Development of antibodies to specific cell surface markers to assess macrophage polarization during Adenovirus 14 and 14p1 infection in the Syrian hamster

开发针对特定细胞表面标记物的抗体，以评估叙利亚仓鼠腺病毒 14 和 14p1 感染期间的巨噬细胞极化

• 批准号: 10725702
• 负责人: Jay R Radke
• 金额: $ 6.23万
• 依托单位: IDAHO VETERANS RESEARCH / EDUCATION FDN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725702
• 关键词: 2019-nCoV; Acute Lung Injury; Acute Respiratory Distress Syndrome; Address; Adenoviruses; Alveolar Macrophages; Alzheimer' s disease model; Animal Model; Anti-Inflammatory Agents; Antibodies; Biological; Biological Models; Bronchoalveolar Lavage; Bronchopneumonia; CD80 gene; CD86 gene; CRISPR/Cas technology; CXCL10 gene; CXCR3 gene; Cell Surface Proteins; Cell surface; Cells; Cessation of life; Communicable Diseases; Comparative Study; Complement; Development; Disease; Disease Outbreaks; Ebola; Edema; Experimental Models; Fatality rate; Flow Cytometry; Gene Expression; Goals; Hamsters; Hantavirus; Hospital Mortality; Human; IL8 gene; Immune; Immune response; Immunocompetent; Immunologics; Incidence; Infection; Infectious Agent; Inflammation; Inflammatory; Inflammatory Response; Influenza; Innate Immune Response; Interleukin-1 beta; Interleukin-6; Lung; Lung infections; MERTK gene; Macrophage; Macrophage Activation; Marburgvirus; Measures; Mediating; Mesocricetus auratus; Methods; Military Personnel; Modeling; Molecular; Mus; Mutation; Natural Immunity; Neutrophil Infiltration; Pathogenicity; Pathway interactions; Phenotype; Play; Population; Positioning Attribute; Predisposition; Production; Proteins; Protocols documentation; Reagent; Reporting; SARS coronavirus; Sampling; Severities; TNF gene; Testing; Time; Transgenic Organisms; Trauma; Variant; Viral Genes; Virulent; Virus; comparative; cytokine; data analysis pipeline; epidemiology study; gene product; human pathogen; immunopathology; immunoregulation; in vivo; lung injury; mortality; neutrophil; novel; novel strategies; permissiveness; protein expression; respiratory; response; single-cell RNA sequencing; targeted treatment; tool; virus genetics

PROJECT SUMMARY Adenovirus (Ad) normally induces mild, self-limited infections in immunocompetent human hosts. A more virulent strain of Ad14, Ad14p1, first emerged in the U.S. military and has since spread globally to civilian populations resulting in severe infections, sometimes leading to acute respiratory distress syndrome (ARDS). The incidence of ARDS in the U.S. is ~200,000 cases annually, with a hospital mortality rate of ~40%. Most ARDS treatment measures target the inflammatory response; however, all have failed to show a mortality benefit. We have shown that the Ad E1B 20K (20K) gene product controls modulation of the macrophage inflammatory response to cells dying from Ad infection (Ad CPE corpses). Absent or low level 20K gene expression generates Ad CPE cells that are pro-inflammatory, whereas normal (wild type virus) 20K gene expression generates anti-inflammatory Ad CPE cells. Ad14p1 expresses only 20% of the 20K expressed by wild type Ad14. This reduced 20K expression induces pro-inflammatory Ad14p1 CPE corpses, whereas wild type Ad14 CPE corpses are anti- inflammatory. The central hypothesis of this application is that this viral genetic change in the immunomodulatory effect of infection with emergent Ad14p1 is the key biological difference through which this emergent virus increases the incidence and severity of acute lung injury (ALI) that can result in ARDS and death. The Syrian hamster, Mesocricetus auratus, is permissive for infection with human adenoviruses. Syrian hamsters are also susceptible to many other human viruses such as influenza, hantavirus, SARS-CoV, SARS-CoV-2, Marburg and Ebola. We have shown that infection of hamsters with Ad14p1 replicates many of the key features of human ALI/ARDS including patchy bronchopneumonia, increased pro-inflammatory cytokine expression, edema and neutrophil infiltration into the lung and airways. A problem with the Syrian hamster model system has been that there are no transgenic hamsters and that there is a lack of immunological reagents available, such as those for the mouse and human. Development of the CRISPR/Cas9 Syrian hamster has removed one of those obstacles. This project addresses the other problem by creating antibodies that are specific for hamster cell surface markers expressed on macrophages. These antibodies will allow identification and isolation of macrophage sub- populations and comparative characterization of macrophage activation in response to Ad14 and Ad14p1 infections. The antibodies will complement the small number of existing hamster-specific antibodies available for flow cytometry studies. These antibodies will allow us to begin to understand the effects of Ad14 and Ad14p1 infection on the innate immune response and to develop mechanistic studies of how these two viruses generate such different immune responses. Understanding those key factors will allow development of targeted therapeutics to treat not only Ad-induced ALI/ARDS but potentially ARDS triggered by other causes of ALI. In addition, these antibodies can be used to study the effects of other human pathogens on macrophages and neutrophils in the Syrian hamster.

