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Researchmaster ‘Infection & Immunity’

Research topics 2017Vs 171115

Research Master Infection & Immunity Research topics for Internships 2 / 98

IMMUNOLOGY – RESEARCH TOPICS


Title: Chronic lymphoproliferations: at the interface between normal and aberrant lymphoid development

Group leader: dr. A.W. Langerak T: 010-704 4089E: [email protected]

W: https://www.erasmusmc.nl/immu/research/mid

Duration Yes/No Available per (date)

6 months Yes December 2017


18 months Yes After further contact

BackgroundV(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) genes is required to establish a broad repertoire of antigen receptors on B and T cells. In chronic lymphoproliferations (CLL, T-LGL) that arise in elderly individuals, these antigen receptors are thought to reflect stimulation by antigens as an early event in the pathogenesis. Insight into the precise role of antigenic stimulation will shed light on the (immune)pathogenesis of chronic lymphoproliferations. This fundamental knowledge can be translated into prognostics and improved treatment stratification, and eventually might also contribute to the identification of novel therapeutic targets.CLL is the most common type of leukemia in elderly in the Western world. It is characterized by a monoclonal expansion of mature B lymphocytes. Based on the somatic hypermutation status of the IGH genes of the B-cell receptor (BcR), CLL can be distinguished into unmutated CLL (U-CLL) and mutated CLL (M-CLL). This division has clinical relevance because U-CLL has an unfavorable prognosis (aggressive clinical disease course), while M-CLL has a more favorable prognosis (indolent form of disease). Over 30% of all CLL cases can be grouped in subsets based on similarities in their BcR. These so-called stereotypic BcR are found in different CLL patients and suggest involvement of highly similar antigens. This implies that antigenic stimulation and BcR specificity play a role in CLL pathogenesis. Also, BcR signaling capacity in human CLL is intrinsically different from normal human B cells, whilst differences exist between CLL subgroups. On top of this, (subclonal) genetic mutations have been identified that associate with particular immunogenetic subgroups with distinct BcR’s.

Research project in the MID group at the Department of Immunology Potential research topics (pending status of project): -To study the role of BCR signalling (including the contribution of antigenic stimulation) in the pathogenesis of (human) CLL.-To study the association of oncogenic aberrations and epigenetic alterations with immunogenetic (U/M/stereotyped) CLL subgroups.-To define prognostic biomarkers (proteomics markers, non-coding RNA markers) in plasma samples in “watch-and-wait” CLL patients prior to treatment and CLL patients under treatment.

The MID group is an (inter)national reference center for chronic lymphoproliferations. Research in the MID group is performed in longstanding collaborations within Erasmus MC (Hematology, Pulmonary Med) and is embedded in several international collaborative networks / clinical trials. In our research projects we use state-of-the-art cellular (multi-color

https://www.erasmusmc.nl/immu/research/mid

mailto:[email protected]


flow cytometry, cell sorting) and molecular (sequencing, spectratyping, qPCR, array, NGS) techniques.

Publications related to the research lines

Agathangelidis A, Darzentas N, Hadzidimitriou A, Brochet X, Murray F, Yan XJ, Davis Z, van Gastel-Mol EJ, Tresoldi C, Chu CC, Cahill N, Giudicelli V, Tichy B, Pedersen LB, Foroni L, Bonello L, Janus A, Smedby K, Anagnostopoulos A, Merle-Beral H, Laoutaris N, Juliusson G, di Celle PF, Pospisilova S, Jurlander J, Geisler C, Tsaftaris A, Lefranc MP, Langerak AW, Oscier DG, Chiorazzi N, Belessi C, Davi F, Rosenquist R, Ghia P, Stamatopoulos K Stereotyped B-cell receptors in one third of chronic lymphocytic leukemia: towards a molecular classification with implications for targeted therapeutic interventions. Blood 2012;119:4467-4475

Goede V, Fischer K, Busch R, Engelke A, Eichhorst B, Wendtner CM, Chagorova T, De la Serna J, Dilhuydy MS, Illmer T, Opat S, Owen CJ, Samoylova O, Kreuzer KA, Stilgenbauer S, Döhner H, Langerak AW, Ritgen M, Kneba M, Asikanius E, Humphrey K, Wenger M, Hallek M. Obinutuzumab (GA101) plus chlorambucil in patients with CLL and comorbidity. New Engl J Med 2014;370:1101-1110 (Epub Jan 8).

Baliakas P, Hadzidimitriou A, Sutton LA, Minga E, Agathangelidis A, Tsanousa A, Scarfo L, Davis Z, Yan XJ, Shanafelt T, Plevova K, Sandberg Y, Vojdeman FJ, Boudjogra M, Tzenou T, Chatzouli M, Chu CC, Veronese S, Gardiner A, Mansouri L, Smedby KE, Bredo Pedersen L, Moreno D, Van Lom K, Giudicelli V, Shkurova Francova H, Nguyen-Khac F, Panagiotidis P, Juliusson G, Angelis L, Anagnostopoulos A, Lefranc MP, Trentin L, Catherwood M, Montillo M, Geisler G, Langerak AW, Pospisilova S, Chiorazzi N, Oscier D, Jelinek D, Darzentas N, Belessi C, Davi F, Rosenquist R, Ghia P, Stamatopoulos K. Clinical effect of stereotyped B-cell receptor immunoglobulins in chronic lymphocytic leukaemia: a retrospective multicentre study. Lancet Hematol 2014;1:e74-e84.

Muggen AF, Kil LP, Yuvaraj S, Van Dongen JJM, Hendriks RW, Langerak AW. High basal Ca2+ signaling in mutated chronic lymphocytic leukemia. Leukemia 2015;29:321-328.

Muggen AF, Pal Singh S, Hendriks RW, Langerak AW. Targeting signaling pathways in chronic lymphocytic leukemia. Current Cancer Drug Targets 2016;16:669-688.


Title: Characterization of acute leukemia

Workgroupleader:Dr. V.H.J. (Vincent) van der VeldenT: 010-704 4253E: [email protected]


6 months Yes 1 jan 2017



BackgroundAcute leukemias are malignancies of immature hematopoietic cells. Depending on the involved cell type, two main categories of acute leukemia can be distinguished: acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Further subdivision can be made, dependent on immunophenotype and genetic aberrations. Such subdivision is necessary because the different subgroups have different clinical outcomes and consequently require different therapeutic approaches. ALL is most prominent in childhood and, using current chemotherapeutic regimens, has a 5-year event free survival up to 85%. For this group of patients it will especially be important to recognize those patients with a high risk of relapse, who may benefit from treatment intensification, and those with a low-risk of relapse, who may benefit from treatment reduction. In contrast, AML is most frequent in adults (especially those >60 years of age) and although chemotherapy induces clinical remission in the vast majority of patients, most of them ultimately relapse, resulting in a 5-year survival of ~40%. It is therefore clear that new therapeutic modalities are required for this type of leukemia. We and others have recently shown that detection of low numbers of leukemic cells during and after treatment, so called detection of minimal residual disease (MRD), is of prognostic value in acute leukemia. In our laboratory, flowcytometric immunophenotyping, PCR analysis of chromosome aberrations, and PCR analysis of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements are used for MRD detection in several patient groups to investigate the kinetics of tumor cell disappearance and thereby define the in vivo treatment effectiveness and their risk of relapse. In addition, MRD information is used to determine the efficacy of new therapies, including antibody-mediated chemotherapy, and to provide insight into the immunobiology of acute leukemia (e.g. Ig/TCR gene rearrangement patterns in leukemia subtypes).

Research topicsThe main aim of our translational research is to improve our understanding on the immunobiology of acute leukemias and to evaluate their response to treatment in order to improve diagnostics and to optimize treatment protocols. To this end, we use start-of-the-art technologies, like 8-color floccytometric immunophenotyping, cell sorting, sequencing, and micro-array technology. The LLD research group coordinates and participates in several national and international networks.

Our specific aims are:1. To obtain insight in the immunobiological characteristics of childhood acute leukemia. This includes

a.o.:a. Analysis of extramedullary acute leukemia for identifying markers that may predict

extramedullary localization and that may be used as new therapeutic targets;b. Analysis of acute leukemia at the myeloid-lymphoid interface for understanding which

molecules play a role in lineage specification and commitment.2. To analyze Minimal Residual disease:

a. Development and standardization of sensitive techniques to measure MRD in acute leukemia patients; this concerns both cellular and molecular methods;

b. Recognition of MRD-based subgroups of patients that may benefit from treatment intensification or treatment reduction.

3. To identify parameters which affect the efficacy of antibody-based chemotherapy in acute leukemia.

Publications related to the research line 1



van Dongen JJ, van der Velden VH, Brüggemann M, Orfao A. Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies. Blood. 2015 Jun 25;125(26):3996-4009. doi: 10.1182/blood-2015-03-580027. Epub 2015 May 21.

van der Velden VH, de Launaij D, de Vries JF, de Haas V, Sonneveld E, Voerman JS, de Bie M, Revesz T, Avigad S, Yeoh AE, Swagemakers SM, Eckert C, Pieters R, van Dongen JJ. New cellular markers at diagnosis are associated with isolated central nervous system relapse in paediatric B-cell precursor acute lymphoblasticleukaemia. Br J Haematol. 2016 Mar;172(5):769-81. doi: 10.1111/bjh.13887. Epub 2015 Dec 21. PubMed PMID: 26898195.

Aalbers AM, van den Heuvel-Eibrink MM, Baumann I, Dworzak M, Hasle H, Locatelli F, De Moerloose B, Schmugge M, Mejstrikova E, Nováková M, Zecca M, Zwaan CM, Te Marvelde JG, Langerak AW, van Dongen JJ, Pieters R, Niemeyer CM, van der Velden VH. Bone marrow immunophenotyping by flow cytometry in refractory cytopenia of childhood. Haematologica. 2015 Mar;100(3):315-23. doi: 10.3324/haematol.2014.107706. Epub 2014 Nov 25.

van der Velden VH, Hoogeveen PG, de Ridder D, Schindler-van der Struijk M, van Zelm MC, Sanders M, Karsch D, Beverloo HB, Lam K, Orfao A, Lugtenburg PJ, Böttcher S, van Dongen JJ, Langerak AW, Kappers-Klunne M, van Lom K. B-cell prolymphocytic leukemia: a specific subgroup of mantle cell lymphoma. Blood. 2014 Jul 17;124(3):412-9. doi: 10.1182/blood-2013-10-533869. Epub 2014 Jun 2.

Szczepanski T, van der Velden VH, Waanders E, Kuiper RP, Van Vlierberghe P, Gruhn B, Eckert C, Panzer-Grümayer R, Basso G, Cavé H, Stadt UZ, Campana D, Schrauder A, Sutton R, van Wering E, Meijerink JP, van Dongen JJ. Late recurrence of childhood T-cell acute lymphoblastic leukemia frequently represents a second leukemia rather than a relapse: first evidence for genetic predisposition. J Clin Oncol. 2011 Apr 20;29(12):1643-9. doi: 10.1200/JCO.2010.30.2877. Epub 2011 Feb 28. PubMed PMID: 21357790.


van der Velden VH, Noordijk R, Brussee M, Hoogeveen PG, Homburg C, de Haas V, van der Schoot CE, van Dongen JJ. Minimal residual disease diagnostics in acute lymphoblastic leukaemia: impact of primer characteristics and size of junctional regions. Br J Haematol. 2014 Feb;164(3):451-3. doi: 10.1111/bjh.12621. Epub 2013 Oct 26. PubMed PMID: 24164448.

Denys B, van der Sluijs-Gelling AJ, Homburg C, van der Schoot CE, de Haas V, Philippé J, Pieters R, van Dongen JJ, van der Velden VH. Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia. Leukemia. 2013 Mar;27(3):635-41. doi: 10.1038/leu.2012.231. Epub 2012 Aug 16. PubMed PMID: 22945774.

Waanders E, van der Velden VH, van der Schoot CE, van Leeuwen FN, van Reijmersdal SV, de Haas V, Veerman AJ, van Kessel AG, Hoogerbrugge PM, Kuiper RP, van Dongen JJ. Integrated use of minimal residual disease classification and IKZF1 alteration status accurately predicts 79% of relapses in pediatric acute lymphoblastic leukemia. Leukemia. 2011 Feb;25(2):254-8. doi: 10.1038/leu.2010.275. Epub 2010 Nov 19.


Bras AE, Beishuizen A, Langerak AW, Jongen-Lavrencic M, Te Marvelde JG, van den Heuvel-Eibrink MM, Zwaan CM, van Dongen JJ, van der Velden VH. CD38 expression in paediatric leukaemia and lymphoma: implications for antibody targeted therapy. Br J Haematol. 2016 Sep 8. doi: 10.1111/bjh.14310. [Epub ahead of print] PubMed PMID: 27604396.

de Vries JF, Zwaan CM, De Bie M, Voerman JS, den Boer ML, van Dongen JJ, van der Velden VH. The novel calicheamicin-conjugated CD22 antibody inotuzumab ozogamicin (CMC-544) effectively kills primary pediatric acute lymphoblastic leukemia cells. Leukemia. 2012 Feb;26(2):255-64. doi: 10.1038/leu.2011.206. Epub 2011 Aug 26.


Title: Laboratory Medical Immunology-Immune regulation diagnostics (IRD)

Groupleader: dr. W.A. Dik (+31 (0)10 70.43528, [email protected]).


6 months Yes/No December 2017



Background The immune system is a complex and tightly regulated network in which several different cell types, signaling molecules (cytokines) and receptors collaborate to fulfill its function. Uncontrolled regulation of the immune system is associated with a variety of diseases and infectious, inflammatory and fibrotic conditions.

Research The main aim is to identify cellular and molecular mechanisms underlying (chronic) infectious, inflammatory and fibrotic processes, with the specific aim to use this information to guide the development of (novel) protective measures to be used in the clinic. The last years there has been a strong focus on ocular immune pathology, for instance Graves ophthalmopathy, but also uveitis associated with mycobacterium tuberculosis infection as well as vitreoretinal disorders such as proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Other diseases of interest are (systemic) autoimmune diseases, sepsis and chronic mucocutaneous candidiasis . Our research is multidisciplinary with an optimal integration of both laboratory and clinical expertise from various departments within the Erasmus MC and the Rotterdam Eye Hospital.

Principal TechniquesAssays and techniques commonly performed within our studies include: multi-color flow cytometry, cell culture, proliferation assays, Western blotting, molecular techniques including RNA sequencing, real-time quantitative PCR and gene expression array, cell transfection and transduction, multiplex immune assay (MIA; Luminex) and ELISA. Within the research lines different types of human tissue is used.

Selected achievements in the past years

Basic FGF and PDGF-BB synergistically stimulate hyaluronan and IL-6 production by orbital fibroblasts. Virakul S, Heutz JW, Dalm VA, Peeters RP, Paridaens D, van den Bosch WA, Hirankarn N, van Hagen PM, Dik WA.Mol Cell Endocrinol. 2016;433:94-104

Histamine induces NF-κB controlled cytokine secretion by orbital fibroblasts via histamine receptor type-1. Virakul S, Phetsuksiri T, van Holten-Neelen C, Schrijver B, van Steensel L, Dalm VA, Paridaens D, van den Bosch WA, van Hagen PM, Dik WA.Exp Eye Res. 2016;147:85-93.

Current perspectives on the role of orbital fibroblasts in the pathogenesis of Graves' ophthalmopathy. Dik WA, Virakul S, van Steensel L.Exp Eye Res. 2016;142:83-91.

Cytokine and viral load kinetics in human herpesvirus 8-associated multicentric Castleman's disease complicated by hemophagocytic lymphohistiocytosis. Zondag TC, Rokx C, van Lom K, van den Berg AR, Sonneveld P, Dik WA, van Doornum GJ, Lam KH, van Laar JA.Int J Hematol. 2016;103:469-72.



Neutrophil CD64 expression is not a useful biomarker for detecting serious bacterial infections in febrile children at the emergency department. van Veen M, Nijman RG, Zijlstra M, Dik WA, de Rijke YB, Moll HA, Neele M, Smit FJ, Oostenbrink R.Infect Dis (Lond). 2016;48:331-7.

Interferon-gamma immunotherapy in a patient with refractory disseminated candidiasis.Buddingh EP, Leentjens J, van der Lugt J, Dik WA, Gresnigt MS, Netea MG, Pickkers P, Driessen GJ.Pediatr Infect Dis J. 2015;34:1391-4

Platelet-Derived Growth Factor-BB Enhances Adipogenesis in Orbital Fibroblasts.Virakul S, Dalm VA, Paridaens D, van den Bosch WA, Mulder MT, Hirankarn N, van Hagen PM, Dik WA. Invest Ophthalmol Vis Sci. 2015;56:5457-64.

Retinal pigment epithelial cells display specific transcriptional responses upon TNF-α stimulation. Korthagen NM, van Bilsen K, Swagemakers SM, van de Peppel J, Bastiaans J, van der Spek PJ, van Hagen PM, Dik WA. Br J Ophthalmol. 2015;99:700-4

The role of thrombin in proliferative vitreoretinopathy. Bastiaans J, van Meurs JC, Mulder VC, Nagtzaam NM, Smits-te Nijenhuis M, Dufour-van den Goorbergh DC, van Hagen PM, Hooijkaas H, Dik WA. Invest Ophthalmol Vis Sci. 2014;55:4659-66.

The tyrosine kinase inhibitor dasatinib effectively blocks PDGF-induced orbital fibroblast activation. Virakul S, Dalm VA, Paridaens D, van den Bosch WA, Hirankarn N, van Hagen PM, Dik WA. Graefes Arch Clin Exp Ophthalmol. 2014;252:1101-9.

Thrombin induces epithelial-mesenchymal transition and collagen production by retinal pigment epithelial cells via autocrine PDGF-receptor signaling. Bastiaans J, van Meurs JC, van Holten-Neelen C, Nagtzaam NM, van Hagen PM, Chambers RC, Hooijkaas H, Dik WA. Invest Ophthalmol Vis Sci. 2013;54:8306-14

Factor Xa and thrombin stimulate proinflammatory and profibrotic mediator production by retinal pigment epithelial cells: a role in vitreoretinal disorders? Bastiaans J, van Meurs JC, van Holten-Neelen C, Nijenhuis MS, Kolijn-Couwenberg MJ, van Hagen PM, Kuijpers RW, Hooijkaas H, Dik WA. Graefes Arch Clin Exp Ophthalmol. 2013;251:1723-33

PDGF enhances orbital fibroblast responses to TSHR stimulating autoantibodies in Graves' ophthalmopathy patients. van Steensel L, Hooijkaas H, Paridaens D, van den Bosch WA, Kuijpers RW, Drexhage HA, van Hagen PM, Dik WA. J Clin Endocrinol Metab. 2012;97:E944-53.

Orbit-infiltrating mast cells, monocytes, and macrophages produce PDGF isoforms that orchestrate orbital fibroblast activation in Graves' ophthalmopathy. van Steensel L, Paridaens D, van Meurs M, van Hagen PM, van den Bosch WA, Kuijpers RW, Drexhage HA, Hooijkaas H, Dik WA. J Clin Endocrinol Metab. 2012;97:E400-8

Training options Interested and motivated students are encouraged to contact us about possibilities to do their lab internship in our team. Together with the student we will define a project that is in line with ongoing projects.

http://www.ncbi.nlm.nih.gov/pubmed/22238384












Title: Guillain-Barré syndrome

Workgroupleaders:Dr. B.C. Jacobs, [email protected]. R. Huizinga, [email protected]


6 months Yes January 2018



BackgroundGuillain-Barré syndrome (GBS) is a post-infectious, immune-mediated neuropathy and the most common cause of acute neuromuscular paralysis. In its most severe form GBS is a life-threatening disease causing rapidly progressive paralysis of arms, legs and the respiratory system for which they are treated at the intensive care. GBS has an intriguing pathogenesis in which preceding infections by molecular mimicry induce cross-reactive antibodies to neural glycolipids (gangliosides) that cause nerve destruction. The predominant cause of this infection is Campylobacter jejuni, a bacterium present in chicken meat and eggs. C. jejuni infections usually cause a mild gastro-enteritis but in 1 in 1000 of infected persons, this infection is followed by GBS. At present, we are investigating why certain persons are susceptible to develop GBS after this infection, and others not. In addition, we also aim to identify the underlying immune mechanisms. GBS is treated with intravenous immunoglobulins, a therapy discovered by chance, but of which we currently do not understand the underlying mechanisms. GBS patients show a highly variable recovery after this treatment, leaving one-fourth of patients severely disabled.

Focus research at Erasmus MCThe aim of our research is to better understand both the pathogenesis of GBS and the working mechanism of the treatment, and to improve the outcome for patients. The Erasmus MC is one of the leading centers in the world for GBS research and we focus on laboratory and clinical studies, in collaboration with international research consortia. Research topicsAt the Department of Immunology we focus on the pathogenesis and diagnosis of GBS, in collaboration with several other Departments within the Erasmus MC (e.g. Neurology, Microbiology, Epidemiology and Pediatrics). There are various specific research projects in which student can participate: 1. Define the relation between infections, the cross-reactive antibody response to nerves

and the clinical manifestations and prognosis of patients with GBS. 2. Define the relation between immunology and clinical features in children with GBS. 3. Develop new diagnostic tests (e.g. glycoarray) for early diagnosis and prediction of

prognosis of GBS and other immune-mediated neuropathies. 4. Identify genetic host factors related to innate immunity that determine whether a

person after an infection with C. jejuni will develop GBS or not. 5. Identification of pathogenic B cells and characterization of the neurotoxic effects of

antibodies to gangliosides in GBS patients. 6. Study the effects of intravenous immunoglobulins on the differentiation and function of

pathogenic B cells in GBS.7. Epidemiological studies on the occurrence of GBS in the Netherlands after exposure to

risk factors such as infections and vaccinations.8. Determine the pharmacokinetics and working mechanisms of treatment with

intravenous immunoglobulins in GBS.




Principal techniquesGeneral epidemiology and descriptive statistics, management of patient cohorts, blood cell isolation, transfection and culture, TLR stimulation assays, ELISA, glycoarray, FACS, antibody purification, immunohistochemistry, Western blotting, quantitative PCR, SNP detection.

Selected publications1 van Koningsveld R, Steyerberg EW, Hughes R A, Swan AV, van Doorn PA and Jacobs BC.

A clinical prognostic scoring system for Guillain-Barré syndrome. Lancet Neurol 2007; 6: 589-594.

2 van Doorn PA, Ruts L and Jacobs BC. Clinical features, pathogenesis, and treatment of Guillain-Barré syndrome. Lancet Neurol. 2008; 7: 939-950.

3 Kuitwaard K, de Gelder J, Tio-Gillen AP, Hop WC, van Gelder T, van Toorenenbergen AW, van Doorn PA and Jacobs BC. Pharmacokinetics of intravenous immunoglobulin and outcome in Guillain-Barré syndrome. Ann Neurol 2009; 66: 597-603

4 Kuijf ML, Samsom JN, van Rijs W, Bax M, Huizinga R, Heikema AP, van Doorn PA, van Belkum A, van Kooyk Y, Burgers PC, Luider TM, Endtz HP, Nieuwenhuis EE and Jacobs BC. TLR4-mediated sensing of Campylobacter jejuni by dendritic cells is determined by sialylation. J Immunol 2010; 185: 748-755.

5 Van den Berg B, van der Eijk AA, Pas SD, Hunter JG, Madden RG, Tio-Gillen AP, Dalton HR and Jacobs BC. Guillain-Barré syndrome associated with preceding hepatitis E syndrome. Neurology 2014; 82: 491-7.

6 Fokkink WJ, Selman MH, Dortland JR, Durmus B, Kuitwaard K, Huizinga R, van Rijs W, Tio-Gillen AP, van Doorn PA, Deelder AM, Wuhrer M and Jacobs BC. IgG Fc N-glycosylation in Guillain-Barré syndrome treated with immunoglobulins. J Prot Res 2014; 13:1722-30.

7. Huizinga R, van den Berg, van Rijs W, Tio-Gillen AP, Fokkink WJ, Bakker-Jonges LE, Geleijns K, Samsom JN, van Doorn PA, Laman JD and Jacobs BC. Innate immunity to Campylobacter jejuni in Guillain-Barré syndrome. Ann Neurol 2015; 78: 343-54.

8. Meyer Sauteur PM, Huizinga R, Tio-Gillen AP, Roodbol J, Hoogenboezem T, Jacobs E, van Rijn M, van der Eijk AA, Vink C, De Wit MC, Van Rossum AM, Jacobs BC (2016). Mycoplasma pneumoniae triggering the Guillain-Barré syndrome: a case-control study. Ann Neurol 80: 566-80.


Title: Immune repertoire in the picture

Workgroupleader:dr. Mirjam van der BurgT: 010-704 3015E: [email protected] https://www.erasmusmc.nl/immu/research/pid


6 months Yes Sept. 2018



BackgroundThe diversity of the immune repertoire of the adaptive immune system determines whether individuals have the potential to cope with invading pathogens. DNA repair plays an important role in generation of the immune repertoire as the three major molecular processes involved in the adaptive immune system are mediated via DNA double strand breaks. The aim of the research project is to unravel the role of DNA repair in the generation of an immune competent repertoire and how DNA repair defects result in immunodeficiency. Immune repertoire diversity is still poorly defined and it is unknown how the immune repertoire develops with increasing age and how diverse the repertoire needs to be to ensure immune competence. Quantitative assessment of repertoire diversity has thus far remained elusive, because molecular techniques were not suited to study repertoire diversity with a depth of >103. Therefore knowledge about the exact role of DNA repair in the generation of an immune competent repertoire is limited. The unique combination of primary immunodeficiencies (PID) and DNA repair disorders as disease model and recent developments in next generation sequencing in this project enables us for the first time to study the exact role of DNA repair in the generation of an immune competent repertoire, which will elucidate new mechanistic principles. In this proposal, we will use our extensive experience in immunoglobulin (Ig) gene analysis to develop next generation (deep) sequencing into an advanced tool for Ig repertoire analysis. For analysis of the massive sequence datasets novel software applications will be developed for interpretation of sequences, pattern recognition, comparisons between patients and controls, and graphical visualization of the results.The immune repertoire of healthy children at different ages and PID patients with known genetic defects will subsequently be analyzed to determine the requirement of an immune competent repertoire and the influence of DNA repair factors. Samples of controls and PID patients are already available in our laboratory, because of our position as PID reference center. In vitro studies will be performed to study the exact role of selected DNA repair factors on immune repertoire formation. Finally, immune repertoire analysis will contribute to the identification of new genetic defects in clinically and immunologically well-defined PID patients. In this project expertise from different fields (immunology, bioinformatics, molecular biology and genomics) will be merged to achieve advances in this field.We anticipate this project will contribute to early recognition of immunological problems in patients with recurrent infections and to identification of new genetic defects in PID. In addition, new insights in immune repertoire development will have impact not only on PID, but also on other immunological disorders with suspected disturbances in the immune repertoire e.g. autoimmune diseases and allergy.

