Rir 10th june_2011_v2

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Rir 10th june_2011_v2

  1. 1. Institut thématique Microbiologie et Maladies infectieuses 3RD INTERNATIONAL RESEARCH MEETING MICROBIOLOGY & INFECTIOUS DISEASES - HÔTEL MARIGNY, PARIS, JUNE 10TH, 2011
  2. 2. Institut thématique Microbiologie et Maladies infectieuses Laurent Abel and Jean-Laurent Casanova................................................................................................................................1,2 Matthew Albert.............................................................................................................................................................................7 Matthieu Allez ............................................................................................................................................................................11 Brigitte Autran............................................................................................................................................................................15 Thomas Baumert .......................................................................................................................................................................21 Monsef Benkirane......................................................................................................................................................................25 Nicolas Blanchard......................................................................................................................................................................29 Stéphanie Blandin......................................................................................................................................................................33 Matteo Bonazzi..........................................................................................................................................................................37 Priscille Brodin...........................................................................................................................................................................41 Bruno Canard ............................................................................................................................................................................45 Béhazine Combadière................................................................................................................................................................49 François-Loïc Cosset. ................................................................................................................................................................53 François Dabis...........................................................................................................................................................................57 Guillaume Duménil.....................................................................................................................................................................61 Gérard Eberl ..............................................................................................................................................................................65 Hidehiro Fukuyama....................................................................................................................................................................69 Benoit Gamain...........................................................................................................................................................................73 Yves Gaudin..............................................................................................................................................................................77 Ivo Gomperts Boneca ................................................................................................................................................................81 Laurent Gutmann.......................................................................................................................................................................85 David Klatzmann........................................................................................................................................................................87 Marc Lecuit................................................................................................................................................................................91 Eric Leroy ..................................................................................................................................................................................95 Elena Levashina ........................................................................................................................................................................99 Yves Lévy................................................................................................................................................................................103 Camille Locht...........................................................................................................................................................................105 Nicolas Manel ..........................................................................................................................................................................109 Robert Ménard.........................................................................................................................................................................113 Tâm Mignot..............................................................................................................................................................................117 Hannu Myllykallio.....................................................................................................................................................................121 Xavier Nassif............................................................................................................................................................................125 Patrice Nordmann....................................................................................................................................................................129 Eric Oswald .............................................................................................................................................................................133 Jean-Michel Pawlotsky.............................................................................................................................................................135 Carole Peyssonnaux................................................................................................................................................................139 Eliane Piaggio..........................................................................................................................................................................143 Marie-Cécile Ploy.....................................................................................................................................................................147 Lluis Quintana-Murci................................................................................................................................................................151 Didier Raoult............................................................................................................................................................................155 Jean-Marc Reichhart................................................................................................................................................................157 Felix Rey..................................................................................................................................................................................161 Human Rezaei.........................................................................................................................................................................165 Carla Saleh..............................................................................................................................................................................169 Philippe Sansonetti ..................................................................................................................................................................173 Olivier Schwartz.......................................................................................................................................................................177 Maria-Isabel Thoulouze............................................................................................................................................................181 Eric Vivier - Sophie Ugolini.......................................................................................................................................................185 Contacts of participants............................................................................................................................................................190
