ENHANCING COLLECTIN MEDIATED HOST DEFENSE

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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.