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.

项目摘要 腺病毒（AD）通常在免疫能力的人类宿主中诱导轻度，自限的感染。更猛烈的 AD14，AD14P1的压力首先在美国军方出现，此后全球范围内传播到平民人口 导致严重的感染，有时导致急性呼吸窘迫综合征（ARDS）。发病率 美国的ARDS每年约20万例，医院死亡率约为40％。大多数ARDS治疗 措施针对炎症反应；但是，所有人都没有表现出死亡率。我们已经显示了 AD E1B 20K（20K）基因产物控制巨噬细胞对细胞的炎症反应的调节 死于AD感染（AD CPE尸体）。缺乏或低水平的20K基因表达会产生AD CPE细胞 促炎性的，而正常（野生型病毒）20K基因表达产生抗炎 AD CPE细胞。 AD14P1仅表示野生型AD14表示20K的20％。这减少了20k 表达诱导促炎AD14P1 CPE尸体，而野生型AD14 CPE尸体是抗 炎症。该应用的中心假设是免疫调节中这种病毒遗传变化 紧急AD14P1感染的影响是这种新兴病毒的关键生物学差异 增加急性肺损伤（ALI）的发病率和严重程度，这可能导致ARDS和死亡。叙利亚人 仓鼠，载质体，允许人类腺病毒感染。叙利亚仓鼠也是 容易受到许多其他人类病毒的影响 埃博拉病毒。我们已经表明，具有AD14P1的仓鼠的感染复制了人类的许多关键特征 ALI/ARDS，包括斑块的支气管瘤，促炎性细胞因子表达，水肿和 中性粒细胞浸润到肺和气道中。叙利亚仓鼠模型系统的问题是 没有转基因仓鼠，并且缺乏可用的免疫试剂，例如 老鼠和人类。 CRISPR/CAS9叙利亚仓鼠的发展消除了其中一个障碍。 该项目通过创建针对仓鼠细胞表面标记的抗体来解决其他问题 在巨噬细胞上表达。这些抗体将允许鉴定和分离巨噬细胞亚 响应AD14和AD14P1的巨噬细胞激活的种群和比较表征 感染。这些抗体将补充可用于现有的仓鼠特异性抗体 流式细胞仪研究。这些抗体将使我们能够开始了解AD14和AD14P1的影响 关于先天免疫反应的感染，并开发有关这两种病毒如何产生的机械研究 如此不同的免疫反应。了解这些关键因素将允许开发目标 治疗药物不仅可以治疗广告诱导的ALI/ARD，而且可能由其他ALI原因触发。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 叙利亚仓鼠中的中性粒细胞。