  3. 3. Institut thématique Microbiologie et Maladies infectieuses Selected publications: • Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease. Bustamante, J., A.A. Arias, G. Vogt, 24 co-authors, M.C. Dinauer, L. Abel, and J.L. Casanova. Nat Immunol: 2011 Mar; 12(3):213-21. • Interferon gamma receptor 2 gene variants are associated with liver fibrosis in patients with chronic hepatitis C infection. Nalpas, B., R. Lavialle-Meziani, S. Plancoulaine, 15 co-authors, F. Matsuda, S. Pol, and L. Abel. Gut. 2010;59:1120-1126. • Two loci control tuberculin skin test reactivity in an area hyperendemic for tuberculosis. Cobat, A., C.J. Gallant, L. Simkin, G.F. Black, K. Stanley, J. Hughes, T.M. Doherty, W.A. Hanekom, B. Eley, J.P. Jais, A. Boland-Auge, P. van Helden, J.L. Casanova, L. Abel, E.G. Hoal, E. Schurr, and A. Alcais. J Exp Med. 2009; 206:2583-2591. • Stepwise replication identifies a low-producing lymphotoxin-alpha allele as a major risk factor for early-onset leprosy. Alcais, A., A. Alter, G. Antoni, M. Orlova, N. Van Thuc, M. Singh, P.R. Vanderborght, K. Katoch, M.T. Mira, V.H. Thai, N.T. Huong, N.N. Ba, M. Moraes, N. Mehra, E. Schurr, and L. Abel. Nat Genet. 2007; 39:517- 522. • Primary immunodeficiencies: a field in its infancy. Casanova, J.L., and L. Abel. Science. 2007;317:617-619. • Human genetics of infectious diseases: a unified theory. J.L. Casanova and L. Abel. Embo J. 2007;26:915- 922. • An autosomal dominant major gene confers predisposition to pulmonary tuberculosis in adults. Baghdadi, J.E., M. Orlova, A. Alter, B. Ranque, M. Chentoufi, F. Lazrak, M.I. Archane, J.L. Casanova, A. Benslimane, E. Schurr, and L. Abel. J Exp Med. 2006; 203:1679-1684. Keywords • Complex genetic predisposition • Infectious diseases • Leprosy • Tuberculosis • Human Herpes virus 8 (HHV-8) • Human T- lymphotropic virus 1 (HTLV-1) • Hepatitis C Virus (HCV) • Human genetics • Genetic Epidemiology Major Grants (managed by Inserm France): • ANRS “Genome-wide association of HCV related phenotypes” • ANR “Genetic predisposition to pulmonary tuberculosis” • NIH 1 U01 AI088685 “Genetic predisposition to tuberculosis in Morocco” • European Community “Host and Mycobacterial molecular dissection of immunity and pathogenesis of tuberculosis” • European Community “Spontaneous clearance in patients acutely infected with HCV” • ERC advanced grants “Human genetics of tuberculosis” Inserm U980 –Paris Descartes University - Necker Medical School Paris The Rockefeller University New York Human genetics of infectious diseases There are many lines of evidence suggesting that the characteristics of an infectious disease in humans depend largely on the genetic background of the individual exposed to the specific microbe. The infectious agent is, of course, necessary, but is generally not sufficient for the development of an infection or the clinical symptoms. The two teams of our laboratory of human genetics of infectious diseases directed by L. Abel and J.L. Casanova, respectively, work together to address the question of genetic susceptibility to rare and common infections, from the perspectives of both complex and single-gene determinism predisposition, to achieve a unified genetic theory of human infectious diseases. In the past decade, our team has gained leading international expertise in this field of human genetics of common infectious diseases, by identifying several genes associated with susceptibility to leprosy, tuberculosis and predisposition to infection by several oncogenic viruses. Our successful achievements led the Inserm and the Rockefeller University to create an International laboratory of Human genetics of infectious diseases with a Necker branch in Paris and a Rockefeller branch in New-York. Laurent Abel, M.D, Ph.D Within the laboratory, our team aims to identify the main genes involved in the determinism of common infectious diseases, mostly in adults. Our studies of infectious diseases focus on infections due to virulent mycobacteria (leprosy and tuberculosis (TB)) and certain oncogenic viruses. The main results obtained during the last years include: 1) Identification by positional cloning of major leprosy susceptibility variants of the PARK2/PACRG and LTA genes, defining new pathophysiological pathways; 2) Mapping of the first major locus conferring predisposition to pulmonary TB, and of the two first major loci controlling TB infection; 3) Identification of the first Medelian cases of severe TB in children; 4) Dissection of intra-familial transmission of HHV-8 (responsible for Kaposi’s sarcoma) in endemic populations, with the detection and mapping of a major gene predisposing to infection by this virus; 5) Demonstration that current Hepatitis C virus (HCV) infection has a strong familial component explained by both specific modes of intra-familial viral transmission and by genetic predisposition to infection; 6) Mapping of two loci conferring predisposition to HTLV-1 infection in childhood. We will continue our work to further dissect these infections, using the precise identification of the loci we have detected and the initiation of new studies focusing on new phenotypes, and on the severe clinical diseases resulting from these infections. This latter aspect is done with the other team of the unit in order to investigate the Mendelian genetic control of the most extreme forms of these infections (e.g. severe TB, Kaposi’s sarcoma, fulminant hepatitis, herpes simplex encephalitis). This research takes advantage of the recent advances in high-throughput genotyping (we are conducting genome-wide association studies in TB, leprosy, and HCV- related phenotypes) and sequencing (we will search for the role of rare variants in these common infectious diseases). The identification of host genes involved in human infectious diseases will provide new keys to understanding the pathogenesis mechanisms underlying disease development, with potentially major practical implications for the control of infectious diseases. 1
  4. 4. Institut thématique Microbiologie et Maladies infectieuses Keywords • Primary immunodeficiencies • Infectious diseases • Herpes simplex encephalitis (HSE) • Mendelian susceptibility to mycobacterial disease (MSMD) • Invasive pneumococcal disease (IPD) • Chronic mucocutaneous candidiasis • Human genetics • Immunology Human genetics of infectious diseases There are many lines of evidence suggesting that the characteristics of an infectious disease in humans depend largely on the genetic background of the individual exposed to the specific microbe. The infectious agent is, of course, necessary, but is generally not sufficient for the development of an infection or the clinical symptoms. The two teams of our laboratory of human genetics of infectious diseases directed by J.L. Casanova and L. Abel, respectively, work together to address the question of genetic susceptibility to rare and common infections, from the perspectives of both single-gene determinism and complex predisposition, to achieve a unified genetic theory of human infectious diseases. In the past decade, our team has gained international expertise in this field of human genetics of infectious diseases, by revealing that single genetic lesions in children confer severe and selective vulnerability to certain illnesses, including herpes simplex encephalitis, mycobacterial diseases, and invasive pneumococcal diseases. Our successful achievements led the Inserm and the Rockefeller University to create an International laboratory of Human genetics of infectious diseases with a Necker branch in Paris and a Rockefeller branch in New-York. Jean-Laurent Casanova, M.D, Ph.D Our team aims to test the hypothesis that life-threatening infectious diseases in children result from single-gene inborn errors of immunity. We hypothesize that a substantial fraction children with severe infectious diseases suffer from novel primary immunodeficiencies, resulting in a specific susceptibility to one or a few microorganisms. During the last years, we have largely validated this hypothesis with the discovery of the molecular genetic basis of : 1) The syndrome of Mendelian predisposition to mycobacterial disease (MSMD) due to mutations in IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, NEMO, CYBB and IRF8 (IL-12-IFN-γ circuit); 2) Invasive pneumococcal disease (IPD) due to mutations NEMO, IKBA, IRAK4, MYD88 (NF-kB-dependent TLR and IL-1R pathways); and 3) Herpes simplex encephalitis (HSE) due to mutations in the UNC93B1, TLR3, and TRAF3 (TLR3 pathway). Using our golden standard complementary approaches, namely candidate gene dissection (hypothesis-based) or genome-wide dissection (hypothesis-free) we have pursued our investigation of these three diseases. Recently, we have discovered mutations in IL17A and IL17F, the first two genetic etiologies of chronic mucocutaneous candidiasis. We have also pioneered whole-exome deep sequencing strategies which, combined with genome-wide linkage have lead to uncovering a mutation in STIM1, responsible for development of lethal Kaposi sarcoma, as well as a mutation in FADD which is, in part, responsible for auto- immune lymphoproliferative syndrome (ALPS). By combining our standard approaches with novel cutting-edge evolving techniques, our studies over the next years will not merely pursue the lines of research followed over the previous years — they will also explore uncharted territories. We intend to demonstrate not only that MSMD, IPD and HSE result from single-gene variations in most children, but also that other pediatric infectious diseases may as a rule result from single- gene inborn errors of immunity. Our single-gene theory of life-threatening pediatric infectious diseases will have many profound medical and biological implications. Selected publications: • Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Puel A, Cypowyj S, Bustamante J, Wright J, Liu L, Lim HK, Migaud M, Israel L, Chrabieh M, Toulon A, El-Baghdadi J, Bodemer C, Whitters M, Paradis T, Brooks J, Collins M, Wolfman NM, Al-Muhsen S, Galicchio M, Abel L, Picard C, Casanova JL. Science. 