Novel AAV-based tools for liver immunology

基于 AAV 的新型肝脏免疫学工具

• 批准号: 8289839
• 负责人: Ian NICHOLAS Crispe
• 金额: $ 23.63万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8289839
• 关键词: Affinity; Albumins; Antigen Presentation; Antigen Presentation Pathway; Antigen-Presenting Cells; Antigens; Apoptosis; Apoptotic; CD4 Positive T Lymphocytes; CD8B1 gene; Capsid; Cause of Death; Cell Death; Cells; Chronic; Clonal Expansion; Cross Presentation; Cross-Priming; Development; Diphtheria Toxin; Disease; Effector Cell; Engineering; Future; Goals; Hepatitis B; Hepatitis C virus; Hepatocyte; Human; Immune response; Immune system; Immunity; Immunology; Infection; Interferon Type I; Interferon-alpha; Light; Liver; Location; Malaria; Mediating; Memory; Microscopy; Mus; Natural Immunity; Outcome; Ovalbumin; Ovum; Peptides; Population; Proteins; Recombinant adeno-associated virus (rAAV); Reporter; Research; Series; Serotyping; Signal Transduction; Site; T cell response; T memory cell; T-Cell Activation; T-Lymphocyte; Techniques; Testing; Tropism; Vaccines; Variant; adeno-associated viral vector; antigen processing; base; burden of illness; cellular transduction; density; diphtheria toxin receptor; fluorophore; gene therapy; improved; in vivo; knock-down; liver infection; novel; pathogen; promoter; small hairpin RNA; tool; uptake; vaccine development; vector

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.

描述（由申请方提供）：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细胞引发结果的影响尚不清楚。在这个探索性/开发性的建议中，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。具体地，我们将修饰rAAV衣壳和启动子以改变细胞嗜性，我们将工程化一系列荧光报告基因，其将与肝自体荧光形成对比，并且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肝细胞的直接抗原呈递，或引起转导的肝细胞死亡，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与交叉呈递对初级CD 8+和CD 4 + T细胞活化，效应细胞发育和记忆的影响。这些研究将创造具有更广泛用途的新工具。特别是，红色荧光团如mCherry被优化用于体内显微镜检查。此外，如果在本研究中我们能够定义用于记忆T细胞引发的优化引发条件，则在未来的研究中，我们将工程化病原体编码的抗原以测试AAV是否可以用作针对肝细胞病原体的疫苗载体。 公共卫生相关性：肝细胞感染在美国和全球都造成了巨大的疾病负担。免疫系统对这种感染的反应方式知之甚少。在该项目中，我们将使用基因治疗领域的技术来创造新的工具，以增加对肝脏感染免疫反应的理解。