项目概要 腺病毒（Ad）通常会在免疫功能正常的人类宿主中引起轻度、自限性感染。更具毒性 Ad14、Ad14p1 菌株首先出现在美国军队中，此后已在全球范围内传播给平民 导致严重感染，有时导致急性呼吸窘迫综合征（ARDS）。发病率 美国每年约有 20 万例 ARDS，医院死亡率约为 40%。大多数 ARDS 治疗 针对炎症反应的措施；然而，所有这些都未能显示出死亡率方面的益处。我们已经展示了 Ad E1B 20K (20K) 基因产物控制巨噬细胞对细胞炎症反应的调节 死于Ad感染（Ad CPE尸体）。 20K 基因表达缺失或水平较低会产生 Ad CPE 细胞 是促炎性的，而正常（野生型病毒）20K 基因表达则产生抗炎性 Ad CPE 细胞。 Ad14p1 的表达量仅为野生型 Ad14 表达的 20K 的 20%。这样减少了20K 表达诱导促炎 Ad14p1 CPE 尸体，而野生型 Ad14 CPE 尸体则具有抗炎作用。 炎症。该申请的中心假设是，这种病毒基因改变在免疫调节 新兴Ad14p1感染的效果是这种新兴病毒的关键生物学差异 增加急性肺损伤 (ALI) 的发生率和严重程度，从而导致 ARDS 和死亡。叙利亚人 仓鼠 Mesocricetus auratus 允许感染人类腺病毒。叙利亚仓鼠也 易感染许多其他人类病毒，如流感、汉坦病毒、SARS-CoV、SARS-CoV-2、马尔堡病毒和 埃博拉。我们已经证明，感染 Ad14p1 的仓鼠复制了人类的许多关键特征 ALI/ARDS，包括斑片状支气管肺炎、促炎细胞因子表达增加、水肿和 中性粒细胞浸润至肺和气道。叙利亚仓鼠模型系统的一个问题是 没有转基因仓鼠，也缺乏可用的免疫试剂，例如用于 老鼠和人类。叙利亚仓鼠 CRISPR/Cas9 的开发消除了这些障碍之一。 该项目通过创建仓鼠细胞表面标记物特异性抗体解决了另一个问题 表达于巨噬细胞。这些抗体将允许识别和分离巨噬细胞亚型 Ad14 和 Ad14p1 响应的巨噬细胞激活的群体和比较特征 感染。这些抗体将补充现有的少量仓鼠特异性抗体 流式细胞术研究。这些抗体将使我们开始了解 Ad14 和 Ad14p1 的作用 感染对先天免疫反应的影响，并对这两种病毒如何产生进行机制研究 如此不同的免疫反应。了解这些关键因素将有助于制定有针对性的 治疗方法不仅可以治疗 Ad 引起的 ALI/ARDS，还可以治疗其他 ALI 原因引发的潜在 ARDS。在 此外，这些抗体可用于研究其他人类病原体对巨噬细胞和 叙利亚仓鼠的中性粒细胞。