Dissecting 3 processing pathways that generate class II-restricted flu epitopes

剖析产生 II 类限制性流感表位的 3 条加工途径

• 批准号: 7392044
• 负责人: Laurence Crane Eisenlohr
• 金额: $ 23.25万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7392044
• 关键词: Affinity Chromatography; Antigen-Presenting Cells; Antigens; Attention; Avian Influenza; Biochemical; Bioterrorism; CD4 Positive T Lymphocytes; Cell Line; Class; Complex; Cytotoxic T-Lymphocytes; Defect; Depth; Early Endosome; Epitopes; Genetic; Glycoproteins; Goals; Hand; Hemagglutinin; Histocompatibility Antigens Class II; Host Defense; Influenza; Influenza Hemagglutinin; Inhibitory Concentration 50; Investigation; Lead; Lymphoma; MHC Class I Genes; MHC Class II Genes; Modeling; Monoclonal Antibodies; Mutagenesis; Neuraminidase; Oxidoreductase; Pathway interactions; Peptides; Play; Process; Reagent; Recycling; Research; Role; Screening procedure; Site; Small Interfering RNA; Solid; Standards of Weights and Measures; System; Vaccine Design; Virus; antigen processing; antigenic peptide transporter; base; cDNA Library; cell type; extracellular; flu; innovation; insight; late endosome; multicatalytic endopeptidase complex; mutant; reconstitution; response; small molecule; small molecule libraries

DESCRIPTION (provided by applicant): CD4+ T cells recognize peptides (epitopes) presented in the context of MHC class II molecules (MHC class II) and play a key role in host defense by guiding and potentiating effector responses. In the classical model, such peptides are derived from extracellular ("exogenous") antigens that are unfolded, digested and loaded onto MHC class II in the late endosomal compartment with the assistance of a molecule termed H2-M. The "Site 1" (S1) epitope within the influenza A/PR/8/34 hemagglutinin (HA) molecule fits this description. Despite investigation for several decades, key aspects of the classical pathway remain unclear. For example, we have shown that the presentation of S1 requires a reductase activity in the late endosome but the specific reductase is not yet known. We have identified two additional pathways that are naturally utilized for the presentation of influenza antigens. First is a recycling pathway in which class II molecules are loaded in the early endosome without participation of H2-M. The "Site 3" (S3) epitope within HA is presented from exogenously provided virus via this pathway. Second is an endogenous proteasome-dependent presentation pathway. Both S3 and NA79 (a neuraminidase [NA]-derived epitope) are presented from biosynthesized HA and NA via a pathway that requires proteasome and TAP (transporter of antigenic peptide) activities, both generally associated with MHC class I- but not class II-restricted presentation. The recycling and endogenous pathways are poorly understood although our results suggest that both could be just as important as the classical pathway in host defense. Our goal is to gain an understanding of all three pathways at a depth that will drive vaccine design to a less empirical process. This is a pressing need even without bioterrorism and avian influenza. To do this, we propose two discovery-based approaches for identification of critical cellular components that are specific to each of these pathways. First, utilizing epitope-specific reagents that are in hand and to be developed we will isolate antigen-presenting cell mutants that are selectively defective in presenting S1, S3 and/or NA79. Using standard genetic approaches we will then determine the basis for any defect. Second, we will interrogate small molecule libraries for compounds that selectively inhibit presentation and then identify the targets of any positive hit compounds using genetic and/or biochemical approaches. We anticipate that the execution of these complementary approaches will lead to the discovery of key processing components that would otherwise remain hidden and that will help pave the way for more rational vaccine design.

描述（由申请人提供）：CD4+ T 细胞识别 MHC II 类分子（MHC II 类）中存在的肽（表位），并通过引导和增强效应反应在宿主防御中发挥关键作用。在经典模型中，此类肽源自细胞外（“外源”）抗原，在称为 H2-M 的分子的帮助下，这些抗原被解折叠、消化并加载到晚期内体区室中的 MHC II 类上。流感 A/PR/8/34 血凝素 (HA) 分子内的“位点 1”(S1) 表位符合此描述。尽管研究了几十年，经典途径的关键方面仍然不清楚。例如，我们已经表明，S1 的呈递需要晚期内体中的还原酶活性，但具体的还原酶尚不清楚。我们已经确定了另外两种天然用于流感抗原呈递的途径。第一个是回收途径，其中 II 类分子在没有 H2-M 参与的情况下加载到早期内体中。 HA 内的“位点 3”(S3) 表位是通过该途径从外源提供的病毒呈递的。其次是内源性蛋白酶体依赖性呈递途径。 S3 和 NA79（神经氨酸酶 [NA] 衍生表位）均由生物合成的 HA 和 NA 通过需要蛋白酶体和 TAP（抗原肽转运蛋白）活性的途径呈递，两者通常与 MHC I 类限制性呈递有关，但与 II 类限制性呈递无关。尽管我们的结果表明，回收途径和内源途径与宿主防御中的经典途径一样重要，但人们对回收途径和内源途径知之甚少。我们的目标是深入了解所有三种途径，从而推动疫苗设计减少经验过程。即使没有生物恐怖主义和禽流感，这也是一个迫切的需要。为此，我们提出了两种基于发现的方法来识别特定于这些途径的关键细胞成分。首先，利用现有的和待开发的表位特异性试剂，我们将分离在呈递 S1、S3 和/或 NA79 方面选择性缺陷的抗原呈递细胞突变体。然后，我们将使用标准的遗传方法来确定任何缺陷的基础。其次，我们将询问小分子库中是否有选择性抑制呈递的化合物，然后使用遗传和/或生化方法识别任何阳性命中化合物的靶标。我们预计，这些补充方法的执行将导致关键处理组件的发现，否则这些组件将被隐藏，这将有助于为更合理的疫苗设计铺平道路。