Research lines of Workgroup Primary ImmunodeficienicesThe Workgroup Primary Immunodeficiencies (PID) of the Dept. of Immunology, Erasmus MC focuses on:

1. V(D)J recombination and its defects in severe combined immunodeficiencies2. Pathophysiological processes in primary antibody deficiencies

https://www.erasmusmc.nl/immu/research/pid



3. Immune repertoire analysis in primary immunodeficiencies

The PID group is a national and internal reference center for agammaglobulinemia and severe combined immunodeficiency (SCID). The field of research of the PID workgroup is normal (precursor) B-cell differentiation and defective precursor B-cell differentiation resulting in antibody deficiencies as well as V(D)J recombination and its regulation, which is affected in patients with T-B- (SCID). The project on antibody deficiencies is embedded in a longstanding collaboration with the Dept. of Pediatrics and Dept. of Internal Medicine of the Erasmus MC and several international research centers.

Publications related to the project

1. Wentink M, Dalm V, Lankester AC, van Schouwenburg PA, Scholvinck L, Kalina T, Zachova R, Sediva A, Lambeck A,

Pico-Knijnenburg I, van Dongen JJ, Pac M, Bernatowska E, van Hagen M, Driessen G, van der Burg M. Genetic

defects in PI3Kdelta affect B-cell differentiation and maturation leading to hypogammaglobulineamia and recurrent

infections.Clin Immunol. 2017;176:77-86.

2. IJpeert H, van Schouwenburg PA, van Zessen D, Pico-Knijnenburg I, Stubbs AP, van der Burg M.

Antigen Receptor Galaxy: A User-Friendly, Web-Based Tool for Analysis and Visualization of T and B Cell Receptor

Repertoire Data.J Immunol 2017; 198:4156-65.

3. IJspeert H, Rozmus J, Schwarz K, Warren RL, van Zessen D, Holt RA, Pico-Knijnenburg I, Simons E, Jerchel I,

Wawer A, Lorenz M, Patiroglu T, Akar HH, Leite R, Verkaik NS, Stubbs AP, van Gent DC, van Dongen JJ, van der

Burg M. XLF deficiency results in reduced N-nucleotide addition during V(D)J recombination.

Blood. 2016;128:650-659.

4. Murray JE,# van der Burg M,# IJspeert H, Carroll P, Wu Q, Ochi T, Leitch A, Miller ES, Kysela B, Jawad A, Bottani A,

Brancati F, Cappa M, Cormier-Daire V, Deshpande C, Faqeih EA, Graham GE, Ranza E, Blundell TL, Jackson AP,

Stewart GS, Bicknell LS. Mutations in the NHEJ component XRCC4 cause primordial dwarfism.

Am J Hum Genet. 2015;96:412-424.

5. IJspeert H, Driessen GJ, Moorhouse MJ, Hartwig NG, Wolska-Kusnierz B, Kalwak K, Pituch-Noworolska A,

Kondratenko I, van Montfrans JM, Mejstrikova E, Lankester AC, Langerak AW, van Gent DC, Stubbs AP, van Dongen

JJ, van der Burg M. Similar recombination-activating gene (RAG) mutations result in similar immunobiological effects

but in different clinical phenotypes. J Allergy Clin Immunol. 2014;133:1124-1133 e1121.

6. Genovese P, Schiroli G, Escobar G, Di Tomaso T, Firrito C, Calabria A, Moi D, Mazzieri R, Bonini C, Holmes MC,

Gregory PD, van der Burg M, Gentner B, Montini E, Lombardo A, Naldini L. Targeted genome editing in human

repopulating haematopoietic stem cells. Nature. 2014;510:235-240.

7. Driessen GJ, Ijspeert H, Weemaes CM, Haraldsson A, Trip M, Warris A, van der Flier M, Wulffraat N, Verhagen MM,

Taylor MA, van Zelm MC, van Dongen JJ, van Deuren M, van der Burg M.

Antibody deficiency in patients with ataxia telangiectasia is caused by disturbed B- and T-cell homeostasis and

reduced immune repertoire diversity. J Allergy Clin Immunol. 2013;131:1367-1375 e1369.

8. Driessen GJ, van Zelm MC, van Hagen PM, Hartwig NG, Trip M, Warris A, de Vries E, Barendregt BH, Pico I, Hop W,

van Dongen JJ, van der Burg M. B-cell replication history and somatic hypermutation status identify distinct

pathophysiologic backgrounds in common variable immunodeficiency. Blood. 2011;118:6814-6823.

9. van Zelm MC, Smet J, Adams B, Mascart F, Schandene L, Janssen F, Ferster A, Kuo CC, Levy S, van Dongen JJ,

van der Burg M. CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency. J

Clin Invest. 2010;120:1265-1274.

10. van der Burg M, Ijspeert H, Verkaik NS, Turul T, Wiegant WW, Morotomi-Yano K, Mari PO, Tezcan I, Chen DJ,

Zdzienicka MZ, van Dongen JJ, van Gent DC. A DNA-PKcs mutation in a radiosensitive T-B- SCID patient inhibits

Artemis activation and nonhomologous end-joining. J Clin Invest. 2009;119:91-98.

11. van Zelm MC, Reisli I, Van der Burg M, Castano D, van Noesel CJ, van Tol MJ, Woellner C, Grimbacher B, Patino PJ,

van Dongen JJM, Franco JL. An antibody-deficiency syndrome due to mutations in the CD19 gene.

N Engl J Med. 2006;354:1901-1912.


Title: Dysregulated myelomonocytic development in inflammatory and metabolic diseaseWorkgroup Myelomonocytic Cells in Inflammation

Workgroupleader: dr. P.J.M. Leenen, workgroup leader Myelomonocytic Cells in InflammationT: 010-704 3171E: [email protected] W: http://www.erasmusmc.nl/immu/research/5302240/aip


6 months Yes 1-1-2018



BackgroundCentral theme in our research group is the developmental biology of myelomonocytic cells in steady state as well as in diseases where they are aberrant and associated with pathological conditions (Fig. 1). Myelomonocytic cells of interest comprise macrophages and dendritic cells in their various forms as well as granulocytes and their precursors in hematopoietic organs. Despite their extensive functional and phenotypic heterogeneity, these cells are related via common developmental pathways as well as shared functions and regulatory mechanisms.Significant progress in the past decade has indicated that tuning of the activation stage of cells of the myelomonocytic lineage is crucial not only for the appropriate function of host defense systems in infection, but for organ and tissue homeostasis in general. In line with this notion, aberrant function of these cells has been shown in a wide variety of diseases, including metabolic and autoimmune diseases such as type 1 and type 2 diabetes, atherosclerosis, obesity, cancer and psychiatric diseases. In particular the inflammatory nature of the myelomonocytic cells in these conditions underlies many of the pathological signs and symptoms. Such changes affect functionality of the cells in many aspects, including their capacity to function as effector cells in infection, and to interact with the vascular wall.

Research focusOur research projects are centered around the working hypothesis that inflammatory changes in monocytes and granulocytes in patients induced by environmental conditions are (i) indicative of the current clinical status of the patient, and (ii) may predict responses to challenges such as infections. Our current focus is on two specific projects: 1. The influence of lipoprotein Lp(a) on the functional interaction between monocytes and endothelial cells in formation and repair of blood vessels in diabetic retinopathy.2. The inflammatory response of myelomonocytic cells after surgery, and the relationship with subsequent clinical complications, such as post-operative infections.

bone marrow blood periphery

DC

M

LCT

B

Fig. 1. Central research theme: developmental biology of myelomonocytic cells and interaction with adaptive immune cells in steady state and pathological conditions.

http://www.erasmusmc.nl/immu/research/5302240/aip



In these projects, we collaborate with various local, national and international research groups and clinical partners.

Selected publicationsMourik BC, Lubberts E, de Steenwinkel JEM, Ottenhoff THM, Leenen PJM. 2017. Interactions between type 1 interferons and the Th17 response in tuberculosis: lessons learned from autoimmune diseases. Front Immunol 8:294. PMID: 28424682

Ozcan B, Leenen PJM, Delhanty PJD, Baldeon-Rojas LY, Neggers SJ, van der Lely AJ. 2017. Unacylated ghrelin modulates circulating angiogenic cell number in insulin-resistant states. Diabetol Metab Syndr 9:43. PMID: 28572856

Baldeon Rojas L, Weigelt K, de Wit H, Ozcan B, van Oudenaren A, Sempertegui F, Sijbrands E, Grosse L, van Zonneveld AJ, Drexhage HA, Leenen PJ. 2016. Study on inflammation-related genes and microRNAs, with special emphasis on the vascular repair factor HGF and miR-574-3p, in monocytes and serum of patients with T2D. Diabetol Metab Syndr 8:6. PMID: 26779287

Baldeon RL, Weigelt K, de Wit H, Ozcan B, van Oudenaren A, Sempertegui F, Sijbrands E, Grosse L, van Zonneveld AJ, Drexhage HA, Leenen PJ. 2015. Type 2 diabetes monocyte microRNA and mRNA expression: dyslipidemia associates with increased differentiation-related genes but not inflammatory activation. PLoS One 10:e0129421. PMID: 26083362

Baldeon RL, Weigelt K, de Wit H, Ozcan B, van Oudenaren A, Sempertegui F, Sijbrands E, Grosse L, Freire W, Drexhage HA, Leenen PJ. 2014. Decreased serum level of miR-146a as sign of chronic inflammation in type 2 diabetic patients. PLoS One 9:e115209. PMID: 25500583

Riepsaame J, van Oudenaren A, den Broeder BJ, van IJcken WF, Pothof J, Leenen PJM. 2013. MicroRNA-mediated down-regulation of M-CSF receptor contributes to maturation of mouse monocyte-derived dendritic cells. Front Immunol 4:353. PMID: 24198819

Drevets DA, Schawang JE, Mandava VK, Dillon MJ, Leenen PJ. 2010. Severe Listeria monocytogenes infection induces development of monocytes with distinct phenotypic and functional features. J Immunol 185:2432-41. PMID: 20631315


Title: Interferon activation in primary Sjögren’s Syndrome and other systemic auto-immune diseases: unravelling a pathogenic pathway

Workgroupleader:Dr. M.A. Versnel, workgroupleader Autoimmune Diseases IIT: 010-7048086E: m.versnel@ erasmusmc.nl W: http://www.erasmusmc.nl/immu/research/aim/aim.n


6 months Yes February 2018



BackgroundSjögren’s Syndrome (SjS) is an autoimmune disease characterized by focal lymphocytic infiltrations in the salivary and lachrymal glands. Patients mainly suffer from dry mouth (xerostomia) and dry eyes (keratoconjunctivitis sicca), while multiple extraglandular manifestations such as severe fatigue, myalgia and arthralgia are frequently present and form a major cause of morbidity. Diagnosis of SjS is difficult due to lack of diseases specific markers and the heterogeneity of the disease.Dendritic cells (DC) play an important role in the initiation of pSjS (van Blokland et al., 2000). Monocytes migrate through the blood to the peripheral tissues, where they can develop into dendritic cells (DC). Using microarray analysis we detected an Interferon (IFN) Type I induced gene expression signature in monocytes of pSjS patients (Wildenberg et al., 2008). The presence of the IFN type I signature in a subgroup of patients points towards a pathogenic pathway contributing to the development of pSjS. We have shown that systemic IFN activation is present in 57% of pSS patients and that the IFN activation is related to disease activity in pSS (Brkic et al., 2013). IFN activation is also present in other systemic autoimmune diseases like systemic lupus erythematosus (SLE). Childhood onset SLE (cSLE) is incurable and more severe than adult onset SLE leading to increased damage and higher mortality. Medication for SLE is by far not effective enough and leaves children with significant side effects, especially due to prednisone. Treatment choices are mainly made on clinical grounds, as current biomarkers cannot predict disease severity, response to therapy or disease flares. Recent research indicates towards a dysbalance of pattern recognition receptors like TLR-7 and 9 and the RIG-like receptors in patients with an activated IFN type I system (Maria et AL., 2016). Unravelling these pathways is of relevance for development of therapeutics that can treat systemic autoimmune diseases.

ResearchThe overall aim of this research is to determine the prevalence of systemic interferon activation, to relate the presence of this activation to clinical symptoms and response to treatment and finally to therapeutically target IFN activation in patients with systemic autoimmune diseases like Sjögren’s Syndrome and SLE.This aim will be approached by the following specific questions:

1. Is the presence of IFN activation in children with SLE related to the clinical manifestation of the disease and is treatment affecting IFN activation?

http://www.erasmusmc.nl/immu/research/aim/aim.n



2. Can IFN activation be used to monitor treatment?3. What is the mechanism leading to systemic IFN activation in autoimmunity?4. What is the role of pattern recognition receptors in the overactivation of the IFN type I system in systemic autoimmune diseases?5. Are there signaling hubs in the IFN type I pathway that can be used to inhibit IFN type I activation?6. Is increased expression of IDO a mechanism trying to induce tolerance in systemic autoimmunity or is it detrimental?

To approach these questions the following techniques will be used: Isolation of monocytes from peripheral blood, RNA isolation, quantitative PCR, cell culture, Western blotting, immunohistochemistry. Highly motivated students are encouraged to contact us and will get depending on their interest a specific research topic.The research is carried out in close collaboration with the Clinical immunology group of the Department of Internal Medicine and the Departments of Rheumatology and Pediatrics. There is also a larger network in national and international research collaborations (Ireland, Sweden, United Kingdom, France, Italy). Dutch speaking students who follow the medical educational track and the Master I&I can get the opportunity to participate in the collection of patient material at the Clinical Immunology outpatient clinic.

Selected publications related to the project

Maria NI, Steenwijk EC, IJpma AS, van Helden-Meeuwsen CG, Vogelsang P, Beumer W, Brkic Z, van Daele PL, van Hagen PM, van der Spek PJ, Drexhage HA, Versnel MA. Contrasting expression pattern of RNA-sensing receptors TLR7, RIG-I and MDA5 in interferon-positive and interferon-negative patients with primary Sjögren’s syndrome. in press, Annals Rheum Dis 2016 Sep 26. doi: 10.1136/annrheumdis-2016-209589.

Maria NI, van Helden-Meeuwsen CG, Brkic Z, Paulissen SM, Steenwijk EC, Dalm VA, van Daele PL, van Hagen PM, Kroese FG, van Roon JA, Harkin A, Dik WA, Drexhage HA, Lubberts E, Versnel MA. Association of increased Tregs with elevated Indoleamine-2,3-dioxygenase activity and an imbalanced Kynurenine pathway in IFNpositive primary Sjögren's syndrome. Arthritis Rheumatol. 2016 Feb 11. doi: 10.1002/art.39629. [Epub ahead of print]

Brkic* Z, van Bon* L, Cossu M, van Helden-Meeuwsen CG, Vonk MC, Knaapen H, van den Berg W, Dalm VA, Van Daele PL, Severino A, Maria NI, Guillen S, Dik WA, Beretta L, Versnel MA** and Radstake T**. The Interferon type I signature is present in Systemic sclerosis before overt fibrosis and might contribute to its pathogenesis through high BAFF gene expression and high collagen synthesis. In press, Ann Rheum Dis. 2016 Aug;75(8):1567-73. doi: 10.1136/annrheumdis-2015-207392. Epub 2015 Sep 14. *,**

authors contributed equally.

Maria NI, Vogelsang P, Versnel MA. The clinical relevance of animal models in Sjögren’s syndrome: the IFN signature from Mouse to Man. Arthritis Research and Therapy 2015, 17 (3):172.

Brkic Z, Corneth OB, Versnel MA, Lubberts E. Response to ‘T-helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus?’ – Authors’ reply. Arthritis Res Ther. 2014;16(3):410.


Brkic, Corneth OB, van Helden-Meeuwsen CG, Dolhain RJ, Maria NI, Paulissen SM, Davelaar N, van Hamburg JP, van Daele PL, Dalm VA, van Hagen PM, Hazes JM, Versnel MA, Lubberts E. T helper 17 cell cytokines and interferon type I: partners in crime in systemic lupus erythematosus? Arthritis Res Ther. 2014 Mar 6;16(2):R62.

Brkic Z, Versnel MA. Type I IFN signature in primary Sjögren’s syndrome. Expert Rev Clin Immunol. 2014 Apr;10(4):457-67. Epub 2014 Jan22.


Title: The role of post-transcriptional regulators in the development of effective cytotoxic T cell responses.

Group leader: Prof. dr. P.D. KatsikisT: 010-704 4188E: [email protected]: http://www.erasmusmc.nl/immu/research/ii/iig.n?lang=en

Duration Yes/No Available per (date)6 months Yes -12 months Yes -18 months Yes -

BackgroundCytotoxic CD8+ T lymphocytes (CTL) are a crucial component of the adaptive immune system’s protection against infectious diseases and cancer. CTL specifically play a key role in the successful elimination of acute viral and intracellular bacterial infections and tumors through the direct killing of infected cells. Similarly, CTL can aid in the control of chronic diseases such as chronic viral infections or cancer. However, these same cells often fail to completely control or clear the virus, such as in HIV-1 infection, or cancerous tumor, such as in patients with metastatic melanoma. This failure of CTL remains a complex and vexing obstacle for the development of effective therapeutics. Our research is focused on understanding the cellular and molecular mechanisms that differ between “successful” versus “failed” antigen-specific CTL, and the identification of molecular regulators that can be systematically targeted to enhance CTL function.

It has been established that intrinsic signaling molecules such as post-transcriptional and transcriptional regulators of gene expression have shown powerful control over the development of effective immune responses, including CTL. Post-transcriptional gene expression is tightly regulated by unique classes of RNA and proteins. Three classes in particular have become a major focus of genome regulation: microRNA (miRNA), long noncoding RNA (lncRNA), and RNA-binding proteins (RBP). Previous work within our group has established that modulation of a single microRNA in CTL can exert a profound impact on CTL-immunity. Ongoing studies aim to establish the cellular and molecular mechanisms that are unique to failed CTL, with a goal of identifying the post-transcriptional regulators responsible for these changes.

Lines of InvestigationThe main aim of this study is to identify role of transcriptional and post-transcriptional regulators and their associated cellular and molecular mechanisms and pathways responsible for CTL failure. The main lines of current investigation are:

1. Identification of differentially expressed post-transcriptional regulators in successful versus failed CTL.

a. Which are the most highly upregulated/downregulated?b. Which post-transcriptional regulators are most essential to an effective

immune response?c. Can overexpression or knockdown of these regulators enhance immunity?

2. Understanding the mechanisms by which deficiency or overexpression of a specific transcriptional regulator promotes protection against influenza viral infection.

a. What are the specific immune cells that are enhanced?b. What cellular/molecular mechanisms are directly affected?

Principal TechniquesAssays and techniques commonly performed for these studies include: multi-color flow cytometry, cell sorting, proliferation assays, DNA/RNA isolation, quantitative PCR, Western blotting, RNA sequencing, cell culture, plasmid cloning, cell transfection and transduction, and genotyping/phenotyping of mouse lines. We perform experiments using both human patient

http://www.erasmusmc.nl/immu/research/ii/iig.n?lang=en



material and mouse models of acute viral infection (Influenza), acute intracellular bacterial infection (Listeria monocytogenes) and a mouse tumor model.

Training OptionsHighly motivated students are welcome to contact us regarding possible internships within our group. Individual projects will be discussed between the student, group leader, and mentor to identify interesting projects that fall under one of the ongoing projects within the group that can be successfully completed in a timely fashion.

Selected PublicationsStelekati E, Shin H, Doering TA, Dolfi DV, Zeigler CG, Beiting D, Liboon J, Wolski D, Katsikis PD, Shen H, Roos DS, W. Haining WN, Lauer G, Wherry EJ. Bystander Chronic Infection Negatively Impacts Development of CD8 T Cell Memory. Immunity. 40:801-13, 2014.

Fraietta JA, Mueller YM, Lozenski KL, Ratner D, Boesteanu AC, Hancock AS, Lackman-Smith C, Zentner IJ, Chaiken IM, Chung S, LeGrice SF, Snyder BA, Mankowski MK, Jones NM, Hope JL, Gupta P, Anderson SH, Wigdahl B, Katsikis PD. Abasic phosphorothioate oligomers inhibit HIV-1 reverse transcription and block virus transmission across polarized ectocervical organ cultures. Antimicrob. Agents Chemother. 58:7056-71, 2014.

Fraietta JA, Mueller YM, Yang G, Boesteanu AC, Gracias DT, Do DH, Hope JL, Kathuria N, McGettigan SE, Lewis MG, Giavedoni LD, Jacobson JM, Katsikis PD. Type I interferon upregulates Bak and contributes to T cell loss during human immunodeficiency virus (HIV) infection. PLoS Pathogens. 9:e1003658, 2013.

Gracias DT, Stelekati E, Hope JL, Boesteanu AC, Doering TA, Norton J, Mueller YM, Fraietta JA, Wherry EJ, Turner M, Katsikis PD.The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling. Nat Immunol. 14:593-602, 2013.

Turner M, Katsikis PD. A new mechanism of gene regulation mediated by noncoding RNA. J Immunol. 189:3-4, 2012.

Dolfi DV, Duttagupta PA, Boesteanu AC, Mueller YM, Oliai C, Borowski AB, Katsikis PD. Dendritic cells and CD28 costimulation are required to sustain virus-specific CD8+ T cell responses during the effector phase in vivo. J. Immunol. 186:4599–4608, 2011.

Borowski AB, Boesteanu AC, Mueller YM, Carafides C, Altman JD, Jennings SR, Katsikis PD. Memory CD8+ T cells require CD28 costimulation. J. Immunol. 179:6494-6503, 2007.

Mueller YM, Petrovas C, Bojczuk PM, Dimitriou ID, Beer B, Silvera P, Villinger F, Cairns JS, Gracely EJ, Lewis MG, Katsikis PD.Interleukin-15 increases effector memory CD8+ t cells and NK Cells in simian immunodeficiency virus-infected macaques. J Virol. 79:4877-85, 2005.

Mueller YM, Bojczuk PM, Halstead ES, Kim AH, Witek J, Altman JD, Katsikis PD. IL-15 enhances survival and function of HIV-specific CD8+ T cells. Blood. 101:1024-9, 2003.

Halstead ES, Mueller YM, Altman JD, Katsikis PD. In vivo stimulation of CD137 broadens primary antiviral CD8+ T cell responses.Nat Immunol. 3:536-41, 2002.

Mueller YM, De Rosa SC, Hutton JA, Witek J, Roederer M, Altman JD, Katsikis PD. Increased CD95/Fas-induced apoptosis of HIV-specific CD8(+) T cells. Immunity. 15:871-82, 2001.


Title: In Vitro Characterization and Enhancement of HIV-Specific CD8+ T Cells

Group leader: Prof. dr. P.D. Katsikis, dr. Y.M. Müller T: 010-70 44188/70 43796E: [email protected] [email protected]: http://www.erasmusmc.nl/immunologie/


6 months Yes12 months Yes18 months Yes

BackgroundAntigen-specific cytotoxic CD8+ T cell responses are considered to be major players in the protective immune response against acute and chronic viral infections. In infections with the human immunodeficiency virus (HIV) the key role of CD8+ T cells was indicated by several studies showing that CD8+ cytotoxic T lymphocytes (CTL) are activated in vivo during the initial clearance of the primary HIV infection, are present during the asymptomatic phase of the disease, HIV-specific CTL precursors are lost during progression to AIDS and an inverse correlation between the frequency of HIV-specific CTLs and plasma RNA viral load. Although the immune response in HIV infected individuals can control HIV replication for several years, ultimately, HIV-specific CD8+ T cells fail to clear or control HIV infection and AIDS develops. When HIV-specific CD8+ T cells were compared with other virus-specific CD8+ T cells in the same patients, HIV-specific CD8+ T cells show a skewed memory phenotype, reduced cytokine release and increases apoptosis sensitivity, leading to impaired effector functions and therefore failure to eradicate or control long-term HIV. These features of HIV-specific CD8+ T cells raises the question whether HIV-specific CD8+ T cells become exhausted. Exhaustion of chronically antigen-stimulated T cells was first described in a mouse model of chronic viral infection and is characterized by loss of proliferation, cytokine production and cytotoxicity, characteristics similar to what is observed in HIV-specific CD8+ T cells. Furthermore, recent studies indicated that upregulation of inhibitory receptors like PD-1, LAG-3, CD244 (2B4), CD160, TIM-3 and CTLA-4 are contributing to the functional loss of virus-specific CD8+ T cells in chronic viral infections. Our research focuses on the question how gene activity regulation alters exhaustion of virus-specific CD8+ T cells. Two principal mechanisms which regulate gene activity are studied in our group: epigenetic modification and post-transcriptional regulations. Epigenetic modification leads to chances in chromatin structure through methylation of DNA and modification of histones. Post-transcriptional regulation plays an important role in controlling the activity of genes. So far, microRNA (miRNA, 19 – 23 nucleotide long non-coding RNA molecules), long noncoding RNA (lncRNA, longer than 100 nucleotides) and RNA-binding proteins (RBP) have been indicated as altering gene expression in a network by mechanism like silencing translation, destabilizing mRNA, preventing degradation and modulating localization. Since restoring and enhancing the functions of HIV-specific CD8+ T cells could have a profound effect on HIV infection, understanding whether it can be reversed through manipulation of epigenetic modification and post-transcriptional regulators would further intervention therapy in HIV infection.

Research focus in the IIG-HIV group at the Department of Immunology

http://www.erasmusmc.nl/immunologie/


Research topics: 1. Examining whether altering epigenetic modification can reduce exhaustion of HIV-specific CD8+ T cells leading to improved effector functions of these cells.We hypothesize that epigenetic modifications control the expression of the exhaustion phenotype of HIV-specific CD8+ T cells. We will test this using epigenetic pathway inhibitor molecules to reverse epigenetic modifications and examine if exhaustion of HIV-specific CD8+ T cells can be reversed.

2. Understanding the role post-transcriptional regulation play in the function and dysfunction of HIV-specific CD8+ T cells Another mode of gene expression control are post-transcriptional regulators like miRNA, lncRNA and RBP. We hypothesize that post-transcriptional regulators are differentially expressed in virus-specific CD8+ T cells which control infections (CMV-specific CD8+ T cells) compared to those that fail to control viruses (HIV-specific CD8+ T cells) in chronic viral infection. To test this we will measure key post-transcriptional regulators in HIV- and CMV-specific CD8+ T cells that we have identified to play a role in virus-specific CD8+ T cell responses in murine models.

Publications related to these research topics

Fraietta JA, Mueller YM, Yang G, Boesteanu AC, Gracias DT, Do HD, Hope JL, Kathuria N, McGettigan SE, Lewis MG, Giavedoni LD, Jacobson JM, Katsikis PD. 2013. Type I interferon upregulates Bak and contributes to T cell loss during human immunodeficiency virus (HIV) infection. PLoS Pathog 9(10): e1003658.

