Mechanisms and optimization of epitope presentation by HIV-infectable cell subset

描述（由申请人提供）：HIV特异性细胞毒性t淋巴细胞（CTL）在急性感染或自发控制的情况下在抑制HIV病毒血症中发挥关键作用，使其成为疫苗策略的有吸引力的候选者。然而，迄今为止开发的疫苗尽管产生了CTL反应，却未能预防艾滋病毒感染。事实上，针对病毒某些区域的CTL反应可能比具有其他特异性的CTL更有效地控制HIV复制，这表明疫苗应该引起与保护相关的选择性CTL反应。hiv感染细胞有效呈递同源表位是保护性CTL反应的关键条件。然而，对于控制CD8 T细胞识别的HIV表位呈递的细胞内机制仍然知之甚少。大多数关于HIV特异性CTL功能的研究利用合成肽形式的同源表位，从而绕过导致表位呈现的所有细胞内蛋白质降解步骤。HIV感染几种表达CD4的亚群（CD4 T细胞、单核/巨噬细胞和树突状细胞），这些亚群将呈现HIV表位。这些亚群是否表现出具有相同动力学的相似表位尚不清楚。不同亚群表位呈现的差异可能影响CTL的抗病毒效果。相反，鉴定所有亚群中有效加工成表位的HIV区域对于鉴定保护性CTL反应和选择免疫原至关重要。在新的表位加工实验的基础上，我们展示了一些HIV表位的优先加工，这一特性依赖于我们用来改变不相关表位产生的基序。我们还发现了一个涉及表位加工效率的新因素，即最佳HIV表位的高度可变细胞内稳定性，也由特定基序驱动。最后，我们发现CD4 T细胞的加工活性比单核细胞低，这影响了HIV蛋白降解产物的动力学和抗原性。这些数据表明，表位的产生是受规则控制的，这些规则可以用来设计定制的免疫原。具体来说，我们建议：1)确定与HIV病毒血症自发控制相关的CTL反应是否能有效识别和杀死所有HIV感染细胞亚群。利用大量控制者和进展者的优势，我们将评估受各种hiv感染亚群刺激的CD8 T细胞的功能。2)鉴定在可感染亚群的不同抗原加工途径中共同产生的肽，这些抗原加工途径有助于自发控制性病毒血症。使用纳米颗粒靶向细胞亚群内的HIV蛋白，我们将识别所有亚群产生的抗原肽。3)设计和测试序列签名，导致保护性HIV表位的选择性呈现。该建议依赖于跨学科的合作方法，包括计算科学，生物工程工具以及为原代细胞设计的表位加工和CTL功能的生化和免疫学分析。hiv特异性CD8 T细胞能够杀死被hiv感染的细胞，因为被感染的细胞显示出被称为表位的病毒片段。破解刺激有效免疫反应的HIV表位的产生和呈递机制是合理设计疫苗的关键。该项目旨在鉴定来自自发无法检测到的病毒血症患者（控制者）的hiv感染细胞亚群所呈现的免疫反应和表位，并确定控制表位加工效率的因素，从而能够在免疫原中选择性地呈现保护性表位。

DESCRIPTION (provided by applicant): HIV-specific cytotoxic T-lymphocytes (CTL) play a critical role in containing HIV viremia in acute infection or in situation of spontaneous control, rendering them attractive candidates for vaccine strategies. However vaccines developed so far have failed to protect against HIV infection, this despite generating CTL responses. The fact that CTL responses against certain areas of the virus may be more effective at controlling HIV replication than CTL with other specificities suggests that vaccine should elicit selected CTL responses associated with protection. Effective presentation of the cognate epitopes by HIV-infected cells is a crucial condition for protective CTL responses. However, there is still surprisingly little understanding of intracellular mechanisms governing the presentation of HIV epitopes recognized by CD8 T cells. Most studies on HIV- specific CTL functions utilize cognate epitopes in the form of synthetic peptides, thus bypassing all intracellular steps of protein degradation leading to the presentation of epitopes. HIV infects several CD4-expressing subsets (CD4 T cells, monocyte/macrophages and dendritic cells) that will present HIV epitopes. Whether these subsets present similar epitopes with identical kinetics is unknown. Variations in epitope presentation between subsets may affect the antiviral efficacy of CTL. Conversely identifying areas of HIV that are efficiently processed into epitopes in all subsets is of highest importance for the identification of protective CTL responses and selection of immunogens. Building on novel epitope processing assays, we showed preferential processing of some HIV epitopes, a property that relies on motifs we used to alter the production of irrelevant epitopes. We also identified a novel factor involved in epitope processing efficiency, namely the highly variable intracellular stability of optimal HIV epitopes, also driven by specific motifs. Finally we show that CD4 T cells have lower processing activities than monocytes, which affects the kinetics and antigenicity of degradation products from HIV proteins. These data suggest that epitope production is controlled by rules that could be exploited to design customized immunogens. Specifically we propose to: 1) Determine whether CTL responses associated with spontaneous control of HIV viremia efficiently recognize and kill all HIV-infectable cell subsets. Taking advantage of a large cohort of controllers and progressors, we will assess the functionality of CD8 T cells stimulated by various HIV-infected subsets. 2) Identify peptides commonly produced in distinct antigen processing pathways of infectable subsets contributing to spontaneous controlled viremia. Using nanoparticles to target HIV proteins inside cell subsets, we will identify antigenic peptides produced by all subsets. 3) Design and test sequence signatures leading to the selective presentation of protective HIV epitopes. This proposal relies on a cross-disciplinary collaborative approach involving computational science, bioengineered tools and biochemical and immunological assays of epitope processing and CTL functions designed for primary cells. HIV-specific CD8 T cells are able to kill HIV-infected cells because infected cells display pieces of virus called epitopes. Deciphering the mechanisms of production and presentation of HIV epitopes stimulating efficient immune responses is key to rational vaccine design. This project seeks to identify immune responses and epitopes presented by HIV-infected cell subsets from persons with spontaneously undetectable viremia (controllers) and to define factors governing the efficiency of epitope processing that will enable selected presentation of protective epitopes in immunogens.