Novel AAV-based tools for liver immunology

描述（由申请人提供）：肝脏是抗原接触可导致T细胞免疫或耐受的部位，但决定T细胞启动结果的影响尚不清楚。在这个探索性/发展性建议中，我们将创建一套基于重组腺相关病毒（rAAV）的新工具来探索这个问题。具体来说，我们将修改rAAV衣壳和启动子来改变细胞的向性，我们将设计一系列荧光报告，与肝脏自身荧光形成对比，我们将使用高亲和力抗原肽或序列变体或对照肽构建载体。其他载体将促进或抑制转导的肝细胞的直接抗原呈递。或者导致转导的肝细胞死亡，从而使它们的抗原可用于交叉呈递。使用所有这些工具，我们将测试抗原密度、亲和力、细胞类型特异性表达和直接递呈与交叉递呈对原代CD8+和CD4+ T细胞活化、效应细胞发育和记忆的影响。这些研究将创造出具有更广泛用途的新工具。此外，如果在本研究中我们能够定义记忆T细胞启动的优化启动条件，在未来的研究中，我们将在病原体编码抗原中进行工程设计，以测试AAV是否可以用作抗肝细胞病原体的疫苗载体。

DESCRIPTION (provided by applicant): The liver is a site at which antigen encounter can result in either T cell immunity or tolerance, but the influences that determine the outcome of T cell priming are not well understood. In this exploratory/developmental proposal, we will create a set of novel tools based on recombinant adeno- associated virus (rAAV) to explore this issue. Specifically, we will modify the rAAV capsid and promoter to alter cell tropism, we will engineer in a series of fluorescent reporters that will contrast with liver autofluorescence, and we will buld vectors using either a high-affinity antigenic peptides, or sequence variants, or a control peptide Other vectors will promote or inhibit direct antigen presentation by the transduced hepatocytes, or cause the death of transduced hepatocytes thus rendering their antigens available for cross-presentation. Using all of these tools, we will test the impact of antigen density, affinity, cell ype-specific expression and direct versus cross-presentation on primary CD8+ and CD4+ T cell activation, on the development of effector cells, and on memory. These studies will create novel tools of wider usefulness. In particular, the red fluorophores such as mCherry are optimized for in vivo microscopy, Furthermore, if in this study we are able to define optimized priming conditions for memory T cell priming, in future studies we will engineer in pathogen-encoded antigens to test whether AAV could be used as a vaccine vehicle against hepatocellular pathogens. PUBLIC HEALTH RELEVANCE: Infections of liver cells impose a large disease burden both in the USA and globally. The way the immune system responds to such infections is poorly understood. In the project we will use techniques from the field of gene therapy to create new tools that will increase understanding of immune responses to infections of the liver.