Enhancing Collectin Mediated Defense Against Influenza

增强收集素介导的流感防御

• 批准号: 8681493
• 负责人: Kevan L Hartshorn
• 金额: $ 40.86万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-12-12 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8681493
• 关键词: Accounting; Amino Acid Sequence; Antiviral Agents; Bacteria; Bacterial Infections; Binding; Binding Sites; Biological Assay; CAP18 lipopolysaccharide-binding protein; Carbohydrate Sequence; Carbohydrates; Cell Line; Chemosensitization; Collaborations; Collagen; Collectins; Complication; Defensins; Development; Electron Microscopy; Epithelial Cells; Failure; Goals; Grant; Hemagglutinin; Host Defense; Human; Immune; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Response; Influenza; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H5N1 Subtype; Influenza A virus; Injury; Interferons; Knowledge; Laboratories; Lectin; Length; Link; Lung; Lung Inflammation; Mass Spectrum Analysis; Mediating; Modeling; Modification; Molecular; Molecular Models; Mus; N-terminal; Neck; Pathogenesis; Pathogenicity; Peptide Sequence Determination; Phagocytes; Phagocytosis; Play; Polysaccharides; Positioning Attribute; Process; Property; Protein Analysis; Proteins; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Protein D; Recombinants; Research; Respiratory Burst; Role; Streptococcus pneumoniae; Surface Plasmon Resonance; Techniques; Testing; Type II Epithelial Receptor Cell; Variant; Viral; Viral Hemagglutinins; Virulence; Virus; Virus Diseases; X-Ray Crystallography; adverse outcome; antimicrobial peptide; base; cathelicidin; crosslink; cytokine; drug resistant virus; glycosylation; in vitro Assay; in vitro testing; in vivo; influenzavirus; inhibitor/antagonist; lung injury; macrophage; molecular modeling; monocyte; mouse model; mutant; neutrophil; novel; pandemic disease; pandemic influenza; resistant strain; respiratory; response; retrocyclin; seasonal influenza

DESCRIPTION (provided by applicant): Surfactant proteins A and D (SP-A and SP-D), defensins and LL-37 are important for innate defense against influenza A virus (IAV). We have recently found, however, that pandemic IAV strains are not inhibited by SP-D and this contributes to the increased virulence of these strains in vivo. We have also found, however, that modifications of the isolated neck and carbohydrate recognition domain (NCRD) of SP-D results in marked increase in neutralization and seasonal IAV as well as neutralization of pandemic IAV. These findings result from collaboration with Drs. Erika Crouch and Barbara Seaton, using structural analysis to predict changes in the trimeric neck and carbohydrate recognition domains (NCRDs) of SP-D that will increase binding to glycans on the viral hemagglutinin (HA). In aim 1 we will analyze of the structural basis of increased viral inhibition by these these NCRDs. We will use purified viral hemagglutinins, novel recombinant viral strains, viral binding assays, and in vitro and in vivo assays of viral inhibition. HA glycans will be determined by Mass Spectroscopy. NCRD binding to viral glycans will be evaluated by molecular modeling, Xray crystallography and glycan array. Based on structural analysis we predict further modifications of the NCRDs of SP-D or SP-A that should result in increased antiviral activity for seasonal or pandemic IAV strains. We will prepare and then study these novel NCRDs and also create full length SP-Ds containing the mutant NCRDs. Further analysis of these proteins should allow us to predict additional changes to increase activity. Excessive lung inflammation is implicated in adverse outcomes with some IAV strains. In aim 2 we will evaluate how SP-D and SP-A modulate inflammatory responses to IAV again with the goal of developing novel constructs that will promote viral neutralization and viral clearance by phagocytes, while minimizing inflammatory responses that could cause injury or promote bacterial super-infection (an important complication of IAV infection). In vitro findings will again be compared to results in mice. We have also made significant recent discoveries regarding the antiviral and immune modulatory effects of antimicrobial peptides with respect to IAV that we will pursue in aim 3. We find that defensins and LL-37 inhibit infectivity of a many seasonal and laboratory IAV strains; however, unexpectedly, some defensins and LL-37 increase infectivity of pandemic H1N1 (2009 strains). We will determine the mechanisms antiviral activity of defensins and LL-37 against seasonal IAV and how they increase infectivity of the pandemic strains. Defensins and LL-37 also modulate interactions of seasonal IAV with neutrophils and monocytes in different ways. The mechanisms of these effects will be determined and compared to effects on pandemic strains. These studies could have important implications for pathogenesis of pandemic IAV. We have also found that novel synthetic defensins have increased antiviral activity that will be characterized further in vitro and in vivo. An important rationale for these studies overall is that novel defensins and collectins have potential for treatment of drug resistant viruses and bacteria.

描述（由申请人提供）：表面活性剂蛋白A和D（SP-A和SP-D）、防御素和LL-37对于针对甲型流感病毒（IAV）的先天防御是重要的。然而，我们最近发现，大流行性IAV毒株不被SP-D抑制，这有助于这些毒株在体内的毒力增加。然而，我们还发现，SP-D的分离的颈部和碳水化合物识别结构域（NCRD）的修饰导致中和和季节性IAV以及中和大流行IAV的显著增加。这些发现来自与Erika Crouch和Barbara Seaton博士的合作，使用结构分析来预测SP-D的三聚体颈部和碳水化合物识别结构域（NCRD）的变化，这将增加与病毒血凝素（HA）上聚糖的结合。在目标1中，我们将分析这些NCRD增加病毒抑制的结构基础。我们将使用纯化的病毒血凝素、新型重组病毒株、病毒结合试验以及体外和体内病毒抑制试验。HA聚糖将通过质谱法测定。将通过分子建模、X射线晶体学和聚糖阵列评价NCRD与病毒聚糖的结合。基于结构分析，我们预测SP-D或SP-A的NCRD的进一步修饰应导致季节性或大流行性IAV毒株的抗病毒活性增加。我们将制备并研究这些新的NCRD，并创建含有突变NCRD的全长SP-D。对这些蛋白质的进一步分析应该使我们能够预测增加活性的其他变化。过度的肺部炎症与某些IAV毒株的不良结局有关。在目标2中，我们将再次评估SP-D和SP-A如何调节对IAV的炎症反应，目的是开发新的构建体，其将促进病毒中和和吞噬细胞的病毒清除，同时最小化可能导致损伤或促进细菌超感染（IAV感染的重要并发症）的炎症反应。将再次将体外结果与小鼠结果进行比较。我们最近还发现了抗微生物肽对IAV的抗病毒和免疫调节作用，我们将在目标3中继续研究。我们发现防御素和LL-37抑制许多季节性和实验室IAV毒株的感染性;然而，出乎意料的是，一些防御素和LL-37增加了大流行性H1N1（2009毒株）的感染性。我们将确定防御素和LL-37对抗季节性IAV的抗病毒活性的机制，以及它们如何增加大流行毒株的感染性。防御素和LL-37也以不同的方式调节季节性IAV与中性粒细胞和单核细胞的相互作用。将确定这些作用的机制，并与对大流行毒株的作用进行比较。这些研究可能对大流行性IAV的发病机制具有重要意义。我们还发现，新的合成防御素具有增加的抗病毒活性，这将进一步在体外和体内表征。总体而言，这些研究的一个重要理论基础是新型防御素和胶原凝集素具有治疗耐药病毒和细菌的潜力。