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的最有效工具。亚基疫苗比传统的杀死或减毒整个生物体疫苗更安全，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基疫苗的疗效，尤其是那些能够指导强细胞介导的免疫力（CMI）的疫苗。该探索性项目将评估新型基于植物凝集素的抗原载体RTB和RTBER的效用，以选择性地根据对亚细胞运输的操纵和相关疫苗抗原的免疫表现来选择所需的免疫反应。 RTB是ricin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透射传递，并引起强抗原特异性抗体抗体介导的免疫。为了特别增强CMI响应的诱导，RTB被修改为包含C末端KDEL内质网（ER）检索基序（称为RTBER）。与内吞作用后主要积累的RTB相比，在内体/溶酶体室（MHC II抗原加工的位置）中，RTBER应重定向与ER/cytosol界面相关的抗原“有效载荷”（MHC I抗原处理CMI的位点）。 RTB和RTBER都促进了跨粘膜表面和免疫反应细胞的相关抗原的积极摄取。该项目检验了以下假设：将RTBER用作疫苗抗原载体将通过逆行ER途径选择性动员抗原，以通过MHC I途径有效地呈现CD8+ T细胞的有效抗原加工，从而导致强大的细胞介导的免疫力。流感A核蛋白（NP）是一种需要强大CMI保护的潜在的“通用”抗原，用作模型抗原和RTB与RTBER在选择性策划NP特异性免疫反应中的影响。该项目将包括1）重组NP的产生：在简单的基于植物的生物生产系统中的RTB融合蛋白，2）重组产物的纯化和表征，3）评估对照组内鼻内施用后，NP：RTB和NP：RTB和NP：RTBER IMMUNEGAN FEMATICS在鼻内施用后的小鼠中引起的免疫反应的评估。在介导强大的NP特异性CMI响应中证明了RTBER功效是未来多局部分流感挑战/疾病保护试验的关键先决条件。这项研究，如果成功的话，将有潜在地确定一种新的策略，以指导强烈的细胞介导的粘膜输送亚基疫苗的免疫力，并为关注流感的通用疫苗开发的后续实验为基础。