2011 Feb 24. • Germline but macrophage-tropic CYBB mutations in kindreds with X-linked predisposition to tuberculous mycobacterial diseases. Bustamante J, Arias AA, Vogt G, 24 co-authors, Dinauer MC, Abel L, Casanova JL. Nature Immunology. 2011 Mar;12(3):213-21. • Human TLRs and IL-1Rs in host defense: natural insights from evolutionary, epidemiological, and clinical genetics. Casanova JL, Abel L, Quintana-Murci L. Annu Rev Immunol. 2011, in press. • Human TRAF3 Adaptor Molecule Deficiency Leads to Impaired Toll-like Receptor 3 Response and Susceptibility to Herpes Simplex Encephalitis. Pérez de Diego R, Sancho-Shimizu V, Lorenzo L, 17 co- authors. Jouanguy E, Zhang SY, Abel L, Casanova JL. Immunity. 2010 Sep 24;33(3):400-1. • Pyogenic bacterial infections in humans with MyD88 deficiency. Von Bernuth H, Picard C, Jin Z, 30 co- authors. Abel L, Li X, Chaussabel D, Puel A, Casanova JL. Science. 2008 Aug 1;321(5889):691-6. • TLR3 deficiency in otherwise healthy children with herpes simplex encephalitis. Zhang, SY, Jouanguy E, Ugolini S, Smahi A, Elain G, Segal D, Sancho-Shimizu V, Lorenzo L, Puel A, Picard C, Chapgier A, Plancoulaine S, Titeux M, Cognet M, von Bernuth H, Ku CL, Casrouge A, Zhang XX, Barreiro L, Hamilton C, Lebon P, Héron B, Vallée L, Quintana-Murci L, Hovnanian A, Rozenberg F, Vivier E, Geissmann F, Tardieu M, Abel L and Casanova JL. Science. 2007; 317:1522-7. • Herpes simplex virus encephalitis in human UNC-93B deficiency. Casrouge A, Zhang SY, Eidenschenk C, Jouanguy E, Puel A, Yang K, Alcais A, Picard C, Mahfoufi N, Nicolas N, Lorenzo L, Plancoulaine S, Senechal B, Geissmann F, Tabeta K, Hoebe K, Du X, Miller RL, Heron B, Mignot C, de Villemeur TB, Lebon P, Dulac O, Rozenberg F, Beutler B, Tardieu M, Abel L, Casanova JL. Science. 2006; 314:308-12. Inserm U980 - Paris Descartes University - Necker Medical School Paris The Rockefeller University New York Major Grants (managed by Inserm France): • March of Dimes “Herpes simplex encephalitis: a novel group of primary immunodeficiencies” • ANR “Herpes simplex encephalitis: a novel group of primary immunodeficiencies” 2
  5. 5. Institut Thématique Microbiologie et Maladies Infectieuses Laurent Abel, M.D, Ph.D and Jean-Laurent Casanova, M.D, Ph.D Human Genetics of Infectious Diseases Objectives: • Why do some exposed individuals (and not others) develop infectious diseases • What are the critical immunological pathways in natural conditions of infection? Immunity to infection Microbial factors Exposure to Microbe Biological Phenotypes Clinical Phenotypes Non microbial factors Non genetic factors Genetic factors Environment Host SampleSample ToolsTools PhenotypePhenotype LargeLargeSmallSmall Genetic EpidemiologyGenetic EpidemiologyMendelianMendelian GeneticsGenetics Milder/chronicMilder/chronic (adults)(adults) Severe/acuteSevere/acute (children)(children) Methods of investigation in humans Rare mutations Strong individual effect Common polymorphisms Modest individual effect Tools: Using the very last genomic technology advances: - High throughput genotyping (Genome-wide association studies) - Deep sequencing (Exome/Genome) 3
  6. 6. Institut Thématique Microbiologie et Maladies Infectieuses Laurent Abel, M.D, Ph.D and Jean-Laurent Casanova, M.D, Ph.D Human Genetics of Infectious Diseases Mendelian genetics Disseminated mycobacterial infections Impaired production of or response to IFN-γγγγ Chronic Mucocutaneous Candidiasis IL12Rββββ1 IFNγγγγR1 IL12p40 IFNγγγγR2STAT1 NEMO Impaired IL-17 immunity C. albicans STAT-3 IK K IL-17A IL-17F IL-17RA IL-17RA Impairment of TIR signaling pathway Mendelian genetics 4
  7. 7. Institut Thématique Microbiologie et Maladies Infectieuses Laurent Abel, M.D, Ph.D and Jean-Laurent Casanova, M.D, Ph.D Human Genetics of Infectious Diseases • Extending Mendelian studies to other extreme infectious phenotypes (eg fulminant hepatitis, severe Flu…). • Searching for rare variants with stronger effect in common infectious diseases (eg Tuberculosis, HCV-related phenotypes). • Extensive use of deep sequencing techniques to achieve these goals • Using iPS technology for functional studies in specific cells/tissues (eg CNS cells). Perspectives Complex predisposition: main studies DISEASEDISEASE (clinical phenotypes)(clinical phenotypes) INFECTIONINFECTION (biological phenotypes)(biological phenotypes) EXPOSUREEXPOSURE M. leprae Mitsuda reaction Leprosy, subtypes, reversal reactions M. tuberculosis TST, IGRAs Pulmonary TB, Disseminated TB HCV serology, RNA Liver fibrosis, Fulminant hepatitis HHV-8 serology Kaposi sarcoma 5
  8. 8. Institut Thématique Microbiologie et Maladies Infectieuses Laurent Abel, M.D, Ph.D and Jean-Laurent Casanova, M.D, Ph.D Human Genetics of Infectious Diseases • Pioneer and leader in the identification of new primary immuno-deficiencies predisposing selectively to a given microbe • Unique strong expertise in human genetics of infectious diseases by the synergic association of genetic immunology and genetic epidemiology teams. • Define a new frontier in infectious diseases 1. Understanding of the pathogenesis, in immunological and genetic terms. 2. Molecular diagnosis of predisposed individuals, genetic counseling of affected kindreds. 3. Definition of the clinical outcome, in terms of morbidity and mortality. 4. Novel treatment to restore immunity (cytokines, etc.), or to circumvent inborn errors (vaccines, etc.). Unique selling points 6
  9. 9. Institut thématique Microbiologie et Maladies infectieuses Keywords • Hepatitis C • Chikyngunya infection • BCG & Bladder Cancer • Dendritic cells • Type I Interferon Major Grants • EURYI (2004) • ERC – Young Investigators (2008) • FP7 – Acute HCV Biomarker Discovery (2009) • ANR – CHIKV infection and Host response (2007, 2010) • ANRS – HCV disease pathogenesis (2004 - present) • Ligue Labelled Research Unit (2005 - present) Host response to infection, Hepatitis C Our laboratory is interested in defining the role of host immunity in disease pathogenesis with a specific concern for how dying cells influence the establishment of anti-viral responses Matthew Albert, M.D, Ph.D Our group’s basic science and clinical research goals are to investigate the cellular and molecular mechanisms underlying the different immunologic outcomes of antigen cross-presentation - cross-priming versus cross- tolerization. These pathways impact many homeostatic and pathological processes. Specifically the lab focuses on questions of viral and tumour immunity. We aim to identify key points of regulation involved in activating cytolytic T lymphocytes (CTLs) as well as key points of dysregulation where viral and tumor proteins interfere with the generation of specific immunity. Our recent studies in HCV pathogenesis have revealed a surprising and new mechanism implicated in limiting immune-mediated viral clearance. Specifically, we have demonstrated that as a result of chronic inflammation, a host protease is upregulated, resulting in the rapid NH2-terminal truncation of a key chemokine called interferon-induced protein 10 (IP-10). As a result of this cleavage event, IP-10 is converted from an agonist into an antagonist, resulting in the perturbation of lymphocyte trafficking. These studies, performed in close collaboration with clinical partners within the Ile-de-France region (led by Pr. Stanislas Pol), have provided new diagnostic tools for predicting response to therapy and established a new therapeutic target for enhancing anti-viral immunity. Institut Pasteur Paris - Inserm U818 Paris Selected publications: • Evidence for an antagonist form of the chemokine CXCL10 in patients chronically infected with HCV. Casrouge A, Decalf J, Ahloulay M, Lababidi C, Mansour H, Vallet-Pichard A, Mallet V, Mottez E, Mapes J, Fontanet A, Pol S, Albert ML. Journal of Clinical Investigation. 2011 Jan 4;121(1):308-17. • Circulating plasmacytoid dendritic cells in acutely infected patients with hepatitis C virus genotype 4 are normal in number and phenotype. Mansour H, Laird ME, Saleh R, Casrouge A, Eldin NS, El Kafrawy S, Hamdy M, Decalf J, Rosenberg BR, Fontanet A, Abdel-Hamid M, Mohamed MK, Albert ML, Rafik M. J Infect Dis. 2010 Dec 1;202(11):1671-5. • Visualizing the functional diversification of CD8+ T cell responses in lymph nodes. Beuneu H, Lemaître F, Deguine J, Moreau HD, Bouvier I, Garcia Z, Albert ML, Bousso P. Immunity. 2010 Sep 24;33(3):412-23. • Harnessing naturally occurring tumor immunity: a clinical vaccine trial in prostate cancer. Frank MO, Kaufman J, Tian S, Suárez-Fariñas M, Parveen S, Blachère NE, Morris MJ, Slovin S, Scher HI, Albert ML, Darnell RB. PLoS One. 2010 Sep 1;5(9). • Biology and pathogenesis of chikungunya virus. Schwartz O, Albert ML. Nat Rev Microbiol. 2010 Jul;8(7):491- 500. • Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies. Alanio C, Lemaitre F, Law HK, Hasan M, Albert ML. Blood. 2010 May 6;115(18):3718-25. • Signal 0 for guided priming of CTLs: NKT cells do it too. Bousso P, Albert ML. Nat Immunol. 2010 Apr;11(4):284-6. • Type I IFN controls chikungunya virus via its action on nonhematopoietic cells. Clémentine Schilte*, Thérèse Couderc*, Fabrice Chretien, Marion Sourisseau, Anton Kraxner, Florence Guivel-Benhassine, Alain Michault, Fernando Arenzana-Seisdedos, Marco Colonna, Olivier Schwartz, Marc Lecuit and Matthew L. Albert. Journal of Experimental Medicine. 2010 Feb 15;207(2):429-42. • Autophagy within the antigen donor cell facilitates efficient antigen cross-priming. Martin Uhl*, Oliver Kepp*, Hélène Jusforgues-Saklani, Jose-Miguel Vicencio, Guido Kroemer and Matthew L. Albert. Cell Death & Differentiation. 2009;16(7):991-1005. • A mouse model for Chikungunya infection: young age and inefficient type-I interferon signaling are risk factors for severe disease. Thérèse Couderc, Fabrice Chrétien, Clémentine Schilte, Olivier Disson, Pierre Roques, Madly Brigitte, Florence Guivel-Benhassine, Michel Huerre, Yasmina Touret, Isabelle Shuffenecker, Philippe Desprès, Fernando Arenzana-Seisdedos, Alain Michault, Matthew L. Albert and Marc Lecuit. Plos Pathogen. 2008;4:2:e29. • Plasmacytoid dendritic cells initiate a complex chemokine and cytokine network and are a viable drug target in chronic HCV patients. Jérémie Decalf, Sandrine Fernandes, Randy Longman, Mina Alhoulay, Françoise Audat, François Lefrerre, Charles M. Rice, Stanislas Pol and Matthew L. Albert. The Journal of Experimental Medicine. 2007;204:1395-403. • Dendritic cell maturation alters intracellular signaling networks enabling differential effects of type I IFNs on antigen cross-presentation. Randy S. Longman, Sandra Pelegrini, Charles M. Rice, Robert B. Darnell and Matthew L. Albert. Blood. 2007;109:1113-22. 7