Gracias DT, Stelekati E, Hope JL, Boesteanu AC, Fraietta JA, Doering T, Norton J, Mueller YM, Wherry EJ, Turner M, Katsikis PD. 2013. MicroRNA-155 controls CD8+ T cell responses by regulating interferon signaling. Nature Immunology. 14:593-602.

Petrovas C, Chaon B, Ambrozak DR, Price DA, Melenhorst JJ, Hill BJ, Geldmacher C, Casazza JP, Chattopadhyay PK, Roederer M, Douek DC, Mueller YM, Jacobson JM, Kulkarni V, Felber BK, Pavlakis GN, Katsikis PD, Koup RA. 2009. Differential association of programmed death-1 and CD57 with ex vivo survival of CD8+ T cells in HIV infection.

Mueller YM, Petrovas C, Do DH, Altork SR, Fischer-Smith T, Rappaport J, Altman JD, Lewis MG and Katsikis PD. 2007. Early establishment and antigen dependence of SIV-specific CD8+ T cell defects. J Virol 81:10861-8

Mueller YM, De Rosa S, Hutton JA, Witek J, Roederer M, Altman JD, Katsikis PD. 2001. Increased CD95/Fas induced apoptosis of HIV-specific CD8+ T cells. Immunity 15: 871-872


Research Line: Investigating the functions of miRNAs in normal and malignant hematopoiesis

Workgroup leader: Dr. Stefan Erkeland; Department: Immunology T: 010-7043796E: [email protected]

Duration Yes/No3-6 months Yes9-12 months Yes18 months Yes

Background:MicroRNAs (miRNAs) are small non-protein coding RNA molecules (~21 nucleotides) and are key players in normal and malignant hematopoiesis by regulation of gene expression (Figure 1) 1,2. Hundreds of miRNAs exist and many of them are evolutionary conserved between species. MiRNA expression is dependent on the cell type and developmental stages. Deletion of miRNAs in hematopoietic stem cells causes complete loss of blood cell development 1,3. Also, deletion at a later stage of hematopoietic cell development causes apoptosis, loss of specific blood cell linages and malfunction of hematopoietic cells, indicating their importance for blood cell development and function 1. MiRNAs are frequently deregulated in leukemia 4. Recent studies have shown that miRNA miR-155 is an oncogenic miRNA (oncomiR). For instance, Eµ/VH-miR-155-transgenic mice, which express miR-155 from the immunoglobulin heavy chain promoter, develop acute lymphoblastic leukemia (ALL) 5. We found that miR-155 induces acute myeloid leukemia in combination with the loss of transcription factor C/EBPA, a critical factor for myeloid cell development which is frequently mutated in leukemia patients (submitted for publication). In a different study, in which we investigated the role for miRNAs in human bone marrow failure, we identified miRNA-199a-3p and miR-139-3p playing critical roles in the balance between the loss of hematopietic stem and progenitors and acute leukemia development under DNA-damage stress conditions (Figure 1B)6. However, most functions of miRNAs in normal hematopoiesis and leukemia development are still largely unknown.Research and technology:To study the functions of miRNAs, we use CRISPR-CAS9-mediated genome editing. We recently generated several miRNA knockout cell lines and mouse models. For miRNA downstream pathway investigation we use cutting-edge technology such as next generation sequencing, proteomics and miRNA-target complex immunoprecipitation. Furthermore, we recently developed retroviral miRNA expression and inhibition libraries for functional screens in hematopoietic cells. We use diverse functional hematopoietic assays to investigate the functions of miRNAs in proliferation, differentiation, cell death and leukemic transformation in vitro and in mice.

If you are interested in fundamental research and want to learn how to investigate what these tiny non-coding RNA species do in hematopoietic cell development and what

their role is in leukemia, please contact me by email to make an appointment to discuss a project. A) B)



Figure 1A) microRNA biogenesis and functions (He L and Hannon GJ, Nat Rev Genet, 2004). B) Micrograph showing the morphology of Cebpa-deficient and miR-199a–overexpressing leukemic blasts in the mouse spleen. See reference 6 for more information.

References1. Alemdehy MF, Erkeland SJ. Stop the dicing in hematopoiesis: what have we learned? Cell Cycle. 2012;11:2799-2807.2. Alemdehy MF, Erkeland SJ. MicroRNAs: key players of normal and malignant myelopoiesis. Curr Opin Hematol. 2012;19:261-267.3. Guo S, Lu J, Schlanger R, et al. MicroRNA miR-125a controls hematopoietic stem cell number. Proc Natl Acad Sci U S A. 2010;107:14229-14234.4. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature. 2005;435:834-838.5. Costinean S, Zanesi N, Pekarsky Y, et al. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci U S A. 2006;103:7024-7029.6. Alemdehy MF, Haanstra JR, de Looper HW, et al. Interstrand cross-link induced miR139-3p and miR199a-3p have opposite roles in hematopoietic cell expansion and leukemic transformation. Blood. 2015.


RHEUMATOLOGY – RESEARCH TOPICS


Title: The role and modulation of the IL-23-IL-17/Th17 immune pathway in inflammatory arthritis and psoriasis

Workgroupleader:Dr. E. Lubberts, workgroup leader Rheumatology Research Lab.Tel. 010-7044309e-mail: [email protected]


6 months Yes12 months Yes18 months Yes

DepartmentThe research of the Department of Rheumatology is focused on early diagnosis of RA and to understand the immunological mechanism(s) critical in the development of chronic destructive arthritis (RA).

BackgroundRheumatoid arthritis (RA) and spondyloarthritis (SpA) are the two most common forms of chronic immune-mediated inflammatory arthritis, and the IL-23–IL-17 axis is thought to have a critical role in both. The discovery of the IL-23–IL-17 immune pathway has boosted our knowledge regarding the immunological mechanisms of many chronic inflammatory diseases including inflammatory arthritis and psoriasis. However, many questions remain and further clarification is needed to fully understand the contribution of this important pathway, including potentially distinct roles of IL-17 and IL-23, and where, when and how this pathway influences the pathogenesis of these inflammation-driven tissue destructive diseases. In addition to targeting TNF activity, novel therapeutic opportunities are available through modulation of the IL-23–IL-17 axis (Lubberts, E. The IL-23-IL-17 axis in inflammatory arthritis. Nat Rev Rheumatology, 11, 415-429 (2015)).

ResearchThe Rheumatology Research lab focuses on four interrelated subjects (see below under research topics) and we are using human material from different patient cohorts: RA, Psoriatic arthritis (PsA), psoriasis, the REACH (Rotterdam Early Arthritis Cohort) and the PARA (Pregnancy Amelioration of Rheumatoid Arthritis) as well as various (humanized) mouse models for RA, PsA, psoriasis and SLE to study these subjects.

The following techniques can be learned:Multi-colour Flow cytometry, FACS-sorting, Q-PCR, ELISA, multi-bead arrays, cell cultures including co cultures assays, genomics/micro-array/transcription profiling, micro-RNA, shRNA, techniques for art9.

Together with the student we will define a project that is in line with ongoing projects in the group.

Research topics

1. The role and modulation of lymphocytes in the development of chronic inflammatory autoimmune-mediated arthropathies (e.g. RA, SLE, psoriatic arthritis, psoriasis) with focus on the IL-23/Th17 immune pathway.

Th17 cells are implicated in human autoimmune diseases, such as rheumatoid arthritis (RA), psoriatic arthritis (PsA), psoriasis and SLE, although it has not been established whether these diseases are driven by Th1 and/or Th17 cells. In this research line we are studying the


role of the IL-23/IL-17 immune pathway in both mouse and human arthritis and are investigating the molecular pathways involved in driving this pathway and the activity of the (memory) IL-17+/Th17 cells.

A major challenge in medicine is how to control pathogenic Th17 cell activity in human autoimmune-mediated diseases. The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been shown to completely suppress the autoimmune collagen-induced arthritis and to affect Th17 cytokine expression and function. In this research line the molecular mechanism of vitamin D as a modulator of Th17 cells is investigated.

2. The role of T cell cytokines/receptors/signaling pathways in the development of chronic inflammatory autoimmune-mediated arthropathies (e.g. RA, SLE, psoriatic arthritis, psoriasis).

The focus is on IL-23R and IL-17R signaling pathways using human cells including RNA-seq and shRNA modulation. In addition, unique transgenic mouse models will be used including cell type specific receptor deficient mice to study the role of IL-17 and IL-23 signaling in specific target cells in the development and progression of autoimmunity.

3. The identification of the immunological profile including plasticity and biomarkers during the development of RA and the inflammatory OA process and the effect of therapy to guide early diagnosis and treatment.

The discovery of the Th17 cell has resulted in a revision of the role of T cells in human diseases. The Th17 population is a heterogeneous population. Further identification of the Th17 subpopulations will be analyzed both at the molecular and functional level.

4. Osteo-immunology: interaction between the adaptive immune system, bone biology, aging, and hormones.

Osteoimmunology is an interdisciplinary research field focusing on the molecular understanding of the interplay between the immune and the skeletal systems. In addition to the immune regulation of osteoclasts, in this research line the molecular interactions between osteoclasts and/or osteoblasts in relation to lymphocytes and/or lymphocyte-derived cytokines are examined. In addition, the influence of aging and hormones are investigated.


Title: T- and B-cell mechanisms underlying brain autoimmunity NeuroImmunology Brain workgroup, Rotterdam MS Center ErasMS

Project leaders:Prof.dr. R.Q. Hintzen and Dr. M.M. van LuijnE: [email protected] and [email protected]: 010-7033780 and 010-7043190W: http://www.erasmusmc.nl/immu/research/nib/


6 months Yes, but preferably 7 or more -

12 months Yes -

18 months Yes -

BackgroundMultiple sclerosis (MS) is the most common neurological disorder affecting young adults. The disease is largely characterized by demyelination and eventually by neuronal loss leading to neurological disability. MS disease course is highly variable with the relapsing remitting form being the most frequent in which episodes of disease are followed by recovery. This phase is often followed by the progressive form characterized by severe paralytic disability. The other disease of interest is neuromyelitis optica (NMO), a proven autoimmune disease against aquaporine-4, again leading to demyelination.The most pivotal players in these diseases are autoaggressive B and T cells in conjunction with genetic traits and environmental factors. Current genetic studies identified approximately 200 new risk loci in MS, revealing a strong overlap with other types of autoimmune diseases and support the key role of B and T cells in MS immunopathogenesis. It is implicated that such genetic factors interact with environmental risk factors in MS, e.g. infection with the Epstein Barr virus (EBV), to trigger disease development. Recent B-cell depletion therapy indicates that T-cell activation and not antibody production probably underlies the pathogenic role of B cells in MS.

ResearchThe work of the Neuroimmunology Brain group aims to 1. understand the pathogenicity of B- and T-cell subsets during brain inflammation and demyelination to improve therapy and 2. to use this knowledge to find better biomarkers for clinical management.

Our workgroup in the Dept. Immunology is supervised by Dr. Marvin van Luijn and Prof. Rogier Hintzen, who is head of MS Center ErasMS (Depts. Immunology and Neurology, Erasmus MC).The main research theme of ErasMS is “Biological determinants of the MS disease course: genetics, microbiology and immunology”. To perform our research, we use state of the art research technology such as quantitative and real-time PCR assays, DNA/RNA sequencing, multicolor flow cytometry, lentiviral RNA interference, confocal microscopy, immunohistochemistry and extensive primary cell cultures. We have a large collection of patient blood samples (often at the very early phase in the disease), which are still collected on a daily basis. For other studies, we have an internationally unique set of fresh and post-mortem MS brain tissue (cells) provided by the Netherlands Brain Bank.



Research lines

1. Targeted genome editing of MS risk loci implicated in B- and T-cell function in MS. Using human cell lines and primary cells from patients and controls, MS risk genotypes will be correlated to phenotype (mRNA levels, protein expression) and function (cell biology, signaling pathways). Candidate MS risk genes will be silenced using lentiviral shRNA constructs and risk SNPs located in protein-coding sequences will be edited using the CRISPR-Cas9 system (expertise by Dr. Stefan Erkeland, Dept. Immunology, Erasmus MC). Currently, we focus on MS risk genes linking to IFN-γ-mediated control of the HLA class II pathway in human antigen-presenting cells. HLA class II is the strongest risk factor in MS, but its regulation during the pathogenesis is poorly understood. This work is performed in collaboration with the group of Prof. Jacques Neefjes (LUMC, Leiden). We will perform similar CRISPR assays to edit MS risk genes linking to the pathogenicity of Th17 subsets (research line 2).

2. Internal and external factors controlling human Th17 cell differentiation during MS development. We recently found that IFN-γ-producing Th17 subpopulations are associated with rapid disease onset in patients with CIS, the earliest clinical presentation of MS . To unravel how these subpopulations are controlled during MS pathogenesis, both genetic and environmental factors (e.g. MS-modifying vitamin D, corticosteroids, sex steroids) will be related to human Th17 pathogenicity. Naive and memory T cells will be isolated from patients and controls and functionally explored under Th17-stimulating conditions using specific pathogens and cytokines. Thirteen-color flow cytometry , quantitative PCR and intracellular FACS and ELISA will be used as read-outs. Functional differences will be correlated to MS risk genotypes and clinical parameters such as time to conversion into clinically definite MS, disease severity (EDSS) and treatment responses. This work is carried out in collaboration with the group of Prof. Erik Lubberts (Dept. Rheumatology, Erasmus MC).

3. The role of the Epstein-Barr virus in local B-cell development and T-cell reactivity to MS autoantigens. We argue that EBV causes selective changes in B-cell development and contributes to T cell activation by mimicking or triggering the expression of MS autoantigens. Memory B-cell subsets from early MS and control blood will be flow cytometrically sorted to analyze differences in EBV DNA load, replication history, somatic hypermutations and autoreactivity using advanced molecular and cellular assays. In addition, primary T-cell lines from autopsy MS white matter are generated and co-cultured with autologous, HLA- and autoantigen-restricted EBV+ B-cell lines (Prof. Georges Verjans, Dept. Viroscience) to assess differences in reactivity. Our ongoing work also shows a prominent role of IFN-γ-induced T-bet in IgG subclass switching, probably enhancing B-cell reactivity to EBV and self antigens. This will be tested for differentiating B cells from patients and controls using an IL-21- and CD40L-based culture system (Dr. Marieke van Ham, Sanquin, Amsterdam) To identify novel B-cell autoantigens in MS, we are currently setting up assays to explore local B-cell specificity in MS and non-demented brain (Netherlands Brain Bank, Amsterdam). These B-cell clones are cultured using recently developed EBV-independent immortalization technology (Prof. Hergen Spits, AIMM, AMC, Amsterdam).


MICROBIOLOGY – RESEARCH TOPICS


Title: Targeting the brain-eating amoeba – efficacy of compounds against Naegleria gruberi

Workgroupleaders: Jaap van Hellemond, Maarten Sarink (Dept. MMIZ)

Duration Yes/No Available per (date)6 months Yes 06-10-201712 months Yes 06-10-201718 months No

BackgroundThe free-living amoeba Naegleria fowleri causes Primary Amoebic Meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). This amoeboflagellate is distributed worldwide in soil and water, growing at temperatures above 25 ˚C. When humans are exposed to the amoeba, it can enter the nose and penetrate the olfactory mucosa, migrate along the olfactory nerve and reach the olfactory bulbs of the CNS. Here it will induce an intense inflammatory response associated with lytic necrosis. At present PAM is reported mainly in developed countries, however, this is probably due to underreporting in developing countries because of diagnostic challenges. Current treatment consists of amphotericin B, fluconazole, rifampin, azithromycin, dexamethasone and miltefosine. Despite this extensive treatment, prognosis is extremely poor with a fatality rate of more than 95%.Research and aimsWe have developed a high throughput growth monitoring assay for a non-infectious relative of N. fowleri : N. gruberi. This species is used as a model system for N. fowleri and the effect of compounds on the growth of N. gruberi can therefore be evaluated to discover new leads for drugs against the extremely deadly infection with N. fowleri. When hits are found, further analysis of mechanism of action and killing properties can be evaluated. Optionally, this research can also be extended to another amoeba, Acanthamoeba castellanii, which causes ocular keratitis that is also difficult to treat.

Principal techniques- Culturing of amoeba (cell culture methods)- Pharmacokinetic/dynamic analysis- Biochemical and cellular analysis to resolve mechanism of action

Selected Publications1. Martínez-Castillo et al (2016) Naegleria fowleri after 50 Years: Is it a neglected

pathogen? J Med Microbiol. 65, 885–896. 2. Stubhaug TT et al (2016) Fatal primary amoebic meningoencephalitis in a Norwegian

tourist returning from Thailand. JMM Case Rep. 3:e005042. 3. Siddiqui R et al (2016) Biology and pathogenesis of Naegleria fowleri. Acta Trop.

164:375-394.

Contact details:Jaap van Hellemond ([email protected]; Tel: 06-22279138)


Title: New “Lights” on the pathogenesis of Biofilm formation of Staphylococcus aureus

Principal investigator: Willem van Wamel





Background: Biofilm-associated Staphylococcus aureus (S. aureus) infections are a big problem in particular in osteomyelitis. Besides being able to resist antibiotic treatment, S. aureus is also able to withstand the natural host defense mechanisms by its accumulation into large cell clusters imbedded in a matrix associated to a surface. By studying the humoral response of 10 osteomyelitis patients we know which bacterial proteins are produced in vivo, but we do not know at which particular stage. Therefor we set up several biofilm assays, including an in vito bone infection model. Initial studies show that we can use an in-house reporter construct based on green fluorescent protein (GFP) to study in vitro transcription of bacterial virulence factors during biofilm formation.

Overall aim: Study the pathogeneses of S. aureus biofilm formation.

Research objectives: Generate promotor/reporter construct of biofilm associated virulence factors of S. aureus and study their transcription in different in vitro biofilm assays.

Techniques: Molecular biology technics like: constructing, checking and cloning of promotor/reporter constructs in to E. coli and in S. aureus. Cellular biology technics like: fluorescence microscopy and FASCan.

Contact details: E: [email protected] T: 0107035820



Title: Mycetoma

Workgroupleader:Dr. W.W.J. van de Sande, [email protected]


6 months Yes depending on project



BackgroundMycetoma is a chronic granulomatous infection of the subcutaneous tissue. It can be caused by both bacteria (actinomycetoma) and fungi (eumycetoma). The most common site of the infection is the foot. Mycetoma can be mainly found in the (sub)tropical countries and was recently added to the Neglected Infectious Diseases list of the World health Organization. The medical management of mycetoma is difficult. Diagnosis is currently done clinically, due to the lack of proper diagnostic tools, and identification of the causative agent is based on culturing, which is time-consuming and results in many misidentification. If not treated, this infection results in massive deformities and loss of function of the limb. For actinomycetoma treatment with antibiotics only, usually gives good results. In contrast, eumycetoma cannot be treated with antifungal agents only and a combination of surgery and antifungal therapy is needed. Even after more than 2 years of therapy, recurrent infections are common. The aim of our research is to develop novel diagnostic tools, applicable in the tropical setting, and to test novel antifungal strategies to improve outcome for patients. We are also part of the first clinical trial for eumycetoma in which the novel antifungal agent fosravuconazole will be tested. This clinical started in 2017 and our laboratory will perform in vitro susceptibility testing, pharmacogenomic testing, betaglucan testing and determine if resistance will develop. For this we also make use of our recently developed mycetoma model in the wax moth Galleria mellonella. The Erasmus MC is one of the leading centers in the world for mycetoma research and as such we have a large network in mycetoma endemic countries, especially with the mycetoma research centre in Sudan.

Research topicsAt the Department Medical Microbiology and Infectious Diseases, several aspects of mycetoma are focused upon. There are various specific research projects in which student can participate. 1. Develop an in vitro grain model to identify novel grain inhibiting agents. In this project,

the student will develop an in vitro grain model by co-culturing mammalian cells with fungal hyphae in 2D culture systems and 3D culture systems. Clinically relevant phenotypes will be generated via RNAi.

2. Test novel antifungal agents both in vitro as in vivo in our recently developed mycetoma model in the wax moth Galleria mellonella. In this project, the student will be testing novel antifungal compounds and derivatives from them via an open source drug discovery program in collaboration with Drugs for Neglected Diseases initiative and the university of Sydney. Testing will be via our in vitro susceptibility assay and our invertebrate model of Galleria mellonella. Testing will be against several causative agents of eumycetoma.

3. Develop an antigen detection system based on recently identified antigens secreted in hemolymph of infected Galleria mellonella larvae. Recently in collaboration with the


university of Wageningen and university of maynooth, Ireland, the transcriptome and proteome of G. mellonella larve infected with M. mycetomatis were analysed. In these analyses six proteins were found to be secreted in the hemolymph of infected larvae and not in that of uninfected larvae. These are ideal candidates for the development of a diagnostic tool. This protein will be recominantly expressed in E. coli, and antibodies or aptamers will be selected against this proteins. These will be used to determine if this protein is also secreted in human serum.

Principal techniquesCell culture techniques, 3D cell culture techniques, in vivo infection models in galleria mellonella larvae (this does not require “artikel 9 or 12”, since insects are not considered to be laboratory animals), cloning, protein expression, aptamer selection. Selected publications1. Van de Sande WWJ (2013) Global burden of human mycetoma: a systematic review and meta-

analysis. PLoS Negl Trop Dis 7: e2550.2. Van de Sande WWJ, Fahal AH, Goodfellow M, Mahgoub el S, Welsh O, et al. (2014) Merits and

pitfalls of the currently used diagnostic tools in mycetoma. PLoS Negl Trop Dis 8: e2918.3. van de Sande WWJ, Mahgoub el S, Fahal AH, Goodfellow M, Welsh O, et al. (2014) The mycetoma

knowledge gap: identification of research priorities. PLoS Negl Trop Dis.4. Kloezen W, Meis JF, Curfs-Breuker I, Fahal AH, Van de Sande WWJ (2012) In vitro antifungal activity

of isavuconazole to Madurella mycetomatis. Antimicrob Agents Chemother 56: 6054-6056.5. van de Sande WW, Janse DJ, Hira V, Goedhart H, van der Zee R, et al. (2006) Translationally

controlled tumor protein from Madurella mycetomatis, a marker for tumorous mycetoma progression. J Immunol 177: 1997-2005.

6. Kloezen W, van Helvert-van Poppel M, Fahal AH, van de Sande WW (2015) A Madurella mycetomatis Grain Model in Galleria mellonella Larvae. PLoS Negl Trop Dis 9: e0003926.


Title: The mycobacterial burden; a new treatment strategy is needed!

Group leaders: Hannelore Bax MD. MSc.T: 010-703 5037E: [email protected]

Jurriaan de Steenwinkel MD. PhD.T: 010-704 4881E: [email protected]


6 months No N/A



BackgroundTuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is still a global health problem of immense proportion. Indeed, TB is considered the second most frequent cause of infectious disease-related death worldwide (after HIV-AIDS). The World Health Organization (WHO) is increasing its efforts to reduce the global threat of TB. Nevertheless, the morbidity and mortality statistics for TB remain shocking. For example, the WHO estimates that the global burden of disease caused by TB in 2010 comprised 8.8 million incident cases.

Additional to these impressive figures is the underlying problem of latent TB infections, with as many as one-third of the world’s population being thought to be latently infected with this bacterium, an immense pool of potential patients around. Furthermore, all current treatment programs tend to be specifically aimed towards the treatment of active TB infection, leaving the huge underlying problem of latent TB infections untouched, essentially meaning that the worldwide elimination of TB is still a distant dream.

Moreover, there is a need to further understand the selection of drug-resistant Mtb mutants and the mechanisms of development of drug resistance. Ultimately, as the current treatment for TB is complicated by its length and complexity, which has led to inadequate response and emergence of MDR-TB and even XDR-TB, there is very urgent need for new powerful TB regimens and strategies to minimize mortality and/or morbidity of TB.

Non-tuberculous mycobacteria (NTM), most frequently Mycobacterium avium, are ubiquitous in the environment. Infection occurs through ingestion, inhalation and direct inoculation. In contrast to Mtb, human-to-human transmission usually doesn’t occur. NTM can cause life-threatening infections in immunocompromised patients, such as patients with AIDS, interferon gamma signaling defects and patients on immunosuppressive therapy. The widespread use of immunosuppressive therapy is a major cause of increasing incidence worldwide. Long-term antimycobacterial treatment is often necessary, which may lead to drug resistance, subsequent therapy failure and high mortality. In fact, current mortality within the Erasmus MC HIV population with disseminated M. avium infections is 65 %. Therefore, new therapeutic strategies are urgently needed aiming at improving treatment efficacy and overall prognosis.

ResearchAt the Department of Medical Microbiology and Infectious diseases (MMIZ) there are three themes of which Emerging Infections of Global Concern and Emerging Antimicrobial Resistance are two, therefore, it is logic that treatment of mycobacterial infections is one of the current research lines. This research line is the result of a close collaboration between the MMIZ department and the Infectious Diseases Section of the Department of Internal Medicine.


We focus on improvement of treatment efficacy using different approaches. These new approaches (e.g. new drugs, new combinations or new dosages of currently given drugs) are tested in different assays in vitro, ex vivo and/or in vivo. With this wide variety of assays, we are able to fill the translational gap in drug development.

The proposed project will consist both in vitro and ex vivo analyses. Given the fact that there are quite some ‘old drugs’ with alleged efficacy against Mtb, further research on these leads is needed. Since these drugs are already approved by the European Medicines Agency (EMA) and US Food and Drug Administration (FDA) the phase two and three studies might be much easier to implement and thus this is a highly pragmatic research approach. For the same reason, one of the chosen strategies to improve anti-NTM drug efficacy is to investigate the role of antibiotics that are already used in clinical practice, but for other indications.

ImplementationThe technical execution of the different assays will include techniques like time-kill kinetic (TTK) assays, the hollow fiber system model of TB (HFS-TB), determinations of Minimal Inhibitory Concentrations (MICs), genetic analysis of resistance and/or microarray analysis. The ex vivo experiments will focus on intracellular infection models using a human macrophage cell line (THP-1) and human monocytes from healthy blood donors. Techniques available under BSL-3 conditions are:

TTK HFS-TB MIC PCR / qPCR Microarray (including data analysis) Macrophage cell culture (possible in combination with confocal and super resolution

fluorescence microscopy or FACS analysis)

Candidate requirement

It’s a well-known statement that techniques can be learned along the way but the correct attitude must be present at the start, this statement holds true for this project. Given the ‘high risk’ research we are proposing, we are searching for a colleague with indefinite interest in translational research. It is considered high risk due to the fact that Mtb is a class thee micro-organism but also because results aren’t promised upfront. Thus, the candidate must be accurate in executing the experiments, willing to explore different techniques and creative in solving (research) questions.

