地协调基于亚细胞运输和相关疫苗抗原的免疫呈递的所需免疫反应。RTB是蓖麻毒素的无毒碳水化合物结合亚基B，已被证明可有效介导相关疫苗抗原的经黏膜递送，并引发强抗原特异性抗体介导的免疫。为了特异性地增强CMI反应的诱导，RTB被修饰为包含c端KDEL内质网（ER）检索基序（称为RTBER）。RTB主要在内噬作用后在内体/溶酶体区室（MHC II抗原加工部位）积累，与之相反，RTBER应该将相关抗原“有效载荷”重定向到内质网/细胞质界面（MHC I抗原加工部位）。RTB和RTBER都能促进相关抗原在粘膜表面的主动摄取并进入免疫应答细胞。本项目验证了RTBER作为疫苗抗原载体，通过逆行ER途径选择性动员抗原，通过MHC I途径高效加工抗原并呈递给CD8+ t细胞，从而产生强大的细胞介导免疫的假设。甲型流感核蛋白（NP）是需要强CMI保护的甲型流感的潜在“通用”抗原，将被用作模型抗原，并将确定RTB与RTBER在选择性协调NP特异性免疫反应中的影响。该项目将包括1)在一个简单的基于植物的生物生产系统中生产重组NP:RTB融合蛋白，2)纯化和表征重组产品，以及3)评估小鼠在鼻内给予对照、NP:RTB和NP:RTBER免疫原制剂后引发的免疫反应。证明RTBER在介导强np特异性CMI反应中的有效性是未来多株流感挑战/疾病保护试验的关键先决条件。这项研究如果成功，将有可能确定一种新的策略，用于指导粘膜递送亚单位疫苗的强细胞介导免疫，并为后续实验提供基础，重点是开发甲型流感通用疫苗。

DESCRIPTION (provided by applicant): Vaccines remain our most effective tool for global prevention of infectious disease. Subunit vaccines are considered safer than traditional killed or attenuated whole organism vaccines but typically lack the immunogenicity to provide long-term protection. Thus, there is an urgent need for new vaccine carriers and adjuvants that enhance the efficacy of subunit vaccines, especially those capable of directing strong cell- mediated immunity (CMI). This exploratory project will assess the utility of novel plant lectin-based antigen carriers, RTB and RTBER, to selectively orchestrate desired immune responses based on manipulation of subcellular trafficking and immune presentation of associated vaccine antigens. RTB, the non-toxic carbohydrate binding subunit B of the ricin toxin, has been shown to effectively mediate transmucosal delivery of associated vaccine antigens and elicit strong antigen-specific antibody-mediated immunity. In efforts to specifically enhance induction of CMI responses, RTB was modified to contain a C-terminal KDEL endoplasmic reticulum (ER) retrieval motif (termed RTBER). In contrast to RTB which predominately accumulates in endosomal/lysosomal compartments (the site of MHC II antigen processing) following endocytosis, RTBER should redirect associated antigen 'payload' to the ER/cytosol interface (the site of MHC I antigen processing for CMI). Both RTB and RTBER facilitate active uptake of associated antigens across mucosal surfaces and into immune responsive cells. This project tests the hypothesis that using RTBER as the vaccine antigen carrier will selectively mobilize the antigen through the retrograde ER pathway for efficient antigen processing and presentation to CD8+ T-cells via the MHC I pathway resulting in strong cell-mediated immunity. The influenza A nucleoprotein (NP), a potential "universal" antigen for influenza A requiring strong CMI for protection, will be used as the model antigen and the impact of RTB versus RTBER in selectively orchestrating NP-specific immune responses will be determined. The project will encompass 1) production of recombinant NP:RTB fusion proteins in a facile plant-based bioproduction system, 2) purification and characterization of the recombinant products, and 3) assessment of elicited immune responses in mice following intranasal administration of control, NP:RTB, and NP:RTBER immunogen formulations. Demonstrating RTBER efficacy in mediating strong NP-specific CMI responses is a key prerequisite to future multi-strain influenza challenge/disease protection trials. This research, if successful, will potentially identify a novel strategy for directing strong cell mediated immunity of mucosally delivered subunit vaccines and provide the foundation for follow-on experiments focused on development of universal vaccines for influenza A. PUBLIC HEALTH RELEVANCE: Vaccines represent one of the most effective approaches for protection against infectious disease agents. The proposed exploratory project will test the potential of the RTB plant lectin (the non-toxic carbohydrate binding subunit B of ricin) to facilitate both the delivery of associated vaccine antigens into immune responsive cells and to differentially "present" vaccine antigens to specific arms of the immune system. If successful, this research could lead to new approaches for 'needle-free' vaccine delivery and efficacy that enhance adaptive cell-mediated immunity, considered critical for protection against many viral disease agents and for the development of broadly effective "universal" flu vaccines.