  10. 10. Institut Thématique Microbiologie et Maladies Infectieuses The paradoxical role of type I interferons in hepatitis C disease pathogenesis and treatment Clinical Investigation Fundamental Human Immunobiology Mouse Models Why study HCV Immunopathogenesis? It offers an example of in vivo cross-priming of T cells and provide an opportunity of uncovering new mechanism of immune evasion. By understanding the role of the immune system as it responds to HCV infection we may be able to identify strategies to stratify patients receiving treatment and propose alternative immune strategies for increasing the likelihood of clearing the virus (= cure). It is a major public health problem – 175M infected & leading cause of liver cancer. Liver / Tissue Lymph organ iDCs MHC-I MHC-II Activatio n Signals mDCs T4 T8 Immune Complex αVβ5 CD36 Maturation Stimulus - Inflammatory cytokines - Pathogen elements (LPS, ssRNA, dsRNA) Apoptotic Cell (infected or tumor) FcR Pathogen Entry x HCV and Antigen Cross-presentation elicits HCV-specific T cell response Matthew L. Albert, M.D, Ph.D Host response to infection, Hepatitis C 8
  11. 11. IFNγ / IFNα / TNFα => CXCL10 CXCL10 (IP-10) is an interferon-induced protein of ~10kDa (Luster, J Exp Med 1987) It is produced by hepatocytes during liver inflammation (Zeremski, Hepatology 2008) CXCR3 expression on NK and T cells induces their migration (Loetscher, J Exp Med 1996) CXCL10 T cells (CXCR3+) = CXCL10 receptor) T cells (CXCR3-) Neutrophils Monocytes B cells T cells cDCs Endothelial cells Keratinocytes Hepatocytes An unexpected role for CXCL10 in HCV disease pathogenesis 2 0 4 12 24 7248 weeks IFN+Rib Observational study: CXCL10 production in response to Peg-IFNα / Ribavirin therapy in chronic HCV patients HCV Paris cohort g1/g4 Stanislas Pol, Vincent Mallet (Cochin) Jean-Michel Pawlotsky, Christophe Hézode (Mondor) Jacques Denis (Sud-Francilien-Site Louise Michel) Philippe Renard (Argenteuil) Lawrence Serfaty (Saint-Antoine) Hervé Hagège, Isabelle Rosa (Créteil) sCXCL10 (3-77) in Chronic HCV patients 58% with evidence of N-terminal truncated CXCL10 Chronic disease Spontaneous resolvers Acute Hepatitis C Responders Non- Responders IFNαααα IP-10 IP-10 / CXCL10 is best predictor for failure to respond to IFN/Riba treatment Chronic Cohort (Paris, Genotype 1b) IP-10 α−2M EGF Ferritin N S H N S H N S H N S H p < 0.01 p < 0.01p = ns H N S H N S N S H N S H Plots (relative expression)* Matthew L. Albert, M.D, Ph.D Host response to infection, Hepatitis C Institut Thématique Microbiologie et Maladies Infectieuses 9
  12. 12. Translational potential of HCV studies New plasma biomarker-based algorithms for predicting response to therapy (required to help manage patients and optimize treatment response) New drug target to regulate lymphocyte trafficking for improving immunotherapy and therapeutic vaccination Chemokine antagonism: a new mechanism of immune regulation that may serve as a tractable drug target for chronic diseases IL-2 CD4+ T cell (*) Chronic inflammation alters T cell trafficking, limiting the probability of ‘antigen’ clearance IL-12 CD4+ Antigen persistence CD8+ CD4+ Dendritic Cell IFN-γ Peripheral Tissue Lymph Node CD40 / CD40L * An unexpected role for IP-10 in HCV disease pathogenesis The cleavage of IP-10 by CD26 results in an antagonist form of the molecule that participates in the disruption of T cells trafficking in the liver, accounting in part for the failure to spontaneously clear the virus and a failure to respond to therapy. IP-10 DPPIV sIP-10 WE PROPOSE TO RESTORE / STRENGTHEN THE CHEMOKINE GRADIENT BY INHIBITING DIPEPTIDYLPEPTIDASE IV IP-10IP-10 DPPIVX Killer T cells (CXCR3+ = IP-10 receptor) Unactivated T cells (CXCR3- ) Killer T cells (CXCR3+ = IP-10 receptor) Unactivated T cells (CXCR3- ) Matthew L. Albert, M.D, Ph.D Host response to infection, Hepatitis C Institut Thématique Microbiologie et Maladies Infectieuses 10
  13. 13. Institut thématique Microbiologie et Maladies infectieuses Keywords • Inflammatory bowel diseases • Crohn disease • Mucosal, immunity • T cell antigen receptor specificity • Antigens, bacterial • NKG2 proteins (NKG2D) • Cytokines: TNF, IL17 Major Grants • AVENIR INSERM (2007-2012) • European grant (IMI, Innovative Medical Initiative): Be the Cure IMI call topic: inflammation – Translational research and adaptive immunity; Academic coordinators: Tom Huizinga (LUMC, Leiden), Lars Klarekog (Karolinska Institutes) Matthieu Allez, M.D, Ph.D Selected publications: • Activation of the Receptor NKG2D Leads to Production of Th17 Cytokines in CD4+ T Cells of Patients with Crohn's disease. Pariente B, Mocan I, Camus M, Dutertre CA, Ettersperger J, Cattan P, Gornet JM, Dulphy N, Charron D, Lémann M, Toubert A, Allez M. Gastroenterology. 2011; in press. • The efficacy and safety of a third anti-TNF monoclonal antibody in Crohn’s disease after failure of two other anti-TNF antibodies. Allez M, et al. Aliment Pharmacol Ther. 2010;31:92-101. • CD4+NKG2D+ T cells in Crohn's disease mediate inflammatory and cytotoxic responses through MICA interactions. Allez M, Tieng V, Nakazawa A, Lemann M, Mayer L, Toubert A. Gastroenterology. 2007; 132:2346-58. • Defects in CD8+ regulatory T cells in the lamina propria of patients with inflammatory bowel disease. Brimnes J, Allez M, Dotan I, Ling S, Nakazawa A, Mayer L. Journal of Immunology. 2005;174:5814-22. • Regulatory T cells: Peace Keepers in the gut. Allez M, Mayer L. Inflamm Bowel Dis. 2004; 10:666-76. • Expansion of CD8+ T cells with regulatory function after interaction with intestinal epithelial cells. Allez M, Brimnes J, Dotan I, Mayer L. Gastroenterology. 2002;123:1516-1526. • Long-term outcome of patients with active Crohn’s disease exhibiting extensive and deep ulcerations at colonoscopy. Allez M, Lémann M, Bonnet J, Cattan P, Jian R, Modigliani R. Am J Gastroenterol. 2002; 97:947-53. Inserm U940 - Hôpital Saint-Louis, APHP - Paris Diderot University Paris Immunopathology of inflammatory bowel diseases (IBD). Focus on the aberrant adaptive immune function in IBD, by investigating phenotype, pathways of activation and function of protective- and inflammatory T cell subsets Our team is focused on the study of mucosal adaptive immunity in the human intestine, on interactions with the microbiota, and on pathogenesis of IBD. We have a strong expertise in isolation and sorting of T and non-T cell subsets (NK, B, DC), and in their phenotypic, molecular and functional analysis. We have access to the mucosal tissue (inflamed and non inflamed areas from surgical specimens or endoscopic biopsies from healthy individuals and IBD patients). Translational research is favoured by a close interaction with our clinical department (Hôpital Saint-Louis, Paris), including an important cohort of IBD patients (>5000 patients) connected with national (GETAID, REMIND) and international (ECCO) networks. We have accesses to cohorts of IBD patients treated with immunosuppressants and/or targeted therapies permits analysis of alterations in T cell biology, as well as products made by these cells, in regards of response to therapies. We coordinate a national biobank on post- operative model (REMIND network). The immune response triggered following pathogen recognition, though required to clear the infection, can be detrimental if it is produced in excess or fails to resolve promptly. Excessive inflammation contributes to infectious and noninfectious pathologies in the gut (such as inflammatory bowel disease, IBD: Crohn’s disease, CD). IBD are characterized by uncontrolled immune responses towards the intestinal microbiota. T cells contribute significantly to pathology during inflammation. Our objective is to improve the scientific understanding of aberrant immune function in IBD by investigating how generation/expansion, phenotype and function of protective and inflammatory T cell subsets can contribute to mucosal inflammation. Scientific expertise: isolation and sorting of T and non-T cell subsets from the intestinal mucosa and the periphery, and their phenotypic, molecular and functional analysis. Functional analyses include examination of antigen specificity of T cells to panel of known antigens in IBD, as well as of antigens of microbial origin. Major data: We have recently identified a subset of CD4+ T cells mediating inflammatory response in CD (Allez et al, Gastroenterology 2007). These cells are characterized by the expression of the stimulatory receptor NKG2D. We have shown that ligand activation of the NKG2D receptor triggers release of Th1 cytokines and induces cytotoxicity. More recently, we have shown that the CD4+ NKG2D+ T cell subset represents a major source of IL17 in CD, and has typical features of Th17 cells (Pariente et al, Gastroenterology 2011). Interactions between NKG2D and its ligands influence IL17 production. Furthermore, we demonstrated that CD4+ NKG2D+ T cells have a highly restricted TCR repertoire (submitted). Perspectives: To better define the phenotype and functional aberrations of pathogenic and regulatory cell subsets; - To develop novel assays to examine antigen-specificity of T cells to panel of microbial antigens; To understand post-operative relapse and failures of targeted therapies; To identify pathways of activation of pathogenic cells and thus specific targets for novel therapies. 11
