Learning from attenuated CMV how to broaden HIV-specific T cell responses

描述(申请人提供)：用携带SIV蛋白的CMV减毒病毒(RhCMV-SIV)接种恒河猴后，50%的接种猕猴SIV获得了前所未有的持久控制和清除，从接种过程中CD8 T细胞反应的大小和感染后淋巴结中的效应T细胞反应来预测这一积极结果。RhCMV-SIV疫苗打破了自然免疫优势，导致广泛的CD4T细胞和CD8应答，覆盖了SIV抗原的许多区域。令人惊讶的是，这种疫苗激发的大多数SIV特异性CD8T细胞反应都受到MHC-II的限制。虽然这些结果是迄今为止艾滋病毒疫苗领域最有希望的结果之一，但使用巨细胞病毒载体进行艾滋病毒疫苗接种引起了健康问题。启动T细胞反应所需的第一个事件是抗原提呈细胞(APC)将MHC-I和MHC-II结合的多肽呈递给T细胞。这些多肽来自于抗原处理机制对蛋白质的多步细胞内降解。考虑到WT CMV改变MHC多肽呈递的能力，我们认为表达HIV蛋白的减毒CMV(HCMV-HIV)为HIV表位的加工和呈递创造了独特的条件，导致广泛的非常规T细胞反应，并且这些条件可以在没有CMV的情况下通过在APC中递送免疫原期间瞬时操纵抗原处理机制来复制。为了揭示在减毒的HCMV-HIV存在下蛋白质降解和表位呈递的这些机制，我们开发了用于测量原代细胞中抗原处理活动的吞吐量分析、基于质谱学的分析以跟踪病毒可能进入的两个细胞隔室中蛋白质或病毒粒子的降解：胞浆和内切溶酶体，以及在HIV感染细胞中识别细胞内表位前体和MHC结合多肽的方法。我们发现，不同细胞亚群之间的多肽酶活性水平的差异在形成为表位呈递而产生的多肽的长度和动力学方面起着关键作用。结合生化、计算、体外和体内免疫学方法，我们建议1)确定减毒的HCMV-HIV对靶细胞亚群中HIV蛋白降解的影响，2)评估在减毒的HCMV-HIV感染背景下T细胞反应的表位呈递和启动，以及3)设计和测试导致广泛的HIV特异性T细胞反应的无CMV抗原递送系统。该项目建立在PI、Heckerman博士(艾滋病毒降解产物的计算分析)、Picker博士(巨细胞病毒专业知识)、Moris博士(HIV特异性T细胞体外启动分析)、Walker博士(HIV特异性T细胞)、Tager博士(人源化小鼠)和Irvine博士(欧文博士)之间的多学科协作方法的基础上，开发用于抗原传递的纳米颗粒。

DESCRIPTION (provided by applicant): Vaccination of Rhesus Macaques with an attenuated CMV virus carrying SIV proteins (RhCMV-SIV) led to an unprecedented durable control and clearance of SIV in 50% of vaccinated Macaques, a positive outcome predicted by the magnitude of CD8 T cell responses during vaccination and effector T cell responses in lymph nodes after infection. RhCMV-SIV vaccination broke natural immunodominance and led to broad CD4 T cells and CD8 responses covering many areas of SIV antigens. Surprisingly most SIV-specific CD8 T cells responses elicited by this vaccine were restricted by MHC-II. Whereas the results are among the most promising to date in the HIV vaccine field the use of CMV vectors for HIV vaccination raise health concerns. The first event required to prime T cell responses is the presentation of MHC-I- and MHC-II-bound peptides by antigen presenting cells (APC) to T cells. These peptides come from the multistep intracellular degradation of proteins by the antigen processing machinery. Considering the capacity of WT CMV to alter MHC-peptide presentation we propose that attenuated CMV expressing HIV proteins (HCMV-HIV) creates unique conditions for processing and presentation of HIV epitopes leading to broad unconventional T cell responses, and that these conditions can be replicated without the use of CMV by transiently manipulating the antigen processing machinery during the delivery of the immunogen in APC. To unveil these mechanisms of protein degradation and epitope presentation in the presence of attenuated HCMV-HIV we have developed throughput assays to measure antigen processing activities in primary cells, mass spectrometry-based assays to follow the degradation of proteins or virions in the two cellular compartments where the virus may enter: cytosol and endo-lysosomes, and methodologies to identify intracellular epitope precursors and MHC-bound peptides in HIV-infected cells. We showed that variations in the levels of peptidase activities among cell subsets play a critical role in shaping the lengths and kinetics of peptides produced for epitope presentation. Using a combination of biochemical, computational, in vitro and in vivo immunological approaches we propose to 1) Determine the effect of attenuated HCMV-HIV on HIV protein degradation in target cell subsets, 2) Assess epitope presentation and priming of T cell responses in the context of attenuated HCMV-HIV infection, and 3) Design and test a CMV-free antigen delivery system leading to broad HIV-specific T cell responses. This project builds on a multidisciplinary collaborative approach between the PI, Dr Heckerman for computational analysis of HIV degradation products, Dr Picker for HCMV expertise in CMV, Dr Moris for in vitro priming assays of HIV-specific T cell responses, Dr Walker for HIV-specific T cells, Dr Tager for humanized mice and Dr Irvine for the development of nanoparticles for antigen delivery.