Inflammation and Therapy for Respiratory Virus Infection

呼吸道病毒感染的炎症和治疗

• 批准号: 7006273
• 负责人: HELENE ROSENBERG
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/7006273
• 关键词: Paramyxovirus; antiviral agents; asthma; chemokine; enzyme linked immunosorbent assay; flow cytometry; high performance liquid chromatography; human tissue; inflammation; influenzavirus A; microarray technology; molecular cloning; plethysmography; polymerase chain reaction; protein purification; respiratory disease /disorder therapy; respiratory disorder chemotherapy; respiratory function; respiratory infections; respiratory syncytial virus; respiratory virus; viral pneumonia; virus diseases

As of this writing, we are still without safe and effective therapeutic strategies for acute respiratory virus infections. Despite considerable efforts over the almost 50 years since its first description, supportive therapy alone remains the standard of care for the treatment of severe cases of respiratory syncytial virus (RSV), a disease with significant morbidity and mortality, particularly among infants born prematurely, and for which there is currently no vaccine. Even when a vaccine is available for a respiratory pathogen, such as for epidemic influenza virus, similar problems of disease prevention and management exist despite seasonal reformulation of vaccine preparations. While progress has been made toward the development of agents with antiviral activity in vitro, the clinical impact of these therapies has been disappointing. At the source of the problem is the observation that, once established, respiratory virus disease results from two concurrent pathologic components: ongoing virus replication and the resulting inflammatory response. Even when antivirals clearly inhibit virus replication, the biochemical and cellular inflammatory responses to the initial infection-related events continue despite diminished virus titer. Prolonged inflammation has been recognized as a significant component contributing to the pathologic sequelae of RSV and influenza virus, and most recently, to the morbidity and mortality of SARS-CoV infection. Our group has developed and characterized a mouse model of severe, acute respiratory infection using the highly virulent natural rodent pathogen, pneumonia virus of mice (PVM, family Paramyxoviridae, subfamily Pneumovirinae, strain J3666). Mice that are inoculated with fewer than 100 plaque forming units (pfu) develop acute lower airway disease characterized by progression to pneumonia, respiratory failure and death (see previous publications, since 2000). We have previously demonstrated that, analogous to influenza and RSV infection, the chemokine MIP-1a is central to this virus-induced inflammatory process, and that interruption of MIP-1a signaling via the receptor CCR1 results in marked reduction in pulmonary inflammation (J Immunol, 2000). Furthermore, we have shown that the inflammatory events associated with pneumovirus infection are not inextricably linked to ongoing viral replication, as replication blockade with ribavirin does not mitigate the associated inflammatory events contributing to the infection-associated morbidity and mortality (J Virol, 2003). In the first of the two manuscripts reported in this year (ref #1 Altered pathogenesis of severe pneumovirus infection in response to combined antiviral and specific immunomodulatory agents), we reported the responses of mice with symptomatic pneumovirus infection to combined antiviral and specific immunomodulatory agents. Mice infected with pneumonia virus of mice, a natural mouse pathogen that replicates the signs and symptoms of severe infection with respiratory syncytial virus (RSV), responded to the antiviral agent ribavirin when it was administered in the setting of endogenous (gene deletion) or exogenous (antibody-mediated) blockade of the MIP-1alpha proinflammatory signaling cascade. Although neither treatment was effective alone, together they offered a dramatic reduction in symptoms and pathology, the most impressive of which was a significant reduction in morbidity and mortality. The findings presented are consistent with the notion of unique and independent contributions of virus replication and ongoing inflammation to the pathogenesis of severe respiratory virus infection, and they provide the impetus for the study of this treatment regimen in RSV-infected humans. In the second (ref #2 Functional antagonism of chemokine receptor CCR1 reduces mortality in acute pneumovirus infection in vivo) we presented an antiviral-immunomodulatory therapeutic strategy involving the chemokine receptor antagonist Met-RANTES, which yielded significant survival in the setting of an otherwise fatal respiratory virus infection. We demonstrated that ribavirin reduced mortality, from 100% to 10 and 30%, respectively, in gene-deleted CCR1 (-/-) mice and in wild-type mice treated with the small-molecule chemokine receptor antagonist, Met-RANTES. As MIP-1alpha-mediated inflammation is a common response to several distantly related respiratory virus pathogens, specific antiviral therapy in conjunction with blockade of the MIP-1alpha/CCR1 inflammatory cascade might ultimately prove to be a useful, generalized approach to severe respiratory virus infection and its pathological sequelae in human subjects. We have also authored four major invited reviews on this subject (refs #5, 8, 9 and 13). Methods: PCR RT-PCR Quantitative RTPCR Molecular Cloning Restriction Digestion DNA sequencing Bacterial protein expression expression Baculovirus protein expression Mammalian cell protein expression Adenovirus expression FACS analysis Northern blotting Southern blotting Fast Protein Liquid Chromatography Affinity Chromatography Western blotting Enzymatic assays ELISA Immunoprecipitation Gene microarray Immunohistochemistry