  14. 14. Objectives: • How generation and function of protective and inflammatory T cell subsets can contribute to mucosal inflammation? • Examine antigen-specificity of T cells to panel of microbial antigens • To understand post-operative relapse and failures of targeted therapies Tools: • Isolation and sorting of T and non-T cell subsets from the intestinal mucosa • Phenotypic (flow cytometry) and molecular analysis (TCR repertoire, array) • Functional analyses include examination of antigen specificity of T cells to antigens of microbial origin. • Organization and access to cohorts of IBD patients (post-operative outcome, biotherapies) Figure 1: Expansion of CD4+ NKG2D+ T cells in CD Bacterial flora IEC Lamina propria CD4+ NKG2D+ T cells TCRαβαβαβαβ CD28- CD4 TCR NKG2D IFNγγγγ TNFαααα IL17 MICA NKG2D CD4 CD4 CD4 CD4 MHC II MICA/B ULBPs Matthieu Allez M.D, Ph.D Immunopathology of inflammatory bowel diseases Institut Thématique Microbiologie et Maladies Infectieuses 12
  15. 15. Figure 2 : IL17 producing CD4+ T Cells from Patients with CD highly express NKG2D 3.1% CD4 IL17 7 66 9.218 CD161 NKG2D CD161 NKG2D 22 1.6 1.874 Figure 3 : CD4+ pathogenic T cells have a restricted repertoire (17.1%) LP CD4+ Relativeexpression(%) Oligoclonality = 8% CDR3length(aa) Relativeexpression(%) Oligoclonality = 5% LP CD4+NKG2D- Vββββ-chain family LP CD4+NKG2D+ Oligoclonality = 24% Vββββ-chain family CD4+NKG2D+ CD4+NKG2D- Vββββ-chain family CDR3length(aa) Matthieu Allez M.D, Ph.D Immunopathology of inflammatory bowel diseases Institut Thématique Microbiologie et Maladies Infectieuses 13
  16. 16. Perspectives Unique Selling Points • Identification of factors involved in the generation of pathogenic CD4+ NKG2D+ T cells • Characterization of cytotoxic properties of pathogenic T cells • Identification of microbial antigens recognized by inflammatory T cell subsets • To examine the role of T cells in post-operative relapse • To analyze inflammatory pathways in failures of targeted therapies (anti-TNF) • Expertise in mucosal immunology, identification and characterization of protective and pathogenic intestinal T cells • Clinical expert in inflammatory bowel diseases (management, clinical trials) • Organization and access to cohorts of IBD patients (biobank, post-operative relapse, response to therapies) • Strong collaborations and participations to national (GETAID) and European networks (scientific officer of ECCO) • Bench to bedside: clinical phenotype, characterization of immune responses at tissue level, sorting of specific subsets, phenotypic and molecular analysis Matthieu Allez M.D, Ph.D Immunopathology of inflammatory bowel diseases Institut Thématique Microbiologie et Maladies Infectieuses 14
  17. 17. Institut thématique Microbiologie et Maladies infectieuses Keywords • Virus-specific T Cell Immunity • HIV • Oncogenic viruses • HCV • Herpesviruses • HPV • Influenza • Immunogenomics • Vaccines Major Grants • Europe (FP5, FP6, FP7) RTD projects Inserm U945 - Pierre and Marie Curie University Paris - APHP Federative Institute of Research on Immunity – Cancer – Infection - IFR113 Paris Immunology, Immunity and Immunogenetics to viruses and vaccines Investigating innovative aspects and biomarkers of immunity and Immunogenetics to viruses and vaccines and developping innovative immune-based interventions for the HIV infection Brigitte Autran, M.D, Ph.D Brigitte Autran, Professor of Immunology, in charge of the Immunology Department of Pitié-Salpêtrière Hospital at UPMC School of Medicine, co-chairs the Federative Institute of Research ‘Immunity-Cancer-Infection”. With a 25 years experience on T cell immunity to HIV, she produced several innovative findings with the first demonstrations of Cytotoxic T Lymphocytes directed against HIV (Nature 1987) and of immune reconstitution with antiretroviral therapies (Science 1997) and gains international recognition in the field of immunotherapy and vaccines for HIV and viruses (Nat.Immunol. Rev 2003; Science 2004). She belongs to the Top 1% researchers in the WOS with an H Index of citations of 49. The B Autran’s research team dedicated to Cellular Immunity and Immunogenetics of viral infections and vaccines » explores tightly interacting domains: 1. Immune responses and Immune-based therapies: Definition of immune correlates of protection against HIV and other viruses (CMV, oncogenic viruses [HHV8 and HCV], influenza) with definition of eQTL in conjunction with the genomics studies; 2. Immunogenomics of chronic viral Infections: with the responsibility of the ANRS genomics platform providing human-genome wide analysis of large cohorts, replacing the prior gene candidate approach in order to define impact of hosts genetic polymorphism on the course of the HIV and HCV infections. 3. Development and evaluation of innovative antiviral immune-based therapies and vaccines in HIV- infected and other immune-suppressed populations, by coordinating national, European and international collaborations and networking. Selected publications: • Long-term nonprogressors and elite controllers in the ANRS CO5 HIV-2 cohort. Thiébaut R, Matheron S, Taieb A, Brun-Vezinet F, Chêne G, Autran B; for the immunology group of the ANRS CO5 HIV-2 cohort. AIDS. 2011 Mar 27;25(6):865-867. • Immune reconstitution after a decade of combined antiretroviral therapies for human immunodeficiency virus. Guihot A, Bourgarit A, Carcelain G, Autran B. Trends Immunol. 2011 Mar;32(3):131-7. • Comprehensive analysis of virus-specific T-cells provides clues for the failure of therapeutic immunization with ALVAC-HIV vaccine. Papagno L, Alter G, Assoumou L, Murphy RL, Garcia F, Clotet B, Larsen M, Braibant M, Marcelin AG, Costagliola D, Altfeld M, Katlama C, Autran B; ORVACS Study Group. AIDS. 2011 Jan 2;25(1):27-36. • Distinct differentiation profiles of HIV-Gag and Nef-specific central memory CD8+ T cells associated with HLA-B57/5801 and virus control. Xie J, Lu W, Samri A, Costagliola D, Schnuriger A, da Silva BC, Blanc C, Larsen M, Theodorou I, Rouzioux C, Autran B; ALT-ANRS-CO15 study group. AIDS. 2010 Sep 24;24(15):2323-9. • Control of vaccinia virus skin lesions by long-term-maintained IFN-gamma+TNF-alpha+ effector/memory CD4+ lymphocytes in humans. Puissant-Lubrano B, Bossi P, Gay F, Crance JM, Bonduelle O, Garin D, Bricaire F, Autran B, Combadière B. J Clin Invest. 2010 May 3; 120(5):1636-44. • Acute hepatitis C in HIV-infected patients: rare spontaneous clearance correlates with weak memory CD4 T- cell responses to hepatitis C virus. Schnuriger A, Dominguez S, Guiguet M, Harfouch S, Samri A, Ouazene Z, Slama L, Simon A, Valantin MA, Thibault V, Autran B; ANRS HC EP21 study group. AIDS. 2009 Oct 23;23(16):2079-89. • Tuberculosis-associated immune restoration syndrome in HIV-1-infected patients involves tuberculin-specific CD4 Th1 cells and KIR-negative gammadelta T cells. Bourgarit A, Carcelain G, Samri A, Parizot C, Lafaurie M, Abgrall S, Delcey V, Vicaut E, Sereni D, Autran B; PARADOX Study Group. J Immunol. 2009 Sep 15;183(6):3915-23. • A step ahead on the HIV collaboratory. Murphy RL, Autran B, Katlama C, Brucker G, Debre P, Calvez V, Clotet B, Clumeck N, Costagliola D, Deeks SG, Dorrell L, Gatell J, Haase A, Klein M, Lazzarin A, McMichael AJ, Papagno L, Schacker TW, Wain-Hobson S, Walker BD, Youle M. Science. 2009 Jun 5;324(5932):1264-5. • Therapeutic vaccines for chronic infections. Autran B, Carcelain G, Combadiere B, Debre P. Science. 2004 Jul 9;305(5681):205-8. Erratum in: Science. 2004 Sep 24;305(5692):1912. • Therapeutic vaccines against HIV need international partnerships. Autran B, Debré P, Walker B, Katlama C. Nat Rev Immunol. 2003 Jun;3(6):503-8. Review. 15
  18. 18. Federative Institute of Researches: Immunity – Cancer – Infection Brigitte Autran - Co-Chair with David Klatzmann* * See scientist specific section Objectives: Control of Infectious Diseases • HIV : - Immunity to HIV and viruses: (Inserm U945) - B Autran et al. : Immunity & Immunogenetics to viruses and vaccines - P Debré & V Vieillard: Innate Immunity to viruses and vaccines - V Appay & A Moris: Immune control of viruses and Immunosenescence - Clinical epidemiology, therapeutic strategies and virology in HIV infection: (Inserm U943) - C Katlama et al.: Innovative Therapeutic strategies - V Calvez & P Flandre: Antiretroviral Resistance - D Costagliola : Clinical epidemiology, complications and treatment • Mycobacteria: V Jarlier et al. (ER 5 UPMC) • Malaria: D Mazier et al. (Inserm U945) • Viruses: H Agut et al. (ER1 UPMC) • Vaccines: B Combadière * et al., B Autran et al. (Inserm U945) D Klatzmann* et al. (Inserm U959) Brigitte Autran et al. Immunity and Immunogenetics to Viruses and Vaccines (Unit Inserm-UPMC 945) Objectives: • Immune Correlates & Biomarkers of Virus Control : HIV, HCV & oncogenic viruses. • Hosts Genetic determinant of chronic viral infections (HIV, HCV…) progression. • Vaccines and Therapeutic control of viruses in Immune-suppressed patients Tools: for Translational Research: • Networking and Collaborations with • Pitié-Salpétrière CRIV (Center for Integrated Researches on HIV : see slides 4,5) • Inserm/UPMC Unit 945 with: • V Appay, A Moris et al.: Mechanisms of T cell-based control of viruses • P Debré, V Vieillard et al: Innate immunity to viruses and new vaccines • B Combadière et al.: Vaccine immunity (see specific presentation) • International Platforms of Immune-Monitoring • Cohort studies and Clinical Trials: • HIV+ : Local File [n=4,000]; Chair of the French LTNP Cohort [ANRS-CO15] • Vaccine recipients, Immune-suppressed patients • Hi-Tech Platforms: Genomics (ANRS), Advanced Flow-cytometry High sensitivity/High thoughput Virus-specific T cell assays Main Achievements (H Index: 49) : see slide N°4 0 10 2 10 3 10 4 10 5 <PE-Cy5-A>: CD107 0 10 2 103 10 4 10 5 <AlexaFluor700-A>:IFNg 0.26 1.45 0.4697.8 Autran et al. J Exp Med 2003, 2005 Appay et al. Nat. Med 2001 JEM 2000,,2007, Blood 2009 I Theodorou et al. Blood 2000 PLosOne2009.. NK CD4 +- NKp44 NKp44L Vieillard et al. PNAS 2008, 09…. B Autran et al. Nat.Rev.Immunol. 2002 Science 1997,2000, 2004, 2009 Brigitte Autran, M.D, Ph.D Immunity – Cancer – Infection Institut Thématique Microbiologie et Maladies Infectieuses 16
