10th MIM Retreat - ethz.ch · MIM retreat Locarno, Ticino August 24th-26th 2017 Welcome and contact information Dear MIM PhD students, We are pleased to welcome you to the 10thMIM

10th MIM Retreat

24th-26th August 2017

Locarno, Ticino

Sponsored by

SUK-Program ‘Doktoratsprogramme‘ ETH Zurich & UZH

With generous additional contributions from

MIM retreat Locarno, Ticino August 24th-26th 2017

Contents

Welcome and contact information 2Travel information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Accomodation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Program schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Guest speakers 5

Talks schedule 7Session I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Session II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Session III . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Session IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Session V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Session VI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Posters schedule 9Poster session I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Poster session II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Talks abstracts 12

Posters Abstracts 27

Participants 51

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Welcome and contact information

Dear MIM PhD students,

We are pleased to welcome you to the 10th MIM PhD retreat, taking place in Locarno from 24th-26th

August 2017.

The MIM retreat is an annual event organized by students of the MIM program and open to MIMmembers at any stage of their PhD. This is a great opportunity to become acquainted with fellowstudents of the program, exchange ideas and set up collaborations in an informal setting, and receivevaluable insights from accomplished guest speakers. Additionally, you will have the chance to spendsome leisure time together, socialize and explore beautiful Locarno!

Please feel free to contact us with any questions or problems before or during the retreat. We lookforward to an eventful and enjoyable retreat with you!

Your MIM Retreat 2017 Organization Team

• Lenka Cernikova +41 786462266 [email protected]

• Markus Furter +41 798223866 [email protected]

• Nicola Häffner +41 786633689 [email protected]

• Markus Huemer +41 786850142 [email protected]

• Leandra Knecht +41 793361438 [email protected]

• Harini Subbaraman +41 763340790 [email protected]

• Sebastian Utz +41 787272808 [email protected]

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Travel information

Thursday, 24th August 2017

Meeting time 07:45 at the group meeting point in Zürich HB

Departure from Zürich 08:09

Arrival in Locarno 10:27

Saturday, 26th August 2017

Departure from Locarno 15:05

Arrival in Zürich 17:28

If you are a MIM member located outside Zürich, you will get a refund for the cost of travel toZürich or directly to Locarno upon submission of your receipt. Please inform the organizers inadvance, as other individual travel arrangements will not be compensated.

Accomodation

All attendees will be hosted at the youth hostel in Locarno.

Schweizer JugendherbergeJugendherberge Locarno PalagiovaniVia B. Varenna 18CH-6600 Locarno

E-Mail: [email protected]

Tel: 091-7561500Fax: 091-7561501

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Program schedule

Thursday, August 24th

7:45 – 10:45 Zurich – Locarno10:45- 11:45 Check in11:45-12:00 Welcome12:00-13:00 Lunch13:00-14:00 Student talks Session I14:00-14:15 Break14:15-15:15 Dr. Jens Fleischer and Dr. Laura Lipka15:15-16:45 Poster Session I16:45-17:30 Student talks Session II17:30-17:45 Break17:45-18:30 Student talks session III18:30 onwards Ice breaker and BBQ

Friday, August 25th

8:00 - 9:00 Breakfast09:00-10:00 Student talks Session IV10:00-10:15 Break10:15-11:00 Student talks Session V11:00-11:15 Break11:15-12:00 Student talks Session VI12:00-13:00 Lunch13:00-13:45 Dr. Andrew Croxford13:45-14:00 Break14:00-14:45 Dr. Ines Matos14:45-16:00 Poster Session II16:00 onward Social activity and dinner

Saturday, August 26th

8:00-9:00 Breakfast9:00-10:00 Check out

10:00-12:00Dr. Andrea DegenWorkshop: Successful funding acquisition

12:00-13:00 Lunch

13:00-14:30Dr. Andrea DegenWorkshop: Successful funding acquisition

14:45-17:30 Locarno-Zurich

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Guest speakers

Dr. Jens Fleischer

Dr. Jens Fleischer pursued his PhD in immunology and cellbiology at the Research Center Borstel and continued withpostdoctoral work at the same facility. He then joined BDas an applications specialist and has been working for thepast 15 years as a high-end instrumentation and applicationconsultant, specializing in sales and marketing, technicalsupport and training programs.

Dr. Laura Lipka

Dr Laura Lipka has extensive experience in industry,working for companies such as Mettler Toledo International,Invitrogen and Affymetrix as an account manager, technicalsales specialist and business development manager. Since2016 she has worked at BD as an account manager andproduct specialist.

Dr. Andrew Croxford

Dr. Andrew Croxford completed his PhD at the JohannesGutenberg University of Mainz with Dr. Ari Waisman,focusing oon IL-17A biology. He then pursued postdoctoralwork on myeloid cells and proinflammatory cytokines withDr. Burkhard Becher at the University of Zurich until May2016, following which he joined Actelion PharmaceuticalsLtd. The company was acquired by Johnson&Johnson in June2017 and Actelion‘s drug discovery operations and early stageclinical development programs were spun off into the newlycreated company Idorsia Ltd, where Dr. Croxford continuesto work as a lab head in Immunology Drug Discovery.

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Dr. Ines Matos

Dr. Ines Matos started her scientific research career focusingon malaria vaccine development. In 2007 she moved to NewYork City to pursue her PhD in dendritic cell biology underthe supervision of Dr. Ralph Steinman at the RockefellerUniversity. Following a two year postdoctoral positionat Columbia University, Dr. Matos started looking foropportunities in industry to follow her passion of workingin cancer, and moved to Zurich as a postdoctoral researchassociate at Roche. After a period of maternity leave in2016, Dr. Matos become a Senior Scientist at Roche andcurrently works on the early development of novel cancerimmunotherapies.

Dr. Andrea Degen

Dr. Andrea Degen is a former clinician and researcherwith over ten years of experience in R&D projects in energy,environment and life sciences. She has been an independentmanagement partner in various R&D ventures since 2006,and is the founder of EUrelations AG, a research managementagency. Her areas of expertise include research management,acquisition of public funding and compilation of researchapplications, and she teaches several courses on projectmanagement in research and third party funding acquisition.

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Talks schedule

Session I

Natalia Krempaska Adaptation of free-living freshwater microbes to a variable substratefield

Lenka CernikovaA family of PX-domain lipid binding proteins as organizers of theendocytic organelle system in Giardia lamblia

Marisa O.D. SilvaPriming of microbial microcystin degradation in biomass-fedgravity driven membrane filtration biofilms

Christian StockerInvestigating mechanism and function of the secreted fusion proteinbetween chorismate mutase and cyclohexadienyl dehydratase fromShewanella baltica

Session II

Harini SubbaramanDissecting viral features leading to broadly neutralizing antibodydevelopment in an HIV-1 infected transmission pair

Eva MüllerIdentification of novel interferon-stimulated genes involved in theantiviral response against influenza A virus

Pengfei LiFeLV in cats: An animal model to study retroviral reservoireradication using a CRISPR/Cas9-based cure strategy

Session III

Yu Xueyang Regulation of alveolar macrophage development and homeostasis

Sebastian Utz TGF β signaling in developing microglia

Hana ZdimerovaExamining the role of MS-associated genetic risk factors in ahumanized mouse model of Epstein-Barr virus infection

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Session IV

Philipp SchineisThe role of CD155 in lymphatic vessel morphology and leukocytemigration through afferent lymphatic vessels

Morgan Hunter Elucidating intralymphatic dendritic cell-T cell interactions

Yun DengRole of CD27/CD70 deficiency in cell-mediated immune control ofthe Epstein Barr virus

Florian Kirchner Th17 cell priming during persistent oral Candida albicans infection

Session V

Veronika HaunerdingerReconstitution of the Innate Immune System after HematopoieticStem Cell Transplantation in Children

Ute SüssbierFunctional and structural dynamics of the bone marrow stromalmicroenvironment after cytoreductive therapies

Bruce WederBCL-2 levels do not predict azathioprine treatment response ininflammatory bowel disease, but inhibition induces lymphocyteapoptosis and ameliorates colitis in mice

Session VI

Rahel Winiger Implementation of nanobodies in the Toxoplasma gondii field-Search for new biomarkers

Emiel ten Buren Development of a non-invasive reporter method to monitor effectorCD4+ effector T cell accumulation in vivo

Semjon SidorovGeneration of in vitro model of IgH/c-myc translocation in EBV-infected cells using CRISPR/CAS9 RNPs

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Posters schedule

Poster session I

Mona FriessInvestigating the role of ACKR4 on leukocyte trafficking withinlymphatic vessels and on lymphatic vessel biology

Markus Furter The gut mucus and its protective role in Salmonella Typhimuriuminfection

Ratchara Kalawong Influence of biosurfactants on Pseudomonas putida membranevesicles

Audrey Kelley The replication capacity of HIV-1 Transmitter/Recipient pairs of theZPHI and SHCS Cohort

Leandra Knecht Identification of Phage receptor on Erwinia amylovora CFBP 1430

Maarja Grünbach

A comparative study on the biology and anti-HIV-1 effect of IFN-α subtype, IFN-α14, IFN-α mutant, YNS, with elevated bindingaffinity to IFNAR and prototype IFN-α subtype, IFN-α 2, in vivoin humanized mice

Annika Hunziker Characterization of entry factors used by seasonal circulating humanand avian Influenza A strains

Marisa LoiInvestigation of the molecular mechanisms regulating recovER-phagy in mammalian cells

Hanna MartiUnderstanding the transmission mechanism of the tetracyclineresistance gene C (tetC) in Chlamydia suis

Barbara Müller Towards understanding stability and function of plant-associatedmicrobiota

Julia Neuhaus In vivo kinetic analysis of N-glycosylation in yeast

Tony Nguyen The role of resident regulatory T cells in long-term graft acceptance

Alaz ÖzcanContribution of neutrophils to the induction of local and systemicpsoriasis inflammation

Michela Perotti Non-classic mouse Th1 cells and the requirements for theirdifferentiation

Benjamin Peschke Therapeutic Efficacy of low molecular weight polysialic acid in EAE

Gabriela Purtschert Pseudomonads contact-dependent competition in mixed biofilms

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Fiona Steiner Assessment of the antiviral activity of MxA against Influenza AVirus

Sereina SutterGlobal gene expression analysis of adeno-associated virus type 2(AAV2)-infected cells

Marjan Verest A novel peptide splicing enzyme from Pleurocapsa. sp. creates ?-amino acid residues

Anja WalkamEstablishment of a molecular toolbox to generate apoptosis inducingvectors for the depletion of distinct cell types in vivo

Mirjam ZündLongitudinal stratification of microbial communities along thegastrointestinal tract

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Poster session II

Ilaria AffolterTime-resolved analysis of the N-glycan processing in the ER andGolgi of CHO cells

Sandra ChaudronCharacterization of HIV superinfection in the Swiss HIV CohortStudy

Stefano GualdiGenome-wide identification of Burkholderia cenocepacia H111molecular responses to cadmium

Nicola Häffner Role of colonizing Staphylococcus aureus on Influenza A Infectivity

Lukas HeebBlocking the human type 2 interleukin 4 receptor and its effects onhuman neutrophil

Markus HuemerStaphylococcus aureus persister formation as an adaptive responseduring infection

Ufuk KarakusDevelopment of Treg cell-stimulating IL-2/antibody complexes andinvestigation of the IL-2 receptor assembly

Umut Karakus Characterization of bat influenza virus entry

Monika Kotur Harnessing autophagy to limit autoimmune CNS inflammationx

Markus KreuzerSignature-Tagged Mutagenesis Screen in Salmonella Typhimuriummurine gastrointestinal colonization

Verena KufnerVirus transmission during kidney transplantation assessed byvirome analysis of living donor and recipient

Anita Meier Cell cycle-dependent gene expression of the adeno-associated virus

Peter RungeInvestigating the role of lymphatic endothelial cell expressedimmunomodulatory molecules (CD112, CD274) in intralymphatictrafficking and cellular immunity

Martin Schäfer Probing bacterial interactions in the A. thaliana phyllosphere

Alessandra VitaleIdentification of the mode of action of peptide antibiotics inPseudomonas aeruginosa

Tobias WeissSynergistic activity of NKG2D-based chimeric antigen receptor(CAR)-T cells and radiotherapy against glioma

Dominik WronaGeneration of a human p47phox-deficient chronic granulomatousdisease cell line using CRISPR/Cas9 for fast gene therapy vectortesting

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Talks abstracts

Session IAdaptation of free-living freshwater microbes to a variable substrate field

N. Krempaska, Dr. K. Horňák, Prof. J. Pernthaler

Department of Plant and Microbial Biology, University of Zürich

[email protected]

Free-living freshwater microbes appear to thrive in unstructured environments withheterogeneous substrate distributions. However, physical, biogeochemical and biologicalfactors superimpose structure onto pelagic habitats at various spatial and temporal scales.This heterogeneity may be relevant for bacterial assemblages, as it mediates theco-existence of genotypes with contrasting growth strategies. For example, motile andnon-motile bacterioplankton populations could have different ecophysiologicaladaptations to variable substrate levels. Motile cells track gradients of organic compounds;they are thus expected to sense rise of substrate concentrations as a signal for increasinglyfavorable growth conditions, and to rapidly undergo general metabolic up-regulation. Bycontrast, non-motile bacteria are passively exposed to stochastic change of the substratefield from which they cannot predict future growth conditions. However, also non-motilebacteria should be well-adapted to thrive in variable substrate fields, e.g., by permanent‘preparedness’ to rapidly incorporate substrates across concentration ranges that matchenvironmental fluctuations. Due to the tight coupling between DFAA consumption andrelease, they qualify as sensitive tracers for exploring the heterogeneous substrate fieldexperienced by bacterioplankton. The aim is to investigate the extent of substratevariability at the meso- to macroscale, and its consequences on growth patterns ofbacterioplankton populations. We shall chart the heterogeneity of DFAA in Lake Zürich atdifferent spatiotemporal scales to provide an ecological framework for performing pulseaddition experiments with DFAA. We shall also obtain metagenomic information fromthese experiments to distinguish between populations with and without genes for motility,and subsequently map metatranscriptomic data onto these populations.

