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.

Public Health Relevance

This research will try to explain how the body defends itself against influenza virus in the first few days after infection by studying some proteins that normally are present in the lung and block the virus from growing. Failure of these proteins to block infection with pandemic influenza virus may explain why pandemic influenza viruses cause more severe illness than usual seasonal strains of influenza. We have been able to develop new, more effective forms of these natural inhibitor proteins in the laboratory that could be used as treatment all types of influenza and possibly bacteria as well.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01HL069031-13
Application #
8681493
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Eu, Jerry Pc
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Boston Medical Center
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02118
Nikolaidis, Nikolaos M; White, Mitchell R; Allen, Kimberly et al. (2014) Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses. Am J Physiol Lung Cell Mol Physiol 306:L1036-44
Tripathi, Shweta; Verma, Anamika; Kim, Eun-Jeong et al. (2014) LL-37 modulates human neutrophil responses to influenza A virus. J Leukoc Biol 96:931-8
White, Mitchell R; Kandel, Ruth; Tripathi, Shweta et al. (2014) Alzheimer's associated ?-amyloid protein inhibits influenza A virus and modulates viral interactions with phagocytes. PLoS One 9:e101364
Goh, Boon Chong; Rynkiewicz, Michael J; Cafarella, Tanya R et al. (2013) Molecular mechanisms of inhibition of influenza by surfactant protein D revealed by large-scale molecular dynamics simulation. Biochemistry 52:8527-38
Hartshorn, Kevan L (2013) Why does pandemic influenza virus kill? Am J Pathol 183:1125-7
Doss, Mona; Ruchala, Piotr; Tecle, Tesfaldet et al. (2012) Hapivirins and diprovirins: novel ?-defensin analogs with potent activity against influenza A virus. J Immunol 188:2759-68
Crouch, Erika; Nikolaidis, Nikolaos; McCormack, Francis X et al. (2011) Mutagenesis of surfactant protein D informed by evolution and x-ray crystallography enhances defenses against influenza A virus in vivo. J Biol Chem 286:40681-92
Seaton, Barbara A; Crouch, Erika C; McCormack, Francis X et al. (2010) Review: Structural determinants of pattern recognition by lung collectins. Innate Immun 16:143-50
White, Mitchell R; Boland, Patrick; Tecle, Tesfaldet et al. (2010) Enhancement of antiviral activity of collectin trimers through cross-linking and mutagenesis of the carbohydrate recognition domain. J Innate Immun 2:267-79
Hartshorn, Kevan L (2010) New look at an old problem: bacterial superinfection after influenza. Am J Pathol 176:536-9

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