Publications related to the research linesColistin as a potentiator of anti-TB drug activity against Mycobacterium tuberculosis. Bax HI, de Steenwinkel JEM, ten Kate MT, van der Meijden A, Verbon A, Bakker-Woudenberg IAJM. J Antimicrob Chemother. 2015 Oct;70(10):2828-37

SILA-421 activity in vitro against rifampicin-susceptible and rifampicin-resistant Mycobacterium tuberculosis, and in vivo in a murine tuberculosis model. de Knegt GJ, Bakker-Woudenberg IA, van Soolingen D, Aarnoutse R, Boeree MJ, de Steenwinkel JE. Int J Antimicrob Agents. 2015 Jul;46(1):66-72

Enhancement of in vitro activity of tuberculosis drugs by addition of thioridazine is not reflected by improved in vivo therapeutic efficacy. Tuberculosis (Edinb). De Knegt GJ, ten Kate MT, van Soolingen D, Aarnoutse R, Boeree MJ, Bakker-Woudenberg IA, de Steenwinkel JE. 2014 Dec;94(6):701-7

Optimization of the rifampin dosage to improve the therapeutic efficacy in tuberculosis treatment using a murine model. de Steenwinkel JE, Aarnoutse RE, de Knegt GJ, ten Kate MT, Teulen M, Verbrugh HA, Boeree MJ, van Soolingen D, Bakker-Woudenberg IA. Am J Respir Crit Care Med. 2013 May;187(10):1127-34.

Rifampicin-induced transcriptome response in rifampicin-resistant Mycobacterium tuberculosis. de Knegt GJ, Bruning O, ten Kate MT, de Jong M, van Belkum A, Endtz HP, Breit TM, Bakker-Woudenberg IA, de Steenwinkel JE. Tuberculosis (Edinb). 2013 Jan;93(1):96-101.


Consequences of noncompliance for therapy efficacy and emergence of resistance in murine tuberculosis caused by the Beijing genotype of Mycobacterium tuberculosis. de Steenwinkel JE, ten Kate MT, de Knegt GJ, Verbrugh HA, Aarnoutse RE, Boeree MJ, den Bakker MA, van Soolingen D, Bakker-Woudenberg IA. Antimicrob Agents Chemother. 2012 Sep;56(9):4937-44.

Drug susceptibility of Mycobacterium tuberculosis Beijing genotype and association with MDR TB. de Steenwinkel JE, ten Kate MT, de Knegt GJ, Kremer K, Aarnoutse RE, Boeree MJ, Verbrugh HA, van Soolingen D, Bakker-Woudenberg IA. Emerg Infect Dis. 2012 Apr;18(4):660-3.

Time-kill kinetics of anti-tuberculosis drugs, and emergence of resistance, in relation to metabolic activity of Mycobacterium tuberculosis. de Steenwinkel JE, de Knegt GJ, ten Kate MT, van Belkum A, Verbrugh HA, Kremer K, van Soolingen D, Bakker-Woudenberg IA. J Antimicrob Chemother. 2010 Dec;65(12):2582-9.

Interferon alpha treatment of patients with impaired interferon gamma signaling. Bax HI, Freeman AF, Ding L, Marciano B, Kristosturyan E, Jancel T, Spalding C, Pechacek J, Oliver KN, Barnhart L, Boris L, Holland SM, Sampaio EP. Interferon alpha treatment of patients with impaired interferon gamma signaling. J Clin Immunol. 2013Jul;33(5):991-1001

A novel STAT1 mutation associated with disseminated mycobacterial disease. Sampaio EP, Bax HI, Hsu AP, Kristosturyan E, Pechacek J, Chandrasekaran P, Paulson ML, Dias DL, Spalding C, Uzel G, Ding L, McFarland E, Holland SM. J Clin Immunol. 2012Aug; 32(4):681-9


Identify the leading risk factors for acquiring multidrug-resistant Acinetobacter baumannii – a systematic review

Project leaders:Prof. dr. Margreet C. Vos Anne F. Voor in ’t holt MSc

T: 0627042465 / 0107033411 / 0107043510 T: 0657381619 / 0107037267E:

[email protected] E: [email protected]


6 months Yes 01-10-201712 months No n.a.18 months No n.a.

BackgroundInfections in patients with multidrug-resistant (MDR) bacteria are difficult to treat, have a high morbidity and mortality and are very costly. In the EU, each year 3.2 million patients get healthcare-associated infections; one in 18 patients every day. Many of these infections could have been prevented. Also, MDR Acinetobacter baumannii are major healthcare-associated pathogens, especially in patients at the intensive care [1]. To prevent these infections in critically ill patients, it is important to know the risk factors leading to these infections. In the Netherlands, a national typing system will be implemented. With the strain to be typed, epidemiological data are needed, to confirm or exclude relationship in genetically related strains. For this, it is important to summarize al available evidence on this subject already available – a systematic review and meta-analysis. In this way, all risk factors already published by other researchers around the world can be identified and aggregated. These risk factors can be ordered into categories of importance. In this way, leading risk factors for acquiring an infection of MDR A. baumannii can be identified.

Research topicThe main aim of this research is identifying risk factors for acquisition of MDR A. baumannii. This knowledge will be useful for the nation-wide typing surveillance and world-wide healthcare centers that are faceting the threat of MDR A. baumannii and it will help in designing strategies to stop the emergence of spread of this pathogen.

Principal techniquesFollowing the checklist detailed in the PRISMA guideline [2]. Including:

- Writing a protocol and publish this in the PROSPERO register- Study selection and data collection- Analyzing the data, data interpretation, statistical analysis (meta-analysis)

Related References1. Kim UJ, Kim HK, An JH, Cho SK, Park KH and Jang HC. Update on the Epidemiology, Treatment, and Outcomes of Carbapenem-resistant Acinetobacter infections. Chonnam Med J 2014;50:37-44

2. Liberati A, Altman DG, Tetzlaff J, et al. The PRISMA statement for reporting systematic reviews and meta-analyses of studies that evaluate health care interventions: explanation and elaboration. J Clin Epidemiol 2009;62:e1-34




Eradication of a VIM2-positive Pseudomonas aeruginosa from hospital wards – an intervention study

Project leaders:Prof. dr. Margreet C. Vos Dr. Juliëtte Severin Anne F. Voor in ’t holt MScT: 0627042465 / 0107033411 T: 0107032879 / 0107033510 T: 0657381619 / 0107037267E: [email protected] E: [email protected] E: [email protected]


6 months Yes 01-10-201712 months Yes 01-10-201718 months No n.a.

BackgroundPseudomonas aeruginosa is a common nosocomial pathogen and the emergence of multidrug-resistant strains of P. aeruginosa is of global concern. Since 2003, a carbapenem-resistant, VIM2-positive clone of P. aeruginosa has emerged in our hospital and became entrenched causing multiple episodes of colonization and/or infection. Sinks are a well-known source of bacteria, but are not specifically designed for use in hospitals. That is why we hypothesized that transmission occurred via sinks since bacteria can survive in a humid environment. Several measures were implemented to stop transmission via this route. One of them was placing a so-called "pop-up" on the drain of sinks of several departments where transmission occurred. To test the efficacy of this intervention several environmental cultures were taken at several time points. However, this was not reported or collected systematically and the data collection is not finished yet. Therefore, in order to conclude if the "pop-ups" worked in preventing transmission we need additional data and evidence.

Research topicThe aim of this study is to measure the effectiveness of this intervention in order to conclude if this intervention helped in terminating the outbreak at the intervention wards.

Principal techniques Data collection: culturing the environment of departments with "pop-ups" on the drains of

their sinks Data analysis Data interpretation Repetitive sequence typing (Diversilab)

Related References

Voor in ´t holt AF, Severin JA, Lesaffre EM and Vos MC. A systematic review and meta-analyses show that carbapenem use and medical devices are the leading risk factors for carbapenem-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 2014;58:2626-37

Duration of carriage and occurrence of infections in patients with a VIM2-positive Pseudomonas aeruginosa: A risk factor analysis

Project leaders:Prof. dr. Margreet C. Vos Dr. Juliëtte Severin Anne F. Voor in ’t Holt MSc


T: 0627042465 / 0107033411 T: 0107032879 / 0107033510 T: 0657381619 / 0107037267E: [email protected] E: [email protected] E: [email protected]



Since electronic patient files will be used to collect information for this study, it is important that you can read and understand Dutch.BackgroundPseudomonas aeruginosa is a common nosocomial pathogen and the emergence of multidrug-resistant strains of P. aeruginosa is of global concern. For years now, a carbapenem-resistant, VIM2-positive clone of P. aeruginosa has emerged in our hospital and became entrenched causing multiple episodes of colonization and/or infection. There are no options for treatment of carriage with P. aeruginosa. As these resistant microorganisms are relatively new in hospitals, not much is known about the duration of carriage. Furthermore, the duration of carriage is possibly different in patients colonized compared to those infected with a VIM2-positive P. aeruginosa. The outcome in terms of infection, kind and outcome, has not been studied yet. Therefore, we want to establish the duration of carriage and the incidence of infection in the hospital and after discharge. Furthermore, the number of cultures needed to establish end of carriage will be studied. We also want to identify the driving forces or risk factors for this carriage and subsequent infections in order to possibly change current guidelines on time and moment of follow-up cultures.

Research topicThe aim of this study is to identify risk factors for duration of carriage in colonized and infected patients with a VIM2-positive P. aeruginosa. For this, we will look for risk factors as known in the electronic patient records. We will also develop and sent a questionnaire to the identified patients to ask for their current condition, their reaction on and experience with acquisition of this strain and use of medication

Principal techniques Writing a study protocol Data collection of patients in the Erasmus MC Development of a questionnaire for carriers and culturing carriers Data analysis; data interpretation

Related References

Voor in ´t holt AF, Severin JA, Lesaffre EM and Vos MC. A systematic review and meta-analyses show that carbapenem use and medical devices are the leading risk factors for carbapenem-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 2014;58:2626-37


Evaluation of the Methicillin-Resistant Staphylococcus aureusSearch-and-Destroy Policy: 2005 to 2015

Project leaders:Prof. dr. Margreet C. Vos Anne F. Voor in ’t holt MScT: 06-27042465 / 010-7033411T: 06-57381619 / 010-7037267E: [email protected] E: [email protected]



Since electronic patient files will be used to collect information for this study, it is important that you can read and understand Dutch.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a leading antibiotic-resistant pathogen causing healthcare-related and community-acquired infections worldwide.(1) In many countries, MRSA proportions have reached at least 20 percent of all clinical isolates of S. aureus, and even exceed 40 percent in some parts of Europe.(2) On the contrary, in the Netherlands and in Scandinavian countries the prevalence of MRSA is low.(1-3)

To control and maintain a low prevalence of MRSA in the Netherlands, a strict antibiotic policy and an active Search-and-Destroy (S&D) policy are practiced. The S&D policy focuses on [1] defining groups at risk for MRSA carriage and screening of both patients and healthcare workers (HCWs) considered at risk, [2] pre-emptive strict isolation based on the defined risk groups, [3] follow-up of carriers, [4] eradication of carriage in both patients and their household members, and [5] disinfection of the environment. These risk categories and isolation measures are outlined in the national MRSA guidelines of the Dutch working Party on Infection Prevention (WIP).(4)

After two decades of stable epidemiological risk factors for acquisition a recent change in epidemiology has resulted in two groups of patients with MRSA: [1] MRSA-positive patients with described risk factors, defined as MRSA of Known Origin (MKO), and [2] MRSA-positive patients without described risk factors, defined as MRSA of Unknown Origin (MUO).(5) Patients at risk for MRSA carriage but without risk factors (MUO) are not screened. Thus, isolation measures are not taken and the MRSA carrier may transmit MRSA undetected to other patients; which may result in outbreaks. When an undetected MRSA carrier is discovered unexpectedly by means of a clinical sample, contact tracing is performed to determine the outbreak and its size. The methods of contact tracing differ per health facility and have changed throughout the years within the Erasmus MC.

Research topicIn this study we aim to evaluate: [1] the number of patients identified with a MUO or MKO from 2005-2015, [2] the burden of MUO and MKO by investigating the results of contact tracing and the total number of isolation days, and [3] the difference between the results of [1] and [2] in comparison to data from 2000-2004 to evaluate the results of changes in the MRSA guidelines and the possible changes of the epidemiology over time.(1) In addition, we want to create a risk profile of patients and HCWs transmitting MRSA to others. We want to do this by comparing patients and HCWs who did transmit MRSA to others to MRSA patients and HCWs who did not.

Principal techniques Writing a study protocol Data collection of patients Data analysis; data interpretation Writing of a manuscript

Related References 1) Vos MC, Behrendt MD, Melles DC, Mollema FP, de Groot W, Parlevliet G, et al. 5 years of experience

implementing a methicillin-resistant Staphylococcus aureus search and destroy policy at the largest university medical center in the Netherlands. Infect Control Hosp Epidemiol. 2009;30(10):977-84.




2) Tiemersma EW, Bronzwaer SL, Lyytikainen O, Degener JE, Schrijnemakers P, Bruinsma N, et al. Methicillin-resistant Staphylococcus aureus in Europe, 1999-2002. Emerg Infect Dis. 2004;10(9):1627-34.

3) Grundmann H, Aires-de-Sousa M, Boyce J, Tiemersma E. Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet. 2006;368(9538):874-85.

4) Dutch Working Group on Preventing Infections (WIP). Ziekenhuizen: Meticilline-resistente Staphylococcus aureus. 2012.

5) Lekkerkerk WS, van de Sande-Bruinsma N, van der Sande MA, Tjon ATA, Groenheide A, Haenen A, et al. Emergence of MRSA of unknown origin in the Netherlands. Clin Microbiol Infect. 2012;18(7):656-61.


Staphylococcal scalded skin syndrome

Project leaders:Prof. dr. Margreet C. Vos Dr. Juliëtte Severin Dr. Nelianne VerkaikT: 0627042465 / 0107033411 T: 0107032879 / 0107033510 T: 0107030423 / 0107033510E: [email protected] E: [email protected] E: [email protected]


6 months Yes 01-01-201812 months No n.a.18 months No n.a.

BackgroundStaphylococcal scalded skin syndrome is a serious skin infection mainly in young infants. The disease is characterized by blistering of the upper layer of the skin, caused by exfoliative toxin a or b (eta or etb) producing strains of Staphylococcus aureus. In 2015, the neonatology ward in Erasmus MC reported on two patients with SSSS within a period of four weeks. The eta positive S. aureus strains of these two patients were similar as determined with molecular techniques. Infection control measures were implemented by a multidisciplinary team consisting of (infection prevention) nurses and doctors from the Dept. of Pediatrics, division of Neonatology, division of Infectiology, Municipal Public Health Service and Dept. of Medical Microbiology. These measures included isolation of patients, surveillance, enhanced environmental cleaning and decolonization of staff carrying the outbreak S. aureus strain. With these measures, the outbreak was successfully halted. However, later on in 2015 and in the early 2016s new patients developed SSSS (with different strains) and again incidental transmission was shown. Outbreaks were halted by implementing infection control measures. Still, infection prevention is preferential above infection control.

Research topicIn order to help to prevent further SSSS outbreaks, the aim of this study is to get a better understanding of the clinical syndrome, the prevalence of exfoliative toxin producing S. aureus strains and SSSS in the Netherlands and, in particular, Erasmus MC and infection control measures that should be implemented.

Principal techniques PCR cgMLST / whole genome sequencing spa typing Data collection of patients in the Erasmus MC Data analysis; data interpretation Questionnaire for prevalence data in centers of neonatal intensive care

Related References Paranthaman K, Bentley A, Milne LM, Kearns A, Loader S, Thomas A, Thompson F, Logan M, Newitt S, Puleston R. Nosocomial outbreak of staphyloccocal scalded skin syndrome in neonates in England, December 2012 to March 2013. Euro Surveill. 2014;19(33):pii=20880. Patel GK, Finlay AY, Staphylococcal scalded skin syndrome: diagnosis and management. Am J Clin Dermatol. 2003;4(3):165.



Title: Immunological mechanisms of HIV infection

Workgroup leaders:Dr. A. Verbon ([email protected])Dr. M.A.A. Claassen ([email protected]) WEG

Investigators: Dr. A. Boonstra (Department of Gastroenterology and Hepatology)


6 months Yes November 2017



BackgroundIn the last 20 years, the treatment of HIV infected patients with combination anti-

retroviral therapies (cART) have greatly improved and effective suppression of viral replication is a realistic goal for the vast majority of patients. Consequently, for many patients who receive cART treatment, HIV has become a chronic disease without progression to AIDS and with a good survival. However, cART-treated patients still experience substantial health issues resulting in ongoing decreased survival. We hypothesize that this impaired health is mainly caused by side effects of antiretroviral therapy, lifestyle factors such as heavy smoking, by low-grade chronic systemic HIV-induced inflammation, or additional immune activation by co-infections such as the hepatitis B or C virus (HBV or HCV).

Also, despite the optimized antiretroviral regimens, the risk of viral resistance remains, especially with poor compliance to therapy. The quality of life of patients is therefore influenced to varying degrees by the fact that they have to take their anti-retroviral medication daily, life-long and conscientiously.

Further improvement of antiretroviral therapies to treat HIV patients is aimed at the development of strategies with less long-term side effects, lower rate of viral resistance and reduced HIV-induced inflammation. We hypothesize that chronic HIV-induced immune activation can be minimized by long-term maximal viral suppression, and can be reactivated by low-grade and repeated HIV replication (blips). Therefore, ultra-deep viral suppression by cART may be an important goal of HIV therapy. The ultimate goal, however, is to find a treatment that leads to final eradication of HIV, which makes that life-long treatment is no longer needed, and patients are cured.

In order to prove our hypotheses and achieve our objectives more knowledge of the host response against HIV and other chronic viral infectious diseases is needed. Currently, the factors that play a role in the persistence of these chronic viral infections and subsequent persistent immune activation are still largely unknown. To get a better understanding of these factors, studies of the host response and pathophysiological processes are necessary, and may lead to the identification of biological markers, the improvement of diagnosis and improvement of anti-retroviral therapies.

The research is aimed at understanding the ongoing immune activation in chronic HIV patients during effective ART therapy. These studies combine clinical and translational research.

Research To examine in detail the immune parameters that comprise the state of chronic

immune activation as observed in cART-treated chronic HIV patients. This will be done by evaluation of the phenotype and function of leukocytes, such as monocytes, NK, MAIT and T cells using immunological and systems biological tools.

To better characterize the effect of chronic immune activation as observed in cART-treated chronic HIV patients on increased morbidity and mortality




The project will be performed in a team of clinicians and immunologists, and consequently will combine clinical and translation studies. Some of the studies are joint projects with the Department of Gastroenterology & Hepatology (Dr. A. Boonstra).

A broad array of immunological as well as molecular techniques are being used in the laboratory, including processing of patient material (blood and biopsies), cell culture, cell purification by sorting, multi-color flow cytometry for surface markers and intracellular staining, various immunoassays, functional NK and T cell assays (cytotoxicity, cytokine secretion), DNA/RNA isolation, qPCR etc.


The detection of bacterial protease activity for the diagnosis of complex infections of the CF lung

Workgroupleaders:Dr. Wendy Kaman and Dr. John HaysT. 010-7033089 (Kaman) / 010-7032177 (Hays)E. [email protected] / [email protected]

Duration Yes/No Available per (date)6 months No nvt12 months Yes 1 Feb 2018/ 1 Sept 201818 months Yes 1 Feb 2018

BackgroundCystic fibrosis (CF) is a genetic disease in which bacterial infections of the airways play a major role in the long-term clinical outcome. In previous studies we showed that the detection of bacterial proteolytic activity can be used in the diagnosis of bacteria-related infections (1,2). In 2013 for example, we designed and evaluated a novel 3xGly Fluorescence Resonance Energy Transfer-peptide substrate (FRET-probe) for the assessment of virulence in Pseudomonas aeruginosa (3), one of the most common pathogens in CF. Detection of bacterial protease activity (from P. aeruginosa but also other CF related pathogens), might thus able us to predict the clinical outcome of CF patients receiving initial antibiotic therapy and rapidly gain information on the character of the bacterial isolate (4).

Research and aimsWithin this project we aim to develop a simple and rapid tool to determine the bacterial composition of the CF lung based on the detection of bacterial protease activity. The project is divided in five objectives:

1. Literature search on which microorganisms are associated with the different stages of CF and which bacterial proteases are involved.

2. Design, development and evaluation of a panel of bacteria-specific FRET-probes.3. Micelle-based protease detection in order to specifically determine the bacterial composition of

the CF lung.4. Development of an internal control to quantify the amount of bacteria/ protease present.5. Screening clinical material on bacterial-protease activity and correlation of this activity with

infection severity.

Principal techniquesThe techniques used within this study are mainly flow cytometry, bacterial culture and proteolytic detection assays.

Selected Publications1. Kaman WE, Hulst AG, van Alphen PT, Roffel S, van der Schans MJ, Merkel T, van Belkum A,

Bikker FJ. Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillus species. Anal Chem. 2011 Apr 1;83(7):2511-7.

2. Kaman WE, Galassi F, de Soet JJ, Bizzarro S, Loos BG, Veerman EC, van Belkum A, Hays JP, Bikker FJ. Highly specific protease-based approach for detection of Porphyromonas gingivalis in diagnosis of periodontitis. J Clin Microbiol. 2012 Jan;50(1):104-12.

3. Kaman WE, Arkoubi-El Arkoubi NE, Roffel S, Endtz HP, van Belkum A, Bikker FJ, Hays JP. Evaluation of a FRET-peptide substrate to predict virulence in Pseudomonas aeruginosa. PLoS One. 2013 Nov 26;8(11):e81428.

4. Kaman WE, Hays JP, Endtz HP, Bikker FJ. Bacterial proteases: targets for diagnostics and therapy. Eur J Clin Microbiol Infect Dis. 2014 Jul;33(7):1081-7.


MAX PLANCK INSTITUTE FOR INFECTION BIOLOGY - RESEARCH TOPICS


Sandra Franch-Arroyo, PhD Student

Department of Regulation in Infection Biology (RIIB)Max Planck Institute for Infection Biology

Phone: +49 176 47613192E-mail: [email protected]: Charitéplatz 1, 10117 Berlin, Germany

RNA-Media ted Adapt ive Immuni ty (CRISPR-Cas)

T o p r o t e c t t h e m s e l v e s f r o m t h e a t t a c k b y i n v a d i n g a l i e n g e n o m e s ( i n p a r t i c u l a r p h a g e s a n d p l a s m i d s ) , b a c t e r i a a n d a r c h a e a h a v e e v o l v e d a n R N A -g u i d e d a d a p t i v e i m m u n e s y s t e m , c a l l e d C R I S P R - C a s ( c l u s t e r e d , r e g u l a r l y i n t e r s p a c e d s h o r t p a l i n d r o m i c r e p e a t s – C R I S P R - a s s o c i a t e d p r o t e i n s ) . T h e s y s t e m u s e s r i b o n u c l e o p r o t e i n c o m p l e x e s c o m p o s e d o f s h o r t C R I S P R R N A s ( c r R N A s ) a n d C a s p r o t e i n ( s ) t o s i l e n c e i n v a d i n g n u c l e i c a c i d s e q u e n c e s i n a s e q u e n c e - s p e c i f i c m a n n e r . W e a r e i n t e r e s t e d i n d e c i p h e r i n g t h e m o l e c u l a r m e c h a n i s m s i n v o l v e d i n t h e a d a p t a t i o n , e x p r e s s i o n a n d i n t e r f e r e n c e p h a s e s o f t h e b a c t e r i a l i m m u n e s y s t e m . W e a r e a l s o f o c u s i n g o n t h e m o l e c u l a r d e t a i l s o f t h e r e c e n t l y d i s c o v e r e d t r a c r R N A : c r R N A - C a s 9 g e n o m e e d i t i n g m e c h a n i s m / t e c h n o l o g y a s w e l l a s o f r e c e n t l y i d e n t i f i e d C R I S P R - C a s s y s t e m s .

R e g u l a t o r y s m a l l R N A s a n d R N a s e s

I n a d d i t i o n t o t h e C R I S P R - a s s o c i a t e d R N A s , b a c t e r i a e n c o d e m u l t i p l e o t h e r s m a l l R N A s ( s R N A s ) t h a t p l a y c r i t i c a l r e g u l a t o r y f u n c t i o n s i n m a j o r b i o l o g i c a l p a t h w a y s . W e h a v e i d e n t i f i e d a n u m b e r o f p u t a t i v e c i s - a c t i n g s R N A s ( e . g . r i b o s w i t c h e s ) a n d t r a n s - a c t i n g s R N A s i n t h e h u m a n p a t h o g e n Streptococcus pyogenes. W e a r e a d d r e s s i n g t h e q u e s t i o n o f h o w s e l e c t e d s R N A s i n t e g r a t e i n t o t h e g e n e r a l r e g u l a t o r y n e t w o r k c o n t r o l l i n g p a t h o g e n e s i s a n d r e l a t e d m e c h a n i s m s i n t h i s p a t h o g e n . W e w a n t t o u n d e r s t a n d t h e r e g u l a t i o n o f s R N A e x p r e s s i o n b y r i b o n u c l e a s e s , a n a l y z e t h e b i o l o g i c a l f u n c t i o n s o f s R N A s , i d e n t i f y t h e i r i n t e r a c t i n g p a r t n e r s a n d d e t e r m i n e t h e i r m o d e s o f a c t i o n a t t h e m o l e c u l a r a n d c e l l u l a r l e v e l .

R e g u l a t o r y P r o t e i n Q u a l i t y C o n t r o l

T h e r a p i d d e g r a d a t i o n o f f u l l y f u n c t i o n a l r e g u l a t o r y p r o t e i n f a c t o r s i n r e s p o n s e t o t h e e n v i r o n m e n t i s a w e l l - e s t a b l i s h e d p o s t - t r a n s l a t i o n a l r e g u l a t o r y m e c h a n i s m i n b a c t e r i a t h a t i s k n o w n t o b e c r i t i c a l f o r b a c t e r i a l v i r u l e n c e . S i n c e p r o t e o l y s i s i s a n i r r e v e r s i b l e a n d c o s t l y p r o c e s s , h i g h l y s p e c i f i c s u b s t r a t e r e c o g n i t i o n i s c r u c i a l t o i m p l e m e n t p r o t e i n d e g r a d a t i o n w i t h i n a c o m p l e x r e g u l a t o r y c i r c u i t . T h u s , w e a r e i n t e r e s t e d i n u n d e r s t a n d i n g h o w b a c t e r i a e n s u r e t h e r e c o g n i t i o n a n d d e g r a d a t i o n o f a r e g u l a t o r y p r o t e i n a t t h e a p p r o p r i a t e t i m e , e n v i r o n m e n t a l c o n d i t i o n ( s ) a n d / o r l o c a l i z a t i o n ( s ) d u r i n g a n i n f e c t i o n p r o c e s s .