  19. 19. HIV : «Clinical epidemiology, therapeutic strategies and virology in HIV infection” (Unit Inserm-UPMC 943) Objectives: D Costagliola (Head of the Research Unit, H Index: 42): Clinical epidemiology of HIV infection, its complications and its treatment : C Katlama (H Index: 64): Innovative Therapeutic strategies against HIV V Calvez (H Index: 38): HIV Resistance to antiretroviral drugs, Main Tools and Resources: D Costagliola: Chair of FHDH (French Hosp. Database on HIV): the world largest (>110 000 patients) ANRS Center for monitoring & statistics of clinical trials. C Katlama : Chair of: Clinical research unit, Pitié-Salpétrière active file (3,000 HIV+ patients), and CRIV ORVACS: International Platform promoting and conducting international clinical trials with N. American & European Univ., supported by the Bettencourt Foundation, ANRS group for Innovative Antiretroviral Strategies V Calvez: Chair of the ANRS group for resistance (www.hivfrenchresistance.org) Main Achievements : see slide N°4 The Pitié-Salpétrière Centre de Recherches Intégrées on HIV (CRIV) achievements - In the Heart of HIV Researches: Brigitte Autran, M.D, Ph.D Immunity – Cancer – Infection Institut Thématique Microbiologie et Maladies Infectieuses 17
  20. 20. HIV: Perspectives: The CRIV Programme HIV Beyond Undetectability UMR-S Inserm / UPMC 943 and 945 Novel Cohort HIV Comorbidities C Katlama et al & D Costagliola et al. Comorbidities Cancers Immune control of HIV & co-infections B Autran, P Debré & V Appay, A Moris et al. Mechanisms of T cell control of HIV Immune control of HIV Reservoirs Antiretroviral resistance mutations Antiretroviral Resistance to HIV & New Drug discovery V Calvez et al. & P Flandre et al. Antiretroviral drug discovery To maintain maximal HIV suppression Innovative Therapies for HIV C Katlama, B Autran, V Calvez, D Costagliola et al. HIV Eradication strategies cART strategies & HIV reservoirs Vincent Jarlier et al. Antibiotics, tuberculosis (TB) and other mycobacterial infection Axis: • Molecular targets, mechanism of action • Mechanism of acquired resistance • In vitro and in vivo evaluation of new antibiotics • New tools of diagnosis of resistant mycobacteria • Rates and characteristics of resistance 3D structure of Mtb DNA gyrase. PLoS One, 2010. Tools: • Enzymatic and structural study of proteins • Genetic studies and cell physiology • Epidemiology of resistance (National Reference Center, 2 networks) • Experimental chemotherapy (animal model) 3D structure of Mtb pyrazinamidase PLoS One, 2011. Evaluation of TMC207 (ATP synthase inhibitor) PLoS One, 2011. Mtb ATP synthase Science, 2005. Selected Bibliography on Mycobacteria: W. SOUGAKOFF, et al. Clin Microbiol Infect. 2004 K. ANDRIES, et al. Science 2005 N. VEZIRIS et al. Antimicrob Agents Chemother. 2005 E. CAMBAU et al. Clin Infect Dis. 2006 N. LOUNIS et al. Antimicrob. Agents Chemother 2006 VEZIRIS N. et al. Am J Respir Crit Care Med. 2009 PITON J. et al. PLoS One. 2010 BROSSIER F. et al. Antimicrob Agents Chemother. 2011 PETRELLA S. et al. PLoS One. 2011 VEZIRIS N. et al. PLoS One. 2011 (UPMC Unit1541), Pitié-Salpêtrière–C.Foix Hosp., Faculté de Médecine P. M. Curie, UPMC) Brigitte Autran, M.D, Ph.D Immunity – Cancer – Infection Institut Thématique Microbiologie et Maladies Infectieuses 18
  21. 21. O Silvie and D. Mazier et al. : Malaria Identification and pre-clinical validation of novel drug and vaccine targets Objectives: • Characterization of host- Plasmodium interactions during liver stages • Identification of novel vaccines and drugs targets against liver stages • Pre-clinical validation of anti-malarial vaccines and drugs Tools: • In vitro assays: primary hepatocytes (mouse, human), cell lines • In vivo assays: mouse models (KO, transgenic, humanized mice) • Human and Rodent parasites (genetically modified transgenic, KO) • Screening approaches: proteomics, transcriptomics, drug assays Main achievements: • Unique expertise for malaria liver stages (including P. falciparum) • First identification of a host molecule required for parasite entry (Silvie Nat Med 2003) • Novel anti-malarial compounds (Carraz PLoSMed 2006, Cosledan PNAS 2008 ; Mazier et al Nat Drug Discov 2009) • Medium/high throughput in vitro screening assays (Gego et al Antimicrob Agents Chemother 2006) • In vitro model for the study of P. vivax malaria relapses (Dembele et al PLoS One 2011) • Pre-clinical validation of a virosomal malaria vaccine (Okitsu et al PLoS One 2007) P Buffet and D Mazier et al. : Malaria: Deformability & circulation of parasitized red blood cells « Laveran » Research group UMRs945 UPMC & Pitié-Salpêtrière Hospital 2. New tools 1. Principle: • Micro-bead layers mimic splenic filters (A&B) (Deplaine et al., Blood 2011) • Spleens filter-out Plasm. Falciparum infected blood cells (Buffet et al.Curr Op Hematol 2009, Blood 2008, 2011) • P. falciparum -infected red blood cells : equally retained in microbead filters & human spleens (A) •« Pitting»: Spleen and filters can remove altered parasites from host red blood cells (C) 3. Application to the control of malaria Patent National Phase 29/11/10 UPMC/Institut Pasteur/APHP I. Screen for transmission-blocking compounds inducing retention of sparasites exual forms (gametocytes) in spleens II. Analyse the resistance of parasites to reference artemisinin derivatives (pitting process) III. Identify the parasite components altering infected red blood cells deformability 4. Specific objectives I. Adapt prototype (A) to medium throughput screening Collaboration with Discovery Biology (Brisbane) II. Assess correlation of parasite resistance with pitting rates in microbead filters Collaboration with Wellcome Trust NIH in South-East Asia I. Assess correlation between retention rates and expression level of candidat genes Collaboration with Institut Pasteur C (Unit Inserm/UPMC 945) Silvie O., et al. 2003. Nat Med.; Carraz M., et al. 2006. PLoS Medecine ; Coslédan F., et al. 2008. PNAS ; Siau A., et al. 2008. PLoS Pathogen ; Safeukui I, et al. 2008. Blood; Yalaoui S., et al. 2008. Cell Host & Microbe; Yalaoui S., et al. 2008. PLoS Pathogen ; Mazier D., et al. 2009. Nature Reviews Drug Discovery; Deplaine G, et al. 2010 Blood.; Buffet PA, et al. 2011 Blood. D Mazier et al.: Selected recent references UMRs945 Inserm/UPMC: Immunity and Infection, & Pitié-Salpêtrière Hospital Brigitte Autran, M.D, Ph.D Immunity – Cancer – Infection Institut Thématique Microbiologie et Maladies Infectieuses A 19
  22. 22. H. Agut et al. Dynamics, Epidemiology, and Therapy of Viral Infections ER1 DETIV Objectives: • Control of hepatitis virus (HBV, HCV), herpesvirus (HSV, VZV, CMV, EBV, HHV-6, HHV-8) • Quantification of viral multiplication in vitro and in vivo • Rate and mechanism of viral transmission in human populations • Analysis, prediction and prevention of resistance to antivirals in treated patients Tools: • Molecular detection, quantitation & genetic analysis of viral genomes in vitro and in vivo • Virus multiplication in cell cultures • Functional enzymatic assays • Molecular imaging • Cohort studies (AIDS, transplant recipients, emerging viral diseases ) Results : • Kinetics of reactivation of betaherpesviruses (CMV, HHV-6) in immunocompromised patients • Characterization of resistance of HBV, HHV-6, HSV, and CMV to DNA polymerase inhibitors • Molecular epidemiology of HSV lung infections in intensive care unit patients • Differential detection and transmission of HBV genotypes in developing countries • Burrel S, et al. Antimicrob Agents Chemother. 2010 • Hannachi N, et al. J. Virol. 2010 • Bonnafous P, et al. H. Antiviral Res. 2010 • Agut H, Future Microbiol. 2009 • Thibault V,et al. J Virol Methods. 2009 • Deback C, et al J Clin Microbiol. 2009 • Boutolleau D, et al. Antiviral Res. 2009 • Bonnafous P, et al. Antiviral Res. 2008 • Schnuriger A, et al J Clin Microbiol. 2006 • Palleau S, et al. Clin Infect Dis. 2006 H. Agut et al.: Selected recent references Dynamics, Epidemiology, and Therapy of Viral Infections ER1 DETIV Brigitte Autran, M.D, Ph.D Immunity – Cancer – Infection Institut Thématique Microbiologie et Maladies Infectieuses 20
