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

描述(由申请人提供)：疫苗仍然是我们在全球预防传染病的最有效工具。亚单位疫苗被认为比传统的灭活疫苗或减毒整体疫苗更安全，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，特别是那些能够引导强大的细胞免疫(CMI)的疫苗。这一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的实用性，以基于对相关疫苗抗原的亚细胞转运和免疫递呈的操纵，选择性地编排所需的免疫反应。RTB是蓖麻毒素的无毒碳水化合物结合亚基B，已被证明能有效地介导相关疫苗抗原的经粘膜递送，并诱导强大的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被修改为包含一个C端KDEL内质网(ER)检索基序(称为RTBER)。与RTB不同的是，RTB主要聚集在内吞作用后的内体/溶酶体室(MHC II抗原加工的部位)，RTBER应将相关抗原“有效载荷”重定向到ER/细胞质界面(CMI的MHC I抗原加工部位)。RTB和RTBER都能促进相关抗原跨粘膜表面和免疫反应细胞的主动摄取。本项目验证了一种假设，即使用RTBER作为疫苗抗原载体，将通过逆行ER途径选择性地动员抗原，从而有效地处理抗原并通过MHC I途径呈递给CD8+T细胞，从而产生强大的细胞免疫。甲型流感核蛋白(NP)是一种潜在的甲型流感“通用”抗原，需要强大的CMI进行保护，将被用作模型抗原，并将确定RTB和RTBER在选择性协调NP特异性免疫反应方面的影响。该项目将包括1)在一个简单的植物生物生产系统中生产重组NP：RTB融合蛋白，2)重组产品的纯化和鉴定，以及3)评估鼻腔注射对照、NP：RTB和NP：RTBER免疫原制剂后在小鼠身上引发的免疫反应。证明RTBER在调节强大的NP特异性CMI反应方面的有效性是未来多毒株流感挑战/疾病保护试验的关键先决条件。如果这项研究成功，将可能确定一种新的策略来引导粘膜递送亚单位疫苗的强大细胞介导免疫，并为专注于开发通用甲型流感疫苗的后续实验提供基础。

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.