A family of PX-domain lipid binding proteins as organizers of the endocytic organellesystem in Giardia lamblia

Lenka Cernikova, Jon Paulin Zumthor, Carmen Faso, Adrian Hehl

Institute of Parasitology, University of Zürich

[email protected]

Giardia trophozoites have a distinctly polarized cellular architecture and are able to attachto the small intestinal epithelium of the host via a unique ventral organelle. Compartmentsof the highly simplified endosome-lysosome organelle system, the peripheral vacuoles(PV), are arrayed in the dorsal cortical region on the opposite side of the cell at ∼50 nmdistance from the plasma membrane (PM). We have recently shown that giardial clathrindoes not produce coated vesicles at the PM, but instead forms unique highly stable arraysat the PV membrane – PM interface. Analysis of the clathrin interactome revealed two

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novel phosphatidylinositol-binding proteins containing conserved PX-domains. Usingdata-mining and ectopic expression of tagged variants we demonstrated that Giardiatrophozoites express a total of 6 PX-domain proteins. All family members localizeexclusively to PVs by confocal fluorescence microscopy, but comprehensive analysis ofreverse co-immunoprecipitation experiments show that only 2 are integrated into stableclathrin arrays via their effector domains. Modeller modelling revealed conservedphospholipid-binding domains showing typical structural elements of6 PX-domainsrequired for lipid binding. In vitro binding assays with purified recombinant PX-domainsshowed distinct phospholipid binding profiles consistent with localization at the PM andmembranes of endocytic organelles. Further characterization of this protein familyincludes direct interference with function to test the hypothesis that PX-domain proteinshave evolved as organizers of the highly specialized endocytic system in Giardia.

Priming of microbial microcystin degradation in biomass-fed gravity driven membranefiltration biofilms

Marisa O.D. Silva, Judith F. Blom, Yana Yankova, Jörg Villiger, Jakob Pernthaler

Limnological Station, Department of Plant and Microbial Biology, University of Zürich

[email protected]

Gravity Driven Membrane (GDM) filtration is a promising tool for low-cost decentralizeddrinking water production. The biofilms on GDM systems are able to remove potentiallyharmful chemical components, in particular toxic cyanobacterial metabolites such asmicrocystins. This is relevant for the application of GDM filtration, because anthropogenicnutrient input and climate change have led to an increase of toxic cyanobacterial blooms.However, removal of microcystins in newly developing GDM biofilms is only establishedafter a prolonged period of time. Since cyanobacterial blooms are transient phenomena, itis important to understand microcystin removal in mature biofilms with or without priortoxin exposure. We studied microbial community composition of GDM biofilms in systemsfed with water from a lake with periodic blooms of microcystin-producing cyanobacteria.Two of three experimental treatments were supplemented with dead biomass of amicrocystin-containing cyanobacterial strain, or of a non-toxic mutant, respectively.Analysis of bacterial rRNA genes revealed that both biomass-amended treatments weresignificantly more similar to each other than to an unamended control. We thushypothesized that biofilms could be potentially ‘primed’ for rapid microcystin removal byprior addition of non-toxic biomass. A subsequent experiment showed that microcystinremoval developed significantly faster in biofilms pre-fed with biomass from the mutantstrain than in unamended controls. Therefore, microcystin degradation was a facultativetrait of bacterial populations in GDM biofilms. The significant enrichment of both, aerobicand anaerobic known microcystin degraders, such as Aeromonas, Pseudomonas andAcidaminobacter, suggested that this process might occur in parallel in differentmicroniches.

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Investigating mechanism and function of the secreted fusion protein betweenchorismate mutase and cyclohexadienyl dehydratase from Shewanella baltica

Christian Stocker, Kathrin Würth-Roderer, Peter Kast

Laboratory of Organic Chemistry, Department of Chemistry and Applied Biosciences, ETHZürich

[email protected]

Chorismate mutase (CM) and cyclohexadienyl (prephenate) dehydratase (CDT) are twoenzymes of the shikimate pathway, required for synthesizing the essential aromatic aminoacids phenylalanine and tyrosine. In the cytoplasm of bacteria, the CM convertschorismate to prephenate that is used by CDT to make phenylpyruvate, the directprecursor of phenylalanine. A secreted fused version of CM and CDT was discovered inShewanella baltica, the predominant H2S-producing strain contributing to fish spoilage at0◦C, and in a few other marine organisms. It was shown that the fusion protein has a highcatalytic efficiency for chorismate conversion (kcat/KM = 5·105 M-1 s-1); the CDT activityhas not been determined yet. Further mechanistic investigations, such as the enzymaticactivity of separated domains, as well as the possibility for substrate channeling from CMto CDT are ongoing. We intend to examine the individual separated domains using adirected evolution approach to identify features essential for catalytic activity. Therefore,robotics-supported automation of directed evolution processes will be implemented.Furthermore, comparing the secreted CM and CDT versions to their cytoplasmiccounterparts is planned. Ultimately, the biological role of secreted CM-CDTs is underinvestigation. Currently, their occurrence is still enigmatic, because neither chorismate norprephenate is generally found outside of the cell.

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Session II

Dissecting viral features leading to broadly neutralizing antibody development in anHIV-1 infected transmission pair

Harini Subbaraman, Peter Rusert, Natacha Espirito Santo, Jacqueline Weber, Alexandra TrkolaInstitute of Medical Virology, University of Zürich

[email protected]

Broadly neutralizing antibodies (bnAbs) targeting the HIV-1 envelope trimer glycoprotein(Env) inhibit diverse heterologous viral isolates; their elicitation via immunization is hencea primary goal for HIV-1 vaccine design. However, only 10-25% of HIV-1 infected patientsdevelop bnAbs due to a complex mechanism involving several rounds of selection andmaturation in response to viral escape variants influenced by host, viral and diseasecharacteristics. Research into the determinants of bnAb development and strategies fortheir induction in HIV-1 naïve individuals is therefore of great interest.

A screen of 4,484 HIV-1 infected individuals from the Swiss HIV Cohort and the ZürichPrimary Infection study conducted by our group identified 239 bnAb producers anddetermined that viral load, viral diversity, infection duration and host ethnicityindependently correlate with the propensity for bnAb development. Additionally, thesubtype of the infecting virus was found to influence the Env target specificity of theresulting bnAb. These findings implicate specific, possibly subtype-dependent viralcharacteristics in triggering and steering bnAb development. One case in particularcomprises a linked transmission pair of individuals, both of whom developed a strongbnAb response with similar neutralization profiles despite having different gender andethnicity. We aim to dissect the Env features and longitudinally track evolution of the viralpopulations in both individuals to identify the shared sequence and structural elementsthat favored bnAb induction.

Identification of novel interferon-stimulated genes involved in the antiviral responseagainst influenza A virus

E. E. Müller, S. Stertz, B. G. Hale

Institute of Medical Virology, University of Zürich

[email protected]

Hundreds of diverse ISGs are known to be upregulated upon IFN treatment. There aresome ISGs characterized to have antiviral activities against IAV, including the MxA,IFITM3, or the recently identified PAI-1. However, only a few have been found to havepotent antiviral function against IAV. Hence, we hypothesise that the currently knownfactors alone cannot explain the strong effect of IFN against IAV. The aim of this PhDproject is to identify novel ISGs with potent antiviral action against IAV. More precisely, asmall interference RNA (siRNA) library targeting 102 ISGs was tested for their potential torescue viral replication efficiency in the presence of IFN. Furthermore, newly identifiedfactors will be further characterized concerning their function, localization and mechanismof regulation of the IAV replication cycle. Potential impact on the IFN production or IFNsignalling pathways by the ISGs were tested using two different reporter cell linesexpressing GFP. Another aspect to this project will be testing the antiviral potential of ISGsagainst novel reporter IAVs that will be developed, including an avian strain (H5N1) which

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is a serious zoonotic threat to humans. It is known, for example, that MxA shows strongerantiviral activity against avian IAVs than against human IAVs. Hence, different IAV strainswill be tested with the aim of identifying specific ISGs that may act as host-rangerestriction factors.

FeLV in cats: An animal model to study retroviral reservoir eradication using aCRISPR/Cas9-based cure strategy

Pengfei Li, A. Katrin Helfer-Hungerbuehler, Salomé LeibundGut-Landmann, Regina Hofmann-Lehmann

Clinical Laboratory, Vetsuisse faculty, University of Zürich

[email protected]

Like HIV in human, feline leukemia virus (FeLV) is a retrovirus that can cause persistentinfection in cats and form provirus by reverse transcription and integration into the hostgenome. We aim to use gene editing tools to aid cats in overcoming FeLV retroviral infectionby lowering provirus loads.

In this project, CRISPR/Cas9 system will be employed for provirus cleavage andsubsequent inactivation. First, efficiencies of different small Cas9 orthologues inintroducing double strand breaks and reducing viral replication are assessed in vitro. Insilicon algorithm prediction and various in vitro assays are used to decrease the off-targetratio, increase the target specificity and finally screen the most promising target siteswithin FeLV provirus. Meanwhile, we will survey the prevalence of pre-existing antibodiesagainst different adeno-associated virus (AAV) serotypes in cats for the subsequent genetherapy vector selection. This is of importance since neutralizing antibodies may reducethe gene therapy efficiency. The optimal AAV vector will be constructed to deliver theCRISPR/Cas9 system into the organism. The safety, tropism and efficiency will beinvestigated by in vitro transduction assay by using various FeLV positive cell lines andPBMCs. The eradication of FeLV provirus will be analysed by real-time qPCR and p27antigen ELISA.

Finally, we will evaluate the vector in vivo by analysing the toxicology in healthy cats andexcision efficiency in persistently FeLV-infected cats. The goal is to find an efficient andsafe gene therapy strategy which specifically causes FeLV abortive infection on geneticlevel.

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Session III

Regulation of alveolar macrophage development and homeostasis

Yu X, Buttgereit A, Lelios I, Cansever D, Utz S, Becher B, Greter M

Institute of Experimental Immunology, University of Zürich

[email protected]

Tissue-resident macrophages are heterogeneous populations that have uniquegene-expression patterns and distinct functions, despite largely overlapping embryonicorigins. It has been shown that lung alveolar macrophages (AMs) are dependent onGM-CSF and PPAR-γ for their development and function. Here we show that cell-intrinsicdeletion of TGF-β receptor signaling leads to their absence in vivo. TGF-β emerged as acritical factor for the differentiation of AMs from fetal monocytes, for the maturation ofAMs after birth, and for their homeostasis in the steady state. Collectively, our findingsidentify a previously unappreciated developmental pathway for AMs.

TGF-β signaling in developing microglia

Sebastian Utz, Anne Buttgereit, Melanie Greter


[email protected]

Microglia are the tissue-resident macrophages of the brain. They arise very early duringdevelopment from the extra-embryonic yolk sack repopulating the brain from day E7.5 on.In the adult microglia are self-maintaining and in the steady state they do not expandclonally. Microglia protect the brain during viral or bacterial infections and clear theinfection via secretion of cytokines, phagocytosis and recruitment of other infiltratingperipheral immune cells. They are further involved in synaptic pruning and seem tointeract with many other CNS associated cell types. Microglia, however, are very distinctfrom other CNS-associated macrophages, such as choroid plexus, meningeal orperivascular macrophages. They strongly depend on CSF1-R signaling for their survivaland maintenance. Little is known about the impact of TGF-β in embryonic microglia.Hence, our research is focusing on investigating the intriguing role of TGF-β in microgliausing a mouse model with a microglia-specific knock out of TGF-β receptor 2. We try tounderstand what impact loss of TGF-β rector in the embryo has on microglia, if microgliashow plasticity and how this affects also other associated cells such as astrocytes, neuronsin the brain.

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Examining the role of MS-associated genetic risk factors in a humanized mouse modelof Epstein-Barr virus infection

Hana Zdimerova1, Bithi Chatterjee1, Liliana Danusia Vanoaica1, Mark Robinson2,

Christian Münz1

1Department of Viral Immunobiology, Institute of Experimental Immunology, University ofZürich

2Institute ofMolecular Life Sciences, University of Zürich

[email protected]

More than one hundred genetic and environmental risk factors have been implicated in theautoimmune disease multiple sclerosis (MS). The presence of the HLA-DR2 MHC Class IIhaplotype confers the strongest genetic risk, however its contribution to disease remainspoorly understood. Of the environmental risk factors, infection with Epstein-Barr virus(EBV) is strongly correlated with increased MS risk, especially in patients who haveexperienced infectious mononucleosis (IM), the severe acute form of EBV infection. IMsynergizes with the HLA locus for a sevenfold increased risk to develop MS, however themechanisms behind this interplay are not known. Our study aims to use humanized miceas an in vivo EBV infection model to investigate T cell responses in animals reconstitutedwith donors positive for genetic MS risk factors.

Using our model of NOD-scid gamma chain-deficient (NSG) mice reconstituted withhuman immune system components (huNSG), animals reconstituted with HLA-DR2+donors showed hyperactivated T cells in the steady state. Furthermore, following EBVinfection, these huNSG mice developed elevated blood viral titers, as well as higherfrequencies of CD3+ and CD8+ T cells. Recently, genome-wide association studies haveidentified multiple single nucleotide polymorphisms (SNPs) that seem to increase the riskof MS. We find that a higher immune activation in HLA-DR2+ donor-reconstituted animalsis associated with certain

SNPs in immune related genes, including signaling molecules and transcription factorsinvolved in antigen processing and differentiation of cytotoxic effector CD8+ T cells.Thus,

MS-associated genetic risk factors could predispose individuals to elevated, poorlyprotective T cell responses after EBV infection, possibly priming autoreactive T cellspecificities in the process.