  23. 23. Institut thématique Microbiologie et Maladies infectieuses Keywords • Antiviral • Functional genomics • Hepatitis C virus • Transplantation • Viral entry • Vaccine Major Grants • ERC-2008-AdG- 233130-HEPCENT • EU INTERREG-IV- FEDER-Hepato-Regio- Net • ANRS • ANR Chaire d’Excellence Thomas Baumert, M.D Inserm U748 – Strasbourg University - Nouvel Hôpital Civil Strasbourg Strasbourg Virology Institute Functional genomics of virus-host interactions, viral hepatitis and liver disease, molecular virology of hepatitis C Functional genomics of virus-host interactions for discovery of antivirals and vaccines Hepatitis C virus (HCV) infection is a major cause of liver disease world-wide. A vaccine is not available and antiviral therapies are limited. Recent advancements in functional genomics, cell culture and animal model systems have allowed rapid progress in the understanding of the molecular and clinical mechanisms of HCV- host interactions. HCV entry is the first step in a cascade of interactions between the virus and its target cell and thus plays a key role for the viral life cycle. Using a functional RNAi screen we have recently identified a network of receptor tyrosine kinases (RTKs) as HCV entry factors. RTKs mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and promoting membrane fusion (Lupberger et al. Nature Medicine 2011 in press). By studying virus-host interactions in unique clinical cohorts we have defined a key role of HCV entry for evasion from host immune responses and viral persistence (Fafi-Kremer et al., J. Exp. Med. 2010; Haberstroh et al. Gastroenterology 2008, Pestka et al. PNAS 2007). Since viral entry is required for the initiation, dissemination and maintenance of infection, it is a promising novel target for antiviral therapies and vaccines (Zeisel et al. J. Hepatol. 2011). Indeed, our recent data in preclinical animal models suggest that targeting host entry factors using receptor-specific monoclonal antibodies or small molecules constitutes a novel antiviral approach for prevention and treatment of HCV infection (Fofana et al. Gastroenterology 2010; Krieger et al. Hepatology 2010; Lupberger et al. Nature Medicine 2011 in press). Taken together, the results define key pathways for pathogenesis of viral disease and open new perspectives for the development of antivirals and vaccines. Selected publications: • EGFR and EphA2 are host factors for hepatitis C virus entry and targets for antiviral therapy. Lupberger J, Zeisel MB, Xiao F, Thumann C, Fofana I, Zona L, Davis C, Mee CJ, Turek M, Royer C, Zahid MN, Lavillette D, Fresquet J, Cosset FL, Rothenberg SM, Pietschmann, T, Patel A, Pessaux P, Doffoël M, Raffelsberger W, Poch O, McKeating JA, Brino L, Baumert TF. Nat. Med. 2011, in press. • Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation. Fafi-Kremer S, Fofana I, Soulier E, Carolla P, Meuleman P, Leroux-Roels G, Patel AH, Cosset FL, Pessaux P, Doffoël M, Wolf P, Stoll-Keller F, Baumert TF. J. Exp. Med. 2010; 207:2019-31. • Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes. Fofana I, Krieger SE, Grunert F, Glauben S, Xiao F, Fafi-Kremer S, Soulier E, Royer C, Thumann C, Mee CJ, McKeating JA, Dragic T, Pessaux P, Stoll-Keller F, Schuster C, Thompson J, Baumert TF. Gastroenterology. 2010;139:953-64. • Inhibition of hepatitis C virus infection by anti-claudin-1 antibodies is mediated by neutralization of E2-CD81- claudin-1 associations. Krieger SE, Zeisel MB, Davis C, Thumann C, Harris HJ, Schnober EK, Mee C, Soulier E, Royer C, Lambotin M, Grunert F, Dao Thi VL, Dreux M, Cosset FL, McKeating JA, Schuster C, Baumert TF. Hepatology. 2010;51:1144-57. • Sustained delivery of siRNAs targeting viral infection by cell-degradable multilayered polyelectrolyte films. Dimitrova M, Affolter C, Meyer F, Nguyen I, Richard DG, Schuster C, Bartenschlager R, Voegel JC, Ogier J, Baumert TF. Proc Natl Acad Sci U S A. 2008;105:16320-5. • Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C. Pestka JM, Zeisel MB, Bläser E, Schürmann P, Bartosch B, Cosset FL, Patel AH, Meisel H, Baumert J, Viazov S, Rispeter K, Blum HE, Roggendorf M, Baumert TF. Proc. Natl. Acad. Sci. U S A 2007;104:6025-30. 21
  24. 24. Institut Thématique Microbiologie et Maladies Infectieuses Thomas Baumert, M.D Functional genomics of virus-host interactions Objectives: • Investigation of molecular mechanisms of virus-host interactions using functional genomics • Identify key pathways of viral pathogenesis as novel targets for antivirals • Preclinical and early clinical development of antivirals Tools: • High-throughput RNAi screening platform • High-throughput HCV infection assay • State-of-the-art molecular virology • HCV human chimeric mouse model • Unique clinical cohorts with well defined clinical isolates Figure 1: Molecular mechanisms Receptor tyrosine kinases are HCV entry factors and antiviral targets A. B. EGFR is a cofactor for HCV entry and antiviral target in human hepatocytes RNAi screen identifies identifies a network of 58 host cell kinases as HCV entry factors Lupberger / Baumert Nature Medicine 2011, in press 22
  25. 25. Figure 2 : Clinical Impact Mechanisms of viral evasion in HCV-infected patients B. A. HCVppentry(Log10RLU) C. Neutralizationtiter Fafi-Kremer et al. J. Exp. Med. 2010, Haberstroh et al. Gastroenterology 2008, Pestka et al. PNAS 2007 HCV variants before transplantation HCV variants 7 days after transplantation • Liver transplantation – unique clinical model to study HCV pathogenesis • Variants re-infecting the liver graft (A) are characterized most efficient viral entry (B) and poor neutralization by autologous antibodies (C) • Viral entry plays a key role for viral evasion in acute and chronic HCV infection • Understanding of virus-host interactions identifies novel targets for antiviral therapy and vaccines Figure 3 : Technology transfer Preclinical development of innovative antivirals and vaccines Fofana et al. Gastroenterology 2010; Krieger et al. Hepatology 2010; Robinet / Baumert unpublished data 2011 Anti-receptor antibodies block infection of highly infectious escape variants that are resistant to patients’ neutralizing antibodies Institut Thématique Microbiologie et Maladies Infectieuses Thomas Baumert, M.D Functional genomics of virus-host interactions 23
  26. 26. Perspectives Unique Selling Points • Unravel the molecular mechanism of virus host-interactions and pathogenesis of virus- induced disease. • Identify key pathways involved in pathogenesis of disease. • Develop innovative antivirals and vaccines for viral infections with a major unmet medical need (viral hepatitis, dengue, HIV). • Pioneer and leader in HCV-host interactions with unique expertise in viral entry and pathogenesis • From molecular tools to clinics : integration and access to all research materials : molecular constructs, large panel of unique recombinant viruses, state-of-the-art cell and animal models including key functional assays • BSL3 high-throughput screening platform, BSL3 animal facility for recombinant viruses, RNAi screening and systems biology platform (IGBMC) • Unique patient cohorts, clinical trials with focus on innovative first-in-class compounds (U Strasbourg Medical Center) • Strong collaborations with rapid and versatile production of unique antibodies and antivirals Institut Thématique Microbiologie et Maladies Infectieuses Thomas Baumert, M.D Functional genomics of virus-host interactions 24
  27. 27. Institut thématique Microbiologie et Maladies infectieuses Keywords • AIDS • HIV • Restriction • Dendritic cells • Innate immunity • Vaccine Major Grants • ERCAdv grant 2010- 2015 • ANRS • SIDACTION • ANR Monsef Benkirane, Ph.D CNRS UPR1142 Montpellier Institut de Génétique Humaine Montpellier Mechanisms of HIV replication Our major aim is to identify cellular factors controlling HIV-1 replication and to understand their mechanism of action Human Immunodeficiency virus type 1 (HIV-1) infects primarily cells of the immune system. The outcome of HIV-1 infection results from complex interactions between viral proteins and host cell factors. In most cases, HIV-1 successfully uses cellular pathways and bypasses cellular restriction factors for optimal replication leading to continuous rounds of infection, replication and cell death. However, in certain situations virus replication can be successfully controlled. First, HAART (Highly Active AntiRetroviral Therapy) treatment revealed the existence of a pool of resting memory CD4+ T cells harbouring integrated but silent HIV-1 provirus. Although this situation occurs in a small number of cells, it suggests that intracellular defence mechanisms can be effective against HIV. This long lived viral reservoir is believed to be the major obstacle against HIV-1 eradication by HAART. Second, HIV-infected individuals who are able to control their virus to undetectable levels for many years in absence of any treatment have been identified and referred to as Elite HIV controllers “EC”. Again, this is a rare situation observed in 0.5% of infected patients. Still, it demonstrates that it is possible to naturally and effectively control HIV replication and disease progression. A major challenge in the HIV field is to identify the host factors and define the molecular mechanisms involved in the control of virus replication. Our lab is interested at identifying cellular factors (chromatin modifiers, microRNA and restriction factors) involved in HIV-1 silencing and restriction. We will present our recent data which lead us to identify the dendritic and myeloid cells-specific HIV-1-restriction factor. Our finding is of crucial importance to both the understanding of the physiopathology of HIV-1 infection and to the design of DC-target vaccines against HIV. Selected publications: • VIP8 is the dendritic and myeloid cells-specific HIV-1-restriction factor counteracted by Vpx. Nadine Laguette, Bijan Sobhian, Nicoletta Casartelli, Mathieu Ringeard, Christine Chable-Bessia, Emmanuel Ségéral, Stéphane Emiliani, Olivier Schwartz, and Monsef Benkirane. Nature. 2011. In press. • Competition between Dicer mRNA, pre-miRNA, viral RNA for exportin-5 binding strikes a new regulatory mechanism for Dicer expression. Yamina Bennasser, Christine Chable-Bessia, Robinson Triboulet, Derrick Gibbings, Carole Gwizdek, Catherine Dargemont, Eric J Kremer, Olivier Voinnet and Monsef Benkirane. Nature Structural & Molecular Biology. 2011 Mar;18(3):323-7. • HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP. Sobhian, B., Laguette, N., Yatim, A., Nakamura, M., Levy, Y., Kiernan, R., and Benkirane, M. Mol Cell. 2010;38,439-45 (Faculty of 1000. FF8). • Suppression of microRNA-silencing pathway by HIV-1 during virus replication. Triboulet, R., Mari, B., Lin, Y.L., Chable-Bessia, C., Bennasser, Y., Lebrigand, K., Cardinaud, B., Maurin, T., Barbry, P., Baillat, V., et al. Science. 2007;315,1579-1582 (Editor choice. Faculty of 1000. FF10). • Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2. Linares, L.K., Kiernan, R., Triboulet, R., Chable-Bessia, C., Latreille, D., Cuvier, O., Lacroix, M., Le Cam, L., Coux, O., and Benkirane, M. Nat Cell Biol. 2007;9,331-338(Faculty of 1000. FF8). • Suv39H1 and HP1gamma are responsible for chromatin-mediated HIV-1 transcriptional silencing and post- integration latency. Du Chene, I., Basyuk, E., Lin, Y.L., Triboulet, R., Knezevich, A., Chable-Bessia, C., Mettling, C., Baillat, V., Reynes, J., Corbeau, P., et al. Embo J. 2007;26:424-435. 25
