Enhancing Collectin Mediated Defense Against Influenza

 描述（由申请人提供）：甲型流感病毒（IAV）仍然是人类健康的主要威胁，因为它能够不断改变其表面蛋白并逃避适应性免疫。该项目涉及先天免疫反应的关键方面，这些免疫反应为肺部提供了针对新型IAV毒株的初始保护。了解这些先天免疫反应可以解释为什么有些人患有严重的IAV感染疾病，或者为什么大流行IAV毒株比季节性毒株更具致病性。例如，我们最近的研究结果表明，大流行性IAV毒株逃避重要的先天免疫介质的中和，包括表面活性蛋白A和D（SP-A和SP-D）和抗微生物肽（AMP），如LL-37，防御素和组蛋白。我们已经试图利用对先天免疫蛋白的作用机制的理解来开发具有增加的抗病毒活性的这些蛋白的修饰形式。迄今为止，我们已经表明SP-D的突变形式和衍生自LL-37的修饰的合成肽获得了抑制大流行性IAV的能力。在目标1中，我们将在分子水平上确定SP-D如何与IAV血凝素（HA）结合，以解释大流行IAV如何逃避中和，以及我们的SP-D新突变形式如何克服这一点并中和IAV。SP-A与SP-D的不同之处在于它通过提供诱饵唾液酸配体供HA结合来中和病毒。在目标2中，我们将确定为什么大流行性IAV对SP-A具有抗性，但对一些其他抑制剂（例如，H-纤维胶凝蛋白）， 具有相似的抗病毒活性机制。在目标3中，将进行类似的研究， AMP的重点是LL-37，它不抑制流行性IAV，和LL-37衍生的肽，它抑制it. Finally，目的4将探讨如何不同的蛋白质调节炎症反应IAV，以减少潜在的有害炎症（SP-D，SP-A或LL-37）或可能增加它（组蛋白）在体外和体内。该提案的关键技术将包括病毒膜糖蛋白的质谱（例如，HA）、X射线晶体学和分子建模、反向遗传学以产生经修饰的病毒株、以及体外和体内病毒感染测定。我们的合作者是糖蛋白组学和蛋白质结构分析方面的杰出专家，我们共同开发了强大的技术，以前所未有的精确度了解先天蛋白中和病毒的分子基础。这些研究应使我们能够通过对胶原凝集素（SP-A和SP-D）或AMP进行结构上知情的修饰来开发其他病毒抑制剂。

 DESCRIPTION (provided by applicant): Influenza A virus (IAV) remains a major threat to human health because of its ability to continually change its surface proteins and evade adaptive immunity. This project addresses key aspects of the innate immune response that provide initial protection of the lung against novel IAV strains. Understanding these innate immune responses may explain why some people suffer severe illness with IAV infection or why pandemic IAV strains are more pathogenic than seasonal strains. For example, our recent findings indicate that pandemic IAV strains evade neutralization by important innate immune mediators, including the surfactant proteins A and D (SP-A and SP-D) and anti-microbial peptides (AMPs) like LL-37, defensins and histones. We have sought to exploit understanding of the mechanisms of action of innate immune proteins to develop modified forms of these proteins with increased antiviral activity. We have thus far shown that mutated versions of SP-D, and modified synthetic peptide derived from LL-37, gain the ability to inhibit pandemic IAV. In Aim 1, we will determine at a molecular level how SP-D binds to the IAV hemagglutinin (HA) in order to explain how pandemic IAV evades neutralization and also how our novel mutated forms of SP-D can overcome this and neutralize IAV. SP-A differs from SP-D in that it neutralizes the virus by providing a decoy sialic acid ligand for the HA to bind to. In Aim 2, we will determine why pandemic IAV is resistant to SP-A but sensitive to some other inhibitors (e.g., H-ficolin) that have similar mechanisms of antiviral activity. In Aim 3, similar studies will be done regarding the AMPs with emphasis on LL-37, which does not inhibit pandemic IAV, and the LL-37 derived peptide which does inhibit it. Finally, Aim 4 will explore how the different proteins modulate inflammatory responses to IAV to reduce potentially harmful inflammation (SP-D, SP-A or LL-37) or possibly increase it (histones) in vitro and in vivo. Key techniques for the proposal will include mass spectroscopy of viral membrane glycoproteins (e.g., HA), xray crystallography and molecular modeling, reverse genetics to generate modified viral strains, and in vitro and in vivo viral infection assays. Our collaborators are prominent experts in glycoproteomics and protein structure analysis and together we have developed powerful techniques to understand the molecular basis for viral neutralization by innate proteins at a level of precision not previously achieved. These studies should enable us to develop additional viral inhibitors through structurally informed modification of collectins (SP-A and SP-D) or AMPs.