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Session IV

The role of CD155 in lymphatic vessel morphology and leukocyte migration throughafferent lymphatic vessels

Philipp Schineis1, Jorge Arasa Aparici1, Javier Montoya2, Peter Runge1, Michael Hertwig1,Cornelia Halin1

1Institute of Pharmaceutical Sciences, ETH Zürich 2Image and Data Analysis Unit @ ScopeM,ETH Zu¨rich [email protected]

CD155, formerly known as Poliovirus Receptor (PVR) or necl-5, is a cell adhesion moleculeof the nectin family, which is expressed on fibroblasts and certain endothelial cell types, butalso on dendritic cells (DCs). CD155 reportedly engages in heterophilic interactions withNectin-3 and alpha-V-beta-3 integrin. Physiologically, its role in fibroblast biology has beeninvestigated and these studies could identify CD155 as a regulator of cell proliferation andcell-cell contact inhibition.

The aim of this project is to validate the expression of CD155 in the lymphatic system and toinvestigate the role of CD155 in lymphatic vessel (LV) function and its impact on lymphaticendothelial cell (LEC) biology.

Transcriptional analysis of LECs isolated from murine ear skin, recently indicated to usthat CD155 is expressed in the lymphatic vasculature. Performing FACS based stainings ofmouse ear skin we confirmed CD155 expression at the protein level on capillary andcollecting LECs of afferent LVs in vivo and on cultured human and murine LECs in vitro.In addition, immunofluorescence-staining of human skin sections showed a vessel-likeexpression pattern of CD155.

In functional in vitro assays, a ligation of CD155 with monoclonal antibodies (clone D171and SKII.4), exhibiting agonistic and blocking functions, increased human LEC proliferationand adhesion to fibronectin and collagen coated plates. Moreover, in tube-formation assays,CD155 ligation enhanced tube network complexity and tube persistence.

Collectively, our results for the first time show the expression of CD155 on LVs, and our invitro results indicate a potential contribution of CD155 to lymphangiogenesis.

Elucidating intralymphatic dendritic cell – T cell interactions

Hunter MC, Teijeira A, Russo E, Proulx ST, Frei T, Debes GF, Coles M, Melero I,

Detmar M, Rouzaut A, Halin C

Institute of Pharmaceutical Sciences, ETH Zürich

[email protected]

Afferent lymphatic vessels fulfill important immune functions by transporting solubleantigen, dendritic cells (DCs) and recirculating T cells from peripheral tissues to draininglymph nodes. Over the past 15-20 years, several molecules involved in DC migration and -to a lesser extent - in T cell migration have been identified. Thanks to live, time-lapseimaging, intralymphatic leukocyte migration is now starting to be unraveled at the cellularlevel. Performing intravital microscopy in murine ear skin, we found that T cells, assimilarly reported for DCs, actively crawl within initial lymphatic capillaries and are onlypassively transported with lymph flow once they reach contracting collecting vessels.Moreover, we reported that intralymphatic T cell motility in lymphatic capillaries was

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increased during contact-hypersensitivity-induced inflammation and dependent onadhesive interactions between ICAM-1 and LFA-1.

The discovery that both dendritic cells and T cells actively crawl and patrol within initiallymphatic capillaries has raised fundamental questions regarding the mode and purpose ofintralymphatic leukocyte migration. Recent intravital microscopy for the first timerevealed interactions occurring between endogenous T cells and adoptively transferreddendritic cells within initial lymphatic capillaries of inflamed ear skin. Furthermore, ininflamed ear skin of a three-color fluorescent reporter mouse, regulatory T cells activelymigrated within initial lymphatic capillaries and frequently interacted with DCs insidelymphatic vessels. While the functional consequences of these interactions are beingfurther explored, these findings suggest that initial lymphatic capillaries might represent anovel immune compartment in which immune modulation occurs.

Role of CD27/CD70 deficiency in cell-mediated immune control of the Epstein Barrvirus

Yun Deng, Bithi Chatterjee, Christian Münz


[email protected]

Epstein Barr virus (EBV) is one of the most successful pathogens in the human population,persistently infecting more than 90% of adult individuals. At the same time it wasdiscovered as the first human tumor virus which is responsible for 1-2% of all cancers inhumans. Primary immunodeficiencies that predispose for EBV induced tumors anduncontrolled virus infection, have identified molecules in the differentiation,co-stimulation and effector function of cytotoxic lymphocytes that seem to be essential tokeep EBV in check. Due to the lack of animal model for EBV infection and immune controlit was previously difficult to address the biology of these molecules during EBV infection.In this project, we are assessing CD27/CD70 interaction EBV specific immune control andinvestigating which immune compartment(s) are compromised in the absence of thisco-stimulatory signal. Humanized mice with reconstituted human immune system areused as animal model to characterize in vivo pathology of EBV under the condition ofCD27 depletion. By addressing the role CD27 in different cell compartments, it shouldallow us to strengthen the respective immune responses with EBV associated diseases andto elicit them by EBV specific vaccination.

Th17 cell priming during persistent oral Candida albicans infection

Florian Kirchner, Salomé LeibundGut

Institute of Virology, Vetsuisse Faculty Zürich

[email protected]

Besides their autoimmune potential, T helper 17 (Th17) cells play an important role in thebody by providing protection against fungal infections such as those caused by Candidaalbicans. Here, we introduce a novel mouse model of persistent oral candidiasis that closelyreflects C. albicans commensalism in humans. In contrast to previously available murinemodels of candidiasis, in which the fungus is rapidly cleared by an acute inflammatory hostresponse, T cells are responsible for fungal control in our new model, as it is the case inhumans. This was evidenced by increased susceptibility of RAG1-deficient mice that couldbe rescued by transfer of C. albicans-experienced CD4+ T cells. The restricted localization

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of persistent C. albicans strains in the stratum corneum of the oral epithelium and theirincapability of triggering an inflammatory response raises the question of how they are stillable to induce a potent and sustained antigen-specific Th17 response. Using a recentlydeveloped C. albicans-specific TCR-transgenic mouse we are currently dissecting the APCsubsets and polarizing cytokines that regulate this antifungal Th17 response in the oralmucosa.

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Session V

Reconstitution of the Innate Immune System after Hematopoietic Stem CellTransplantation in Children

Veronika Haunerdinger, Claudia Möller, Tayfun Güngör, Mathias Hauri-Hohl

Pediatric Stem Cell Transplantation, University Children’s Hospital Zürich

[email protected]

Allogenic Hematopoietic Stem Cell Transplantation remains the only curative option for avariety of malignant diseases as well as inborn errors of the immune system or metabolism.Although the procedure has markedly improved over the past decades, patients in theearly post-transplant phase are still at high risk of transplant-related complications such asinfectious diseases, febrile neutropenia, vasculopathy or graft-versus-host syndrome. Theclinical symptoms of these complications are very similar at onset and thus present adiagnostic and therapeutic challenge for the treating physician.

After Hematopoietic Stem Cell Transplantation, the cells that first become detectable inthe peripheral blood belong to the innate immune system. These cells might therefore besuitable as biomarkers for various inflammatory conditions in the patient. We developed a13-colour flow cytometry panel for the evaluation of most innate immune cell subsets in theblood which can be performed in one single tube.

So far, we have longitudinally followed several patients during the early post-transplantphase and collected data on average on 6 to 8 time points. Comparison to results fromroutine hematology testing revealed that our flow cytometry approach can identify thesame cell populations at similar levels. In addition, we are able to detect additional cellpopulations and we can detect rare cell populations much more precisely. Furthermore,our flow cytometry panel gives information about the activation and maturation status ofdistinct cell populations which will be very valuable in the assessment of these cellpopulations as suitable biomarkers.

Functional and structural dynamics of the bone marrow stromal microenvironment aftercytoreductive therapies

Ute Süssbier, Hui Chyn Wong, Alvaro Gomariz, Stephan Isringhausen, Patrick Helbling,

Takashi Nagazawa, Antonia MS Müller, Markus G Manz, César Nombela-Arrieta

Experimental Hematology, University Hospital Zürich

[email protected]

Bone marrow (BM) cavities are the primary sites of high throughput, continuous andtightly regulated production of mature blood cells during adulthood. Hematopoiesis issustained by the proliferation and differentiation of hematopoietic stem and progenitorcells (HSPCs), which are maintained by signals emanating from stromal networks ofmesenchymal, endothelial and neural origin. BM tissues are highly sensitive tomyeloablative treatments and the induced cytotoxic effects and killing of rapidly cyclinghematopoietic progenitors have been extensively characterized. However, it is still largelyunknown whether and to what extent these treatments target BM stromal cells.

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Here we show the structural and functional alterations triggered in the BM stromalinfrastructure upon myeloablation by combining conventional flow cytometry protocolsand advanced 3D-imaging.

As previously reported, cytoreductive treatments led to a severe loss of HSPCs. Notably,hematopoietic defects were accompanied by a similar profound decrease in sinusoidalendothelial and mesenchymal, CXCL12-abundant reticular (CAR) cells. Decline in stromalcell numbers was apparent 7 days after treatment and encompassed a major loss ofstructural integrity of the BM microenvironment. 3D imaging revealed massive sinusoidaldilation followed by appearance of ruptures in vessel walls. Partial restoration of tissueintegrity was observed 14 days post-treatment and was almost completely achieved by day28 after induction of tissue damage. We observed massive de novo differentiation ofmesenchymal progenitors into adipocytes leading to adipogenic infiltration of large regionsof the BM during the phases of acute damage and tissue regeneration. This process wasfully reversible as virtually almost all adipocytes were cleared 56 days aftertreatment.

BCL-2 levels do not predict azathioprine treatment response in inflammatory boweldisease, but inhibition induces lymphocyte apoptosis and ameliorates colitis in mice

Bruce Weder1 and Mahdi Mozaffari1, Luc Biedermann1, Céline Mamie1, Lichun Wang2, StephenH. Clarke2, Gerhard Rogler1, Bradford L. McRae2, Candace L. Graff2, Pedro A. Ruiz1, MartinHausmann1

1 Department of Gastroenterology and Hepatology, University Hospital Zürich, University ofZürich

2 AbbVie Bioresearch Center, AbbVie Worcester, MA, USA

[email protected]

Introduction: In the pathogenesis of inflammatory bowel disease (IBD), inflammation issustained by an exaggerated response of T-lymphocytes. This results from an enhancedexpression of anti-apoptotic BCL-2 and BCL-XL. Here, we investigated whether the BCL-2family mRNA expression pattern could be used to predict treatment response toazathioprine in IBD patients. We also sought to determine whether the BCL-2 inhibitorA-1211212 effectively diminishes accumulated lymphocytes and ameliorates inflammationin a murine model of spontaneous colitis.

Methods: BCL-2 family expression pattern in azathioprine patients was determined bynext generation sequencing and qPCR. A-1211212 was administered orally to Il-10-/- mice.Haematological analyses were performed with an ADVIA 2120 flow cytometer.

Results: We determined similar expression levels of members of the BCL-2 family in patientswith remission and patients refractory to treatment. A-1211212 initiated cell death in CD4+T cells from patients refractory to azathioprine and reduced lymphocyte count in Il-10-/-mice (p < 0.05). CyTOF analysis of PBLs revealed diminished CD8+ T and B cells uponA-1211212 treatment in Il-10-/- mice. This finding was confirmed by increased apoptosisupon A-1211212 treatment in splenocytes and LPMNCs, together with a decrease in pro-inflammatory cytokines. A-1211212 positively altered the colonic mucosa at macroscopicand microscopic level and ameliorated intestinal inflammation in Il-10-/- mice.

Conclusions: The expression pattern of BCL-2 family genes does not predict azathioprinetreatment response in IBD patients. Pro-apoptotic BCL-2 inhibitor A-1211212 diminishes

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accumulated lymphocytes and ameliorates colitis in Il-10-/- mice. BCL-2 inhibition couldbe a new therapy option for the treatment of IBD.

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Session VI

Implementation of nanobodies in the Toxoplasma gondii field- Search for newbiomarkers

Rahel Winiger

Institute of Parasitology, Laboratory of Molecular Parasitology, University of Zü[email protected]

Camelids have evolved antibodies composed only of heavy chains. Nanobodies are derivedfrom the variable regions of these heavy chain only antibodies. Due to their small size andan unusually long complementarity determining region 3, they have access to cavities andclefts on antigens that are not approachable by conventional antibodies. Despite the factthat they consist only of a single monomeric variable antibody domain, they harbor fullantigen-binding capacity. These properties are exploited for different researchapplications.

The apicomplexan parasite Toxoplasma gondii secretes a wide range ofexcretory-secretory-proteins (ESP) in order to develop its niche in the host cells. Almost allEPS are released from three highly specialized secretory organelles in a sequential mannerfollowing a cascade mode. The micronemal proteins, rhoptry proteins and dense granuleproteins have specialized functions in host cell recognition, attachment, formation of theparasitophorous vacuole and the formation of the cyst wall, a process that ensures parasitepersistence and transmission.

There is only a limited number of ESP markers available and therefore a tool is needed totrack the proteins in a spatial and temporal manner to better understand their functionsin the context of host cell invasion and persistence. In this project, we aim to generateand identify specific nanobodies to profile the subcellular localization of ESP, to track thesecretion pathways, followed by the investigation of possible roles of the ESP in general andon the molecular structure of the cyst wall of Toxoplasma gondii.

