Development of novel anti-infectives for treatment of respiratory infections in patients with Cystic Fibrosis.This project aims to develop a novel peptide-based antimicrobial drug that can be used to treat respiratory (persistant) infections in CF-patients more effectively as compared to currently applied conventional antibiotics. Based upon our knowledge about antimicrobial peptides that exhibit distinct killing activity against a wide range of bacterial pathogens, we study which peptide appears most promising based upon its antibacterial properties assessed against CF-relevant pathogens using CF-patient-derived samples. Ultimately, this novel anti-infective will be developed (a) as therapeutic drug to treat pulmonary infections at later stages of CF in older children and adults and (b) as prophylactic drug in younger children. Both strategies will help to reduce the emergence of chronic bacterial infections and pulmonary inflammation at later stages of CF-disease and cut down antibiotic use. The combined use of this peptide-based antimicrobial drug and novel drugs that aim to restore CFTR function, will provide an innovative approach to prevent and treat infectious disease and respiratory tissue damage in CF-patients.Main research project collaborators:Cystic Fibrosis Center Utrecht, University Medical Center, Utrecht, The Netherlands; Microbiology & Institute of Biomembranes, Department of Biology, Utrecht University, The Netherlands; TNO, Microbiology and Systems Biology, Zeist, The Netherlands.Funded by ZonMw and NCFS.

Development of novel antivirals based on recombinant surfactant protein DInfluenza epidemics continue to occur annually causing excess morbidity and mortality in humans. The continuous threat of pandemic outbreaks requires novel means of protection since vaccination strategies (time-consuming) and currently available antiviral drugs (viral resistance) can not provide adequate protection against pandemics. This project aimed to develop a novel class of antiviral drugs, based upon a recombinant variant of ‘Surfactant Protein D’ (SP-D), that helps to protect against infections caused by a broad-range of influenza A viruses. SP-D is a naturally occuring protein, involved in innate defense in mammalian species. It is involved in early defense against pulmonary infections caused by many pathogens, including influenza A virus. The interactions between SP-D and a broad panel of IAVs from different host species as well as influenza B viruses were investigated. Ultimately, a mutant form of human SP-D (hSP-D) was developed that, due to several innovative molecular modifications is much more effective in neutralizing a much broader range of influenza A virus strains as compared to wildtype hSP-D. In vitro studies were supported by an in vivo model in mouse, showing protection against pandemic H1N1 (2009). A patent application regarding this technology was filed ("Novel polypeptide and uses thereof", PCTNL2014/050720). Main research project collaborators:Department of Viroscience, Erasmus Medical Centre, Rotterdam, The Netherlands; Departments of Physiology and Biophysics and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, U.S.A.; U-Protein Express B.V., Utrecht, The Netherlands. Funded by Technology Foundation STW (Open Technology Program).

Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. Focus of this project was to identify the immunodominant and virus-specific epitopes on Erns and E2 by the use of 3D-structured 'CLIPS'-peptides. These findings helped to gain insights into the molecular basis for the development of a novel DIVA vaccine against classical swine fever.

EU consortium Research project "Antimicrobials through Immune Stimulation" (AMIS).Design, production, and characterization of novel recombinant innate immune-based (hybrid) proteins to develop innovative innate immune-based strategies. Investigate their potential as a more effective alternative means for currently available antibiotics/antivirals and aimed to fight microbial infections in humans more effectively.

Research on porcine collectins to dissect their role in respiratory antiviral defense against influenza A viruses (IAVs) in this animal species, known for its mixing vessel potential as regards IAVs and the emergence of novel pandemic strains. Keywords: C-type lectins, influenza, innate immunity, pandemics, sialic acids, N-glycosylation, hemagglutinin, protein modification, antivirals.

Study interactions between porcine collectins and influenza A viruses in vitro.Pigs are (among the animal species studied so far) the only species that have adistinct glycosylation profile present on SP-D. Since these animals are consideredimportant for interspecies transmission of IAV, the anti-influenza activity of porcine SP-D wasextensively studied in vitro on a variety of IAV strains by the use of several (functional)assays. Although SP-A is in general less potent than SP-D in neutralizingIAV, we also included studies on porcine SP-A. The activity of both porcinelung collectins was compared to the activity of those from other species, with focus on thecontribution of the sialylated CRD-linked oligosaccharide to the antiviral activity.To further elucidate the role of SAs in IAV-neutralization, the linkage pattern of theSA moieties (either α(2,3) or α(2,6)) present on the complex sugar in the CRD of pSP-Dwas analyzed and enzymatically modified. It was determined by HAAinhibition analysis to what extent this SA-linkage pattern is important for the neutralizationof IAV by pSP-D, and whether this is dependent on the SA-linkage specificity of the HAreceptor of a particular strain.

Cloning, characterization and chromosomal localization of porcine pulmonary surfactant protein D (SP-D)cDNA cloning and sequence analysis of the full-length cDNA revealed that the predicted protein sequence contains unique features that are absent in the SP-D of other species previouslycharacterized. Amongst these features was the presence of an N-glycosylation motifsequence in the CRD of pSP-D, potentially important for interactions with respiratory pathogens. We also analyzed the tissue distribution of this protein as determined by Northern blot analysis and also performed chromosomal assignment of the SP-D (and SP-A) genes by somatic cell hybrid analysis. At a later stage, a more precise localization of both genes was performed by fluorescent in situ hybridization.

Pulmonary surfactant research, including studies on the structure and function of surfactant proteins, their interactions with surfactant lipids, and composition analysis of surfactant-based products and preparations derived from bronchoalveolar lavage. Developed a broad range of methods and skills on protein and lipid (bio)chemistry, immunology, biophysics, (electron, fluorescence) microscopy and primary cell culturing techniques.