Novel Pathogen Recognition Pathways and Control of Hepatitis A Virus

新的病原体识别途径和甲型肝炎病毒的控制

• 批准号: 9024432
• 负责人: Stanley M. Lemon
• 金额: $ 40.77万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9024432
• 关键词: Acute; Acute Hepatitis; Antibodies; Antiviral Agents; Antiviral Therapy; Appearance; Biochemical; Blood; CD4 Positive T Lymphocytes; Categories; Cell Line; Cells; Coculture Techniques; Collaborations; Complex; Data; Dendritic Cells; Endocytosis; Family Picornaviridae; Gene Expression; Goals; Hepatitis A; Hepatitis A Virus; Hepatitis C virus; Hepatocyte; Hepatovirus; Human; Immune; Immune response; Immune system; In Vitro; Infection; Infection Control; Inflammatory; Integration Host Factors; Interferons; Kinetics; Life; Ligands; Liver; Measurable; Mediating; Membrane; Molecular; National Institute of Allergy and Infectious Disease; Nucleic Acids; Pathway interactions; Pattern; Peptide Hydrolases; Preparation; Production; Proteins; Proteomics; Publishing; RNA; RNA Binding; RNA Viruses; Recombinants; Research; Resistance; Resolution; Role; Signal Pathway; Signal Transduction; Site; T cell response; TLR7 gene; Time; Vaccines; Variant; Viral; Virion; Virus; Virus Diseases; Virus Replication; base; cell type; cytokine; density; exosome; extracellular vesicles; hepatoma cell; human morbidity; human mortality; insight; liver injury; macrophage; microvesicles; monocyte; neutralizing antibody; novel; pathogen; programs; receptor; receptor binding; response; trafficking; tripolyphosphate; viral RNA

Virus infections of the liver continue to cause substantial human morbidity and mortality worldwide despite advances in vaccines and antiviral therapy. Hepatitis A virus (HAV), a positive-strand RNA virus classified within the genus Hepatovirus of the family Picornaviridae, is a common cause of acute hepatitis and a Category B NIAID Priority Pathogen. Our previously published research reveals that HAV infects the liver in an extraordinarily stealthy fashion, evoking little type l/lll interferon (IFN)-stimulated gene expression despite robust replication within hepatocytes over a period of several weeks. The onset of acute hepatic injury 3-4 weeks after infection is marked by impressive increases in pro-inflammatory cytokines simultaneous with the initial appearance of virus-specific antibodies and a predominantly CD4+ T cell response that correlates with resolution ofthe infection. Our goal in this project is to better understand how HAV is recognized by the innate immune system and how this may contribute to ultimate control ofthe infection. The proposed studies build on our recent discovery that HAV, classically considered a non-enveloped virus, is released from infected hepatocytes and circulates in the blood of infected humans completely enveloped in host membranes. These novel 'enveloped HAV virions (eHAV) are fully infectious but completely resistant to neutralizing antibodies. Our preliminary data show that gradient fractions containing purified eHAV uniquely induce human pDCs to secrete IFN-a and stimulate pro-inflammatory cytokine and NLRPS inflammasomedependent IL-1B expression by THP-1 cells, while highly concentrated, standard, non-enveloped virions do not. In Aim 1, we will determine host factor requirements for activation of pDCs when co-cultured with HAV infected cells, and the RNA and protein composition of pDC-activating microvesicles in collaboration with Core B. We will also quantify pDC recruitment to the liver in hepatitis A. Aim 2 will study how eHAV is sensed by THP-1 cells and primary human monocyte/macrophages, and in collaboration with Projects 2 and 3 identify the origin of signals leading to inflammasome activation by HAV. These studies will exploit unique capabilities of Core C to provide purified recombinant NLRs for quantitative in vitro studies of their potential as direct receptors of pathogen-associated molecular patterns. Aim 3 will define innate immune responses and the molecular basis of inflammasome activation by HAV in hepatocytes. These studies support the goals of this multi-project U19 application by leveraging unique biochemical capabilities to investigate the role of NLRs as novel pathogen recognition receptors (PRRs), applying cutting-edge quantitative proteomic approaches to identify paradigm-shifting signaling pathways involved in sensing and control of human viruses, examining cross-talk between PRR pathways in the human response to a NIAID Category B Priority Pathogen, investigating intracellular trafficking of a viral ligand to the site of engagment by a PRR, and taking maximal advantage of opportunities to validate experimental findings with primary human materials.

尽管疫苗和抗病毒疗法进展，但肝脏的病毒感染仍会在全球引起大量的人类发病率和死亡率。丙型肝炎病毒（HAV）是一种在picornaviridae家族肝病属内分类的阳性RNA病毒，是急性肝炎的常见原因，也是B NIAID优先病原体的常见原因。我们先前发表的研究表明，HAV以一种非常隐秘的方式感染了肝脏，尽管在几周内肝细胞内强良好的复制，但仍引起了很少的L/LLL干扰素（IFN）刺激的基因表达。感染后3-4周的急性肝损伤的发作以与病毒特异性抗体的初始外观同时出现和主要的CD4+ T细胞反应同时出现，与感染分辨率相关。我们在这个项目中的目标是更好地了解HAV如何被先天免疫系统认可，以及这可能如何有助于对感染的最终控制。拟议的研究是基于我们最近发现的，即经典认为是一种非发育的病毒，是从感染的肝细胞中释放出来的，并在被感染的人的血液中循环，完全包裹在宿主膜上。这些新型的“包围HAV病毒粒子（EHAV）完全具有感染性，但完全抵抗中和抗体。我们的初步数据表明，含有纯化的EHAV的梯度分数独特地诱导人PDC分泌IFN-A并刺激THP-1细胞的促炎性细胞因子和NLRP炎性疾病依赖性IL-1B表达，而高度浓缩的，标准的，非发育的病毒体则没有。在AIM 1中，我们将确定与HAV感染细胞共培养PDC的宿主因子的要求，以及与CoreB合作的PDC激活微泡的RNA和蛋白质组成。我们还将在肝炎中量化PDC募集到肝脏中的PDC募集。导致HAV激活炎症体激活的信号的起源。这些研究将利用核心C的独特能力提供纯化的重组NLR，以定量在体外研究其作为病原体相关分子模式的直接受体的潜力。 AIM 3将定义先天免疫反应和HAV在肝细胞中激活炎症的分子基础。 These studies support the goals of this multi-project U19 application by leveraging unique biochemical capabilities to investigate the role of NLRs as novel pathogen recognition receptors (PRRs), applying cutting-edge quantitative proteomic approaches to identify paradigm-shifting signaling pathways involved in sensing and control of human viruses, examining cross-talk between PRR pathways in the human response to a NIAID Category B Priority Pathogen,通过PRR调查病毒配体的细胞内贩运到接近的部位，并利用机会最大的优势来验证用原始材料验证实验发现。