Development of a non-invasive reporter method to monitor effector CD4+ effector T cellaccumulation in vivo

EBJ ten Buren, Thorsten Buch, Johannes vom Berg

Institute of Laboratory Animal Science, Schlieren, University of Zürich

[email protected]

Glioblastoma (GB) is a primary brain cancer, which induces an immunosuppressivemicroenvironment, with an abundance of regulatory T cells (Treg cells). Thismicroenvironment dampens infiltration and activation of effector cells, including adiminished number of CD4+ effector T cells (CD4+Teff) in human patients. Vice versa,tumour infiltration by CD4+ and CD8+ Teffs has been correlated to positive prognosis ofsurvival. During local IL-12 immunotherapy of established ortothopic murine tumours wepreviously observed a significant increase in tumour infiltration by CD4+Teffs(CD4+FoxP3-IFNγ+) at the expense of Tregs (CD4+FoxP3+IFNγ-). Accumulation of Teffspreluded reversal of local immunosuppression into inflammation, leading to advancedstage GB rejection. Usually, tumour-infiltrating lymphocytes (TILs) are analysed viaendpoint analyses, generally including flow cytometry or histology, which is time and

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resource intensive. Additionally, these current methods are unable to harness theprognostic value of a specific cell subset, as longitudinal (non-invasive) analysis of GBinflammation development during immunotherapy is not supported. Here we present anapproach to integrate the activity of the CD4 and the IFNγ promoter to confine luciferaseexpression to only the CD4+ Teffs. As only the CD4+Teffs emit bioluminescence, thiswould allow us to monitor intratumoural accumulation of this subset in a longitudinalfashion. In principle any other cell subset defined by the expression of up to threepromoters can be highlighted with this approach. The high temporal resolution of themethod will allow us to optimize treatment administration with regards to amount, orderand combination.

Generation of in vitro model of IgH/c-myc translocation in EBV-infected cells using

CRISPR/CAS9 RNPs

Semjon Sidorov, Michele Bernasconi, David Nadal, Simone Bürgler

Experimental Infectious Diseases and Cancer Research, University Children’s Hospital of

Zürich

[email protected]

Burkitt lymphoma (BL) is an aggressive B cell cancer which is characterized by theIgH/cmyc translocation. However, co-factors, such as additional mutations and infectionsare also required for BL development. Epstein Barr virus (EBV) is one of the infectiousagents associated with BL. While there is a clear statistical link between EBV and BL, therole of EBV in BL pathology is still unclear. It was hypothesised that EBV promotessurvival of pre-

BL cells during early stages of BL development, but later acquisition of additional mutationsmake EBV-derived support redundant.

In order to better understand early BL development, we are establishing a novel approachto precisely model the effect of the IgH/cmyc translocation in EBV-infected cells. For thispurpose, we are introducing dsDNA breaks near c-myc and IgH genes in in vitroEBVinfected cells (LCLs) using CAS9 Ribonucleoprotein (RNPs), and promote thetranslocation by adding a GFP-encoding DNA template with homology regions to both IgHand c-myc.

Our model offers a number of advantages for studying BL over existing models that rely onstable transfection with c-myc/IgH containing plasmids. Firstly, in our model we inducetranslocation between endogenous c-myc and IgH genes, rather than introducing a thirdcopy of the c-myc gene. Secondly, the existing epigenetic landscape is maintained. Finally,our model ensures that all relevant enhancers/regulators are in place and at physiologicaldistance from c-myc gene. Thus, the approach more precisely models effect of IgH/cmyctranslocation and will be useful for study of early events in EBV-associated BLdevelopment.

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Posters Abstracts

Session I

Investigating the Role of ACKR4 on Leukocyte Trafficking Within Lymphatic Vessels andon Lymphatic Vessel Biology

Mona Friess1, Steven Proulx1, Antal Rot2, Cornelia Halin1

1Institute of Pharmaceutical Sciences, ETH Zürich, 8093 Zürich

2Center for Immunology and Infection, Department of Biology, University of York, York, [email protected]

Atypical chemokine receptors (ACKRs) are chemokine receptors that are uncoupled fromGproteins. ACKRs function as scavengers, internalizing and degrading the chemokinesthey bind. ACKR4, which binds the chemokines CCL19, CCL21 and CCL25, was shown tobe expressed by lymphatic endothelial cells (LECs) lining the subcapsular sinus (SCS)ceiling of lymph nodes (LNs). As such, ACKR4 is important for the formation of a CCL21gradient across the SCS and for enabling DC migration into LNs. In this project, we set outto investigate ACKR4 expression by LECs in other parts of the lymphatic vasculature andto further study its function in the lymphatic system. Whole mount stainings inACKR4-GFP knock-in mice (ACKR4GFP/GFP and ACKR4GFP/+) revealed strongexpression of ACKR4 in large lymphatic collectors, while only some of the smallerpre-collectors and no capillaries in the dermis showed GFP signal. These findingsprompted us to investigate the functionality of ACKR4 in collector LECs and to study itseffect on leukocyte trafficking. Performing an in vivo chemokine uptake assay withfluorescently labelled CCL19 injected into foot we found that ACKR4 expressed in LECs ofthe large afferent collectors is functional; i.e. it bound

CCL19 in ACKR4GFP/+ mice while no CCL19 signal was detected in ACKR4GFP/GFPmice. In a next step, we are currently investigating the effect of ACKR4 expression on T cellmigration through lymphatic collectors, using adoptive transfer experiments, whole mountstainings and intravital microscopy. Overall, we show that ACKR4 is expressed bylymphatic collectors and aim to further elucidate its impact on leukocyte trafficking.

The gut mucus and its protective role in Salmonella Typhimurium infection

Markus Furter1, Mikael E. Sellin1, Gunnar C. Hansson2, Wolf-Dietrich Hardt1

1Institute of Microbiology, ETH Zürich

2Department of Medical Biochemistry, University of Gothenburg, Sweden

[email protected]

The gut is covered by a protective layer of mucus that separates the tissue from the intestinalmicrobiota. So far, it was unclear how intestinal pathogens such as Salmonella infect thehost tissue despite this mucus barrier. Therefore, we analyzed the mucus morphology andbacterial loads along the entire intestine in a mouse model for oral Salmonella Typhimurium(S. Tm) infection. Strikingly, bacterial tissue loads were highest in the cecum, where themucus layer was not continuous. The colon tissue was covered by a continuous mucus

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layer and significantly less colonized, despite equal gut luminal loads. We performed time-lapse microscopy of S. Tm swimming on gut tissue explants and found two mechanismshow the mucus in the colon protects from motile bacteria. The viscous mucus redirectsincoming bacteria, promoting circular near-surface swimming. In addition, motile S. Tmare trapped at the mucus surface and thus sequestered from the host tissue. In contrast, thececal tissue was accessible for S. Tm and near-surface swimming as well as trapping wereabsent. Chemical disruption of the mucus layer in the colon rendered the tissue accessiblefor S. Tm, emphasizing the importance of an intact mucus layer in controlling S. Tm tissueinvasion.

Influence of biosurfactants on Pseudomonas putida membrane vesicles

Ratchara Kalawong, Masanori Toyofuku, Leo Eberl

Department of Plant and Microbial Biology, University of Zürich

[email protected]

An increasing number of Gram-negative bacteria have been demonstrated to releasemembrane vesicle (MVs) which play important roles in biological processes includingvirulence, stress response, quorum sensing, gene and protein transfer and biofilmformation. Despite their importance for various functions the biogenesis of MVs is onlypoorly understood. Pseudomonas putida IsoF is a plant growth promoting strain which wasisolated from the rhizosphere of a tomato plant. This bacterium produces the signalmolecule 3-oxo-C10HSL, which in turn positively regulates the production of thebiosurfactant putisolvin. Biosurfactants are amphiphilic compounds that reduce theinterfacial tension of two phases, and are known to dissolve membranous materials such asvesicles. In P. putida IsoF, putisolvin is primary associated with the cell, presumablyadsorbed to the cellular membrane. It has been shown that putisolvin is required forswarming motility, but given its association with the producing cell, it is not a commongood and thus is not shared among swarm cells. In this study we examined the ability of P.putida to produce MVs under different conditions, including DNA damaging stressconditions that was previously reported to induce explosive cell lysis in P. aeruginosa. Ourresult suggests that P. putida IsoF produces MVs, albeit stress conditions did notsignificantly enhance MV production, much in contrast to many other bacteria. However,we observed that the production of putisolvin affects MV formation, possibly because of itsaffinity for the outer membrane. The role of biosurfactants on of MV biogenesis will bediscussed.

The Replication Capacity of HIV-1 Transmitter/Recipient pairs of the ZPHI and SHCSCohort

Audrey Kelley1,2, Karin J. Metzner1,2, Huldrych F. Günthard1,2

1Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zürich

2Institute of Medical Virology, University of Zürich

[email protected]

When HIV-1 is transmitted, it has been thought that only a few viral variants aretransmitted and only one viral variant establishes infection due to the homogenous

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population in 60-90% of acute patients. Therefore, we would expect to see the transmitterand recipient viruses to have similar characteristics. In order to study the replicationcapacity characteristics of transmitter and recipient pairs identified within the Swiss HIVCohort Study (SCHS) and the Zürich Primary HIV Infection study (ZPHI), we have startedto establish a replication capacity assay that is high-throughput, robust and reproduciblethat can be used with unmodified patient primary virus isolates. First, we have startedtesting parallel and dual competition infection assays along with different quantitativemethods to determine the best assay to use when measuring replication capacity of thevirus. In order to assess the replicative capacity of the test virus strains, NL4-3H andJR-CSF, we use the wild type (WT) of each virus, as well as, the less fit mutant of each virusstrain that harbors the M184V mutation (MUT). So far, we have characterized the test virusstrains using the p24 ELISA assay, obtaining the 50% tissue culture infectious dose(TCID50) and quantifying the virus titer using RT-qPCR. Parallel infection and dualcompetition infection assays have been performed using all viruses. It has been seen thatonly one round of infection has been happening due to the initial input of virus being toohigh. Since we would like to have an assay with multiple rounds of infection, differentMOI’s are being tested to determine the appropriate amount for multiple rounds ofinfection. The replication assay experiments are still ongoing for both virus strains.

Identification of Phage receptor on Erwinia amylovora CFBP 1430

Leandra Knecht1,2, Katja Felder2, Yannick Born1, David Vinzent1, Jonas Hofmänner1, MartinJ. Loessner2, Lars Fieseler1

1Institute of Food and Beverage Innovation; Zürich University of Applied Sciences,Wädenswil

2Institute of Food; Nutrition and Health; ETH Zürich

[email protected]

Even though bacteriophages are a promising alternative to conventional antibiotics, phageresistance can be observed after exposure. In order to circumvent resistance development,a mixture of different phages could prolong the therapeutic effect. Hence, an optimalcombination of phages is crucial for an effective treatment. We therefore established aneasy to handle high throughput screening assay to identify phage receptors. A Tn5transposon mutant library of the plant pathogen Erwinia amylovora was constructed.Library screening was performed for strictly lytic Erwinia specific phages Y2, Bue1, M7, S6,S2 and L1 and transposon insertion site were identified for validated mutants.

The screen revealed that both phages Y2 and Bue1 seem to be dependent on LPSstructures. The phages M7 and S6 appeared to be ineffective against Erwinia mutants withtransposon insertion sites in the bacterial cellulose synthesis operon (bcs). The screenfurther revealed that mutants with transposon insertion sites in most genes of the amsoperon (except amsF, amsI, amsG), responsible for amylovoran production, are involved inmediating resistance to L1 and S2. Furthermore, mutation of the genes topB1, rfaE and pgmwere identified to mediate resistance against multiple phages independent of their hostreceptor. None of these genes code for a surface receptor suggesting an intracellularresistance mechanism resulting in phage resistance. Overall, this method allows quickidentification of genes involved in mediating phage resistance. Aside from theidentification of phage receptors on the host surface, this assay helps further understandthe predator prey relationship of phages.

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A comparative study on the biology and anti-HIV-1 effect of IFN-α subtype, IFN-α14,IFN-α mutant, YNS, with elevated binding affinity to IFNAR and prototype IFN-αsubtype, IFN-α2, in vivo in humanized mice Grunbach, M., Schlaepfer, E., Grindberg, R.,Schreiber, G., Skerra, A., Speck, RF University Hospital Zü[email protected]

The type I Interferons (IFN-α family) are the most potent anti-viral cytokines. They arereleased subsequent to encounter with pathogens and orchestrate the immune response tofight the invaders. IFN-α comprises of 12 subtypes which all bind to the sameinterferon-α/β receptor (IFNAR) made of two chains, IFNAR1 and IFNAR2 but withdifferent affinity. Currently, a controversy exists whether the difference in biologicalactivity of IFN-α subtypes is quantitative or qualitative, i.e., is it only a function of doseand binding affinity or activation of different signaling cascades all together? Data we havegenerated in vitro show that IFN-α subtypes with high affinity to IFNAR display significantanti-HIV effects already at low doses while those with low affinity need higher doses(manuscript in preparation). Notably, the transcriptomics of tissue specimens treated withIFN-α14 and -α2 were rather identical consistent with identical biological properties. Inthe latter work, we also found that the IFN-α mutant, YNS, with extremely high affinity tothe IFNAR displayed potent anti-HIV activities.

Our data about the biological properties of IFN-α subtypes have been generated usingprimary human cells treated ex vivo with various IFN-α subtypes. However, to take intoaccount the complex activities of IFNs on the variety of cells of the lympho-reticularsystem, and in turn the interplay of the various cells with each other, we need a thoroughwork-up of IFN-α subtypes’ biological properties in vivo. The aim here is to assess thebiological properties of human IFN-α subtypes in vivo using humanized mice.

Characterization of entry factors used by seasonal circulating human and avianInfluenza A strains

Annika Hunziker1, Emilio Yángüez1, Silke Stertz1

1Institute of Medical Virology, University of Zürich [email protected]

The aim of this project is to expand our knowledge about signaling pathways that areactivated upon influenza A virus (IAV) entry into its host cell and how key proteins ofthese cascades influence viral replication. In a previous proteome-wide SILAC-basedquantitative phosphoproteomic screen with A/WSN/1933 (H1N1) strain conducted in ourgroup we quantified around 3000 phosphosites from 1300 different proteins and identifiedinfection-induced changes in the phosphorylation of an important subset. In the currentproject, we would like to perform similar assays to compare the changes in host-cellphosphorylation induced by the infection with the laboratory adapted A/WSN/1933(H1N1) strain with two seasonal circulating IAV strains, namely A/Brisbane/10/07 (H3N2)and A/Brisbane/59/07 (H1N1). In addition, we aim to characterize the signaling pathwaysinduced by avian IAV entry into the host cell by using two low pathogenic avian influenzastrains (A/duck/Ukraine/1/1963 (H3N8) and A/duck/Alberta/35/1976 (H1N1)). To do so,we are establishing binding and infectivity assays in order to exclude differences in bindingefficiency of IAV strains. Proteins that are differentially phosphorylated upon entry ofthese IAV strains will be selected for further studies to address their involvement in viralreplication.

