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

描述（由申请人提供）：CD 4 + T细胞识别在MHC II类分子（MHC II类）背景下呈递的肽（表位），并通过引导和增强效应子应答在宿主防御中发挥关键作用。在经典模型中，这样的肽来源于细胞外（“外源性”）抗原，所述抗原在称为H2-M的分子的帮助下被解折叠、消化并加载到晚期内体区室中的MHC II类上。甲型流感/PR/8/34血凝素（HA）分子内的“位点1”（S1）表位符合这一描述。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的呈递需要在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呈递流感抗原的途径。第一种是再循环途径，其中II类分子在没有H2-M参与的情况下装载在早期内体中。HA内的“位点3”（S3）表位通过该途径由外源提供的病毒呈递。其次是内源性蛋白酶体依赖性呈递途径。S3和NA 79（一种神经氨酸酶[NA]衍生表位）均通过需要蛋白酶体和TAP（抗原肽转运蛋白）活性的途径由生物合成的HA和NA呈递，两者通常与MHC I类限制性呈递相关，但与II类限制性呈递无关。回收和内源性途径知之甚少，虽然我们的研究结果表明，这两个可能是一样重要的经典途径在主机防御。我们的目标是深入了解所有三种途径，从而将疫苗设计推向一个不那么经验的过程。即使没有生物恐怖主义和禽流感，这也是一项迫切需要。为此，我们提出了两种基于发现的方法，用于识别特定于这些途径的关键细胞组分。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NA 79方面有选择性缺陷的抗原呈递细胞突变体。使用标准的遗传学方法，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递的化合物，然后使用遗传和/或生物化学方法鉴定任何阳性命中化合物的靶标。我们预计，这些互补方法的执行将导致发现关键的处理组件，否则将保持隐藏，这将有助于铺平道路，更合理的疫苗设计。

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.