Enhancing Collectin Mediated Defense Against Influenza

描述(由申请人提供)：表面活性蛋白A和D(SP-A和SP-D)、防御素和LL-37对于对抗甲型流感病毒(IAV)的天然防御非常重要。然而，我们最近发现，大流行性IAV毒株不被SP-D抑制，这有助于提高这些毒株在体内的毒力。然而，我们还发现，SP-D分离的Neck和碳水化合物识别结构域(NCRD)的修饰导致中和和季节性IAV以及大流行IAV的中和显著增加。这些发现源于与Erika Crouch和Barbara Seaton博士的合作，他们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，这些变化将增加与病毒血凝素(HA)上的糖链的结合。在目标1中，我们将分析这些NCRD增强病毒抑制作用的结构基础。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。羟基磷灰石多糖将通过质谱学进行测定。NCRD与病毒多糖的结合将通过分子建模、X射线结晶学和糖链阵列进行评估。基于结构分析，我们预测SP-D或SP-A的NCRD的进一步修饰应该会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，并创建包含突变NCRD的全长SP-D。对这些蛋白质的进一步分析应该能让我们预测增加活性的额外变化。过度的肺部炎症与某些IAV毒株的不良后果有关。在目标2中，我们将评估SP-D和SP-A如何再次调节对IAV的炎症反应，目的是开发新的结构，以促进吞噬细胞对病毒的中和和清除，同时最大限度地减少可能导致损伤或促进细菌重叠感染(IAV感染的一个重要并发症)的炎症反应。在体外的研究结果将再次与在老鼠身上的结果进行比较。在目标3中，我们还在抗菌肽对IAV的抗病毒和免疫调节作用方面取得了重大发现。我们发现防御素和LL-37抑制了许多季节性和实验室IAV毒株的传染性；然而，出人意料的是，一些防御素和LL-37增加了大流行性H1N1病毒(2009年毒株)的传染性。我们将确定防御素和LL-37针对季节性IAV的抗病毒活性的机制，以及它们如何增加大流行毒株的传染性。防御素和IL-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。将确定这些影响的机制，并将其与对大流行毒株的影响进行比较。这些研究可能对大流行性IAV的发病机制有重要的意义。我们还发现，新的合成防御素具有增强的抗病毒活性，这将在体外和体内进一步表征。总体而言，这些研究的一个重要理论基础是，新的防御素和集合素具有治疗耐药病毒和细菌的潜力。

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.