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

描述(由申请人提供)：CD4+T细胞识别在MHC II类分子(MHC II类)背景下呈现的多肽(表位)，并通过引导和增强效应器反应在宿主防御中发挥关键作用。在经典模型中，这类多肽是从细胞外(“外源”)抗原衍生而来的，这些抗原在称为H2-M的分子的帮助下，被展开、消化并加载到内体晚期的MHC II类分子上。流感A/PR/8/34血凝素(HA)分子中的“位点1”(S1)表位符合这一描述。尽管进行了几十年的研究，但经典途径的关键方面仍然不清楚。例如，我们已经证明，S1的呈现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条天然用于呈递流感抗原的途径。第一种是循环途径，即在没有H2-M参与的情况下，II类分子被加载到早期的内体中。HA内的S3位表位是由外源提供的病毒通过这一途径呈现的。第二个途径是内源性蛋白酶体依赖的提呈途径。S3和NA79(一种神经氨酸酶[NA]衍生的表位)都是由生物合成的HA和NA通过一条需要蛋白酶体和TAP(抗原肽转运体)活性的途径提呈的，两者通常与MHC I类递呈相关，但不与II类限制性递呈相关。尽管我们的结果表明，在宿主防御中，这两种途径可能与经典途径一样重要，但对循环和内源途径的了解很少。我们的目标是深入了解所有这三条途径，从而推动疫苗设计进入一个较少经验性的过程。即使没有生物恐怖主义和禽流感，这也是一个紧迫的需求。为了做到这一点，我们提出了两种基于发现的方法来识别这些途径中每一个特定的关键细胞成分。首先，利用已有的和即将开发的表位特异性试剂，我们将分离在呈递S1、S3和/或NA79方面存在选择性缺陷的抗原提呈细胞突变体。使用标准的遗传方法，我们将确定任何缺陷的基础。其次，我们将询问小分子文库中选择性抑制呈现的化合物，然后使用遗传和/或生化方法确定任何阳性化合物的靶标。我们预计，这些互补方法的实施将导致发现关键的加工成分，否则这些成分将被隐藏起来，这将有助于为更合理的疫苗设计铺平道路。

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.