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

RTB 介导的递送：协调抗原运输以增强细胞免疫

• 批准号: 8526364
• 负责人: CAROLE L. CRAMER
• 金额: $ 19.18万
• 依托单位: ARKANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8526364
• 关键词: Adjuvant; Affinity Chromatography; Agrobacterium; Antibodies; Antibody Formation; Antigen Presentation; Antigen Presentation Pathway; Antigens; Attenuated; Avian Influenza; Binding; Biological Assay; C-terminal; C57BL/6 Mouse; CD8B1 gene; CTL assay; Carbohydrates; Cells; Cellular Immunity; Chimeric Proteins; Cholera Toxin; Column Chromatography; Communicable Diseases; Cytosol; DNA; Development; Disease; Drug Formulations; Effectiveness; Endocytosis; Endoplasmic Reticulum; Endotoxins; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Foundations; Future; Genes; Genetic; Genetic Drift; Genome; Goals; I-antigen; Immune; Immune response; Immune system; Immunity; Influenza; Influenza A Virus, H1N1 Subtype; Intranasal Administration; Ion Exchange; Lead; Lectin; Mediating; Modeling; Mus; Needles; Nicotiana; Nucleoproteins; Parasites; Pathway interactions; Plant Lectins; Plants; Prevention; Process; Production; Proteins; Protocols documentation; Recombinant Proteins; Recombinants; Research; Research Support; Retrieval; Ricin; Route; Serum; Site; Subunit Vaccines; Surface; Surface Antigens; System; T memory cell; T-Lymphocyte; Techniques; Technology; Testing; Toxin; United States National Institutes of Health; Vaccination; Vaccine Antigen; Vaccines; Viral; Virus Diseases; Western Blotting; Whole Organism; Work; antigen processing; arm; base; cross reactivity; cytokine; cytotoxicity; design; expression vector; immune activation; immunogenicity; influenza virus vaccine; killings; novel; novel strategies; novel vaccines; pandemic disease; pathogen; public health relevance; research study; response; swine flu; tool; trafficking; uptake; vaccine delivery; vaccine efficacy; vector

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.

描述（由申请人提供）：疫苗仍然是我们全球预防传染病最有效的工具。亚单位疫苗被认为比传统的灭活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功效，特别是那些能够指导强细胞介导免疫（CMI）的疫苗载体和佐剂。该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，以基于亚细胞运输和相关疫苗抗原的免疫呈递的操作选择性地协调所需的免疫反应。 RTB 是蓖麻毒素的无毒碳水化合物结合亚基 B，已被证明可以有效介导相关疫苗抗原的跨粘膜递送，并引发强烈的抗原特异性抗体介导的免疫。为了专门增强 CMI 反应的诱导，RTB 被修改为包含 C 端 KDEL 内质网 (ER) 检索基序（称为 RTBER）。与胞吞后主要在内体/溶酶体区室（MHC II 抗原加工位点）中积累的 RTB 相比，RTBER 应将相关抗原“有效负载”重定向至 ER/胞质溶胶界面（CMI 的 MHC I 抗原加工位点）。 RTB 和 RTBER 均有助于跨粘膜表面主动摄取相关抗原并进入免疫反应细胞。该项目测试了这样的假设：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，以实现有效的抗原加工并通过 MHC I 途径将抗原呈递给 CD8+ T 细胞，从而产生强大的细胞介导的免疫。甲型流感核蛋白 (NP) 是甲型流感的潜在“通用”抗原，需要强大的 CMI 保护，将被用作模型抗原，并且将确定 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) 在简便的基于植物的生物生产系统中生产重组 NP:RTB 融合蛋白，2) 重组产品的纯化和表征，以及 3) 评估鼻内施用对照 NP 后在小鼠中引发的免疫反应:RTB 和 NP:RTBER 免疫原制剂。证明 RTBER 在调节 NP 特异性 CMI 反应中的功效是未来多毒株流感挑战/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，并为专注于开发甲型流感通用疫苗的后续实验奠定基础。