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Investigation of the molecular mechanisms regulating recovER-phagy in mammaliancells

Marisa Loi1,2,4, Fiorenza Fumagalli1,2,3, Maurizio Molinari1,2,5

1Institute for Research in Biomedicine, Bellinzona, Switzerland

2Università della Svizzera italiana, Lugano, Switzerland

3Graduate School for Cellular and Biomedical Sciences, Universität Bern, Switzerland

4Department of Biology, Swiss Federal Institute of Technology, Zürich, Switzerland

5École Polytechnique Fédérale de Lausanne, School of Life Sciences, Lausanne, Switzerland

The endoplasmic reticulum (ER) is an intracellular compartment where one third of theeukaryotic cell’s proteome is synthetized. Correctly folded polypeptides leave the ER alongthe secretory pathway while misfolded proteins are retained. In response to anaccumulation of un- or misfolded proteins in the ER lumen, cells activate unfolded proteinresponse programs (UPR) that maintain ER homeostasis by decreasing cargo proteintranslation and by enhancing transcription and translation of molecular chaperones andenzymes involved in protein folding, degradation and quality control. After resolution ofan ER stress, the intracellular concentration of stress-induced ER proteins returns tophysiological levels.

Recently, we identified a novel function of the translocon component SEC62 as anER-resident LC3-II receptor, which ensures delivery of select ER subdomains toautolysosomes for clearance in a series of events that we named recovER-phagy and thatre-establish pre-stress, physiologic ER volume and content

Here, we investigate the molecular mechanisms of recovER-phagy to identify theregulatory gene products that intervene in this process and to clarify similarities anddifferences between recovER-phagy and canonical autophagic programs operating inmammalian cells.

Understanding the transmission mechanism of the tetracycline resistance gene C (tetC)in Chlamydia suis

Hanna Marti, Timothy D. Read, Deborah Dean, Nicole Borel

Institute of Veterinary Pathology, Vetsuisse-Faculty, University of Zürich

[email protected]

An efflux protein-encoding tetracycline resistance class C gene (tetC) confers tetracyclineresistance in Chlamydia (C.) suis. It is the only example of an obligate intracellular bacterialspecies attaining antibiotic resistance through horizontal gene transfer to date. The aim ofthis research project is to advance our understanding regarding the transmission oftetracycline resistance within the Chlamydia species. Comparative whole-genome analysisof both tetracycline sensitive and resistant C. suis strains has shown that the tetC gene ispart of a genomic island inserted at an identical site within the C. suis chromosome.Therefore, among C. suis, tetC is likely transferred through recombination. Co-cultureexperiments in vitro will give insights into the site of insertion, potential biological barriersbetween chlamydial species and the genomic island structure necessary for successfultransmission. Additionally, the influence of selective pressure will be elucidated by

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comparing antibiotic free conditions to the presence of sub-inhibitory concentrations oftetracycline. Briefly, murine McCoy or LLC-MK2 cells (rhesus monkey kidney cells) areco-infected with a tetracycline resistant C. suis and a tetracycline sensitive Chlamydia strain(C. trachomatis, C. suis, C. pecorum). Emerging recombinants are identified and picked bythe use of selective antibiotics. First experiments indicate that tet(C) transfer within C. suisoccurs through double crossover homologous recombination. Interestingly, recombinationevents were not observed at typical permissive sites for recombination such as invertedrepeats, chi sites and direct target repeats. Instead, long homologous DNA may serve astargets for recombination. Subinhibitory concentrations of tetracycline seem to promotetetC transfer.

Towards understanding stability and function of plant-associated microbiota

Barbara Müller, Daniel B. Müller, Julia Vorholt

Institute of Microbiology, ETH Zürich

[email protected]

The phyllosphere of plants harbours an abundant and diverse bacterial microbiota. Most ofthese microorganisms are not pathogenic and play an important role in plant health, asthey can promote plant growth through production of hormones and are involved in plantprotection, by interacting with pathogens either directly by producing antibioticcompounds or indirectly by competing for resources. Additionally, the bacteria mayprotect plants by inducing systemic acquired resistance, priming the plants for a pathogenattack. Though playing an important role for plant health, the interactions the microbiotaand their host and in-between members of the microbiota are not well understood. To getinto depth of this, a gnotobiotic system was established in our lab. Strains of themicrobiota of native Arabidobsis thaliana were isolated and genome sequenced, covering55% of culture-independent taxonomic diversity. It was shown that a synthetic microbiota,composed of these strains, is able to protect A. thaliana against the foliar pathogenPseudomonas syringae DC3000 luxCDABE. In an approach to understand the stability of themicrobiota, the interaction and dynamics of its members, a synthetic microbiota of A.thaliana will be passaged through several plant generations with or without increasingpathogen load in a gnotobiotic system to investigate differences in loss or increase of plantprotection.

In vivo kinetic analysis of N-glycosylation in yeast

Julia Neuhaus


[email protected]

Asparagine-linked protein glycosylation is an essential, highly conserved posttranslationalmodification in all three domains of life. In eukaryotes, the pre-assembled oligosaccharidecomposed of two N-acetylglucosamines, nine mannoses and three glucoses is transferred enbloc to an asparagine residue in the conserved sequon N-X-S/T (where X can be any aminoacid except of proline) of a nascent polypeptide.

The transfer is catalyzed by the oligosaccharyltransferase (OST). OST is localized in theendoplasmic reticulum (ER), where it catalyzes the transfer of the oligosaccharide on thepolypeptide sequon as soon as the nascent chain reaches at least 13 amino acids deep into

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the ER lumen. Directly after translation however, the amino acids of the nascentpolypeptides chain also start to interact with each. The attachment of oligosaccharides bythe OST on sequons in nascent polypeptide chains thus competes with the self-assemblyfolding process of proteins.

The goal of my project is to establish a method for in vivo kinetic analysis ofN-glycosylation in yeast. The in-house designed mass spectrometry based analysis methodparallel-reaction monitoring (RPM) joint with stable isotope labelling of amino acids in cellculture (SILAC) enables to determine differences in glycosylation velocity. For thisapproach the two model proteins ’protein disulfide isomerase’ (PDI) and ’carboxypeptidaseY’ (CPY) will be used throughout this study and their different glycosylation sites will bestudied in a time-resolved manner. The variance of the glycosylation site positions in thestructure of PDI and CPY enable a structure-based analysis of the glycosylationvelocity

The role of resident regulatory T cells in long-term graft acceptance

T. Nguyen, O. Boyman

Department for Immunology, University Hospital Zürich

[email protected]

Regulatory T cells (Tregs) are highly suppressive and are important to maintain theimmunological self-tolerance by suppressing self-reactive lymphocytes. Interleukin-2(IL-2) is an important cytokine for survival and proliferation of different lymphocytessubsets including CD8 T cells, natural killer (NK) cells and CD4 Tregs. IL-2 receptors(IL-2R) are expressed differently on lymphocytes subsets. CD8+ T and natural killer cellsexpress high levels of dimeric IL-2Rs comprising IL-2Rβ (CD122) and IL-2Rγ (γc). Incontrast, Treg cells express high IL-2Rα (CD25) level together with moderate level ofCD122 and γc. Using a formulation of IL-2 together with specific antibodies, one can eitherstimulate Treg or effectors cells. Accordingly, a cytokine antibody complex of IL-2 andJES6-1 antibody (JES6- 1/IL-2 complex), can be used to induce an increase of Tregs by 10-to 20-fold. In a model of pancreatic islets transplantation in mice, this treatment has beensuccessfully used to induce long-term acceptance of over 100 days, notably without usingany immunosuppressants. However, how Tregs prevent the graft rejection remains unclear,as the number of circulating Tregs decline within two weeks. One possible explanation isthe formation of a localized population of Tregs surrounding the graft, which remainsstable over a long period. In this study, our main aim is to understand how induced Tregsprevent graft rejection and to decipher the mechanism of actions tissue resident regulatoryT cells. Secondly, we want to assess if JES6-1/IL-2 complex treatment could be used toinduce similar long-term acceptance in the model of kidney transplantation in mice.

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Contribution of neutrophils to the induction of local and systemic psoriasisinflammation

Alaz Özcan

Immunology Clinic, University Hospital Zürich

[email protected]

Psoriasis is a chronic inflammatory skin disorder that affects 2-3% of the world population.Disease manifestation occurs upon environmental triggers in individuals with geneticsusceptibility. Recent evidence suggests that both the innate and adaptive immune systemsare involved in psoriasis pathogenesis. Moreover, studies have revealed that mainly innateimmune cells are crucial in initiating psoriasis. Best characterized among these is theinvolvement of dendritic cells, which induce the stimulation of naïve T cells. The adaptivecompartment, mainly T cells of the T helper 1 (Th1) or T helper 17 (Th17) phenotype,dominates the maintenance stages of the disease, inducing keratinocyte proliferation andfurther immune cell recruitment. These extensive cell-to-cell interactions are mediatedlargely by cytokines and chemokines.

Neutrophils are immediate responders to infection and inflammation. They have a long listof effector functions, including but not restricted to, phagocytosis, reactive oxygen species(ROS) production, and neutrophil extracellular trap (NET) formation. Neutrophils areknown to contribute multiple autoimmune and auto inflammatory diseases however, theircontribution to the psoriasis pathogenesis is not yet well understood. The aim of this studyis to understand the possible role of neutrophils in the initiation and establishment ofpsoriasis. In order to answer our research question we make use of relevant animal modelsand blood samples from patients and healthy individuals.

Non-classic mouse Th1* cells and the requirements for their differentiation

Michela Perotti1,2, Federica Sallusto1,2

1Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzon

2Institute of Microbiology, ETH Zürich

[email protected]

CD4+ T cells have pivotal roles in orchestrating immune responses to different microbialpathogens. This is accomplished through the differentiation of CD4+ T helper cells tospecialized subsets in response to microbial pathogens with distinct patterns of geneexpression and profiles of cytokine production. Although classically viewed as distinctlineages, it has become clear that not all of these T cells are terminally differentiated, butthe majority is plastic and can acquire different properties and functions during secondaryimmune responses. Data in humans have now provided evidence that different microbialpathogens can induce distinct types of Th1 and Th17 cells, classically imprinted forexpression of IFN-γ and IL-17, by upregulated expression of T-bet and retinoic acid-relatedorphan receptor γt (RORγt), respectively. Indeed, IFN-γ producing T cells in response toMycobacterium tuberculosis infection were found almost exclusively in the Th1* subset, acell type that has been characterized by co-expression of T-bet and RORγt, whereas IFN-γproducing T cells in response to influenza virus were primarily found in the classic Th1subset. The Th1* developmental requirements, relationship with classical Th17 and Th1cells and the acquisition of different characteristics in secondary immune responses are notwell understood. Using a murine model of Bacillus Calmette–Guérin (BCG) infection, we

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aim to define the origin and lineage relationship of these phenotypically and functionallydistinct T cell populations.

Therapeutic Efficacy of low molecular weight polysialic acid in EAE

Benjamin Peschke1, Christian W. Keller1, Jens Kopatz3, Paul Crocker4, Harald Neumann3, JanD. Lünemann1, 2

1Institute of Experimental Immunology, Laboratory of Neuroinflammation, University ofZürich,

2Department of Neurology, University Hospital Zürich

3Institute of Reconstructive Neurobiology, University Hospital Bonn, University of Bonn,Germany

4Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, [email protected]

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) withinflammatory and neurodegenerative immunopathological characteristics. Currentlyavailable disease modifying therapies show modest efficacies in halting disease progressionand are associated with severe side effects due to immunosuppression, reflecting the unmetmedical need for therapeutic strategies that target both inflammatory andneurodegenerative mechanisms with negligible toxicity. Sialic acid bindingimmunoglobulin-like lectin (Siglec) receptors regulate innate and adaptive immune cellfunction through the recognition of their sialic acid containing glycan ligands. Polysialicacids (polySia) as endogenous Siglec ligands comprises repeating sialic acid monomerswith α2,8-linkages which has primarily been studied in the developing CNS and upon CNSinjury where polySia promotes repair mechanisms. The efficacy and mechanisms ofpolySia to inhibit immunemediated CNS tissue injury in appropriate preclinical in vivomodels of MS has not been investigated so far. CNS-invading monocyte-derived DCs(moDCs), the progeny of Ly6ChiCCR2+ monocytes, drive tissue inflammation duringexperimental autoimmune encephalomyelitis (EAE). We report that Siglec-E, the murineinhibitory receptor for polySia, is strongly expressed and upregulated on Ly6ChiCCR2+inflammatory monocytes and on CNS-infiltrating moDCs during EAE. Therapeuticadministration of the Siglec-E ligand α2.8-linked polySia stopped disease progression inEAE. Our data indicate that inhibitory Siglec signaling can be harnessed throughtherapeutic administration of polySia in order to limit CNS inflammation and tissuedamage in a preclinical model of MS.

Pseudomonads contact-dependent competition in mixed biofilms

Gabriela Purtschert1, Gerardo Cárcamo-Oyarce1,2, Claudio Aguilar1 and Leo Eberl1

1Department of Microbiology, Institute of Plant and Microbial Biology, University ofZürich

2Biological Engineering Department, Massachusetts Institute of Technology, Cambridge MA,USA

[email protected]

Bacterial biofilms represent the most relevant form of bacterial growth in the environment.Since multiple bacterial species coexist within most natural biofilms, the competition for

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resources and space has triggered the evolution of competitive strategies to survive andpersist in such multispecies communities.