E t e r n a l C e l l – L i f e w i t h o u t R e p l i c a t i o n

D e c i p h e r i n g t h e p h y s i o l o g i c a l f u n c t i o n s o f f u n d a m e n t a l c e l l u l a r p r o c e s s e s h a s u n t i l r e c e n t l y b e h i n d e r e d b y t h e l a c k o f m o l e c u l a r t o o l s t o m a n i p u l a t e e s s e n t i a l g e n e s . R e c e n t b r e a k t h r o u g h s i n g e n o m e e n g i n e e r i n g , s u c h a s t h e C R I S P R - C a s 9 t e c h n o l o g y , h a v e f a c i l i t a t e d t h e c h a r a c t e r i z a t i o n o f t h e s e u n d e r - s t u d i e d p r o c e s s e s . I n t h e f r a m e o f Eternal Cell: life without replication, w e p r o p o s e t o d e v e l o p t h e C R I S P R - C a s 9 t e c h n o l o g y f o r t h e c r e a t i o n o f a c e l l t h a t l o o s e s t h e a b i l i t y o f D N A r e p l i c a t i o n d u r i n g a n y p o i n t o f t h e b a c t e r i a l l i f e c y c l e . W i t h t h i s a p p r o a c h , w e i n t e n d t o c h a r a c t e r i z e t h e b a s i c p r i n c i p l e o f



D N A r e p l i c a t i o n a n d l e a r n m o r e a b o u t i t s r o l e f o r l i f e . U s i n g m o d e r n – o m i c s , w e w i l l c h a r a c t e r i z e t h e b i r t h , l i f e a n d d e a t h o f t h e s e c e l l s u n d e r n o r m a l a n d s t r e s s c o n d i t i o n s a n d u t i l i z e o u r eternal cell p o p u l a t i o n f o r a p p l i e d b i o t e c h n o l o g i c a l a n d m e d i c a l h y p o t h e s e s .

To x i n - A n t i t o x i n S y s t e m s

T o x i n - a n t i t o x i n ( T A ) s y s t e m s c o n s i s t o f a s t a b l e t o x i n t h a t c a u s e s g r o w t h a r r e s t o r c e l l d e a t h b y i n h i b i t i n g e s s e n t i a l c e l l u l a r p r o c e s s e s a n d a l a b i l e a n t i t o x i n , w h i c h c o u n t e r a c t s t h e t o x i n . I n i t i a l l y , T A s y s t e m s w e r e d e s c r i b e d a s a s t a b i l i z a t i o n e l e m e n t o f p l a s m i d s , b u t t h e s e t w o g e n e m o d u l e s a r e a l s o u b i q u i t o u s i n p r o k a r y o t i c g e n o m e s . W h e n e n c o d e d o n t h e g e n o m e , t h e y o f t e n f u n c t i o n a s a r e g u l a t o r y s w i t c h t h a t c o n v e r t s b a c t e r i a i n t o p e r s i s t e r s , a d o r m a n t s t a t e t h a t i s a s s o c i a t e d w i t h i n c r e a s e d a n t i b i o t i c t o l e r a n c e . W e h a v e r e c e n t l y i d e n t i f i e d s e v e r a l p u t a t i v e T A l o c i i n t h e G r a m - p o s i t i v e h u m a n p a t h o g e n Streptococcus pyogenes i n a b i o i n f o r m a t i c s s e a r c h . W e a i m t o c h a r a c t e r i z e t h e s e l o c i , e l u c i d a t e t h e m o l e c u l a r m e c h a n i s m s o f t h e s e T A s y s t e m s , a n d u n r a v e l t h e i r b i o l o g i c a l r o l e s , i n c l u d i n g t h e t r i g g e r s f o r a c t i v a t i o n o f t h e t o x i n s a n d t h e i r s i g n i f i c a n c e f o r p e r s i s t e r c e l l f o r m a t i o n .

B a c t e r i a l a n d Ve s i c u l a r I n t e r a c t i o n s w i t h H o s t I n n a t e I m m u n i t y

B a c t e r i a m u s t s e n s e a n d r e s p o n d t o t h e i r e n v i r o n m e n t i n o r d e r t o a d a p t t o t h e e v e r - c h a n g i n g e n v i r o n m e n t . W e a r e i n t e r e s t e d i n o n e m e c h a n i s m a l l o w i n g b a c t e r i a t o r e s p o n d t o h o s t i m m u n e - m e d i a t e d s t r e s s c o n d i t i o n s v i a e x p o r t o f d e f i n e d p r o t e i n , l i p i d a n d n u c l e i c a c i d l a d e n e x t r a c e l l u l a r m e m b r a n e - d e r i v e d v e s i c l e s ( M V s ) . M V s a c t i n m u l t i p l e w a y s t o e x e r t a d e c i s i v e i m p a c t o n h o s t -p a t h o g e n i n t e r a c t i o n s d u r i n g i n f e c t i o n b y p a t h o g e n i c b a c t e r i a . W e e m p l o y a n a r r a y o f m o l e c u l a r a n d c e l l u l a r t e c h n i q u e s t o u n d e r s t a n d t h e f o r m a t i o n o f M V s a n d t h e i r r o l e s d u r i n g i n f e c t i o n a n d i m m u n i t y t o f u l l y c o m p r e h e n d t h e i m p a c t o f E V s o n b a c t e r i a l v i r u l e n c e a n d t h e h o s t i m m u n e r e s p o n s e a n d t o p o s s i b l y h a r n e s s t h e i d e n t i f i e d m e c h a n i s m s i n t o p o t e n t i a l l y n o v e l a n t i - i n f e c t i v e p r i n c i p l e s .


CARDIOLOGY– RESEARCH TOPICS


Title: Sonobactericide: developing a novel ultrasound-mediated treatment for infective endocarditis, and possibly other biofilm infections

Project leaders: Klazina Kooiman, PhD Kirby R. Lattwein, MScT: 010-7044036 T: 010-7044633E: [email protected] E: [email protected]: https://www.erasmusmc.nl/thoraxcenterbme/?lang=en


6 months Yes ~ Jan 1, 2018

12 months Yes ~ Jan 1, 2018

18 months Yes After further contact

BackgroundInfective endocarditis (IE) is a life-threatening infection where abnormal growths, referred to

as vegetations that contain collections of bacteria, form on the heart valves. It remains among the deadliest of infectious diseases because it is difficult to diagnose and treat. Vegetations formed in IE predominately consist of a thrombus-like mesh of platelets, fibrin, and bacteria secreting polymeric matrix (biofilm material), which the immune system and antibiotics cannot easily penetrate. As a result, embedded bacteria are 100 - 1000 times less susceptible to antibiotics than non-embedded bacteria, effectively lowering treatment success and increasing the length of antibiotic treatment. Therefore, a novel non-invasive strategy, such as sonobactericide, is desperately needed.

Sonobactericide uses the unique combination of ultrasound, ultrasound contrast agents, and antibiotics to treat IE, and possibly other biofilm infections. Ultrasound contrast agents consist of gas microbubbles (1-10 μm) that are stabilized by a coating. When exposed to ultrasound, microbubbles vibrate volumetrically as their gas cores respond to the ultrasound pressure variations. This vibration increases cell permeability through microbubble-cell interactions, which induce cell membrane perforations, or sonoporation. Vibrating microbubbles stimulate the uptake of drugs that are normally not taken up by cells (demonstrated in mammalian cells). Furthermore, specific, violent microbubble vibration can directly induce death in mammalian cells. These vibrating ultrasound-activated microbubbles can also enhance sonothrombolysis: the dissolution of a thrombus by ultrasound. Since IE vegetations chiefly consist of the same components of a thrombus, we hypothesize that microbubbles can also lead to the dissolution of IE vegetations, exposing the bacteria to antibiotics. Employing the concepts of both sonoporation and sonothrombolysis, we have preliminary results that vibrating contrast agents could kill bacteria and potentially augment antibiotic therapy.

Research In this project, we want to investigate the potential of sonobactericide as an innovative approach to treating (and diagnosing) infective endocarditis (or other biofilm infections) using ultrasound contrast agents.

Potential research topics: -To study and evaluate sonoporation/killing of different species of gram-positive and gram-negative bacteria (clinical specimens) using vibrating microbubbles

https://www.erasmusmc.nl/thoraxcenterbme/?lang=en




-To produce microbubbles specifically targeted to bacteria/biofilm and investigate their potential to adhere to bacteria under flow for use in diagnostic and therapeutic applications

Additionally, we are open to discuss potential variations of these and other research topics.

General informationThe Therapeutic Ultrasound Contrast Agent Group is an international reference center for the therapeutic use of ultrasound contrast agents. These agents are characterized using both acoustical and optical measurement techniques. Gained knowledge is used to improve the activation and detection of the agents in the human body, and to make these agents suitable for therapeutic use. Special attention in our group is paid to drug delivery and molecular targeting/imaging using ultrasound activated agents. This group has collaborations within Erasmus MC (Thorax Center, Medical Microbiology and Infectious Diseases) as well as several (inter)-national collaborative networks. Our research projects (in vitro, ex vivo, and in vivo studies) use state-of-the-art imaging techniques (confocal, Brandaris ultra-high speed camera (25 million frames per second recordings), TIRF, high resolution ultrasound), alongside cellular (cell culture, organ-on-a-chip, histology, immunohistochemistry) and molecular sequencing techniques.

Related References van Rooij, T. et al. Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of

targeting, oscillation, and displacement of microbubbles. J Control Release 238, 197-211, doi:10.1016/j.jconrel.2016.07.037 (2016)

Kooiman, K., Vos, H. J., Versluis, M. & de Jong, N. Acoustic behavior of microbubbles and implications for drug delivery. Adv Drug Deliv Rev 72, 28-48, doi:10.1016/j.addr.2014.03.003 (2014)

van Rooij, T. et al. Targeted ultrasound contrast agents for ultrasound molecular imaging and therapy. Int. J. Hyperthermia 31, 90-106 (2015)

Werdan, K. et al. Mechanisms of infective endocarditis: pathogen-host interaction and risk states. Nat Rev Cardiol 11, 35-50, doi:10.1038/nrcardio.2013.174 (2014)

Cahill, T. J. & Prendergast, B. D. Infective endocarditis. Lancet 387, 882-893, doi:10.1016/S0140-6736(15)00067-7 (2016)


VIROLOGY – RESEARCH TOPICS


Title: Genomics of the virus-host interaction

Workgroup leader: Arno Andeweg ([email protected])

Duration Yes/No Available per (date)6 months Yes Open/after consultation12 months Yes Open/after consultation18 months Yes Open/after consultation

Background:The virus-host interaction is highly dynamic and complex. The host responseto a virus infection involves a series of interactions of many cells, receptorsand ligands. Many host genes are orchestrating this response. Genomics toolsoffer the opportunity to study the virus-host response at the molecular levelfor all genes in a parallel fashion.

We use genomics tools to study viral pathogenesis, to characterize host responses to (experimental) vaccines and to discover biomarkers for infection and/or disease. In a range of in vitro and in vivo model studies as well as in clinical studies mRNA profiling tools are combined with specific proteomics and metabolomics approaches. We have many different projects, so if you are interested, contact us!

Aim:The identification and characterization of gene interacting networks controllingthe host response to virus infections.

Techniques:Gene expression profilingLinux, R scriptingPrinciple component & cluster analysisOrthology Gene set analysis

Suitability for type of project:Students should be available for at least 6 months and should have experience or affinity withcomputer assisted data analysis, preferably using command line tools.

Contact details:Dr. Arno Andeweg, Viroscience, [email protected]. Henk-Jan van den Ham, Viroscience, [email protected]





Workgroup leader: Bernadette van den Hoogen ([email protected]) Department of Viroscience, Room Ee1767-A, tel: 010-70443330

Internship available for 1 person for at least 12 months


6 months No NO

12 months Yes Open/after consultation

18 months Yes Open/after consultation

Research in my group:1. Interaction between respiratory viruses and the innate immune system.2. Development of viro-immunotherapy with NDV for treatment of pancreatic

tumors

1. Interaction between respiratory viruses and the innate immune system.Rapid induction of type I interferon expression is a central event in the establishment ofthe innate immune response against viral infection, and requires the activation of multipletranscriptional proteins following engagement and signalling through Toll-like receptor-dependent and –independent pathways. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Within our group, we study how respiratory viruses,especially the human metapneumovirus (HMPV), subvert the innate immune system. In this study we generate and use recombinant viruses with deletions for particular genes, genomic tools, molecular tools, microscopic tools and bioassays to study the interaction between the virus and the innate immune system. Several research lines are explored to address this research question. Interested and motivated students are invited to contact me to discuss the different possibilities for their lab internship. Together, we will define a project that is in line with the interest and experience of the student.

2. Development of viro-immunotherapy with NDV for treatment of pancreatic tumorsOncolytic virus infection triggered immune responses are a critical component of viro-immunotherapy: T-cells are stimulated to respond to tumour-specific antigens derived from infected and dying tumour cells, thereby adding to the tumour killing capacity of OVs. For maximal viro-immunotherapy effect, a robust oncolytic effect is needed, which in itself provides anti-tumour benefit but also induces an abundant release of tumour antigens and pro-inflammatory cytokines, which ultimately provides the immunotherapeutic effect. As such, incorporation of immune-modulatory genes can enhance the immune effect of viro-immunotherapy. In this project, the effect of oncolytic NDVs on the immune response of pancreatic tumor cells will be determined. These cytokine profiles will aid to a rational design of recombinant viruses armed with immune-modulatory genes. As NDV poses a possible threat to poultry, we are generating attenuated viruses specifically for poultry while maintaining oncolytic efficacy for humans. In this project, these viruses will to be tested for oncolytic efficacy on tumor cells, and tested for attenuation in embryonated-chicken eggs.

During these internships

You will learn a wide range of techniques:



Cloning (parts) of viral genomes, mutagenesis, RT-PCR, sequencing, cell culture, virus culture, reverse genetics, transfection, (immune)-blotting, facs analysis

You will participate in:-weekly lab meetings with whole department of Virosciene-weekly lab meetings with the influenza/HMPV/Oncoltyic virus workgroup

References

HMPVvan den Hoogen BG, van Boheemen S, de Rijck J, van Nieuwkoop S, Smith DJ, Laksono B, Gultyaev A, Osterhaus AD, Fouchier RA.Excessive production and extreme editing of human metapneumovirus defective interfering RNA is associated with type I IFN induction.J Gen Virol. 2014 Aug;95(Pt 8):1625-33

Schildgen V, van den Hoogen B, Fouchier R, Tripp RA, Alvarez R, Manoha C, Williams J, Schildgen O. Human Metapneumovirus: lessons learned over the first decade.Clin Microbiol Rev. 2011 Oct;24(4):734-54

Oncolytic viruses

Buijs P, van Nieuwkoop S, Vaes V, Fouchier R, van Eijck C, van den Hoogen B.Recombinant Immunomodulating Lentogenic or Mesogenic Oncolytic Newcastle Disease Virus for Treatment of Pancreatic Adenocarcinoma.Viruses. 2015 Jun 11;7(6):2980-98

Buijs PR, Verhagen JH, van Eijck CH, van den Hoogen BG.Oncolytic viruses: From bench to bedside with a focus on safety.Hum Vaccin Immunother. 2015;11(7):1573-84.


Title: Identification and characterization of paramyxovirus cross-reactive T-cell responses

Department of Viroscience, group leader:Dr. Rik L. de SwartT: 010-7044280E: [email protected]: http://virosciencelab.org

Co-supervisor: Dr. Rory D. de Vries (postdoc)In collaboration with the department of Immunology (Prof. P. Katsikis, B. Bartol)


6 months No From Sept. 2018

9- 12 months Yes From Sept. 2018

18 months Potentially From Sept. 2018

BackgroundHendra virus (HeV) and Nipah virus (NiV) are zoonotic bat viruses that have been associated with disease outbreaks with high mortality in Australia and South-East Asia, respectively. Both viruses are members of the family Paramyxoviridae and cause severe neurological disease associated with case-fatality rates up to 75%. Surprisingly, few studies have been performed to characterize host immunity to henipavirus infections, and no vaccines are licensed for human use. The family Paramyxoviridae includes a significant number of other important human pathogens, including measles virus (MV), respiratory syncytial virus (RSV), human metapneumovirus (HMPV), mumps virus (MuV) and the parainfluenzaviruses (PIV1-4). Although serological cross-reactivity between these viruses is limited, we have preliminary evidence for cross-reactive memory T-cells targeting conserved regions of the viral proteins. Here, we propose to identify cross-reactive paramyxovirus epitopes, isolate paramyxovirus-specific T-cells, and characterize their potential to recognize and kill target cells infected with different viruses. This knowledge could be further developed into prophylactic (peptide vaccination) or therapeutic (adoptive T-cell transfer) applications.

Preliminary data

We have screened a panel of MV-specific human T-cell clones for cross-reactivity with different human and zoonotic paramyxoviruses. We identified a cross-reactive HLA-B*1501-restricted CD8+ T cell clone, and mapped the epitope to the viral fusion protein. More specifically, the epitope resided in the highly conserved viral fusion peptide, and was conserved in several other paramyxoviruses, including NiV and HeV. We generated HLA-B*1501 tetramers that were able to bind to the T-cell clone, and were used to stain and sort tetramer-specific T-cells from the blood of HLA-B*1501-positive donors. In addition, we also identified a cross-reactive HLA-DQ*0603-restricted CD4+ T-cell clone, which recognized a partially overlapping epitope in the same region of the viral fusion peptide. Using the tetramer, we were able to isolate tetramer-specific human T-cells from PBMC of a healthy human volunteer. These T-cells recognized MV-infected target cells. However, avidity testing with epitope variants from different paramyxoviruses suggested that the clones had not been induced by measles infection or vaccination, but by natural infection with PIV2 or PIV3.

http://virosciencelab.org/



Besides these fusion peptide-specific T-cells, based on paramyxovirus alignments, we have also identified another potential cross-reactive T-cell epitope in the viral nucleoprotein, which is restricted by HLA-A*0201. Peptides have been ordered, but specific cells directed against this epitope still need to be isolated.

These data led us to the hypothesis that the history and order of childhood paramyxovirus infections within an individual can be important in determining the level of cross-immunity to henipavirus (or potentially other paramyxovirus) infections in humans, provided they have an adequate HLA haplotype. Project overview

1. Identification of cross-reactive T-cell responses in HLA-B*1501 human donorsBased on our preliminary data, we propose test the frequency of tetramer-specific T-cells in a number of HLA-B*1501-positive donors. We have tetramers for six minor variants representing six different human paramyxoviruses of the previously identified CD8 epitope available. Tetramer-positive populations will be sorted by single cell sorting and the resulting clones will be tested for their avidity to the different epitope variants.

2. Identification of cross-reactive T-cell responses in HLA-A*0201 human donorsBased on our prediction data, we anticipate the presence of a cross-reactive T-cell epitope in the paramyxovirus N protein. Using synthetic peptides, tetramers and single cell sorting we will isolate specific T-cells and characterize their ability to recognize the minor variants of this epitope that are found in the different human paramyxoviruses.

3. Characterization of cross-reactive T-cellsAlthough initial screening of the reactivity of virus-specific T-cells is often performed using peptide stimulation and read-out of interferon-gamma production, the ultimate evidence for virus specificity must come from viral clearance or killing assays. We will evaluate the capacity of different paramyxovirus-specific T-cells to recognize and kill HLA-matched virus-infected cells.

Literature1. Haanen JB, Wolkers MC, Kruisbeek AM, Schumacher TN. Selective expansion of cross-reactive CD8(+) memory T cells by viral variants. J Exp Med. 1999 Nov 1;190(9):1319-28.2. Brehm MA, Pinto AK, Daniels KA, Schneck JP, Welsh RM, Selin LK. T cell immunodominance and maintenance of memory regulated by unexpectedly cross-reactive pathogens. Nature Immunology. 2002 Jul;3(7):627-34.3. Sewell AK. Why must T cells be cross-reactive? Nat Rev Immunol. 2012 Sep;12(9):669-77.4. Chiu C, McCausland M, Sidney J, Duh FM, Rouphael N, Mehta A, et al. Broadly reactive human CD8 T cells that recognize an epitope conserved between VZV, HSV and EBV. PLoS Pathog. 2014 Mar;10(3):e1004008.


Title: Susceptibility of innate lymphoid cells to measles virus infection

Department of Viroscience, group leader:Dr Rik L de SwartT: 010-7044280E: [email protected]: http://virosciencelab.org

Direct supervisor: Bri Laksono (PhD student)

In collaboration with:- Department of Pulmonary Medicine (Prof R Hendriks)


6 months No9- 12 months Yes 2017 - 2018

18 months No

BackgroundMeasles virus (MV) is a highly contagious virus that transmits via the respiratory route. It utilises the cellular receptors CD150 (expressed on subsets of immune cells) and nectin-4 (expressed on the basolateral side of epithelial cells) for cell entry. Hallmark of measles is the transient immune suppression that may last up to 3 years. At the same time, the disease is paradoxically associated with the induction of strong virus-specific immune responses that last for a lifetime. Previous studies in macaques infected with recombinant MV expressing the fluorescent protein EGFP showed that MV preferentially infects CD4+ and CD8+ memory T-cells, which have higher relative CD150 expression levels than their naive counterparts. Additionally, our current studies suggest that naive and memory B-cells are also highly susceptible to MV infection. We hypothesise that MV infects and subsequently depletes pre-existing CD150+ lymphocytes, which are replaced by MV-specific and bystander lymphocytes. This leads to a temporary immunological amnesia. We aim to study the susceptibility of additional human immune cell subsets to MV infection.Project overview Innate lymphoid cells have been described to play an important role in mediating and regulating immune responses. They belong to the lymphoid lineage but lack a B- or T-cell receptor. Their expression of CD150 and their possible susceptibility to MV infection have never been described. In collaboration with the Department of Pulmonary Medicine, we want to assess the susceptibility of these cells to MV infection, as an indicator of their possible role in the pathogenesis of measles. Multicolour flow cytometry will be performed to assess infection percentages.Literature1. de Vries RD, de Swart RL. Measles immune suppression: Functional impariment or numbers game?

PLoS pathogens. 2014;10(12):e1004482.2. Laksono BM, de Vries RD, McQuaid S, Duprex WP, de Swart RL. Measles Virus Host Invasion and

Pathogenesis. Viruses. 2016;8(8).3. de Vries RD, McQuaid S, van Amerongen G, Yuksel S, Verburgh RJ, Osterhaus AD, et al. Measles

immune suppression: lessons from the macaque model. PLoS pathogens. 2012;8(8):e1002885.4. Spits H, Cupedo T. Innate lymphoid cells: emerging insights in development, lineage relationships,

and function. Annual review of immunology. 2012;30:647-75.


Title: How does the innate immune system see measles virus?

Department of Viroscience, group leader:Dr Rik L de Swart T: 010-7044280E: [email protected]: http://virosciencelab.org

Direct supervisor: Bri Laksono (PhD student)

In collaboration with Dr B G van den Hoogen


6 months No9- 12 months Yes 2017 - 2018

18 months No

BackgroundIn spite of the success of existing live-attenuated measles vaccines in decreasing the number of measles cases globally, little is known about what makes them so effective and in which cells they replicate in vivo. Wild-type and attenuated measles virus (MV) strains utilise the cellular receptors CD150 (expressed on subsets of immune cells) and nectin-4 (expressed on epithelial cells) for cell entry. In addition to these two receptors, vaccine and laboratory-adapted MV strains can also utilise the molecule CD46 (expressed on every nucleated cell) as a cellular receptor. The efficient replication of attenuated MV strains in multiple cell types in vitro is a striking contrast with the limited replication of the same viruses in vivo. Studies in macaques infected with wildtype and vaccine MVs suggest that neither the wildtype nor the vaccine virus induces the production of type I IFNs, suggesting that the innate responses to MV are based on other mechanisms. We aim to study how the innate immune response recognises wild-type and attenuated MV strains.

Project overview In collaboration with the group of Bernadette van den Hoogen, we want to assess the infection efficiency of wildtype and attenuated MV in IFN-deficient and –competent cells, including primary cells and immortalized cell lines. Multicolour flow cytometry will be performed to assess infection percentages. The primary objective is to identify cell types that respond differently to wild-type and attenuated MV strains, and subsequently elucidate the molecular mechanism underlying this difference.

Literature

1. de Vries RD, Lemon K, Ludlow M, McQuaid S, Yuksel S, van Amerongen G, et al. In vivo tropism of attenuated and pathogenic measles virus expressing green fluorescent protein in macaques. Journal of virology. 2010;84(9):4714-24.

2. Shivakoti R, Hauer D, Adams RJ, Lin WHW, Duprex WP, de Swart RL, et al. Limited in vivo production of type I or type III interferon after infection of macaques with vaccine or wild-type strains of measles virus. JInterfCytokRes. 2015;35(4):292-301.

3. Rennick LJ, de Vries RD, Carsillo TJ, Lemon K, Van Amerongen G, Ludlow M, et al. Live-attenuated measles virus vaccine targets dendritic cells and macrophages in muscle of nonhuman primates. Journal of virology. 2015;89(4):2192-200.


Title: Norovirus: Determinants of antigenicity, tropism, and replication.Group leader: Prof. Dr. Marion Koopmans, Department of Viroscience

Dr. Miranda de Graaf, Department of Viroscience, room Ee1771T: +31 10 7043161E: [email protected]


6 months No12 months Yes December, 2017

18 months Yes December, 2017

Human noroviruses cause the majority of all non-bacterial gastroenteritis. Worldwide epidemics occur every 3 to 4 years and novel antigenically divergent strains frequently emerge. Despite the significant impact of noroviruses on global health and economy, surprisingly little is known about the ecology and epidemiology of noroviruses in wild and domestic animals. The Norovirus genus belongs to the family of Caliciviridae and is divided at least six (GI-GVI) different genogroups. These genogroups can be found in a wide range of hosts, such as humans, rodents, feline species, dogs, sea mammals, pigs, sheep, bats, and cattle.

Transmission can occur via direct contact with infected individuals, through inhalation of droplets produced by vomiting, and via contaminated food or water. Studies on human noroviruses were hampered due to the lack of cell-culture systems, but a novel cell culture model has recently been developed for norovirus.

To to study antigenic evolution a protein microarray for the detection of norovirus antibodies was developed. This protein microarray contains antigens (p-particles) representing human noroviruses belonging to the GI, II and IV genogroups and using this array we can distinguish antibodies specific to different norovirus genotypes.

Organoid cultures can be used to culture viruses and investigate replication kinetics. We are currently investigating differences in replication properties of different norovirus genotypes.

