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

描述（由申请方提供）：用携带SIV蛋白的减毒CMV病毒（RhCMV-SIV）接种恒河猴，在50%接种疫苗的恒河猴中实现了前所未有的持久控制和SIV清除，通过接种疫苗期间CD 8 T细胞应答的幅度和感染后淋巴结中的效应T细胞应答预测了阳性结果。RhCMV-SIV疫苗接种打破了天然免疫优势，并导致广泛的CD 4 T细胞和CD 8应答，覆盖了SIV抗原的许多区域。令人惊讶的是，该疫苗引起的大多数SIV特异性CD 8 T细胞应答受到MHC-II的限制。尽管这些结果是迄今为止在艾滋病毒疫苗领域最有希望的结果之一，但使用CMV载体进行艾滋病毒疫苗接种引起了健康问题。引发T细胞应答所需的第一个事件是抗原呈递细胞（APC）向T细胞呈递MHC-I-和MHC-II-结合肽。这些肽来自抗原加工机制对蛋白质的多步细胞内降解。考虑到WT CMV改变MHC-肽呈递的能力，我们提出减毒CMV表达HIV蛋白（HCMV-HIV）为加工和呈递HIV表位创造了独特的条件，导致广泛的非常规T细胞应答，并且这些条件可以在不使用CMV的情况下通过在APC中递送免疫原期间瞬时操纵抗原加工机制来复制。为了揭示在减毒HCMV-HIV存在下蛋白质降解和表位呈递的这些机制，我们开发了通量测定法来测量原代细胞中的抗原加工活性，基于质谱的测定法来跟踪病毒可能进入的两个细胞区室中蛋白质或病毒体的降解：胞质溶胶和内-溶酶体，以及鉴定HIV感染细胞中的细胞内表位前体和MHC结合肽的方法。我们发现，细胞亚群之间的肽酶活性水平的变化在形成用于表位呈递的肽的长度和动力学方面起着关键作用。使用生物化学、计算、体外和体内免疫学方法的组合，我们提出1）确定减毒HCMV-HIV对靶细胞亚群中HIV蛋白降解的影响，2）评估减毒HCMV-HIV感染背景下T细胞应答的表位呈递和引发，和3）设计和测试导致广泛的HIV特异性T细胞应答的无CMV抗原递送系统。该项目建立在PI、Heckerman博士（用于HIV降解产物的计算分析）、Picker博士（用于CMV中的HCMV专业知识）、Moris博士（用于HIV特异性T细胞应答的体外引发试验）、步行者博士（用于HIV特异性T细胞）、Tager博士（用于人源化小鼠）和Irvine博士（用于开发抗原递送纳米颗粒）之间的多学科合作方法的基础上。

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.