In this study, we evaluate competition ability of the plant-associated bacterium Pseudomonasputida strain IsoF in mixed species biofilms. Using different experimental approaches, weshow how IsoF is able to outcompete various soil and plant associated Pseudomonas speciesin a contact-dependent manner. Moreover, IsoF antagonism is shown to be particularlyeffective in mixed-species biofilms. Using flow-through chambers we demonstrate that IsoFis capable of invading and displacing a pre-established biofilm of P. putida strain KT2240.By screening of a mini-Tn5 transposon insertion library we identified a gene cluster that isresponsible for contact-dependent killing by the strain. Bioinformatic analyses revealed thatthe locus encodes a putative type IV secretion system (T4SS). Inactivation of genes encodedby this cluster resulted in mutants that were no longer able to kill other bacteria nor werethey able to invade existing biofilms, in contrast to the wild-type strain. In conclusion, weprovide evidence that the competition strategy of P. putida is based on a T4SS and show thatthis system is not only used for self-defense but also as an offensive weapon for the invasionof biofilms.

Assessment of the Antiviral Activity of MxA against Influenza A Virus

F. Steiner, S. Spirig, M. Crameri, E. Moritz and J. Pavlovic

Institute of Medical Virology, University of Zürich, Zürich, Switzerland

[email protected]

Human MxA protein belongs to the family of dynamin-like large GTPases and exertsantiviral activity primarily against negative-stranded RNA viruses, including influenza A(IAV). MxA is able to form higher order oligomeric structures. However, the mode of actionof MxA remains unknown. There is increasing evidence that MxA targets the IAVnucleoprotein (NP) in conjunction with UAP56, a cellular DEAD-box RNA helicase, whichis required for efficient IAV replication due to its chaperone activity for viral NP. In orderto better understand the molecular mechanism of action of MxA we analyzed in detail theinterplay between MxA, UAP56 and the viral target NP.

By performing co-immunoprecipitation (co-IP) as well as split-GFP experiments we wereable to show that NP binds (the dimeric form of) MxA as well as UAP56. In order tonarrow down the interaction domains of MxA and UAP56 with NP, we introduced N-and/or C- terminal deletions and point mutations to identify the binding regions of thetrimeric complex. Furthermore, we were able to identify the residues in NP important forbinding the MxA-UAP56 complex.

From a mechanistic point of view, we are investigating the importance of the oligomericstate of MxA as well as its GTPase function. We have evidence that a functional G-domain isimportant for proper localization of MxA but is dispensable for its binding to UAP56. Theoligomeric state on the other hand is crucial for the binding of MxA to NP since this bindingis increased if MxA is rendered dimeric.

We are currently testing whether binding of MxA to UAP-NP dimers leads to thesequestration of UAP56 and hence inhibition of IAV replication.

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Global gene expression analysis of adeno-associated virus type 2 (AAV2)-infectedcells

Sereina O. Sutter, Kurt Tobler, Mathias Ackermann, and Cornel Fraefel

Institute of Virology, University of Zürich, Zürich, Switzerland

[email protected]

To investigate the global effect of AAV2 infection on host cells, a global transcriptomeanalysis by next generation sequencing (NGS) of wild-type (wt) AAV2-infected ormockinfected normal human fibroblasts (NHF) was performed, revealing 872 differentiallyexpressed genes between wtAAV2 and mock-infected cells. The downstream analysis of theNGS data identified the histone expression as one of the most important hits with amaximum fold difference of 5.4 between wtAAV2 and mock-infected cells. Moreover,KEGG pathway analysis indicated that the top biological pathway, most affected bywtAAV2 infection, is cell cycle regulation. To validate the NGS data and to furtherinvestigate the effect of wtAAV2 infection on cell cycle regulation, we determined theexpression of selected cellular genes by RT-qPCR. In general, the NGS and RT-qPCR datacorrelated well; for example, both assays revealed that RB1 and TP53 were up-regulatedwhile CCNA2 as well as CDK1 were down-regulated by wtAAV2 infection.

Next, we compared the expression profiles of selected genes involved in cell cycleregulation in NHF cells infected with wtAAV2, UV-irradiated wtAAV2 (UV-wtAAV2) orselfcomplementary AAV2 (scAAVGFP). Interestingly, RT-qPCR revealed that CCNA2 andCCNB2 expression was up-regulated in cells infected with scAAVGFP, while it wasdownregulated in cells infected with either wtAAV2 or UV-wtAAV2. By contrast, CDKN1Aexpression was down-regulated in cells infected with scAAVGFP, while it was up-regulatedupon infection with any virus. Regarding the expression profile TP53 an up-regulation wasobserved in cells infected with wtAAV2 or UV-wtAAV2, while the expression was onlyslightly affected by scAAVGFP.

A novel peptide splicing enzyme from Pleurocapsa. sp. creates β-amino acidresidues

Authors: Marjan Verest1, Brandon I. Morinaka1, Edgars Lakis1, Maximilian J. Helf1,2, ThibaultScalvenzi3, Anna L. Vagstad1, Shinichi Sunagawa1, Muriel Gugger3, Jörn Piel1

1Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zürich

2Boyce Thompson Institute, Cornell University, NY, USA.

3Institut Pasteur, Collection des Cyanobactéries, Département de Microbiologie, Paris, France

[email protected]

Despite being limited to the canonical amino acids as building blocks, ribosomallysynthesized post-translationally modified peptides (RiPPs) show great diversity due to theaction of a variety of modifying enzymes. One such modifying enzyme is the recentlydiscovered radical S-adenosylmethionine enzyme PlpX from Pleurocapsa sp., whichperforms an unprecedented splicing reaction to create α-keto-β-amino acids by tyramineexcision. Homologues of PplX are widespread and can be found in cyanobacteria,proteobacteria and actinomycetes. By producing variants and mutants of one of theNif11-like precursor peptides, we have shown the minimal core sequence to be eleven

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residues long and produced seven novel β-amino acid residues. In addition, we havedemonstrated that the keto group created by the excision can be used to further modify thepeptide via oxime or hydrazone linkage, potentially enabling the use of PlpX or relatedenzymes for a range of biotechnological applications.

Establishment of a molecular toolbox to generate apoptosis inducing vectors for thedepletion of distinct cell types in vivo

Anja M. Walkam, Rashel V. Grindberg, Simon Bredl, Roberto F. Speck

Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zürich

[email protected]

The prospective study was designed to establish a molecular toolbox to generate apoptosisinducing constructs, which will be active only in defined cell types and thus achievetargeted elimination of designated immune cells, e.g. B-, T- cells, dendritic cells ormacrophages. For the generation of the distinct apoptosis inducing vectors we will makeuse of the inducible Caspase9(iCasp9)/AP1903 system developed by Di Stasi et al. in 2011,which consists of a caspase 9 fused to a modified FK-binding protein. The system has beenestablished for human use and has already been explored in a phase I clinical trial withpromising results. The expressed transgene induces cell specific death upon exposure tothe drug AP1903, which upon dimerization activates the intrinsic apoptotic pathway. Wewill modify the system using different cell type-specific promoters to target apoptosis indistinct subsets of immune cells. The generated vector variants will then be tested in vitrousing specific cell lines to evaluate depletion efficiency. As the construct contains atruncated CD19 it permits the monitoring of the disappearance of gene engineered cellsafter adding the dimerizing drug AP1903 by staining for CD19. Additionally, we willconstruct vector variants containing different reporter genes, e.g. GPF or luciferasewidening options for methods of analyses and enabling the use of flow cytometry or in vivoimaging. After successful in vitro establishment, we aim for the application of the systemto in vivo or investigation of targeted gene therapy studies.

Longitudinal stratification of microbial communities along the gastrointestinaltract

Zünd Mirjam, Sunagawa Shinichi


[email protected]

Background: Human and mice harbor complex microbial communities in theirgastrointestinal tract, where the community plays an essential role in host health anddisease. Analyzing the microbial community composition in healthy hosts has shown thatthe overall community structure remains stable over long periods.

Methods: Mouse feces and intestinal mass from different site along the intestinal tract arecollected to extract bacterial DNA and RNA. DNA are used for 16S rRNA ampliconsequencing to detect difference in site-specific microbial community composition. RNAinstead, is used for meta-transcriptomic to compare gene activity pattern of the differentmicrobial communities.

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Results/Conclusion: The phylogenetic diversity comparison between different site alongthe gastrointestinal tract shows a shift in the community composition. This deeperunderstanding of the community composition and its specific transcription activity canhelp to better understand the functional role of the microbiota along the intestinaltract.

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Session II

Time-resolved analysis of the N-glycan processing in the ER and Golgi of CHO cells

Ilaria Affolter1, Chia-wei Lin1, David Brühlmann2, Jonathan Souquet2, Hervé Broly2, andMarkus Aebi1

1 Institute of Microbiology, Department of Bilogy, ETH Zürich

2 Merck-Serono S.A., Corsier-sur-Vevey, Biotech Process Sciences, ZI B, 1809 Fenil-sur-Corsier,Switzerland

[email protected]

N-linked protein glycosylation is a crucial step in the biosynthesis of glycoproteins. Thesynthesis of the glycan portion is not template-driven as for the protein backbone, but itdepends on a multitude of factors. As a result, the glycan structure found on a specificasparagine residue of a protein is not always the same, but rather a distribution of differentstructures generated by the glycosylation enzymes located in the Golgi. This becomes anissue in biosimilar production, when a glycosylation profile needs to be imitated. A kineticmodel of N-glycan processing can help to get insights into the reasons responsible thisheterogeneity.

The goal of this project is to measure the kinetic parameters of the N-glycosylation processin vivo in a time-resolved manner. The use of newly developed, mass spectrometry(MS)-based methods in gylcoproteomics enables on one side to gain site-specific glycanstructure information of the protein of interest. On the other side, by applying apulse-chase regime using stable isotope labelling of amino acids in cell culture (SILAC)time-resolved information on the glycosylation pathway can be obtained.

Data showing the N-glycan distribution of an IgG (model protein) and the time-resolvedvariations observed in the intracellular pool if IgG will be presented.

These data will enable a more precise characterization and quantification of theN-glycosylation flux. A quantitative description of the intracellular glycosylationprocessing pathway will be the basis for the design of productions conditions that result ina defined N-glycoprotein product

Characterization of HIV Superinfection in the Swiss HIV Cohort Study

Sandra Elisabeth Chaudron

Department of Infectious Diseases and Hospital Epidemiology, University Hospital ofZürich

[email protected]

The human immunodeficiency virus (HIV) is responsible for the HIV pandemic with 37million of infected persons worldwide. To date, there is neither cure nor vaccine and thevirus is always ahead of the immune system in infected individuals. Despite all the meansagainst HIV, the transmission is still ongoing in developing and developed countriesresulting in about 2.5 million new infections each year. Our project aims to study type 1HIV superinfection in the Swiss HIV Cohort Study (SHCS) meaning being infected by asecond HIV-1 strain following an already established initial infection. Using the 25378HIV pol sequences of the drug resistance database (DRDB) of the SHCS, we performed aphylogenetic analysis to identify superinfected patients in the cohort. To confirm these

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cases, HIV-1 full-length sequencing using the next generation sequencing (NGS) Illuminasystem will be performed on longitudinal plasma samples from the patients. In parallel, tocircumvent the fact that population sequencing is not accurate for low-frequency viralvariants, a high throughput method combining single genome amplification (SGA) withNGS to amplify and sequence HIV-1 full envelope, will be developed. Finally, we willstudy recombination events during superinfection; assess the incidence of thisphenomenon in the SHCS and identify behavioral risk factors associated with theacquisition of superinfection. So far, with the phylogeny, we identified 330 potentialsuperinfected patients from which plasma samples will be ordered and processed asdescribed above.

Genome-wide identification of Burkholderia cenocepacia H111 molecular responses tocadmium

Stefano Gualdi, Dr. Steven Higgins, Prof. Dr. Leo Eberl Department of plants and microbialbiology, University of Zürich

[email protected]

Burkholderia cenocepacia H111 is a member of the Burkholderia cepacia complex (Bcc), whichis a group of at least 20 closely related bacterial species colonizing a wide variety ofecological niches including water, soil and the rhizosphere of some plants. Having emergedduring the 1980s as opportunistic pathogens, the members of Bcc are known for theirremarkable phenotypic versatility with a high level of intrinsic resistance towardsantibiotics and chemical compounds allowing them to survive in challengingenvironments. Recent studies have shown that members of Burkholderia sp. have beenisolated in cadmium-contaminated soil and are potentially able to promotebioremediation. The aim of the study is to identify the molecular mechanisms exploited byBurkholderia cenocepacia H111 to cope with the presence of stress agents such as cadmiumsulphate. This can be achieved by using a high-density transposon library combined withnext generation sequencing otherwise known as Tn-Seq. Our results confirmed thatintracellular concentration of cadmium is regulated mostly through the induction of ahighly conserved P1-Type ATPase. The data is also showing how cadmium is affecting thecell at multiple levels, by direct DNA damaging, increase ROS production, denaturation ofproteins and through direct depletion of essential components required for cell’sgrowth

Role of Colonizing Staphylococcus aureus on Influenza A Infectivity

Nicola Häffner

Department of Infectious Diseases, University of Zurich

[email protected]

The Gram-positive bacterium Staphylococcus aureus is one of the most frequent causes ofboth hospital and community acquired infections. It is an opportunistic pathogen causingnot only superficial skin infections, but also severe diseases like bacteremia andosteomyelitis. Over the last decades, several studies revealed the ability of S. aureus toinvade and remain inside professional as well as non-professional phagocytes such asepithelial cells. S. aureus is also known as a human commensal, since 30 percentindividuals worldwide carry S. aureus asymptomatic predominantly in the nares and onthe skin. It has been shown by several studies that an influenza infection followed by asecondary S. aureus infection leads to a more severe outcome of the disease. Therefore, a

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synergistic action of the two pathogens is assumed leading to a more severe outcome of thedisease. During my PhD, I will investigate in the interaction between colonizing S. aureusand influenza virus during infection of lung epithelial cells. The setup I am going to usewill be vice versa of the “conventional” infection studies as showed by Warnking andcolleagues in 2015. I will infect lung epithelial cells with well characterized S. aureusstrains followed by a secondary influenza virus infection. The primary infection of theepithelial cells with the bacteria will reflect the invasion as it also occurs duringcolonization. This way, the influence of intracellular S. aureus, as it not only occurs duringdisease but also during colonization, on a secondary influenza infection can beinvestigated.