Objectives for internships To complement protein microarrays with P-particles and other norovirus antigens to

screen for “novel” noroviruses at the human-animal interface To investigate norovirus replication using organoid models

During these internships You will learn a wide range of techniques:Cell culture, virus culture, cloning, sequencing, western blotting, PCR, transfection, fluorescence microscopy, FACS, protein arrays, protein expression, protein arrays.You will participate in:

-weekly labmeetings with whole department of Virosciene-weekly labmeetings with the COMPARE group and Norovirus group-monthly labmeetings with Prof. M. Koopmans

Publication related to this research lineEmergence of a novel GII.17 norovirus – End of the GII.4 era?de Graaf M, van Beek J, Vennema H, Podkolzin AT, Hewitt J, Bucardo F, Templeton K, Mans J, Nordgren J,Reuter G, Lynch M, Rasmussen LD, Iritani N, Chan MC, Martella V, Ambert-Balay K, Vinjé J, White PA,Koopmans MP.Emergence of a norovirus GII.4 strain correlates with changes in evolving Blockade epitopes. Lindesmith LC, Costantini V, Swanstrom, J, Debbink K, Donaldson EF, Vinjé J, Baric RS.Serological evidence of influenza A viruses in frugivorous bats from Africa.Freidl GS, Binger T, Müller MA, de Bruin E, van Beek J, Corman VM, Rasche A, Drexler JF, Sylverken A, OppongSK, Adu-Sarkodie Y, Tschapka M, Cottontail VM, Drosten C, Koopmans M.


Examining changes in norovirus T -cell epitopes that accumulate in chronic infection

Matthew Cotten (direct supervisor), Georges Verjans, Marion Koopmans, EMC Viroscience Department

Studies on the immune responses that control human norovirus infections have focused on humoral responses to the virus with less attention on the cell-mediated immune responses. To explore the cellular immune selective pressure that might occur, we will examine the protein changes that accumulate in norovirus-specific T-cell epitopes encoded by norovirus during long-term chronic infection. Epitope changes that occur and persist are suggestive of T-cell mediated immune selective pressure.

A rich next generation sequencing (NGS) data set of sequential norovirus samples from chronic infections in 16 immunosuppressed patients is available. We will document changes in all predicted T-cell epitopes in all described CD4 and CD8 norovirus proteins that occur during chronic infection.

Steps of the project including the following:1. Generate a catalog of all predicted described CD4 and CD8 T-cell epitopes found in the noroviruses known to infect humans (GI, GII and GIV).

2. Document all described CD4 and CD8 T-cell epitopes present in the NGS norovirus data set from patients with chronic norovirus infections.

3. Document changes that occur in these T-cell epitopes during the observation period for each patient to test the initial hypothesis "Changes in T-cell epitopes occur during chronic norovirus infection".

The candidate needs good English skills, and the project requires a basic knowledge of virology and molecular biology and sufficient Python coding skills to run and manipulate existing code. Some knowledge of T-cell immunology, especially epitope databases and epitope predicting algorithms, is essential. A Python coding test will be given to all interested candidates as part of the selection process.


Title: Functional characteristics of human Zika virus-specific IgG responses

Workgroupleader: Dr. Barry Rockx, principal investigator Exotic virus workgroup, dept. of ViroscienceT: 010 70 44 279E: and [email protected] W: https://www.erasmusmc.nl/viroscience/research/fields-expertise/exotic-virus/

Supervisor: Gijsbert P. van Nierop, E: [email protected], T: 010 70 440 99

Duration Yes/No Available per (date)6 months No n.a.12 months Yes To be discussed18 months After consultation To be discussed

BackgroundZika virus (ZIKV) is a mosquito-borne flavivirus, and the cause of an epidemic of unprecedented scale in Middle/South America and the Caribbean. In the majority of cases, ZIKV results in a self-limiting disease with fever, rash and arthralgia/arthritis. However, severe complications have been observed including congenital abnormalities such as microcephaly, subcortical calcifications as well as GBS and other neurological complications in adults. Currently, no ZIKV vaccines or therapeutics are available.

The emergence of ZIKV in regions where a related flavivirus, dengue virus (DENV), is already endemic, presents an additional challenge regarding the potential effect of pre-exisiting immunity on disease severity. Sub-neutralizing concentrations and low-affinity cross-reactive IgG specific for structural ZIKV and DENV proteins can enhance infection, termed antibody-dependent enhancement (ADE). The dominant neutralizing targets for ZIKV-specific IgG (ZIKV-IgG) are conformational epitopes in the structural envelope protein (E). ZIKV-E and DENV-E are 54-59% homologous and most DENV-E-IgG cross-react with ZIKV-E, albeit in part with decreased binding affinity which promotes ADE and hampers vaccine development against ZIKV. What proportion of IgG has ADE properties, how ADE develops over time, differs between patients and affects clinical outcome is not known.

Combined with DENV-E, also the structural proteins pre-matrix (prM) and capsid (C), as well as West Nile virus, yellow fever virus (YFV) infection and YFV vaccination, need to be considered as potential ADE agents. Humoral responses towards non-structural (NS) target Ag and their functional relevance in ZIKV infected patients are ill defined. Characterizing ZIKV-NS-IgG responses is of added value as these Ag are likely unreceptive for ADE and may offer effective and safe vaccine targets, especially when ZIKV-NS-IgG trigger antibody-dependent cellular cytotoxicity (ADCC).

We hypothesize that ADE and ADCC is, apart from the IgG specificity and binding affinity, dependent on the glycosylation of the Fc tail. Previously defined recombinant human DENV-E-IgG clones are produced in exogenous cells. The Fc tail may therefore not be physiologically relevant due do cell type-specific post-translational protein modifications, limiting the relevance of functional characterization of these clones. Detailed knowledge of the antigenic breadth of ZIKV and functional characteristics of ZIKV-IgG may aid to identify safe candidate ZIKV vaccine targets, enable the development of therapeutic ZIKV-specific IgG and identify potential biomarkers for risk of severe disease.

Research aims:1. Determine the antigenic repertoire (E, prM, C, NS1, NS2A/B, NS3, NS4A/B and NS5) of human ZIKV-IgG over time using longitudinal patient blood samples, including naïve and flavivirus pre-immune samples in serum and subsequently at the clonal level.2. Determine the functional characteristics of ZIKV-IgG endogenously expressed by B-cells of infected patients and controls. These characteristics include virus (cross-)neutralizing potential, ADE, ADCC potential, binding affinity and Fc-tail glycosylation. Assayed flaviviruses include ZIKV, DENV, WNV and YFV.

Principal methods (pending status of the project): We will develop a platform to produce endogenous human IgG by immortalizing memory B-

cells using EBV or by overexpressing the human B-cell transcription factors Bcl-6 and Bcl-xL. A modular lenviral vector sytem to rapidly generate vectors for gene expression will be setup to

stably transduce human cells. Flavivirus Ag with molecular tags will be produced and purified to isolate Ag-specific B-cells

using flow cytometry.


https://www.erasmusmc.nl/viroscience/research/fields-expertise/exotic-virus/



B-cell receptor spectratyping will be used to assay the clonal composition of flavivirus-specific B-cell lines over time.

For the characterization of structural flavivirus-specific IgG responses ZIKV, DENV, WNV and YFV virus-like reporter particles (VRP) will be produced. Plaque reduction neutralization tests (PRNT), ADE, ADCC, and binding affinity assays will be setup.

For the characterization of non-structural protein specific IgG, ADCC, ELISA and ELISpot protein arrays and binding affinity assays will be setup.

The glycosylation of the Fc-tail of flavivirus-specific IgG will be determined using mass-spectrometry. Glycosylases will be used to functionally characterize the glycosylation of the Fc-tail.

Selected publications:

1. Bardina S V, Bunduc P, Tripathi S, et al. Enhancement of Zika virus pathogenesis by preexisting antiflavivirus immunity. Science (80-. ). 2017;356(6334):175–180.

2. Dejnirattisai W, Supasa P, Wongwiwat W, et al. Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat. Immunol. 2016;17(9):1102–8.

3. Dai L, Song J, Lu X, et al. Structures of the Zika Virus Envelope protein and its complex with a flavivirus broadly protective antibody. Cell Host Microbe 2016;19(5):696–704.

4. Kwakkenbos MJ, Diehl SA, Yasuda E, et al. Generation of stable monoclonal antibody – producing B cell receptor – positive human memory B cells by genetic programming. Nat. Med. 2009;16(1):123–128.

5. Geiling B, Vandal G, Posner AR, et al. A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo. PLoS One 2013;8(10):e76279.


NEUROSURGERY – RESEARCH TOPICS (ONCOLYTIC VIRUSES)


Title: Development of viro-immunotherapy strategies for treatment of malignant brain tumors

Group Leader: Martine LamfersDept of Neurosurgery, [email protected]

Co-supervisor: Jeroen de Vrij

BackgroundPatients with the primary malignant brain tumor glioblastoma (GBM) have a poor prognosis despite multimodal treatment with surgery, chemotherapy and radiation therapy. Research at the department of Neurosurgery is focused on the development of novel therapeutics for these highly aggressive tumors. One strategy is the application of oncolytic viruses (OV) that infect and replicate specifically in tumor cells. Clinical phase I/II trials testing the oncolytic adenovirus Delta24-RGD in recurrent GBM were recently completed at our department as well as at MD Anderson Cancer Center in Houston, Texas. Published data from our group shows that Delta24-RGD can efficiently lyse glioma cells (Lamfers et al). Apart from the direct oncolytic effect of Delta24-RGD, our group showed that Delta24-RGD therapy induces a specific T-cell mediated anti-tumor response in immune-competent glioma-bearing mice. However, this response was not elicited in 100% of the virus treated animals (Kleijn et al). The source of variation in response to OV therapy is related to two aspects: First, the susceptibility of tumor cells to virus-induced oncolysis by Delta24-RGD varies enormously. Intrinsic characteristics of the tumor are found to play a role in this susceptibility. Second, the immune response to the viral treatment was found to vary immensely. Both in preclinical animal models as well as in patients (unpublished data) treated with this virus, high variation in virus-associated (anti-tumor) immune parameters was detected. Current ongoing projects address both aspects with the intent of designing new strategies to improve viro-immunotherapy.

Research projectAt the dept of Neurosurgery a patient-derived glioma cell culture repository has been built, which now contains over 400 cell cultures. Drug screening protocols have been optimized to allow efficient and reproducible screening of compounds and viruses (see eg Berghauser Pont). Moreover, extensive molecular characterization of in total 80 cases from our database has been performed, including whole genome copy number analysis, RNA expression analysis and mutational profiling of 2000 cancer-related genes. We will employ a large panel of these patient-derived GBM cultures from our biobank to chart the variability in response to Delta24RGD-induced oncolysis and expand these studies with a panel of other clinically applied viral strains (based on HSV1, Reovirus, NDV and measles virus). With these experiments, we expect to define predictor profiles that allow identification of the most optimal viral strain for a specific tumor in terms of oncolytic potency. In addition, we will chart the innate response of each tumor in the panel to each of the viruses by determining the induction and secretion of a panel of cytokines and chemokines by ELISA. These results will allow us to gain insight into inter-tumoral virus susceptibility and variability in virus-induced release of immune activating cytokines. Whether specific cytokine/chemokine responses lead to improved anti-tumor efficacy will be studied in a syngeneic mouse model for glioma (see Kleijn et al)

Duration Yes/No Available per (date)6 months no9 months Yes To be discussed12/18 months Yes To be discussed

http://www.hersentumorcentrum.nl/



The results obtained in these studies will help identify profiles that predict response to a specific virus as well as molecules involved in enhancing this response. Such molecules may also form targets for incorporation in oncolytic viruses in order to develop a next generation of more potent OVs. In collaboration with Rob Hoeben, Leiden University we will design and construct improved oncolytic adenoviruses. We are seeking motivated students that have affinity with (oncolytic) viruses, immunology, (neuro) oncology and bioinformatica.

Techniques learned during internship: Patient-derived tumor (stem) cell culture, drug /virus screening, viability and cell death/apoptosis assays, virus infection and production techniques, viral engineering techniques, Western blotting, qPCR, transfection, fluorescence microscopy, FACS, protein arrays/ELISAs, and animal experiments (depending on duration traineeship)

You will participate in:-weekly meeting with the Neurosurgery Lab-weekly meetings with Cancer Institute (JNI meetings)-bi-weekly meetings with the neuro-oncology group of the dept of Neurology

Collaborations outside the institute:Leiden University (Rob Hoeben, Jacques van Dongen)Brigham and Women’s Hospital, Harvard Medical School, BostonMD Anderson Cancer Center, Houston Texas

Related publicationsLamfers ML, Grill J, Dirven CM, Van Beusechem VW, Geoerger B, Van Den Berg J, Alemany R, Fueyo J, Curiel DT, Vassal G, Pinedo HM, Vandertop WP, Gerritsen WR.Potential of the conditionally replicative adenovirus Ad5-Delta24RGD in the treatment of malignant gliomas and its enhanced effect with radiotherapy. Cancer Res. 2002 Oct 15;62(20):5736-42. PubMed PMID: 12384532.

Kleijn A, Kloezeman J, Treffers-Westerlaken E, Fulci G, Leenstra S, Dirven C, Debets R, Lamfers M. The in vivo therapeutic efficacy of the oncolytic adenovirus Delta24-RGD is mediated by tumor-specific immunity. PLoS One. 2014 May 27;9(5):e97495. doi: 10.1371/journal.pone.0097495. PubMed PMID: 24866126; PubMed

Balvers RK, Belcaid Z, van den Hengel SK, Kloezeman J, de Vrij J, Wakimoto H, Hoeben RC, Debets R, Leenstra S, Dirven C, Lamfers ML. Locally-delivered T-cell-derived cellular vehicles efficiently track and deliver adenovirus Delta24-RGD to infiltrating glioma. Viruses. 2014 Aug 12;6(8):3080-96. doi:10.3390/v6083080. PubMed PMID: 25118638

Berghauser Pont LM, Kleijn A, Kloezeman JJ, van den Bossche W, Kaufmann JK, deVrij J, Leenstra S, Dirven CM, Lamfers ML. The HDAC Inhibitors Scriptaid andLBH589 Combined with the Oncolytic Virus Delta24-RGD Exert Enhanced Anti-TumorEfficacy in Patient-Derived Glioblastoma Cells. PLoS One. 2015 May18;10(5):e0127058. doi: 10.1371/journal.pone.0127058


RIVM – LABROTATIONS & RESEARCH TOPICS

Will be completed later.


PULMONARY MEDICINE – LAB ROTATIONS & RESEARCH TOPICS


Immunity of the lung – General project overviewDepartment of Pulmonary Medicine

Further informationProf. dr. Rudi Hendriks, Head of the Research Laboratory, Tel. 010-7043700, e-mail: [email protected]

Duration Yes/No Available per (date)6 months Yes/No See individual projects12 months Yes/No See individual projects18 months Yes/No See individual projects

BackgroundThe adaptive immune response provides humans with B and T lymphocytes that carry immunoreceptors that are highly specific for antigens that these cells have not encountered before. Dendritic cells (DCs) are professional antigen-presenting cells (APC) that are located at sites where maximal microbial encounter occurs. They are essential for the transport of antigens (from the airway mucosa and interstitium to the draining lymph nodes) and thereby initiate the activation of lymphocytes. In this way, the adaptive immune system has the capacity to strengthen and to regulate the innate defense mechanisms and to build specific immunological memory so that subsequent challenges with the same pathogen are efficiently overcome. The lung is continuously exposed to the outside world and is a portal of entry for viral, bacterial, and fungal infection. Throughout evolution, an extensive defense mechanism has been developed that protects humans from these potentially lethal assaults and at the same prevents reactivity to harmless antigens. Nevertheless, the incidence of lung diseases such as asthma and sarcoidosis has risen dramatically over the last 50 years and pneumonia is a leading cause of death among young children and the elderly worldwide.

Research The research aims in the Department of Pulmonary Medicine comprise:

To elucidate the role of DCs in directing and maintaining an acute or chronic localized immune response in the lung, e.g. in influenza virus infection, in asthma or in sarcoidosis and other interstitial lung diseases.

To develop DC-based immunotherapy with tumor-pulsed autologous DCs to prevent mesothelioma recurrences. This approach is based on the observation that DCs are able to induce protective immunity in a mouse model of mesothelioma. In this context, we also aim to investigate how the effects of negative regulators of these responses, including myeloid-derived suppressor cells and regulatory T cells can be dampened.

To study the role of effector T cells and innate lymphoid cells in various pathological conditions, including asthma, sarcoidosis, influenza infection and pneumonia.

To identify the molecular mechanisms involved in B cell repertoire selection and B cell activation, both in autoimmunity, such as SLE and Sjogren syndrome and in infection, e.g. with influenza virus.

Molecular approaches become increasingly important to unravel the pathogenesis of pulmonary diseases. Over the last years technical expertise has been acquired in mouse models for various diseases, including asthma, influenza, interstitial lung disease and lung berylliosis. Numerous animal models have been generated with specific immune defects (in DC, or activation or development of B, T or innate lymphocytes). In addition, several projects in the lab include human studies, using patient material. We make use of various assays for humoral and cellular immunity, multi-color flow cytometry and cell sorting, immunohistochemistry, molecular biology, genome-wide expression and epigenetic analyses, chromatin immunoprecipitation, protein biochemistry and retroviral transduction.


CollaborationsWe have fruitful collaborations with various research groups at the Erasmus MC (Departments of Cell Biology, Genetics, Virology, Immunology, Pediatrics) and outside the Erasmus MC (University of Ghent, Belgium; University of Freiburg, Germany; Univeristy hospital Ulm, Germany; Medical University of Vienna, Austria; Institut Pasteur, Paris, France).

ProjectsProjects are available in various research groups in the laboratory, although many of these only from 2018 onwards.


Pulmonary hypertension: a decisive role for the A20 enzyme in dendritic cells

Department of Pulmonary Medicine, project 1

Workgroup leaderDr. M. KoolRoom Ee2224Tel: 010-70437884e-mail: [email protected]

Duration Yes/No Available per (date)6 months Yes Autumn 201712 months Yes Autumn 201718 months Yes Autumn 2017

BackgroundPulmonary arterial hypertension (PAH) comprises a grouping of orphan diseases historically associated with a poor prognosis. A number of diseases can result in PAH, however, in a significant minority there is no cause found and these patients are labelled idiopathic pulmonary arterial hypertension (IPAH). Histologically, IPAH is characterized by arteriopathy of the distal pulmonary arteries, associated with muscularization, concentric intimal thickening, and areas of disordered neovascularisation termed plexiform lesions. IPAH is a debilitating disease characterized by an increase in pulmonary vascular resistance that leads to right ventricle (RV) failure and eventually death. Despite the impressive recent track record of new therapies, the impact on patients’ long-term outcomes has been modest at best and with currently available treatment the 5-year survival is still only 61.2% for newly diagnosed patients.

The strong association of PAH with dysregulated immunity and inflammation has been long established with auto-immune diseases, most prominently scleroderma, but also notably rheumatoid arthritis, SLE, and mixed connective tissue disease. Auto-immune diseases in particular are therefore recognised as causally associated with PAH, and the presence of auto-antibodies has been recorded in IPAH with 40% of patients demonstrating antinuclear antibody and another 10–15% of those patients may express anti-phospholipid antibodies. Pathologically, within the pulmonary vascular lesions, there is accumulation of inflammatory cells including dendritic cells (DCs), T and B cells with altered T regulatory cell function. Tertiary lymphoid organs (TLO) are found around pulmonary arteries in patients with IPAH- evidence of altered immune regulation. TLOs are speculated to be important inductive sites for self-reactive T and B-cells. The presence of TLOs in models of chronic autoimmunity and their association with tissue destruction has led to the suggestion that they are important inductive sites for self-reactive T cells and autoantibodies that contribute to pathology.

DC activation is essential for their effector functions, e.g. antigen presentation and cytokine/chemokine production. Manipulating DC activation is important for the delicate balance between (auto) immunity and tolerance. We recently demonstrated that the A20-enzyme, an essential negative regulator of the NF-κB pathway, in DCs critically controls inflammation. The contribution of DCs and its activation regulator A20/TNFAIP3 to the development in IPAH is poorly examined and warrants further investigation. We investigate how the activation status of DCs influences immunity/tolerance through A20/TNFAIP3 expression in the development of pulmonary hypertension as well as in other autoimmune diseases.


ResearchIn this project, we want to investigate the role of NF-κB signalling in DCs in pulmonary hypertension, which could be essential for directing personalized therapeutic approaches.Our research lines are:

1) Characterizing blood DCs of different types of PAH patients and investigate activation status, T cell priming capacity, and A20 expression/NF-κB activation

2) Studying the phenotype of hyper-activated A20/Tnfaip3-deficient conventional DCs and their T cell priming capacity in the context of pulmonary hypertension.

3) Examine how DCs affects blood vessel remodelling, the start of development of pulmonary hypertension.

Master of science project (6/12/18 months)Students interested in fundamental/translational research who would like to support us in gaining more insight into the role of A20 in DC biology, its function in pulmonary hypertension physiology and pathology.

Master students interested in a 12-month or 18-month Master of Science project can take an animal course (Art. 9). As a Master student, you will be working at the Department of Pulmonary Medicine on the research projects outlined above, together with a PhD student or technician, under the supervision of Mirjam Kool. As these research projects comprise multiple research (sub) questions, you will be focusing during your project mostly on a well-defined part of the research line.

As the project demands an experimental approach that is both fundamental and disease-related, you will have the opportunity to learn and use a whole variety of techniques, including techniques for RNA analysis (RT-PCR), protein analysis, ex vivo analysis of DCs, B, T and innate lymphocytes using advanced flow cytometry (staining of extracellular markers, intracellular molecules such as cytokines, etcetera), immunohistochemistry on lymphoid organs and lungs as well as confocal analysis, and quantification using different assays (ELISA), T cell culture systems, and disease models. Furthermore, the project can contain cell culture systems, working with laboratory animals and/or patient material. Concerning this research project, there is a close collaboration with the group of prof. B. Lambrecht (VIB, Ghent, Belgium) and prof. R. Beyaert /G. van Loo (VIB, Ghent, Belgium) and dr. Mark Toshner and prof Nick Morrell (Cambridge University, UK).


PDE3 inhibition by enoximone suppresses allergic airway inflammation in a house dust mite asthma model. What is the underlying mechanism?


Contact informationDr. A. KleinJane-mail: [email protected]

Workgroup leaderProf. Dr. R.W. Hendriks (Head of research department)Telephone 010-7043700e-mail: [email protected]

Duration Yes/No Available per (date)6 months Yes December 201712 months Yes September 201818 months Yes Autumn 2017

BackgroundAllergic asthma is a chronic lung disease characterized by airway hyperreactivity (AHR) and inflammation in response to inhalation of airborne allergens, such as house dust mite (HDM), molds or animal dander. Although asthma is a complex disease, studies in patients and animal models have shown that enhanced activation of T helper-2 (Th2) cells and their cytokines explain many disease hallmarks. These include immunoglobulin E (IgE) synthesis (IL-4), airway eosinophilia (IL-5), mast cell accumulation (IL-9), goblet cell hyperplasia (IL-4, IL-13) and AHR (IL-13).

Interestingly, a recent status asthmaticus case report showed the beneficial use of the phosphodiesterase III (PDE 3) inhibitor enoximone (Beute, Br. J. Anesthesia 2014). Enoximone is a generic drug and is still in use as a therapy for heart failure to improve cardiac output. One side effect is relaxation of smooth muscle cells that results in an immediate bronchodilatation. Although enoximone clearly improves allergic complaints, the underlying mechanism of the beneficial use of PDE3 inhibition in asthma remains unknown.

Structural cells of the lung and immune cells express PDE3. PDE3 inhibition has various effects including relaxation of smooth muscle cells including bronchodilation. However, PDE3 inhibition appears to go further than a mere inhibitory activity on bronchial smooth muscle cells. It also affects structural cells and, more importantly, it creates an improved barrier function of epithelial and endothelial cells. Enoximone therefore strengthens the immune barrier, but in addition it may influence cells of the immune system, because these also express PDE3. Therefore, many aspects of asthma related pathophysiology seem to be affected by PDE3 inhibition.

ResearchThe effects of PDE3 inhibition on human immune cells will be analysed in vitro. Human peripheral blood mononuclear cells obtained from house dust mite (HDM)-sensitized patients and non-sensitized patients will be stimulated with HDM in the absence and presence of PDE3 inhibitors and other compounds that influence the intracellular levels of cAMP. Analysis of cytokine production (ELISA’s on supernatant and intracellular FACS stainings) will be performed as readouts.

Mast cells and basophils are key cells in allergic inflammation responsible for the immediate allergic reaction in allergic patients including allergic asthma. Mast cells bear IgE on their surface. When mast cells bind an allergen via allergen specific IgE on their surface, cross-linking of the IgE receptors occurs, resulting in mast cell degranulation. Mast cell degranulation is a Ca 2+ dependent effect and is associated with the activation of small G proteins that cause actin polymerization and the relocalisation of actin. So far, little is known about the role of the second messenger cAMP in mast cells and the breakdown inhibition of cAMP. cAMP is a intracellular messenger, the signal is truncated by cAMP degradation by PDE3. In the current projects we want to investigate the role of the cAMP pathway and the


involvement of PDE3 in mast cell degranulation. Intervention will be performed with a PDE3 inhibitor. The role of PDE3 in the barrier function of epithelial cells and endothelial cells will be investigated in culture systems, and in vivo experiments that include a HDM mouse model for allergic airway inflammation in wild-type and various KO mice, including KO for PDE3a and PDE3b. The available project will currently largely focus on experiments in human.

Master of Science projects (6/12/18 months)Students interested translational research who would like to support us in gaining more insight into the mechanism of action of PDE3 inhibition in human and mouse are invited to participate. As a Master student, you will be working at the Department of Pulmonary Medicine on the research projects outlined above, together with A. KleinJan, PhD. As these research projects comprise multiple research (sub)questions, you will be focusing during your project mostly on a well-defined part of the research line. The project demands a translational experimental approach, and therefore you will have the opportunity to learn and use a whole variety of techniques, including techniques for ex vivo analysis of many cells of the immune system using advanced multi-color flow cytometry (staining of extracellular markers, intracellular molecules such as cytokines, etc.), immunohistochemistry on lymphoid organs and lungs and quantification using different assays (ELISA), cell culture systems, and induced disease models, in particular HDM-induced allergic asthma.


Modulation of the immune system for personalized cancer treatment in patients with thoracic malignancies


Contact information:

Workgroup leaderProf. Dr. R.W. Hendriks (Head of research department)Telephone 010-7043700e-mail: [email protected]

Duration Yes/No Available per (date)6 months Yes December 201812 months Yes September 201818 months Yes To be discussed

BackgroundImmunotherapy is a very promising approach in the treatment of cancer. It tries to harness the potency and specificity of the immune system to attack cancer cells, aiming for a non-toxic treatment with minor side-effects and a long-lasting immunological memory. One approach of immunotherapy uses dendritic cells (DC) to present tumour-associated antigens (TAA) and thereby generate tumour-specific immunity. DC are extremely potent antigen-presenting cells specialized for inducing activation and proliferation of CD8+ cytotoxic T lymphocytes (CTL) and helper CD4+ lymphocytes.

