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.

项目总结 腺病毒(Ad)通常在具有免疫能力的人类宿主中诱导轻微的自限性感染。一种更具毒性的 AD14毒株Ad14p1最早出现在美国军方，此后在全球平民中传播 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病例，住院死亡率约为40%。大多数ARDS治疗 针对炎症反应的措施；然而，所有措施都未能显示出对死亡率的好处。我们已经展示了 AdE1B 20K(20K)基因产物调控巨噬细胞对细胞的炎症反应 死于Ad感染(Ad CPE身体)。20K基因缺失或低水平表达可诱导Ad CPE细胞 是促炎的，而正常的(野生型病毒)20K基因表达会产生抗炎 AD CPE细胞。Ad14p1仅表达野生型AD14表达的20K的20%。这减少了20万 表达诱导促炎症的Ad14p1 CPE身体，而野生型AD14 CPE身体是抗炎的 煽动性的。这一应用的中心假设是这种病毒基因在免疫调节中的变化 感染新出现的Ad14p1的影响是这种新出现的病毒的关键生物学差异 增加可导致ARDS和死亡的急性肺损伤(ALI)的发生率和严重性。叙利亚人 仓鼠，即金黄地鼠，允许感染人腺病毒。叙利亚仓鼠也是 易感染许多其他人类病毒，如流感、汉坦病毒、SARS冠状病毒、SARS冠状病毒2、马尔堡和 埃博拉病毒。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 中性粒细胞渗入肺和呼吸道。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。CRISPR/Cas9叙利亚仓鼠的开发消除了其中一个障碍。 这个项目通过创造针对仓鼠细胞表面标志物的抗体来解决另一个问题 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群 巨噬细胞对AD14和Ad14p1激活的群体和比较特征 感染。这些抗体将补充现有的少数仓鼠特异性抗体，可用于 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的机制进行研究 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 治疗学不仅治疗Ad诱导的ALI/ARDS，而且可能治疗由其他原因引发的ARDS。在……里面 此外，这些抗体可用于研究其他人类病原体对巨噬细胞和 叙利亚仓鼠体内的中性粒细胞。