Blocking the human type 2 interleukin-4 receptor and its effects on human neutrophilexpansion and migration

Lukas Heeb

Laboratory of Applied Immunobiology, University Hospital Zurich

[email protected]

Neutrophils are the predominant leukocytes in human blood and are well known for beingone of the first responders to invading pathogens. Patients suffering from neutropenia arethus particularly susceptible to bacterial and fungal infections. It has recently beendiscovered that signaling via the type 2 interleukin (IL)-4 receptor (IL-4R) in murineneutrophils abrogates their expansion and egress from the bone marrow (BM), leavingaffected animals vulnerable to L. monocytogenes and S. aureus infections. Similar resultshave been obtained in human neutrophils. These findings make the type 2 IL-4R anattractive new target for the treatment of conditions where neutrophil count or activity isdecreased, especially in patients with elevated IL-4 levels.

The overall aim of this thesis is the validation and confirmation of the type 2 IL-4R as a targetand the generation of a candidate drug for possible future application in humans.

The first part of the project addresses the generation of a blocking monoclonal antibody(mAb) against human IL-13Rα1 (hIL-13Rα1), a subunit exclusive to the type 2 IL-4R. Thesecond part of the project is dedicated to the characterization of the mAb. Both in vitro andin vivo experiments will serve to assess functionality, activity, and performance of the mAbin various conditions and settings. The third part of the project is intended to be a proof ofconcept clinical study where patients will be administered an approved mAb targeting bothtypes of IL-4Rs and the intervention/s effect on neutrophil count, activity, and mobility willbe determined.

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Staphylococcus aureus persister formation as an adaptive response during infection

Markus Huemer

Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich,University of Zurich

[email protected]

Staphylococcus aureus (SA) is a leading cause of bacterial infections worldwide and a majorthreat to public health. SA has a high potential to acquire resistances against antibiotics asreflected by methicillin-resistant SA (MRSA) strains. In contrast to antibiotic resistance,antibiotic susceptible SA are able to survive antibiotic treatment by hiding in immuneprivileged sides, e.g. abscesses or inside host cells, and by switching to a persister state.Persisters are dormant bacteria, tolerant to antibiotic killing and associated with chronicinfections and treatment failure. A clinically relevant example for this behavior is theso-called small colony variants (SCVs). These phenotypic variants are characterized byproducing small and non-hemolytic colonies growing on solid agar. Clinical SCVs typicallyshow an unstable phenotype (non-stable SCVs), quickly switching between a fast- andslow-growing form. One reversible mechanism that could facilitate such regulatorydynamics is protein phosphorylation mediated by serine/threonine (Ser/Thr) kinases andphosphatases. Recently a new signaling pathway involved in dormancy in Bacillus subtilishas been shown. The Ser/Thr kinase YabT (PknB homologue) phosphorylates theelongation factor EF-Tu leading to inhibition of protein synthesis. This mechanism enablescells to quickly down-regulate one of the most energy-demanding metabolic processes in areversible way, as EF-Tu can be de-phosphorylated by the Ser/Thr phosphatase Stp. Herewe investigate the role of the kinase/ phosphatase pair PknB/ Stp in the formation ofnon-stable SCVs and persistence in SA in vitro as well as in in vivo models.

Development of Treg cell-stimulating IL-2/antibody complexes and investigation of theIL-2 receptor assembly

Ufuk Karakus, Juwela Lam, Onur Boyman

Department of Immunology, University Hospital Zurich, University of Zurich

[email protected]

IL-2 is crucial for the homeostasis and activation of the immune system by playing anessential role in immune tolerance and ensuring optimal T cell responses. In particular,IL-2 is necessary for CD4+ regulatory T (Treg) cells, which are controlled by thetranscription factor forkhead box P3 (FoxP3) and govern peripheral immune tolerance.The current project deals with the generation and characterization of specific anti-humanIL-2 antibodies, which are able to deliver IL-2 specifically to cells that express the highaffinity IL-2 receptor such as Treg cells. Treg cells are characterized by high expressionlevels of the IL-2 receptor alpha (also termed CD25) in addition to the dimeric IL-2receptor consisting of IL-2Rβ (also termed CD122) and the common gamma chain (γc). Inmice, injections of such CD25-directed IL-2 complexes massively expand Treg cells in vivowhereas effector lymphocytes including natural killer (NK) cells and memory CD8+ T cellsexpressing the dimeric receptor barely respond. In order to dissect the underlyingmechanism of the selective action of these IL-2 complexes we are currently fluorescentlylabeling the IL-2R subunits. In this way we will study binding dynamics of IL-2 complexesto cells ectopically expressing dimeric versus trimeric IL-2 receptors. Furthermore, we planon studying the IL-2 receptor assembly process by fluorescence resonance energy transfer(FRET) and whether anti-IL-2 antibodies are able to interfere with this process.

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Characterization of bat influenza virus entry

Umut Karakus1, Emilio Yángüez1, Eugenia Corrales Aguilar2, Carles Martinez-Romero3,

Adolfo Garcia-Sastre3, Martin Schwemmle4, Silke Stertz1

1 Institute of Medical Virology, University of Zurich, Switzerland

2 Faculty of Microbiology, University of Costa Rica, San José, Costa Rica

3 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, USA

4 Institute of Virology, University Medical Center Freiburg, Freiburg, Germany

[email protected]

Two novel influenza A-like virus sequences have recently been isolated from two SouthAmerican bat species. Initial studies revealed that these bat influenza viruses, classified asH17N10 and H18N11, are highly divergent from known influenza A virus (IAV) strains.Unlike conventional IAVs, hemagglutinins (HAs) of the bat derived influenza strains do notbind the canonical IAV receptor sialic acid. Moreover, their neuraminidases (NAs) seem tolack any neuraminidase activity. Little is known about target cells and the entrycharacteristics of the bat influenza viruses. In order to identify cell lines susceptible to batinfluenza virus, we have established a flow cytometry-based system to detect binding ofrecombinant H17 and H18 proteins on a panel of different bat and mammalian cell linesincluding those derived from Sturnira lilium, the bat species from which the H17N10sequences originate. Furthermore, we have developed reporter assays to investigate entryof virus-like particles (VLPs) pseudotyped with the bat influenza virus glycoproteinsH17N10 or H18N11 on different cell lines. Aiming at the identification of the cellularreceptor of H18N11, we performed a comparative transcriptome analysis of two differentMDCKII clones differing in susceptibility to H18N11 entry. Thus, we can test potentialreceptor candidates by silencing or ectopic expression experiments. Altogether, we want tostudy bat influenza virus entry and uncover their cellular receptor to get a deeper insightinto the biology of the newly discovered IAV subtypes.

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Harnessing autophagy to limit autoimmune CNS inflammation

Monika Kotur1, Christian W. Keller1, Christina Sina1,2, Giulia Ramelli1, Sarah Mundt3, IsaakQuast1,4, Patrick Weber1, Burkhard Becher3, Christian Münz5, Jan D. Lünemann1,6

1 Institute of Experimental Immunology, Laboratory of Neuroinflammation, University of Zurich,Zurich,

Switzerland; 2 Brain Research Institute, University of Zurich, Zurich, Switzerland; 3 Instituteof

Experimental Immunology, Laboratory of Inflammation Research, University of Zurich, Zurich,Switzerland;

4 Department of Immunology and Pathology, Central Clinical School, Monash University,Melbourne,

Australia; 5 Institute of Experimental Immunology, Laboratory of Viral Immunobiology,University of Zurich

[email protected]

Reactivation and expansion of autoreactive CD4+ T cells within the central nervous system(CNS) are considered to play a key role in the pathogenesis of multiple sclerosis (MS) andits animal model experimental autoimmune encephalomyelitis (EAE). Autophagy-relatedproteins (ATGs) shuttle antigens into MHC class II loading compartments (MIICs) forrecognition by CD4+ T cells. We could show that mice deficient in the autophagy proteinATG5 in CD11c+ dendritic cells (DC-Atg5-/-) are resistant to EAE development followingadoptive transfer of myelin-specific CD4+ T cells. Moreover, reactivation ofmyelin-specific CD4+ T cells by dendritic cells loaded with apoptotic oligodendroglialcells was abrogated in absence of ATG5 indicating that the autophagic machineryaugments T cell-mediated CNS injury. The cardiac glycoside neriifolin, an inhibitor of theNa+, K+-ATPase α1 subunit has been identified as a potent inhibitor of autophagy. We arenow investigating whether autophagy-dependent antigen presentation can be modulatedby neriifolin in vitro and harnessed for the prevention and treatment of T cell-mediatedCNS autoimmunity in vivo. It is our hope that the proposed research leads to a betterunderstanding how antigen presentation contributes to autoimmune CNS inflammationand reveals new therapeutic targets to treat MS.

Signature-Tagged Mutagenesis Screen in Salmonella Typhimurium murinegastrointestinal colonization

Markus Kreuzer, Bidong Nguyen, Miguel Cuenca, Sandra Wotzka, Lisa Maier, Wolf-DietrichHardt


[email protected]

Mice harboring a complex microbiota are inherently resistant to infection with SalmonellaTyphimurium (S. Tm). This colonization resistance can be broken by treatment withantibiotics or a change in diet that cause a shift in microbiota composition during which S.Tm is thought to occupy liberated niches and feed on unused nutrient sources. A genomewide transposon mutagenesis screen was conducted to determine the genes underlying thegastrointestinal colonization. Mouse microbiota was disrupted either by streptomycintreatment or by a dietary change including administration of one of the following

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substances: arabinose, lactulose, or oleic acid, while still being on normal chow diet (4.5 %fat) or a high-fat diet (36 % fat). Subsequently, mice were orally infected with a pool of S.Tm transposon mutants combined with dilution sets of already characterized mutants tocontrol for bottleneck effects. Random loss of mutants due to inflammation andsubsequent killing by granulocytes started around day 2 post infection and thereforemutants showing a phenotype in later stages of infection were selected according to thecorrectness of control mutant distribution. Mutants with a fitness disadvantage were rarelyattributable to a single treatment but instead seen over several different treatments.Therefore, S. Tm possibly feeds on multiple different nutrients that are temporarilyliberated due to a microbiota shift rather than utilizing a specific compound introduced bythe change in diet. This scavenger mechanism enables S. Tm to outcompete the microbiotain the phase of transient remodeling.

Virus transmission during kidney transplantation assessed by virome analysis oflivingdonor and recipient

Verena Kufner, Peter W. Schreiber, Kerstin Hübel, Osvaldo Zagordi, Stefan Schmutz,

Cornelia Bayard, Michael Greiner, Andrea Zbinden, Riccarda Capaul, Jürg Böni, Thomas

Müller, Nicolas J. Mueller, Alexandra Trkola, Michael Huber

Institute of Medical Virology, University of Zurich

[email protected]

Before kidney transplantation, donor and recipient are routinely screened for a number ofactive or latent viral infections using specific tests. Little is known about other,apathogenic viruses a donor might be infected with and likely will transmit. Here, weaimed to characterize the viral metagenome of donor and recipient using metagenomicsequencing at time of transplantation and up to one year after.

Recipients of kidney grafts and the corresponding donors were enrolled at the time oftransplantation. Follow-up study visits for recipients were scheduled 4-6 weeks and 1 yearthereafter. At each visit, plasma and urine samples were collected and patients wereevaluated for signs of infection or transplant-related complications. For metagenomicsanalysis, blood and urine samples were enriched for viruses, amplified using an anchoredrandom PCR system and sequenced using high-throughput metagenomic sequencing.Viruses detected by sequencing were confirmed using real-time PCR.

Metagenomic sequencing of a total of 14 living kidney donor/recipient pairs detected JCpolyomavirus in the urine of 5 recipients as well as all corresponding donors. Phylogeneticanalysis so far confirmed that donor and recipient were infected with the same strain in 2cases, suggesting a transmission from transplant donor to recipient. Moreover, Torque tenovirus was found frequently in time points after transplantation, as expected in patientsunder immunosuppression. Future studies within larger cohorts may help to define therelevance of the donor’s virome for the recipient, by enabling the analysis of possibleassociations with relevant transplant outcomes, such as rejection, graft loss or death.

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Cell cycle-dependent gene expression of the adeno-associated virus

Anita F. Meier, Sereina Sutter, Cornel Fraefel

Insitute of Virology, University of Zurich

[email protected]

The adeno-associated virus (AAV) is a small, non-enveloped ssDNA virus. It belongs to thegenus Dependoparvovirus and as the name suggests only replicates in presence of a helpervirus (adenovirus (AdV), herpes simplex virus (HSV) or human papilloma virus (HPV)).For replication viruses hijack cellular components and machineries (e.g. NTPs, aminoacids, ribosomes, DNA polymerase) and thus co-infecting viruses compete for limitedresources. We recently reported an interesting strategy of HSV and AAV to overcomecompetition. In absence of AAV, HSV can replicate in each phase of the cell cycle.However, in co-infected cells, HSV DNA replication becomes limited to G1 whereas AAVDNA replication/gene expression occurs preferentially in the S/G2 phases of the cell cycle.The aim of the present project is to identify the licensing factor that supports AAV2 geneexpression in S/G2 phases or the inhibitor that blocks it in G1. For example, wehypothesized that key players of the cellular DNA damage response (DDR) pathways areinvolved in the observed cell cycle dependent AAV gene expression. However, chemicalinhibition of the three main kinases in DDR, ATM, ATR and DNA PKcs, did not affect thecell cycle preference of AAV gene expression. In order to identify further candidateproteins, a pull down of viral DNA from infected cells will be conducted and interactingcellular proteins determined by mass spectrometry.