We study malignant pleural mesothelioma (MM) as a model. This fatal disease has a median survival of < 1 year. The incidence of mesothelioma is closely associated with the inhalation of airborne asbestos fibers. Although in most developed countries the production and usage of asbestos is prohibited, due to the long interval between exposure to asbestos and the onset of disease, incidences of mesothelioma is still increasing. For the Netherlands and most other European countries, the peak is predicted > 2020. Currently 750 patients are diagnosed each year in the Netherlands.

Earlier we investigated the effect of DC-based immunotherapy on the outgrowth of mesothelioma in a murine model. It was established that DC-based immunotherapy induced strong tumour-specific CTL responses leading to prolonged survival. However, the effect of immunotherapy was dependent of the tumour load. The most beneficial effects were established at early stages of tumour development. This is in agreement with our current knowledge on the effects of immunotherapy in other tumour types.

On the basis of these preclinical animal studies, we completed a clinical trial of the intradermal and intravenous administration of autologous tumour lysate-pulsed DC in mesothelioma patients after cytoreductive therapy with chemotherapy. Patients received 50x106 DC pulsed with tumour lysate and KLH every two weeks for a total of three injections. It was shown that DC vaccination in patients was safe with moderate fever as the only side effect. There were no grade 3 or 4 toxicities associated with the vaccines or any evidence of autoimmunity. Local accumulations of infiltrating T cells were found at the site of vaccination. Importantly, distinct immunological responses on the surrogate marker KLH were induced by the vaccinations, both in vitro as in vivo as well as anti-tumour activity against autologous tumour cells in the blood of some patients after the vaccinations. Currently we are finalizing a dose-finding study in patients using DC-immunotherapy and are preparing for a large phase II/III trial with this established safe dose.

ResearchTumour cells create an immunosuppressive environment that can lead to a down-regulation of the anti-tumour immunity. Our research lines focus on different types of suppressive cells present in the tumour microenvironment of mesothelioma and lung cancer tumours; that seems instrumental in allowing a growing tumour to evade immunological attack. For this, we investigate regulatory T cells (Tregs), myeloid derived suppressor cells (MDSC) and in


particular tumour-associated macrophages (M2 macrophages), both locally within the tumor as well as in peripheral blood. Changes in these cell numbers and their activity may predict tumor progression or Measures that can overcome the suppressive function of these cells might be used in combination with DC immunotherapy to increase the success rate of tumour eradication. For this we use state of the art techniques (e.g. multi-colour flow cytometry, in vivo imaging using luciferase), both in-vitro (cell culture) and in-vivo (mice and human) studies; immunohistochemical techniques, Q-PCR and ELISA; amongst others.

Master of science project (6/12/18 months)Students interested in direct translational research who would like to support us in gaining more insight into the different aspects of cancer (in particular the immunosuppression and immunotherapy treatment) are kindly invited to participate. Different aspects and procedures for patient tailored therapy with autologous cell products will also be explained. Currently, there is a MSc student on this project, and we expect new possibilities for a research project from September 2018 onwards.


B cell receptor (BCR) signaling in auto-immune disease


Contact informationOdilia CornethEmail: [email protected]

Workgroup leaderDr. R.W. Hendriks (Head of research department)Room Ee2251a Telephone 010-7043700Email: [email protected]


BackgroundB cells are key players in the pathogenesis of rheumatic auto-immune diseases including rheumatoid arthritis (RA), Sjögren’s syndrome (SjS) and systemic lupus erythematosus (SLE). Upon differentiation into plasma cells, B cells contribute to auto-immune pathology in these diseases by producing highly pathogenic auto-antibodies directed against IgG (rheumatoid factor), citrullinated proteins (-ccp), and nuclear antigens (ANAs). In addition, B cells augment auto-immunity by presenting self-antigens to T cells and by producing cytokines that polarize and enhance autoreactive T cell responses. The central role of B cells in establishing auto-immune diseases has overtly been demonstrated by the highly effective treatment of patients with B cell depletion therapy (Rituximab). In healthy individuals, the appearance of fully differentiated B cells that express self-reactive B cell receptors (BCRs) is mostly prohibited by means of negative selection at multiple stages during B cell differentiation. In RA, SjS and SLE patients however, differentiating self-reactive B cells fail to be removed or regulated at these counterselection checkpoints, leading to increased numbers of mature autoreactive B cells and plasma cells that constitute auto-immune disease. Apart from defects in the counterselection and regulation of autoreactive B cells, it has become increasingly clear that circulating B cells in patients with systemic autoimmunity exhibit enhanced BCR signaling. Both in transgenic SLE mouse models and in patients similar genetic defects have been identified that impair the signaling of several inhibitory receptors in B cells, leading indirectly to B cell hyperresponsiveness upon antigen binding to the BCR. Remarkably, it is largely unknown whether genetic defects that directly affect signaling molecules downstream of the BCR are involved in the pathogenesis of SLE, SjS or RA. Especially the possible effects of overexpression of Bruton’s tyrosine kinase (BTK), a critical BCR signaling molecule, on systemic auto-immunity remain to be elucidated.

Research

The effect of BTK overexpression on auto-immune disease:To study the effects of B cell specific overexpression of BTK on mature B cell differentiation and on the development of auto-immunity, transgenic mice have been generated that overexpress BTK either throughout B cell differentiation or only during final B cell maturation. Mature B cells that overexpress BTK display enhanced Ca2+ influx and increased membrane expression of activation markers upon BCR stimulation in vitro. Moreover, BTK-transgenic mice exhibit increased numbers of activated B cells, germinal center B cells and plasma cells, and aging BTK-transgenic mice spontaneously develop an SLE/SjS-like auto-immune disease characterized by high anti-nuclear auto-antibody levels, immune complex mediated glomerulonephritis, and systemic vasculitis affecting lungs, salivary glands and kidneys.


Master of Science projects (6 months)As a MSc student, you will be working at the Department of Pulmonary Medicine on the research projects outlined above under the supervision of a post-doc (see ‘Contact details’ below). As these research projects comprise multiple research (sub)questions, you will be focusing during your research project mostly on a well-defined part of the project. Because the project on BCR signaling in auto-immune disease demands an experimental approach that is both fundamental and disease-related, you will have the opportunity to learn and use a whole variety of techniques, including techniques for DNA analysis, RNA analysis (RT-PCR), protein analysis, ex vivo analysis of B lymphocytes using advanced flowcytometry (staining of extracellular markers, intracellular molecules such as cytokines, measuring of Ca2+

influx upon B cell stimulation, etcetera), immunohistochemistry on lymphoid organs and other tissues (kidneys, lungs), (auto-)antibody detection and quantification using different assays (HEp2 cell stainings, ELISA), B cell culture systems, and induced disease models such as CIA. In the fields of B cell immunology and rheumatic diseases, close collaborations of the department exist with research groups at local, national and international level, to ensure the exchange of the most up-to-date knowledge and techniques. Concerning this research project, there is a close collaboration with the Department of Rheumatology (with regular joint meetings). In addition, we collaborate with research groups in the Departments of Immunology and Cell Biology at the Erasmus MC, the Max-Planck-Institute for Immunology in Freiburg (Germany) and the Medical University of Vienna (Austria).Currently, there is a MSc student on this project, and we expect new possibilities for a research project from September 2018 onwards.


Innate lymphoid cells: identification of their role in allergic asthma and exacerbations upon respiratory tract infection


Contact information

Prof. Dr. R.W. Hendriks (Head of research department)Telephone 010-7043700e-mail: [email protected]. Ralph Stadhouders; [email protected]


BackgroundAsthma is a chronic lung disease characterized by reversible airway obstruction, hyperresponsiveness and pulmonary inflammation. Classically, asthma is thought to arise from a T helper 2 (Th2) immune response to allergens, such as house-dust mite (HDM). Th2 cells produce vast amounts of cytokines that induce IgE (interleukin-4; IL-4), recruit eosinophils and mast cells (IL-5 and IL-9) and cause smooth muscle hyperreactivity and goblet cell hyperplasia (IL-13). An important aspect of asthma that has never been adequately addressed by the Th2 paradigm is the observation that respiratory pathogens, which can engender robust innate and/or Th1 responses, can initiate and promulgate pulmonary inflammation and airway obstruction in asthma. Rhinovirus, RSV and influenza virus and Mycoplasma pneumoniae bacteria are common causes of asthma exacerbations. Interestingly, a novel non-T/non-B lymphoid cell population, named group 2 innate lymphocytes (ILC2s), producing high amounts of IL-5 and IL-13 was recently discovered. Unlike Th2 cells, ILC2s are not antigen-restricted. ILC2s are activated by epithelial cell-derived cytokines IL-25 and IL-33 and were recently shown to accumulate in the lung after influenza infection in mice, thereby inducing hyperreactivity and promoting tissue repair, dependent on the epidermal growth factor-family member amphiregulin. In human, ILC2s were reported to be enriched in nasal polyps of chronic rhinosinusitis patients. This project is based on our recent finding that ILC2s are involved in allergic airway inflammation in mice. Intranasal administration of IL-25 or IL-33 induced an asthma phenotype and the accumulation in the lungs of ILC2s producing large amounts of IL-5 and IL-13. Importantly, in HDM or ovalbumin-induced asthma the contribution of ILC2s to the total population of IL-5+ or IL-13+ cells in the lung was in the same range as for classical Th2 cells. We also found increased ILC2s in bronchoalveolar lavage at day 18 after infection with H3N2 influenza virus in mice. Using Affimetrix gene chip expression profiling of total lungs during influenza infection, we observed induction of IL-13, IL-33R and amphiregulin. Interestingly, amphiregulin has the capacity to upregulate mucin expression and in increased in sputum of chidren with asthma.

ResearchThe findings described above provide a solid basis to test the hypothesis that ILC2 play an essential role in asthma pathogenesis and may comprise the long sought for link between respiratory tract infection and asthma exacerbation. In this project, we will investigate in a HDM-induced allergic airway inflammation model in mice (1) how ILC2s collaborate with Th2 cells, using cell transfer approaches and confocal microscopy and (2) how an influenza virus infection affects ILC2 activity and asthma symptoms. In these experiments we will determine the effects of treatment with antibodies to the IL-25 and IL-33 receptors and determine genome-wide expression profiles of ILC2s. (3) We also plan to investigate whether ILC2 are increased in bronchial mucosal biopsies or peripheral blood of asthma patients. Collectively, these experiments address the involvement


of ILC2s in asthma and exacerbations and may result in the identification of new therapeutic targets or biomarkers.

Finally, we recently generated epigenomic profiles of in vivo activated ILC2s and Th2 cells from mice with allergic airway inflammation and found largely overlapping patterns of H3K4Me2 at loci controlling type-2 immunity. The few Th2-specific gene regulatory elements (GREs) predominantly regulate genes implicated in T cell receptor signaling and co-stimulation. The surprisingly richer ILC2-specific active epigenome consists of distal GREs and novel superenhancers near genes involved in allergy, metabolism, immune signaling and cellular plasticity. Moreover, transcription factor binding landscapes at GREs were markedly different between ILC2s and Th2 cells. Importantly, shared and ILC2-specific - but few Th2-specific - GREs reside in asthma-associated loci in human and murine cells, prioritizing GREs potentially relevant for asthma and supporting a pathogenic role for ILC2s in human asthma.

Master of Science projects (6/12/18 months)Students interested in fundamental/translational research who would like to support us in gaining more insight into the different aspects of asthma exacerbation and/or physiological relevance of the newly identified population of innate lymphocytes are kindly invited to participate. Master students interested in a 12-month or 18-month Master of Science project can take an animal course (Art. 9). As a Master student, you will be working at the Department of Pulmonary Medicine on the research projects outlined above, together with a post-doc and the head of the research department (see ‘Contact details’). As these research projects comprise multiple research (sub)questions, you will be focusing during your project mostly on a well-defined part of the research line.

As the project demands an experimental approach that is both fundamental and disease-related, you will have the opportunity to learn and use a whole variety of techniques, including techniques for RNA analysis (RT-PCR), protein analysis, ex vivo analysis of B, T and innate lymphocytes using advanced multi-color flow cytometry (staining of extracellular markers, intracellular molecules such as cytokines, etcetera), confocal immunofluorescence microscopy, immunohistochemistry on lymphoid organs and lungs and quantification using different assays (ELISA), T cell culture systems, and induced disease models, in particular HDM-induced allergic asthma and lung influenza virus infection. Moreover, the project may include molecular analyses in the field of epigenetics and bioinformatics, both on human and on mouse ILC2 or Th2 cells.

We have obtained mouse models to specifically identify ILC2 using a YFP reporter through a collaboration with Dr. J. Fehling (Ulm, Germany). Human ILC2 work is done in collaboration with prof. J. di Santo (Paris, France).

Currently, there is a MSc student on this project, and we expect new possibilities for a research project from September 2018 onwards


PAEDIATRIC GASTRO-ENTEROLOGY – LABROTATIONS & RESEARCH TOPICS


Title: Research line: Mucosal immune regulation

Workgroup leader: Janneke Samsom (Department Pediatrics division Gastroenterology and nutrition)Email addresses: [email protected]


6 months Yes/No12 months Yes/No18 months Yes/No

The aim of the research performed in the Samsom lab is to unravel the mechanisms that underlie chronic intestinal disease such as celiac disease and inflammatory bowel disease.The mucosal surfaces of our gastrointestinal tract are continuously exposed to a large variety of foreign antigens, such as pathogens that enter the body at these sites but also harmless antigens including food proteins and constituents of commensal bacteria. Although inflammatory immune responses are needed to eliminate transient encounter with invasive bacteria and protect the body from subsequent infection, active immunity to frequently encountered harmless substances would be detrimental due to chronic tissue damage. To avoid such damage the default immune response to harmless soluble proteins that are encountered via the mucosa is mucosal tolerance. Loss of mucosal tolerance leads to chronic inflammatory disorders of the intestine such as celiac disease (CD) caused by an intolerance to the dietary protein gluten and inflammatory bowel diseases (IBD), that are driven by an aberrant response to bacterial components. Both CD and IBD are characterized by the activation of inflammatory effector T-cell (Te-cell) responses in the lamina propria of the gut causing loss of barrier integrity and severe tissue destruction leading to loss of gut-function. Unfortunately little is known of why these diseases occur. By combining basic science with patient research we aim to identify initial immune mechanisms that are causative for the loss of mucosal tolerance.

The following techniques are in general use: - Flow cytometry- Real-time PCR- Immunohistochemistry- Cell-culture - Magnetic and fluorescent based cell purification- ELISA- Animal models for intestinal disease

Students will participate in the weekly research meetings of the department of Pediatrics, the mucosal immunology group meetings and the pathology meetings.

Master students doing a 6, 12 or 18 month Master of Science project will be able to choose from different research topics, including: - Understanding celiac disease pathogenesisCeliac disease (CD) is an inflammatory disease of the small intestine caused by intolerance to gluten-proteins that are constituents of wheat and rye. CD is a frequent disorder as it affects between 0.5 and 1% of the western population. Due to severe inflammation small intestinal villi, that are required for nutrient uptake, regress leading to malnutrition. Although CD symptoms can be treated by avoiding gluten, the disease has increasingly recognized co-morbidities such as diabetes and cancer leaving patients and clinicians with new therapeutic problems. Although CD has a clear genetic predisposition: virtually all patients are HLADQ2 it is unclear what causes CD development. Using patient biopsies and a unique murine model for CD we are elucidating the first immune events that characterize CD. There is room to


choose an individual subject within this project. The project involves all techniques mentioned above.

- mucosal immune regulation in IBDThe two main forms of inflammatory bowel disease (IBD) are Crohn’s disease (CD) and ulcerative colitis (UC). Both diseases are characterized by a dysregulated mucosal immune response to microbial products leading to chronic inflammation of the intestinal tract. My lab has focused on identifying aberrancies within the intestinal immune network that control early immune responses in pediatric IBD. This research has led to the discovery that inflammatory T cells that infiltrate early intestinal lesions abundantly secrete interleukin-21 (IL-21). This happens not only in IBD, but also in pediatric celiac disease, a chronic small intestinal inflammation driven by an aberrant T cell response to gluten peptides (van Leeuwen et al 2013). The cytokine IL-21 has dual functions as it has potent immune-regulatory function by inhibiting antigen presenting cells and eliciting regulatory cytokine (IL-10) secretion but can also potently augment inflammatory T cell cytokine (IFN-γ/IL-17) production and humoral immunity. However, it is unclear which pathways determine whether the inflammatory activity or regulatory activity of IL-21 prevails. We hypothesize IL-21-driven responses play a major role in intestinal tolerance and inflammation, and that deregulation of IL-21 is an initiating event in IBD. Currently it is unclear whether there is heterogeneity in IL-21 production in the intestine amongst patients during onset of IBD. Moreover, it is unclear whether IL-21 production is related to the localization of disease, severity of disease or response to therapy. The principal aim of this study is to determine IL-21 production in biopsies of IBD patients at time of diagnosis and identify whether a particular disease profile is associated with enhanced IL-21 release. There is room to choose an individual subject within this project. Students will participate in the weekly research meetings of the department of Pediatrics, the mucosal immunology group meetings and the pathology meetings.


Title: Respiratory tract infections in children

Workgroupleader:Dr. WWJ. Unger, [email protected] (Laboratory of Pediatrics)

Duration Yes/No Available per (now)



18 months No depending on project

BackgroundIn children, pneumonia is the most common invasive infection of the respiratory tract and the number one cause of mortality in children worldwide. For proper treatment of patients, it is important to rapidly define the pneumonia-causing pathogen. However, current non-invasive diagnostics tests, including microbiological tests and imaging technologies have no or limited value as they do not reveal whether the pneumonia is of bacterial or viral origin. Since these tests merely demonstrate the presence of a pathogen they also do not distinguish between pathogens that are infective and those that harmlessly exist in the airways without invading and inducing pneumonia (i.e. “carriage”). Streptococcus pneumoniae (Sp) and Mycoplasma pneumoniae (Mp) are the major causes of childhood bacterial pneumonia4,5.Clinicians thus have to rely on non-specific clinical symptoms to decide whether to prescribe antibiotics. However, the majority of lung infections are caused by viruses, for which antibiotics are ineffective. This not only has significant consequences for patients as inaccurate treatment prolongs hospital stay and risk for post-infection sequelae (including neurological complications), but also causes widespread incorrect use of antibiotics, resulting in an alarming increase in antibiotic-resistant bacteria worldwide.

Vaccination or the use of antimicrobial peptides are attractive alternatives to antibiotics in the combat against infections with Mp and Sp as well as other respiratory pathogens. Apart from respiratory tract infections, extrapulmonary manifestations, as a result of either direct infection or immune-mediated events, occur in up to 25% of Mp and Sp infections. Nervous system disease is one of the most common and severe complications. If Mp actually accesses the central nervous system is unknown. We recently found that Mp infections are associated with the Guillain-Barré syndrome, an acute post-infectious immune-mediated polyneuropathy caused by antibodies that cross-react with glycolipid structures on myelin sheets of peripheral nerve cells. Our recent data suggest that the development of such cross-reactive antibodies can be elicited by Mp infections.

Research aims and current researchThe research of our group is aimed at finding novel methods for diagnosis of pneumonia as well finding targets for treatment. Hereto in vivo, in vitro and in situ models are used.

1. The development of a more accurate method to demonstrate the causative agent of pneumonia as well as to discern infection from carriage;

2. Unravel which pathogenic structures and events during Mp infections result in normal vs pathological B cell activation and antibody responses;

3. Notch signaling mediating the barrier function of brain endothelial cells in homeostasis and inflammation;

4. Development of protective strategies and explore non-parenteral administration routes

The very direct link with the clinic is of great value. Together with Dr. A.M.C. van Rossum, Pediatrician/Infectiologist, we work in a bidirectional approach: key questions from the clinic concerning pathology, diagnosis and treatment of pneumonia are translated to an experimental setting. (Dr. A.M. van Rossum, [email protected] ; Pediatrics,




Infectious Diseases). Additionally, we work closely together with national and international collaborators to facilitate these basic and translational studies.

Research topics for internshipsThere are various specific research projects in which students can participate. 1. Development of a new flow cytometry-based diagnostic test to discern bacterial airway colonization from infection by profiling contents of circulating monocytes-macrophages. Set up detection of S.pneumoniae infections.2. Define the antigenic glycolipid structures in M. pneumonia that evoke protective immunity versus autoimmunity (in collaboration with R. Huizinga and B. Jacobs, dept Immunology and Neurology).3. Investigate the induction of glycolipid-specific B cell responses in-vitro.

Principal techniquesM.pneumoniae en S.pneumoniae culture, cell culture of (primary) immune cells and airway epithelial cells, ELISA, flow cytometry, immunohistochemistry, Western blotting, quantitative PCR, Thin Liquid Chromatography, in-vivo studies.

Selected publications1. De Groot RCA, Meyer Sauter PM, Unger WWJ, van Rossum AMC. (2017) Things that could be Mycoplasma pneumoniae. J Infect.;74 Suppl 1:S95-S100.

2. Meyer Sauteur PM, Unger WW, Nadal D, Berger C, Vink C, van Rossum AM. (2016) Infection with and Carriage of Mycoplasma pneumoniae in Children. Front Microbiol.; 7:329.

3. Perdicchio M, Streng-Ouwehand I, Engels S, Verstege MI, Kalay H, Ambrosini M, IlarreguiJ, Geerts D, Veninga H, den Haan JM, van Berkel LA, Samsom JN, Sparwasser T, Berod L, van Kooyk Y*, W.W.J. Unger* (2016). Regulated expression of sialic acids on antigen or tumor cells alters dendritic cells to impose tolerance on T cells. PNAS. 113(12):3329-34.

4. Meyer Sauteur PM, Huizinga R, Tio-Gillen AP, Roodbol J, Hoogenboezem T, Jacobs E, van Rijn M, van der Eijk AA, Vink C, de Wit MY, van Rossum AM, Jacobs BC. Mycoplasma pneumoniae triggering the Guillain-Barré syndrome: A case-control study. Ann Neurol. 2016.

5. Meyer Sauteur PM, Unger WW, Nadal D, Berger C, Vink C, van Rossum AM. (2016) Infection with and Carriage of Mycoplasma pneumoniae in Children. Front Microbiol. Mar 23;7:329.

6. Streng-Ouwehand I, Ho NI, Litjens M, Kalay H, Boks MA, Cornelissen LA, Kaur Singh S, Saeland E, Garcia-Vallejo JJ, Ossendorp FA, van Kooyk Y*, Unger WW*. (2016) Glycan modification of antigen alters its intracellular routing in dendritic cells, promoting priming of T cells. Elife;5.pii: e11765.

7. Unger WW*,Boks MA*, Engels S, Ambrosini M, van Kooyk Y, Luttge R. (2015) Controlled release of a model vaccine by nanoporous ceramic microneedle arrays. Int J Pharm.;491(1-2):375-83

8. Sauteur PM, Hackenberg A, Tio-Gillen AP, van Rossum AM, Berger C, Jacobs BC.(2015) Intrathecal Anti-GalC Antibodies in Bickerstaff Brain Stem Encephalitis. Neuropediatrics.;46(6):428-30

9. Meyer Sauteur PM, Roodbol J, Hackenberg A, de Wit MC, Vink C, Berger C, Jacobs E, van Rossum AM, Jacobs BC. (2015) Severe childhood Guillain-Barré syndrome associated with Mycoplasma pneumoniae infection: a case series. J Peripher Nerv Syst.;20(2):72-8

10. Spuesens EB, Fraaij PL, Visser EG, Hoogenboezem T, Hop WCJ, van Adrichem LNA, Weber F, Moll HA, Broekman B, Berger MY, van Rijsoort-Vos T, van Belkum A, Schutten M, Pas SD, Osterhaus ADME, Hartwig NG, Vink C, van Rossum AM. (2013) Carriage of Mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study. PLoS Med;10:e1001444.



https://www.ncbi.nlm.nih.gov/pubmed/27047456




LABORATORY OF GASTROENTEROLOGY AND HEPATOLOGY - LABROTATIONS & RESEARCH TOPICS

The Laboratory of Gastroenterology and Hepatology offers I&I-research projects on three different subjects in liver and intestinal diseases. In this lab, about 60 researchers (principal investigators, post-docs, PhD-students, technicians, undergraduate students) work in a challenging scientific environment on infection and/or immunity-related subjects and other subjects, and you are invited to join. More information about the laboratory and on these research lines can be found on the website of the lab: www.gastrolab.nl.


Research line: Development of immunotherapy for liver cancer and colorectal cancer

Workgroup leader: Jaap Kwekkeboom, PhD. Room Na-1009. Email: [email protected].


6 months Yes September 2018


18 months No

DescriptionHepatocellular carcinoma (HCC) is the 3rd most common cause of cancer-related death worldwide. The only curative therapies are surgical resection or liver transplantation, but these can only be offered in early stages of the disease. Unfortunately, most patients present with advanced disease and cannot be cured, because chemotherapy and targeted therapy are only marginally effective in HCC. Colorectal cancer (CRC) is the 3rd most common cancer worldwide. Liver metastases develop in about 50% of patients and are the leading cause of death of CRC patients. Curative treatment options for metastasized CRC are limited.

For both types of cancer immunotherapy may be an attractive alternative option, because of the exquisite specificity and memory generation of the adaptive immune response. However, for both types of cancer no effective immunotherapeutic therapies are available yet. Our research aims to identify promising targets for immunotherapy for HCC and CRC, and follows two complementary approaches:

A: Identification of co-inhibitory and co-stimulatory pathways that can be targeted in HCC and CRCWe observed that in both types of cancer the functionality of tumor-infiltrating T cells (TIL) is impaired. Apparently, the tumor micro-environment is immunosuppressive, and inhibits effective anti-tumor T cell responses that can eradicate the tumor cells. Therefore, we try to unravel which intra-tumoral mechanisms suppress T cell functionality. For this purpose, we isolate tumor-infiltrating leukocytes from freshly resected human tumors, and study in ex vivo cultures which immunosuppressive cells and pathways suppress effector T cells in these tumors and how we can prevent their inhibitory effects. We study whether antibody that block co-inhibitory interactions and/or agonistic antibodies against co-stimulatory receptors can boost TIL-responses in ex vivo cultures. These studies have resulted and will result in the identification of ‘immune checkpoint molecule’ targets for antibody therapy for HCC and CRC. B: Development of a therapeutic vaccination protocol for HCCIn the second approach, we aim to develop a therapeutic vaccination strategy for HCC which will effectively stimulate systemic anti-tumor T-cell responses. We intend to apply a novel vaccination technique, in which in vitro transcribed mRNA that encodes full-length tumor antigens is administered. In contrast to classical short peptide vaccines, such mRNA vaccine is expected to boost a broad repertoire of anti-tumor CD4+ and CD8+ T-cell responses. To develop such vaccination approach, we investigate which are the most immunogenic tumor antigens expressed in HCC tumor cells. Using RT-PCR and immunohistochemistry, we determine which tumor antigens are prevalently expressed in human HCC tumors. In addition, we determine the in vivo immunogenicity of these tumor antigens by analysis of peripheral blood CD4+ and CD8+ T-cell as well as antibody responses to these tumor antigens. Since cancer patients often mount poor systemic immune responses to tumor antigens in steady state, we study peripheral anti-tumor T-cell and antibody responses after local tumor destruction by Radio Frequency Ablation, which is a regular ablative treatment and results in release of tumor antigens. After identification of the most immunogenic tumor antigens, we will start a clinical trial in which in vitro transcribed mRNA that encodes for these tumor antigens will be tested as a therapeutic vaccine.