在撰写本文时，我们仍然没有针对急性呼吸道病毒感染的安全和有效的治疗策略。尽管自首次描述以来近50年来做出了相当大的努力，但仅支持疗法仍然是治疗呼吸道合胞病毒(RSV)严重病例的标准护理方法。RSV是一种发病率和死亡率很高的疾病，特别是在早产婴儿中，目前还没有针对这种疾病的疫苗。即使有针对呼吸道病原体的疫苗，例如针对流行性流感病毒的疫苗，也存在类似的疾病预防和管理问题，尽管对疫苗制剂进行了季节性调整。虽然在体外开发具有抗病毒活性的药物方面取得了进展，但这些疗法的临床影响一直令人失望。问题的根源是观察到，一旦确诊，呼吸道病毒疾病是由两个同时发生的病理成分引起的：正在进行的病毒复制和由此产生的炎症反应。即使当抗病毒药物明显抑制病毒复制时，尽管病毒滴度降低，但对最初感染相关事件的生化和细胞炎症反应仍在继续。持续的炎症被认为是导致RSV和流感病毒的病理后遗症，以及最近导致SARS-CoV感染的发病率和死亡率的重要组成部分。 我们小组利用高毒力的天然啮齿动物病原体--小鼠肺炎病毒(PVM，副粘病毒科，肺炎病毒亚科，J3666株)建立并鉴定了一种严重急性呼吸道感染的小鼠模型。接种不到100个斑块形成单位(PFU)的小鼠会发展成急性下呼吸道疾病，其特征是进展为肺炎、呼吸衰竭和死亡(见自2000年以来的先前出版物)。我们先前已经证明，类似于流感和RSV感染，趋化因子MIP-1a是病毒诱导的炎症过程的中心，并且通过受体CCR1阻断MIP-1a信号导致肺部炎症显著减少(J免疫，2000)。此外，我们已经证明，与肺炎病毒感染相关的炎症事件与正在进行的病毒复制并不是千丝万缕的联系，因为用利巴韦林阻断复制并不能减轻导致感染相关发病率和死亡率的相关炎症事件(J Virol，2003)。 在今年报道的两篇论文中的第一篇(参考文献1)中，我们报道了有症状的肺炎病毒感染小鼠对联合抗病毒和特定免疫调节剂的反应。小鼠肺炎病毒是一种复制呼吸道合胞病毒(RSV)严重感染的体征和症状的自然病原体，当给予抗病毒药物利巴韦林时，在内源性(基因缺失)或外源性(抗体介导)阻断MIP-1α促炎信号级联反应的情况下，对该药产生反应。虽然两种治疗方法都不是单独有效，但它们一起显著减少了症状和病理，其中最令人印象深刻的是发病率和死亡率的显著降低。这些发现与病毒复制和持续炎症对严重呼吸道病毒感染发病机制的独特和独立贡献的概念是一致的，并为研究RSV感染人类的这种治疗方案提供了动力。 在第二项研究中(参考文献#2趋化因子受体CCR1的功能拮抗作用降低了体内急性肺炎病毒感染的死亡率)，我们提出了一种使用趋化因子受体拮抗剂Met-RANTES的抗病毒免疫调节治疗策略，该策略在其他致命的呼吸道病毒感染的情况下获得了显着的存活。我们证明，利巴韦林降低了CCR1(-/-)基因缺失小鼠和使用小分子趋化因子受体拮抗剂Met-RANTES治疗的野生型小鼠的死亡率，分别从100%降至10%和30%。由于MIP-1α介导的炎症是几种远亲呼吸道病毒病原体的共同反应，特异性抗病毒治疗结合阻断MIP-1α/CCR1炎性级联反应最终可能被证明是治疗人类严重呼吸道病毒感染及其病理后遗症的一种有用的、普遍的方法。 我们还撰写了四篇关于这一主题的主要应邀评论(参考文献5、8、9和13)。 方法：聚合酶链式反应、RT-PCR、分子克隆、限制性内切酶、DNA测序、细菌蛋白表达、杆状病毒蛋白表达、哺乳动物细胞蛋白表达、腺病毒表达、流式细胞仪分析、Northern blotting、Southern blotting、Fast Protein LLC、亲和层析、Western blotting、酶分析、ELISA、免疫沉淀、基因芯片、免疫组织化学