ENHANCING COLLECTIN MEDIATED HOST DEFENSE

胶原凝集素是一组血清和肺表面活性物质相关的 被认为参与抗体非依赖性宿主的蛋白质 的防御合作. 肺表面活性蛋白A和D（SP-A 和SP-D），具有抗病毒和抗菌特性。 然而，在这方面， 初步研究表明，基因改变的版本， 这些由分子生物学技术产生的蛋白质 可能具有增强的对抗特定呼吸道病原体的活性。 该提案的目标是产生重组的突变的聚集蛋白 具有改善的抗甲型流感病毒（IAV）活性， 肺炎链球菌（肺炎球菌）与野生型相比 收藏品。 选择这些病原体是因为它们代表了 个别严重呼吸道感染的主要原因，因为 肺炎双球菌重叠感染是IAV重要并发症 感染 我们已经生产出一种嵌合集合体， 与碳水化合物融合的SP-D的N-末端和胶原结构域 识别结构域（CRD）。 这种分子具有更大的 比天然或重组体凝集IAV和细菌的能力 以及更强的抑制IAV血凝的能力 活性高于连接蛋白或野生型SP-D。 基于这些 有希望的发现，我们提出了其他几个嵌合体的建设， 具有结合有利的功能性质的目标的集合素 将野生型聚集蛋白转化为单个分子。 两个另外 含有SP-D的N-末端和胶原结构域融合的构建体 与SP-A或血清凝集素的CRD，甘露糖结合 凝集素（MBL）。 因为胶原蛋白结构域 SP-A参与与吞噬细胞受体的结合， 构建体将含有SP-A的该结构域和SP-D（或MBL）的CRD。 我们将比较这些重组凝集素结合， 聚集，抑制感染性，并作为调理素，一组 临床上重要的IAV和肺炎球菌菌株。 接下来，具体 参与碳水化合物结合的残基将在其中一个 构建体，以确定是否可以与IAV或肺炎球菌结合， 增强 聚集蛋白构建体的体内活性最初将 通过在IAV之前将它们鼻内滴注到小鼠中进行测试，或 肺炎球菌感染 其中一个突变体构建体（选择用于 抗IAV和/或肺炎球菌的最佳活性）， 在转基因小鼠的气道，以确定是否在体内抵抗 感染增强。 这些实验将产生重要的 深入了解基本的收集生物学，并证明这种增强 胶原凝集素介导的肺宿主防御可以在体内实现 通过合理改变野生型collectins。

The collectins are a group of serum and pulmonary surfactant-associated proteins which are believed to participate in antibody-independent host defenses. The pulmonary collectins, surfactant proteins A and D (SP-A and SP-D), have antiviral and antibacterial properties. However, preliminary research has indicated that genetically altered versions of these proteins produced by molecular biological techniques would be likely to have enhanced activity against specific respiratory pathogens. The goal of this proposal is to generate recombinant, mutant collectins with improved activity against influenza A viruses (IAVs) and Streptococcus Pneumoniae (pneumococci) as compared to the wild type collectins. These pathogens are chosen because they represent individually major causes of serious respiratory infection, and because superinfection with pneumococci is an important complication of IAV infection. We have produced a chimeric collectin which incorporates the N-terminus and collagen domain of SP-D fused with the carbohydrate recognition domain (CRD) of conglutinin. This molecule has a greater ability to agglutinate IAV and bacteria than native or recombinant conglutinin, as well as greater ability to inhibit IAV hemagglutination activity than either conglutinin or wild type SP-D. Based on these promising findings we propose construction of several other chimeric collectins with the goals of combining favorable functional properties of the wild type collectins into single molecules. Two further constructs containing the N-terminus and collagen domain of SP-D fused to the CRDs of either SP-A or the serum collectin, mannose-binding lectin (MBL), will be made. Since it is likely that the collagen domain of SP-A participates in binding to phagocyte receptors another chimeric construct will contain this domain of SP-A and the CRD of SP-D (or MBL). We will compare the ability of these recombinant collectins to bind to, aggregate, inhibit infectivity of, and act as opsonins for, a panel of clinically important IAV and pneumococcal strains. Next, specific residues involved in carbohydrate binding will be mutated in one of the constructs to determine if binding to IAV or pneumococci can be enhanced. In vivo activity of the collectin constructs will initially be tested by instilling them intranasally in mice prior to IAV or pneumococcal infection. One of the mutant constructs (chosen for optimal activity against IAV and/or pneumococci) will then be expressed in the airway of transgenic mice to determine if in vivo resistance to infection is enhanced. These experiments should yield important insights into basic collection biology and demonstrate that enhancement of collectin-mediated pulmonary host defense can be achieved in vivo through rational alteration of wild type collectins.