In these projects we collaborate closely with oncological surgeons and hepatologists of Erasmus MC. Student projects within these research lines will be supervised on a daily basis by postdoctoral fellows or experienced PhD-students.

Techniques:In every project a variety of cellular, immunological, and molecular techniques will be used, such as cell culture, RNA transfection, immunomagnetic cell sorting, multi-colour flow cytometry, ELISA, Western blotting, Q-PCR.

Meetings:You will participate in the several research meetings:

- Seminars Laboratory of Gastroenterology and Hepatology (weekly) - Intestinal Oncology research meetings (weekly)


- Tumor Immunology Platform meetings (weekly meetings of tumor immunology researchers from several Erasmus MC departments)

Selected recent publications1. Zhou, G., Sprengers, D., Boor, P. P. C., Doukas, M., Schutz, H., Mancham, S., Pedroza-Gonzalez, A.,

Polak, W. G., de Jonge, J., Gaspersz, M., Dong, H., Thielemans, K., Pan, Q., JNM, I. J., Bruno, M. J., and Kwekkeboom, J. (2017) Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-infiltrating T cells in Hepatocellular Carcinomas. Gastroenterology, press. DOI 10.1053/j.gastro.2017.06.017

2. Sideras, K., Biermann, K., Verheij, J., Takkenberg, B. R., Mancham, S., Hansen, B. E., Schutz, H. M., de Man, R. A., Sprengers, D., Buschow, S. I., Verseput, M. C., Boor, P. P., Pan, Q., van Gulik, T. M., Terkivatan, T., Ijzermans, J. N., Beuers, U. H., Sleijfer, S., Bruno, M. J., and Kwekkeboom, J. (2017) PD-L1, Galectin-9 and CD8+ tumor-infiltrating lymphocytes are associated with survival in hepatocellular carcinoma. Oncoimmunology 6, e1273309

3. Sideras K, Bots SJ, Biermann K, Sprengers D, Polak WG, JN IJ, de Man RA, Pan Q, Sleijfer S, Bruno MJ, Kwekkeboom J. Tumour antigen expression in hepatocellular carcinoma in a low-endemic western area. Br J Cancer 2015;112(12): 1911-1920.

4. Pedroza-Gonzalez A, Zhou G, Vargas-Mendez E, Boor PP, Mancham S, Verhoef C, Polak WG, Grunhagen D, Pan Q, Janssen H, Garcia-Romo GS, Biermann K, Tjwa ET, JN IJ, Kwekkeboom J, Sprengers D. Tumor-infiltrating plasmacytoid dendritic cells promote immunosuppression by Tr1 cells in human liver tumors. Oncoimmunology 2015;4(6): e1008355.

5. Pedroza-Gonzalez A, Zhou G, Singh SP, Boor PP, Pan Q, Grunhagen D, et al. GITR engagement in combination with CTLA-4 blockade completely abrogates immunosuppression mediated by human liver tumor-derived regulatory T cells ex vivo. Oncoimmunology. 2015;4(12):e1051297.

6. Pedroza-Gonzalez A, Verhoef C, Ijzermans JN, Peppelenbosch MP, Kwekkeboom J, Verheij J, Janssen HL, Sprengers D. Activated tumor-infiltrating CD4+ regulatory T cells restrain antitumor immunity in patients with primary or metastatic liver cancer. Hepatology 2013;57(1): 183-194.


Research line: Establishment of a donor-specific immunity profile that identifies tolerant liver transplant recipients

Workgroup leader: Jaap Kwekkeboom, PhD. Room Na-1009. Email: [email protected].




18 months No

DescriptionTo prevent graft rejection, organ transplant recipients are lifelong treated with immunosuppressive drugs (IS). Unfortunately, prolonged treatment with IS causes serious adverse effects (SAE), such as recurrent infections, cardiovascular diseases and malignancies. These SAE substantially impair the quality of life and survival after transplantation. Discontinuation of IS after kidney, heart or lung transplantation inevitably leads to graft rejection, whereas discontinuation of IS after liver transplantation (LTx) does not lead to graft rejection in some recipients. These recipients have developed an immunological tolerance to their graft. Previous attempts to predict tolerance or the risk of graft rejection during IS withdrawal based on gene expression and phenotyping of immune cells have failed. Therefore, IS discontinuation has not been introduced into standard LTx patient health care.

In this project we aim to establish a tolerance-predictive profile for LTx patients based on donor-specific immune parameters. By studying LTx patients who discontinued IS for urgent medical reasons, we will assess which combinations of assays can predict immunological tolerance. The predictive power of this donor-specific immune profile will be validated in a prospective IS withdrawal study. The clinical benefits of IS withdrawal will be investigated as well.

Techniques:A variety of cellular, immunological, and molecular techniques are used, such as cell culture, RNA transfection, immunomagnetic cell sorting, multi-colour flow cytometry, ELISA, Western blotting, Q-PCR.

Meetings:You will participate in the several research meetings:

- Seminars Laboratory of Gastroenterology and Hepatology (weekly) - Transplantation research meeting together with other Erasmus MC organ transplantation research

groups (weekly)- LTx research meeting (3-monthly)- Literature meeting Immunology in Transplantation (monthly)

Selected recent publications1. Shi XL, Mancham S, Hansen BE, de Knegt RJ, de Jonge J, van der Laan LJ, Rivadeneira F, Metselaar

HJ, Kwekkeboom J. Counter-regulation of rejection activity against human liver grafts by donor PD-L1 and recipient PD-1 interaction. J Hepatol 2016;64(6): 1274-1282.

2. de Mare-Bredemeijer EL, Shi XL, Mancham S, van Gent R, van der Heide-Mulder M, de Boer R, Heemskerk MH, de Jonge J, van der Laan LJ, Metselaar HJ, Kwekkeboom J. Cytomegalovirus-Induced Expression of CD244 after Liver Transplantation Is Associated with CD8+ T Cell Hyporesponsiveness to Alloantigen. J Immunol 2015;195(4): 1838-1848

3. Shi XL, de Mare-Bredemeijer EL, Tapirdamaz O, Hansen BE, van Gent R, van Campenhout MJ, Mancham S, Litjens NH, Betjes MG, van der Eijk AA, Xia Q, van der Laan LJ, de Jonge J, Metselaar HJ, Kwekkeboom J. CMV Primary Infection Is Associated With Donor-Specific T Cell Hyporesponsiveness and Fewer Late Acute Rejections After Liver Transplantation. Am J Transplant 2015;15(9): 2431-2442

4. Moroso V, van Cranenbroek B, Mancham S, Sideras K, Boor PP, Biermann K, de Vogel L, de Knegt RJ, van der Eijk A, van der Laan LJ, de Jonge J, Metselaar HJ, Joosten I, Kwekkeboom J. Prominent HLA-G Expression in Liver Disease But Not After Liver Transplantation. Transplantation 2015;99(12): 2514-2522


II. Research line:

Viral hepatitis; Immunology of persistent viral infections (HBV, HCV, HEV and HIV)

Workgroup leader: Andre Boonstra, PhD. Room Na1011, Email: [email protected]

Investigators: Thomas Vanwolleghem, Jun Hou


6 months Yes Jan 2018



Description

The hepatitis B virus (HBV) and hepatitis C virus (HCV) are examples of human viral pathogens that are difficult to control by the immune system. As a result of this, about 80% of individuals infected with HCV become chronically infected. In the majority of cases, HBV infection results in self-limiting, acute hepatitis, which confers protective immunity and causes no further disease. The long-term consequences of chronic HBV/HCV infections can be severe, since patients are at increased risk for developing liver fibrosis, cirrhosis and/or hepatocellular carcinoma. Moreover, In HIV-infected individuals, co-infection with HBV and especially with HCV is common because of shared modes of transmission. It is known that HIV accelerates progression of liver disease and results in increased morbidity and mortality associated with viral hepatitis. The human hepatitis E virus (HEV) is an emerging zoonotic pathogen in the West associated with consumption of undercooked pork meat. It generally induces a self-limited hepatitis, but can be associated with severe morbidity in pregnant women and immunosuppressed patients leading to chronic hepatitis and cirrhosis. Currently, apart from ribavirin, no specific antiviral treatments are available for HEV infections.

The research in our laboratory is aimed at understanding why the immune response to HBV/ HCV/HEV is insufficient to clear the virus in chronically infected patients, and what the immunological consequences are of co-infection with HIV. This knowledge is important and will be used to improve therapeutic strategies to treat chronic HBV/HCV/HEV patients. Our studies combine clinical and fundamental research.

You can participate in one of the ongoing research lines as described below:

The innate and adaptive immune response to HBV, HCV, HEV and HIV/HCV co-infections: NK and virus-specific T cells

Our previous studies have shown that NK cells from chronic HBV patients are functionally impaired and that demonstrated that HBV/HCV-specific T cell response in chronic HBV/HCV patients is not potent enough to eradicate the virus. The project is aimed at characterizing the functional defect of NK cell and T cell responses in patients in more detail, with special focus on the mechanisms that regulate and suppress these responses. During the project peripheral blood lymphocytes and also responses in the liver compartment will be assessed using flow cytometry with HBV/HCV/HIV tetramer-specific multimers and functional markers. Furthermore, highly sensitive assays to determine the function of NK cells and HBV/HCV-specific CD4+ and CD8+ T cells will be conducted in order



to identify specific markers and mechanisms that initiate and maintain the chronicity of viral hepatitis infections.

Study on the mechanism of action of current and novel therapies for HBV, HCV and HIVThe therapeutic strategies to treat chronic HBV/HCV patients as well as patients with co-infections have dramatically improved in the last decade. However, still not all patients are responsive to therapy or relapse, and severe adverse effects are frequently observed. A major focus of our laboratory, in close interaction with the clinical branch of the Rotterdam Liverunit, is to examine the mechanisms of action of the current therapy based on virus replication inhibitors and pegylated-interferon in patients with chronic HBV and HCV. In addition, we perform investigator-initiated studies to assess the immunological effect of novel HBV/HCV inhibitors and immune modulators in numerous clinical studies, which are performed to evaluate the efficacy and safety of novel therapeutic strategies to treat HBV or HCV. The studies combine classical immunological studies with transcriptomics/proteomics to identify biomarkers that predict the response to therapy.

Mouse models: intrahepatic immunology and viral hepatitisOur understanding of how the immune system fails to clear HBV and HCV is limited due to the lack of suitable model systems. HCV infects only men and chimpanzees, and none of the available chimeric mouse models allows the complete study of its immunopathogenesis. This severely hampers the development of candidate vaccines and antiviral drugs. As an alternative approach we apply 2 mouse model systems to study virus-host interactions:1/ Mice with chimeric livers reconstituted by human hepatocytes. In these animals, genuine viral hepatitis B, C and E replication, antiviral treatment, viral entry and innate intracellular immune responses are studied. Adaptive or innate cellular immune responses in these animals are lacking due to their immune-deficient background to prevent rejection of the human hepatocyte graft.2/ The natural mouse pathogen lymphocytic choriomeningitis virus (LCMV) induces, apart from a systemic viral infection, chronic liver inflammation that is cleared after a variable period of several weeks to months. We study the role of Kupffer cells and inflammatory monocytes during the post-acute or early chronic viral hepatitis phase, to understand their contribution to liver pathology and viral clearance. In addition, we study the effect of treatment with immunomodulatory agents to overcome T cell exhaustion in vivo.Our research sets out to make use of both mouse models to study fundamental aspects of viral replication and the interaction of the innate immune system with HBV, HCV, HEV or LCMV as a surrogate virus. The ultimate goal is to identify critical viral or immune components, which tip the balance towards chronicity instead of self-resolving acute infection.

Techniques:A broad range of immunological as well as molecular techniques are being used in the laboratory, including cell culture, cell purification by cell sorting, multi-color flow cytometry, various immunoassays, functional NK and T cell assays (cytotoxicity, cytokine secretion), DNA/RNA isolation, Q-PCR, etc

Some of the studies related to co-infections with HIV are joint projects with the Dept of Infectious Diseases (prof. A. Verbon)

The following meetings will take place weekly: - Department of Gastroenterology and Hepatology seminars - Viral Hepatitis meetings - Literature meetings


Note: at the start of the research project, the student needs to be vaccinated and have protective antibody titers to HBV.

Key publications:

1. Spaan M, Hullegie SJ, Beudeker BJB, Kreefft K, van Oord GW, Groothuismink ZMA, van Tilborg M, Rijnders B, de Knegt RJ, Claassen MAA, Boonstra A. Frequencies of circulating MAIT cells are diminished in chronic HCV, HIV and HCV/ HIV co-infection and do not recover during therapy. PlosOne. 2016.11(7): e0159243.

2. Van de Garde MDB, Pas SD, van der Net G, Osterhaus ADME, Haagmans BL, Boonstra A, Vanwolleghem T. Hepatitis E virus genotype three infection of human liver chimeric mice as a model for chronic HEV infection. J Virol. 2016. 90: 4394-4401.

3. Spaan M, van Oord G, Kreefft K, Hou J, Hansen BE, Janssen HLA, de Knegt RJ, Boonstra A. Immunological analysis during interferon-free therapy for chronic hepatitis C virus infection reveals modulation of the natural killer cell compartment. J Inf Dis. 2016. 213(2): 216-23.

4. Vanwolleghem T, Hou J, van Oord G, Andeweg AC, Osterhaus ADME, Pas SD, Janssen HLA, Boonstra A. Re-evaluation of HBV clinical phases by system biology identifies unappreciated roles for the innate immune response and B cells. Hepatology. 2015. 62(1): 87-100.

5. De Groen RA, Boltjes A, Hou J, Liu B-S, Mcphee F, Friborg J, Janssen HLA, Boonstra A. IFNλ-mediated IL-12 production in macrophages induces IFNγ production in human NK cells. Eur J Immunol. 2015. 45(1): 250-259.

6. Hou J, van Oord G, Groothuismink ZMA, Claassen MAA, Kreefft K, Zaaraoui-Boutahar F, van IJcken WFJ, Osterhaus ADME, Janssen HLA, Andeweg AC, de Knegt RJ, Boonstra A. Gene expression profiling to predict and assess the consequences of therapy-induced virus eradication in chronic HCV. J Virol. 2014. 88(21): 12254-12264.

7. Claassen MAA, Janssen HLA, Boonstra A. Role of T cell immunity in hepatitis C virus infections. Curr Opin Virol. 2013. 3: 461-467.


III. Research line:

Viral hepatitis; Immunology of hepatitis B virus infection

Workgroup leader: Andrea Woltman, PhD. Room Na1006Email: [email protected]





Description:

Hepatitis B virus (HBV) is a hepatotrophic DNA virus that has infected 1/3 of the world population. In the majority of cases, HBV infection results in self-limiting, acute hepatitis, which confers protective immunity and causes no further disease. However, in a minority of individuals infected as adults and in the vast majority of vertically infected children, chronic infection occurs. The long-term consequences of chronic HBV infections can be severe, since patients are at increased risk for developing liver fibrosis, cirrhosis and/or hepatocellular carcinoma (HCC), resulting in about 1 million deaths worldwide per year.

In individuals that clear HBV a broad and strong HBV-specific T cell response can be detected. In chronic patients though, less, weaker or more exhausted T cells response are found. This suggest an important role for T cells responses in the clearance of HBV. In our group we focus on understanding the mechanisms that underlie the induction of such effective HBV-specific adaptive immune responses but also we would like to know why they are absent in chronic HBV patients.

The induction of immunogenic T cell responses against virus infected cells or tumors requires antigen presentation of HBV or tumor antigens in HLA molecules on both professional antigen presenting cells such as dendritic cells (DCs) as well as on infected/ transformed hepatocytes. T cell epitopes that are presented well on hepatocytes are possible targets for immunotherapy using vaccines containing long peptide antigens. This type of therapy is based on the unique ability of DCs to process long peptides and extract HLA epitopes for presentation to T cells triggering their activation. In this way adaptive responses against HBV epitopes or tumor antigen may be initiated or boosted. To develop such therapy for chronic HBV and/or HCC we need to know how patients HBV specific T cells can be (re)activated and what factors may enhance or impair this activation. To this end we in depth study the function and phenotype of DCs, T cells and hepatocytes from chronic HBV patients, individuals that effectively cleared HBV as well as from healthy controls.

The research in our laboratory has a strong translational character, combines fundamental research questions with the use of patient material, and aims to provide basic and applied knowledge to develop novel therapeutic strategies to combat chronic viral hepatitis as well as HBV related HCC.

Techniques:Diverse Immunological (e.g. flow cytometry, ELISA, Immune cell isolation and culture, intracellular cytokine staining), molecular (e.g. q-PCR), proteomic (e.g. Immunoprecipitation, Western Blot, mass spectrometry) and virological (e.g. infection assays) techniques are operational in the lab.



The following meetings will take place weekly: - Department of Gastroenterology and Hepatology seminars - Viral Hepatitis Lab meetings - Journal club- Liver cancer meetings (in case of an HCC related topic only)- Tumor immunology meetings (in case of an HCC related topic only)

Note: at the start of the research project, the student needs to be vaccinated and have protective antibody titers to HBV.

For specific questions about projects, contact Sonja Buschow, Postdoctoral researcher in the laboratory. Email : [email protected]

Key publications:

1. van der Aa E, Buschow SI, Biesta PJ, Janssen HL, Woltman AM. The Effect of Chronic Hepatitis B Virus Infection on BDCA3+ Dendritic Cell Frequency and Function.PLoS One. 2016 Aug 16;11(8):e0161235. doi: 10.1371/journal.pone.0161235. eCollection 2016.

2. van Montfoort N, van der Aa E, van den Bosch A, Brouwers H, Vanwolleghem T, Janssen HL, Javanbakht H, Buschow SI, Woltman AM. Hepatitis B Virus Surface Antigen Activates Myeloid Dendritic Cells via a Soluble CD14-Dependent Mechanism.J Virol. 2016 Jun 24;90(14):6187-99.

3. Buschow SI, Sprengers D, Woltman AM. To target or not to target viral antigens in HBV related HCC? J Hepatol. 2015 Jun;62(6):1449-50.

4. Boltjes A, van Montfoort N, Biesta PJ, Op den Brouw ML, Kwekkeboom J, van der Laan LJW, Janssen HLA, Boonstra A, Woltman AM. Kupffer cells interact with hepatitis B surface antigen in vivo and in vitro, leading to proinflammatory cytokine production and natural killer cell function. J. Infect Dis. 211(8):1268-1278, 2015

5. Boltjes A, Movita D, Boonstra A, Woltman AM. The role of Kupffer cells in hepatitis B and hepatitis C virus infections. J Hepatol. 2014;61(3):660-671. Review.

6. van Montfoort N, van der Aa E, Woltman AM. Understanding MHC class I presentation of viral antigens by human dendritic cells as a basis for rational design of therapeutic vaccines. Front Immunol. 2014; 23;5:182. Review.

7. van de Laar L, van den Bosch A, Boonstra A, Binda RS, Buitenhuis M, Janssen HL, Coffer PJ, Woltman AM. PI3K-PKB hyperactivation augments human plasmacytoid dendritic cell development and function. Blood. 2012 13;120(25):4982-91.

8. van de Laar L, Coffer PJ, Woltman AM. Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune homeostasis and therapy. Blood. 2012;119(15):3383-93. Review.

9. Tjwa ET, van Oord GW, Biesta PJ, Boonstra A, Janssen HL, Woltman AM. Restoration of TLR3-activated myeloid dendritic cell activity leads to improved natural killer cell function in chronic hepatitis B virus infection. J Virol. 2012 86(8):4102-9.

10. Tjwa ETTL, Van Oord GW, Hegmans JP, Janssen HLA, Woltman AM. Viral load reduction improves activation and function of natural killer cells in patients with chronic hepatitis B. J. Hepatol. 54(2): 209-18, 2011.


2015:Title: Immunological mechanisms of HIV infection

Workgroup leaders:Dr. A. Verbon ([email protected])Dr. M.A.A. Claassen ([email protected])

Investigators: Dr. A. Boonstra (Department of Gastroenterology and Hepatology)





BackgroundIn the last 20 years, the treatment of HIV infected patients with combination anti-

retroviral therapies (cART) have greatly improved and effective suppression of viral replication is a realistic goal for the vast majority of patients. Consequently, for many patients who receive cART treatment, HIV has become a chronic disease without progression to AIDS and with a good survival. However, cART-treated patients still experience substantial health issues resulting in ongoing decreased survival. We hypothesize that this impaired health is mainly caused by side effects of antiretroviral therapy, lifestyle factors such as heavy smoking, by low-grade chronic systemic HIV-induced inflammation, or additional immune activation by co-infections such as the hepatitis B or C virus (HBV or HCV).

Also, despite the optimized antiretroviral regimens, the risk of viral resistance remains, especially with poor compliance to therapy. The quality of life of patients is therefore influenced to varying degrees by the fact that they have to take their anti-retroviral medication daily, life-long and conscientiously.

Further improvement of antiretroviral therapies to treat HIV patients is aimed at the development of strategies with less long-term side effects, lower rate of viral resistance and reduced HIV-induced inflammation. We hypothesize that chronic HIV-induced immune activation can be minimized by long-term maximal viral suppression, and can be reactivated by low-grade and repeated HIV replication (blips). Therefore, ultra-deep viral suppression by cART may be an important goal of HIV therapy. The ultimate goal, however, is to find a treatment that leads to final eradication of HIV, which makes that life-long treatment is no longer needed, and patients are cured.

In order to prove our hypotheses and achieve our objectives more knowledge of the host response against HIV and other chronic viral infectious diseases is needed. Currently, the factors that play a role in the persistence of these chronic viral infections and subsequent persistent immune activation are still largely unknown. To get a better understanding of these factors, studies of the host response and pathophysiological processes are necessary, and may lead to the identification of biological markers, the improvement of diagnosis and improvement of anti-retroviral therapies.

The research is aimed at understanding the ongoing immune activation in chronic HIV patients during effective ART therapy. These studies combine clinical and translational research.

Research To examine in detail the immune parameters that comprise the state of chronic

immune activation as observed in cART-treated chronic HIV patients. This will be done by evaluation of the phenotype and function of leukocytes, such as monocytes, NK, MAIT and T cells using immunological and systems biological tools.




To better characterize the effect of chronic immune activation as observed in cART-treated chronic HIV patients on increased morbidity and mortality

The project will be performed in a team of clinicians and immunologists, and consequently will combine clinical and translation studies. Some of the studies are joint projects with the Department of Gastroenterology & Hepatology (Dr. A. Boonstra).

A broad array of immunological as well as molecular techniques are being used in the laboratory, including processing of patient material (blood and biopsies), cell culture, cell purification by sorting, multi-color flow cytometry for surface markers and intracellular staining, various immunoassays, functional NK and T cell assays (cytotoxicity, cytokine secretion), DNA/RNA isolation, qPCR etc.


Workgroupleader : Maikel Peppelenbosch / head of the laboratory for gastroeneterology and hepatology.

Room Na-1007. ([email protected])

Internships are available for talented students for any period of timeTitle: The final frontier: the gut epithelium the gatekeeper to our body.

Introduction: Ever since Claude Bernard formulated the concept of the milieu interieur versus the milieu exterieur, it has been clear that our body needs defences and barriers to hostility of the outer world. The mechanisms underlying these defences form the main topic of the Master Infection and Immunity. The most extreme example of where these mechanisms are tested can be found in the intestine: here microbiological load reaches astronomical proportions, in the large intestine > 1014 bacteria per litre can be found, in addition to viral load and protozoal threads to body. At the same time, however, the intestine needs to absorb food and water and thus the epithelium is only one cell layer thick. In my laboratory we examine how the intestine pulls off this Augean task. In addition, we want to examine the mechanisms which underlie disease when these systems go awry, in particular during inflammatory conditions like Crohn’s disease or cancer. We have always place for talented and motivated students to contribute to our quest here.

What can we offer? The laboratory has available a pool of highly talented post-doctoral researchers able to teach and guide students during their internships and transfer meaningful technical and academic skills. Furthermore the availability of large biobanks and the strong link to the clinical departments of digestive disease and surgery means that fresh material can always be collected. The department has various core facilities available, including those for FACS analysis, histochemistry, and kinomics, and is supported by a small but professional facilitatory unit. Finally, the availability of our own diagnostic laboratory offers the possibility of rapid implementation of results in clinical practice, which can be very gratifying to students.

Examples: although we are always happy to discuss the own proposals of students, possible interesting project could include acute diarrheal diseases. These remain one the largest global health challenges, being the most significant cause of infant mortality worldwide and having huge economic impact. ORS with oral zinc therapy has successfully reduced mortality but does not combat diarrhea per se and use is declining. Importantly, increased insight into the biology of diarrhea has led to the realization that blanket anti-diarrheal therapy is possible and that it can be delivered using genetically-modified bacteria (cheap to produce and resistant to supply chain problems). Lactic acid bacteria can be genetically modified to produce (uro)guanylin antagonists. Such bacteria are superior to all existing anti-diarrhea medication, are very cheap to produce (they grow in milk) and are resistant to supply line problems, common to developing nations. We propose to create such an organism, do safety testing in The Netherlands in volunteers and do an actual field trial (probably in Soweto, RSA). We feel potential impact could be huge.

Alternatively, we could investigate the role of p21Rac activation in the epithelium of the intestine and investigate its modulation in inflammatory bowel disease. We recently showed (Parikh K et al. Suppression of p21Rac signalling and increased innate immunity mediate remission in Crohn's disease. Science Transl Med. 2014 Apr 23;6(233):233ra53) that IBD patients exhibit excessive Rac activation, that this effect correlates with reduced innate immunity, and that its inhibition provokes remission in patients. We now feel that studying the consequences of Rac activation in the intestinal epithelium on innate immunity function of these cells, as well as the relation of the microbiome and intestinal epithelial Rac activation warrants further study.Finally, we are always open to specific research proposals of students themselves –if pertinent to translational or experimental gastroenterology. We are happy to discuss the possibilities.

Interested? – Contact Maikel Peppelenbosch


thema x - msc infection & immunity researchtopics... · web viewifn activation is also present...

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