Investigating the role of lymphatic endothelial cell expressed immunomodulatorymolecules (CD112, CD274) in intralymphatic trafficking and cellular immunity

Peter Runge1, Cornelia Halin1

1Institute of Pharmaceutical Sciences, ETH Zurich

[email protected]

Recent findings from our group have revealed that immune cells, such as dendritic cells(DCs) and recirculating effector T cells, actively crawl on the intraluminal surface oflymphatic endothelial cells (LECs). These immune cells spend several hours withinafferent lymphatic capillaries making us speculate that this specialized vascular bed couldrepresent a new immunological compartment, in which immune responses and immunemodulation can take place. Transcriptional analysis of FACS-sorted LECs identified theexpression of immunomodulatory molecules PDL1 (CD274) and Nectin-2 (CD112) ondermal LECs. Each molecule binds to its correspondent immuneinhibitory receptorexpressed on T cells (PDL1 to PD1 and Nectin-2 to CD112R respectively). In this project,we will further investigate the role of LEC-expressed PDL1 and Nectin-2 in intralymphaticimmune cell migration and cellular immunemodulation.

Protein expression of PDL1 and Nectin-2 on LECs was verified in vitro and in vivo by FACSand immunofluorescent stainings. To investigate a functional impact of the two molecules,we will perform several in vitro experiments (transmigration, adhesion, flow chambercrawling, co-culture (LECs with T cells; T cells with DCs). Since it was shown thatregulatory T cells (Tregs) represent the main subset of effector T cells exiting the skinduring inflammation, we will focus on elucidating the influence of LEC-expressed PDL1and Nectin-2 on Treg function. Regarding in vivo experiments, we plan to perform

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adoptive transfer studies in different mouse models (i.e. lymphatic specificCD112-/-/CD274-/- mice) and subsequently analyze the phenotype of migrated cells inthe draining lymph node.

Probing bacterial interactions in the A. thaliana phyllosphere

Martin Schäfer, Miriam Bortfeld-Miller and Julia A. Vorholt


[email protected]

The phyllosphere is a vast microbial habitat, which is exposed to various, fast changingenvironmental conditions. Metagenomic studies and culture-independent communityprofiling have shown that the Arabidopsis thaliana phyllosphere is reproducibly colonizedby a phylogenetically conserved set of bacteria. The availability of a representative straincollection covering most of the phylogenetic diversity found in the A. thaliana phyllospheremakes it now possible to address questions on community assembly and dynamics undercontrolled conditions with predefined synthetic communities. With this resource, we arecurrently studying bacteria-bacteria and bacteria-plant interactions in a gnotobiotic A.thaliana system. With this approach, we study binary and complex bacterial communitiesand test different perturbations and their effect on the community structure and stabilityto understand mechanisms that are important for a stable community structure andcontribute to plant health. In this context, we will also focus on aspects of bacterialcompetition for limited resources and strategies that evolved to defend the niche byproducing secondary metabolites.

Identification of the mode of action of peptide antibiotics in Pseudomonasaeruginosa

Alessandra Vitale1, Gabriella Pessi1, Steven Higgins1, Katja Zerbe2, John Robinson2 and LeoEberl1

1 Institute of Plant and Microbial Biology, UZH Zürich

2 Department of Chemistry, UZH Zürich

[email protected]

One of the biggest challenges of our century is the increasing antibiotic resistance inpathogenic bacteria such as the opportunistic human pathogen Pseudomonas aeruginosa [1].New antibiotic treatments are urgently needed and this challenge could be overcome withnew peptidomimetic antimicrobial compounds, which have been shown to be especiallyactive against many Gram-negative bacteria [2,3].

In this study, we investigated the mechanisms of action of those new antibiotics by usingthe transposon sequencing method [4]. A saturated library of transposon mutants (approx.1 million) was constructed in P. aeruginosa PA14 and challenged with a sub-inhibitoryconcentration of the well-known cyclic peptide antibiotics polymyxins B and E. Weperformed a fitness based data analysis and established a list of candidate genes, which areneeded for autonomous growth or provide a fitness advantage in the presence of theantimicrobial compounds. Among them, three genes involved in outer-membranebiosynthesis and one coding for a multidrug resistance efflux pump were found to beessential in the presence of both polymyxins (B and E). In addition, we are currently

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challenging individual mutants with polymyxins in order to confirm the importance ofspecific proteins, as for example the multidrug resistance efflux pump.

In conclusion, we have constructed a highly saturated library of mutants in P. aeruginosaPA14 and identified new potential mechanisms of polymyxin resistance. In the next step,we will apply this approach to characterize the mode of action of novel peptidomimeticantibiotics.

Synergistic activity of NKG2D-based chimeric antigen receptor (CAR)-T cells andradiotherapy against glioma

Tobias Weiss1, Michael Weller1, Martin Pruschy3, Charles Sentman2, Patrick Roth1

1Department of Neurology and Brain Tumor Center, University Hospital Zurich and Universityof Zurich

2Department of Microbiology & Immunology, Geisel School of Medicine, New Hampshire,USA

3Department of Radiation Oncology, University Hospital Zurich and University of Zurich

Glioblastoma is the most common primary brain tumor in adults and virtually alwayslethal despite a multimodal treatment regimen including surgery, chemotherapy andradiotherapy. Therefore, novel treatment modalities are needed. Adoptive immunotherapywith genetically engineered T cells that express a chimeric antigen receptor (CAR) torecognize and eliminate tumors in a MHC-independent manner is an emerging strategythat has led to remarkable responses in hematologic malignancies. CARs that target thenatural-killer group 2-member D (NKG2D) system elegantly use the promiscuous bindingproperties of the NKG2D receptor that binds to several tumor-associated ligands.NKG2D-based CAR T cells have never been investigated against glioma and it is unknownif this strategy can overcome the challenges of this tumor entity and if it could even beimplemented in established conventional treatment regimens.

Methods

CAR T cells were generated by retroviral transduction of splenocytes derived fromC57BL/6 or VM/Dk mice to express a synthetic receptor with the extracellular domain ofNKG2D, a transmembrane domain and the intracellular CD3ζ domain. As a control,splenocytes were transduced with wildtype-NKG2D (wtNKG2D). For in vitro studies,murine glioma cells (GL-261, SMA-560, SMA-540, SMA-407) were co-cultured withchNKG2D or wtNKG2D T cells and we assessed the cytolytic activity and IFN-? productionby flow cytometry. For in vivo studies, we used GL-261 cells syngeneic to C57BL/6 miceand monitored tumor growth by magnetic resonance imaging. For in vivo tracking of CART cells, we used fluorescence molecular tomography (FMT), flow cytometry andimmunohistochemistry. Long-term survivors were re-challenged with another tumorimplantation and tumor-infiltrating and peripheral lymphocytes were analyzed by flowcytometry. To study the combination of radiotherapy with CAR T cells, mice received asingle subtherapeutic dose of local irradiation.

Results

In all murine glioma cell lines, chNKG2D T cells had a significantly higher specific cytolyticactivity compared to wtNKG2D T cells. Furthermore, chNKG2D T cells produced more

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IFN. In vivo, intravenously injected chNKG2D T cells migrated to the orthotopic tumor site,were tolerated without toxicities, prolonged the survival and cured a fraction of tumor-bearing mice. This anti-tumor effect was even more pronounced in case of intratumoralCAR T cell administration. Survivors were long-term protected against tumor re-challenge.Mechanistically, this was not the result of a classical immune memory response, but ratherdue to local persistence of chNKG2D T cells. Radiotherapy, as part of the standard treatmentregimen against glioblastoma, augmented the effect of chNKG2D T cell therapy already aftera single application of a subtherapeutic dose. We identified two underlying mechanismfor this synergistic activity. First, a direct tumor-cell related effect as demonstrated by anincreased cytolysis and IFN-??production after co-culture of low-dose pre-irradiated GL-261 cells with chNKG2D or wtNKG2D T cells and second an indirect migration-relatedmechanism as demonstrated by increased accumulation of CAR T cells within irradiatedtumors in vivo.

Conclusion

We provide the first systematical preclinical assessment of NKG2D-based CAR T cellsagainst glioma. This strategy was effective, tolerated without toxicities, conferredlong-term protection and was synergistic with radiotherapy. These findings could providea rationale to test this immunotherapeutic strategy also in human glioma patients.

Generation of a human p47phox-deficient chronic granulomatous disease cell line usingCRISPR/Cas9 for fast gene therapy vector testing

Dominik Wrona, Ulrich Siler, Janine Reichenbach

Division of Immunology, University Children’s Hospital Zurich

[email protected]

Chronic granulomatous disease (CGD) comprises a group of monogeneticimmunodeficiencies characterized by defective respiratory burst and microbicidal activityof phagocytes leading to recurrent life-threatening infections. Mutations in any of fivesubunits of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH)oxidase may result in CGD. The gp91phox subunit is most frequently mutated (65%) andthe mutations are scattered throughout the gp91phox gene. Conversely, thep47phox-deficiency that is the second most common CGD form (25%) is almost exclusivelycaused by a single GT-dinucleotide deletion (∆GT) in exon 2 of neutrophil cytosolic factor1 (NCF1) gene. This mutation causes a frameshift and premature translation interruption.The ∆GT is shared with two pseudogenes present on the same chromosome. Homologousrecombination between them leads to the predominance of the ∆GT mutation amongp47phox-deficient CGD patients.

Development of gene therapy vectors for p47phox-deficient CGD is hampered by theabsence of human cell lines allowing for robust gene therapy vector testing. Maintenanceand differentiation of existing ∆GT p47phox-deficient iPSC-based lines is laborious and inmany aspects impractical. Here, we established a novel model of ∆GT p47phox-deficientCGD based on a human acute myeloid leukemia PLB-985 cell line. We utilized CRISPRtechnology to introduce the ∆GT mutation in NCF1. The PLB-985 NCF1 ∆GT cells reflect∆GT p47phox-deficiency genetically and functionally. The cells can be differentiated togranulocytes in seven days and are correctable by γ-retroviral vectors. The PLB-985 NCF1∆GT cell line creates an attractive alternative to currently used iPSC-based models fortesting of novel gene therapy approaches.

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Participants

Last Name First Name Email address Affiliation Institute

Affolter Ilaria [email protected] ETH MicrobiologyCernikova Lenka [email protected] UZH Parasitology

Chaudron Sandra [email protected] UZHInfectious Diseases andHospital Epidemiology

Deng Yun [email protected] UZH Experimental ImmunologyFriess Mona [email protected] ETH Pharmaceutical SciencesFurter Markus [email protected] ETH Microbiology

Grünbach Maarja [email protected] UZH University Hospital ZürichGualdi Stefano [email protected] UZH Plant and Microbial BiologyHäffner Nicola [email protected] UZH Infectiology

Haunerdinger Veronika [email protected] UZHUniversity Children‘s

Hospital ZurichHeeb Lukas [email protected] UZH Immunology

Huemer Markus [email protected] UZHInfectious Diseases andHospital Epidemiology

Hunter Morgan [email protected] ETH Pharmaceutical SciencesHunziker Annika [email protected] UZH Medical VirologyKalawong Ratchara [email protected] UZH Plant and Microbial BiologyKarakus Ufuk [email protected] UZH ImmunologyKarakus Umut [email protected] UZH Medical VirologyKelley Audrey [email protected] ETH Infectious Diseases

Kirchner Florian [email protected] UZH Virology

Knecht Leandra [email protected] ETHZürich University

of Applied SciencesKotur Monika [email protected] UZH Experimental Immunology

Krempaska Natalia [email protected] UZH Plant and Microbial BiologyKreuzer Markus [email protected] ETH MicrobiologyKufner Verena [email protected] UZH Medical Virology

Li Pengfei [email protected] UZH Vetsuisse FacultyLoi Marisa [email protected] ETH Research in Biomedicine

Marti Hanna [email protected] UZH Veterinary PathologyMeier Anita [email protected] UZH VirologyMüller Eva [email protected] UZH Medical VirologyMüller Barbara [email protected] ETH Immunology and Microbiology

Neuhaus Julia [email protected] ETH MicrobiologyNguyen Tony [email protected] ETH ImmunologyOzcan Alaz [email protected] UZH ImmunologyPerotti Michela [email protected] ETH Research in Biomedicine

Peschke Benjamin [email protected] UZH Experimental ImmunologyPurtschert Gabriela [email protected] UZH Plant and Microbial Biology

Runge Peter [email protected] ETH Pharmaceutical SciencesSchäfer Martin [email protected] ETH MicrobiologySchineis Philipp [email protected] ETH Pharmaceutical Sciences

Sidorov Semjon [email protected] UZHExperimental Infectiology

and Cancer Research

Silva Marisa [email protected] UZHPlant Biology

Limnological StationSteiner Fiona [email protected] UZH Medical VirologyStocker Christian [email protected] ETH Laboratory of Organic Chemistry

Subbaraman Harini [email protected] UZH Medical VirologySüssbier Ute [email protected] UZH Experimental HematologySutter Sereina [email protected] UZH Virology

ten Buren Emiel [email protected] UZH Laboratory Animal ScienceUtz Sebastian [email protected] UZH Experimental Immunology

Verest Marjan [email protected] ETH MicrobiologyVitale Alessandra [email protected] UZH Plant and Microbial Biology

Walkam Anja [email protected] UZH University Hospital Zürich

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Weder Bruce [email protected] UZH Gastroenterology and HepatologyWeiss Tobias [email protected] UZH Molecular Neurooncology

Winiger Rahel [email protected] UZH Parasitology

Wrona Dominik [email protected] UZHUniversity Children‘s

Hospital ZürichYu Xueyang [email protected] UZH Experimental Immunology

Zdimerova Hana [email protected] UZH Experimental ImmunologyZünd Mirjam [email protected] ETH Microbiology

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Documents

10th MIM Retreat - ethz.ch · MIM retreat Locarno, Ticino August 24th-26th 2017 Welcome and contact information Dear MIM PhD students, We are pleased to welcome you to the 10thMIM