  28. 28. Institut Thématique Microbiologie et Maladies Infectieuses Monsef Benkirane, Ph.D Infection of Dendritic cells: Problem solved Understanding Interactions between HIV and its host Objectives: • Deciphiring the molecular mechanisms involved in HIV-1 latency • Role of RNAi in HIV-1 replication • Identification of host cell factors involved in HIV-1 replication Tools: • Transcriptional analyses • Chromatin Immunopecipitation (ChIP) • ChIP combined with deep sequencing and RNA-seq • Biochemistry • Cellular and Molecular imaging • Primary cells isolated from HIV-patients Identification of Dendretic and myeloid cells specific HIV-1 restriction factor Vpx interacts and induces proteasomal degradation of VIP8 M W F/H-VpxCtrl  ¡¡ ¢¢£ ¤¥ ££ ¦¦ §¥ §¢  ¢ ¢¨ © VIP8 ¢ !¢ #¨ ¢ VIP8 F/H-Vpx F/H-Vpx: + Flag-IP DDB1 Cul4A - R.S.I. 1 0.05 0.7 VIP8 Tubulin DDB1 Mg132: - +- VLP-Vpx: + +- 26
  29. 29. Institut Thématique Microbiologie et Maladies Infectieuses VIP8 expression is cell type specific and correlates with HIV-1 susceptibility N.I. HIV-LUC-G VLP-Vpx: - + VIP8 DDB1 Tubulin THP-1 FoldIncrease 6 0 4 8 10 12 2 VIP8 DDB1 Tubulin - + MDDC b 14 6 0 4 8 10 12 2 VIP8 DDB1 Tubulin - + MDM 6 0 4 8 10 2 shRNA Scr VIP8- 3 VIP8- 4 FoldIncrease THP-1 x12 x4 0 2 4 6 8 10 12 14 FoldIncrease shRNA Scr VIP8 VIP8R - - + VIP8 THP-1 0 5 10 15 20 shRNA Scr VIP8 Foldincrease Vpx/noVpx N.I. HIV-LUC-G THP-1 0 2 4 6 U937 WT mut-VIP8 FoldIncrease 0 0.2 0.4 0.6 0.8 1 1.2 1.4 VIP8 is an HIV-1-restriction factor counteracted by Vpx Monsef Benkirane, Ph.D Infection of Dendritic cells: Problem solved 27
  30. 30. Institut Thématique Microbiologie et Maladies Infectieuses VIP8 restricts HIV-1 infection of Dendritic cells Knock-down of VIP8 renders Dendritic cells permissive to HIV-1. Scr 1 2 HD3 siRNA HIV-G Foldincrease (p24+cells) Scr 1 2 HD4 0 10 20 30 40 VIP8 is the dendritic and myeloid cells-specific HIV-1-restriction factor counteracted by Vpx • How VIP8 expression is regulated both at the transcriptional and protein stability levels? • What is the natural function of VIP8 in Macrophages and Dendritic cells? • Does VIP8 bind to viral nucleic acid or proteins? • Does VIP8 play role in DC-mediated innate immunity? Perspectives Monsef Benkirane, Ph.D Infection of Dendritic cells: Problem solved 28
  31. 31. Institut thématique Microbiologie et Maladies infectieuses Keywords • Toxoplasma gondii • Intracellular parasite • Antigen presentation Major Grants • ATIP-Avenir team Inserm U1043 - CNRS UMR5586 - Toulouse University Toulouse Immunology, cell biology, parasitology Using recently identified natural T cell epitopes from intracellular parasites such as Toxoplasma gondii, we study the molecular and cell biological mechanisms controlling antigen processing and presentation by MHC class I molecules Nicolas Blanchard, Ph.D Immune cells invaded by infectious microorganisms present fragments of the pathogens (antigens) on their surface. This leads to stimulation of T lymphocytes expressing receptors that specifically recognize the cognate antigens. These processes are paramount to eliminate or contain the infection. Our team focuses on infections caused by intracellular parasites. One fascinating example is the protozoan parasite Toxoplasma gondii. Toxo replicates in a vacuole inside infected cells and persists definitively within an individual’s brain. Toxo is the causative agent of toxoplasmosis, an opportunistic disease which can trigger serious neurological disorders in immunocompromised subjects and can have dramatic consequences on the fetus if infection occurs during pregnancy. Furthermore, Toxo constitutes an attractive model to better understand Plasmodium the parasite responsible of malaria. Using a model of toxoplasmosis in the mouse, we take advantage of our previous discoveries, like the identification of several natural Toxo antigens, among which an immunodominant antigen inducing large populations of protective CD8 T cells. Our project addresses 3 main goals: 1. Understand how Toxo proteins are transported and degraded by infected cells 2. Analyze how host cell-parasite interactions influence parasite growth and innate recognition 3. Dissect the first steps of T cell priming in the intestine Our results may suggest strategies to optimize presentation of antigens from intracellular parasites. Hence they may facilitate the creation of Toxo vaccines in humans and participate in improving vaccines against other parasites. By studying inflammation induced by Toxo in the mouse gut, our project may also help to better understand mechanisms leading to human chronic inflammatory bowel diseases (IBD). Selected publications: • Topological journey of parasite-derived antigens for presentation by MHC class I molecules. Blanchard N, Shastri N. Trends Immunol. 2010 Nov;31(11):414-21. Review. • Endoplasmic reticulum aminopeptidase associated with antigen processing defines the composition and structure of MHC class I peptide repertoire in normal and virus-infected cells. Blanchard N, Kanaseki T, Escobar H, Delebecque F, Nagarajan NA, Reyes-Vargas E, Crockett DK, Raulet DH, Delgado JC, Shastri N. J Immunol. 2010 Mar 15;184(6): 3033-42. • Immunodominant, protective response to the parasite Toxoplasma gondii requires antigen processing in the endoplasmic reticulum. Blanchard N, Gonzalez F, Schaeffer M, Joncker NT, Cheng T, Shastri AJ, Robey EA, Shastri N. Nat Immunol. 2008 Aug;9(8):937-44. • Coping with loss of perfection in the MHC class I peptide repertoire. Blanchard N, Shastri N. Curr Opin Immunol. 2008 Feb;20(1):82-8. Review. • ERAAP synergizes with MHC class I molecules to make the final cut in the antigenic peptide precursors in the endoplasmic reticulum. Kanaseki T, Blanchard N, Hammer GE, Gonzalez F, Shastri N. Immunity. 2006 Nov; 25(5): 795-806. 29
  32. 32. Antigen processing and parasite immunity Main parasite model : Toxoplasma gondii (Toxo) Objectives: • How are parasite proteins transported and degraded by infected cells ? • How does parasite vacuole trafficking affect host-parasite interactions ? • How are T cells primed in the intestine after Toxo oral infection ? Tools: • Toxo : a genetically tractable model of protozoan parasite • Focus on natural T cell epitopes (as opposed to model antigens) • Reporter T cell hybridomas to measure antigen presentation levels • MHC I and MHC II tetramer reagents to track antigen-specific T cells • Differential susceptibility of inbred and knock-out mouse strains to toxoplasmosis • shRNA library targeting host vesicular trafficking (collaboration) • Flow cytometry, confocal imaging, RP-HPLC, antigen presentation assays Macrophage infected by fluorescent Toxo Figure 1: Approach to identify natural T cell epitopes from Toxo Toxo-specific CD8 T cell hybridomas expressing the NFAT- inducible   -galactosidase reporter gene were used to screen a cDNA library from Toxo parasites and identify the cognate antigen(s) Institut Thématique Microbiologie et Maladies Infectieuses Nicolas Blanchard, Ph.D Antigen processing and parasite immunity 30
  33. 33. Figure 2 : Toxo protein GRA6 is an immunodominant and protective CD8 T cell antigen (A) Antigen : protein from Toxo dense granules (GRA6), secreted in parasite vacuole Final epitope : decamer peptide (HF10) presented by H2-Ld MHC I (B) Large CD8 T cell populations respond to GRA6-derived HF10 peptide after infection (C) Immunization with HF10 protects against lethal challenge with Toxo MHC I tetramer CD8 36% 0.33% 1.7%0.09% Brain HF10 (GRA6) SM9 (GRA4) IF9 (ROP7)YL9 (ctrl) 6 days Toxo LD100 Immunization Challenge Peptide-pulsed BMDCs Protection A B C Figure 3 : Processing in the host cell endoplasmic reticulum is required for GRA6 presentation GRA6 presentation by Infected BM-macrophages 0 0.75 1.5 3 6 12 0.0 0.1 0.2 0.3 MOI CTgEZ.4response(A595) +/– ERAAP –/– HF10-specificCD8+ (%) GRA6-specific CD8 T cell response in spleen Toxo 12 days 0 1 2 3 * ERAAP+/+ +/– ERAAP–/– Test the role of ER aminopeptidase associated with antigen processing (ERAAP) In vitro In vivo Institut Thématique Microbiologie et Maladies Infectieuses Nicolas Blanchard, Ph.D Antigen processing and parasite immunity 31
  34. 34. Perspectives Unique Selling Points Figure 4 : Fusion between parasite vacuole and host ER favors presentation of parasite-derived antigen Blockade of ER-vacuole fusion in dendritic cells was achieved by transduction of shRNA targeting a host ER SNARE protein (A)IF detection of TAP2 in shRNA-transduced DCs after infection with OVA/YFP+ Toxo (B) Presentation of OVA secreted by OVA-expressing or control Toxo 8h post-infection was evaluated by measuring the proportion of CD69+ OT-I TCR-transgenic T cells Collaboration with A. Savina, Curie Institute, Paris and L.F. Moita, Institute of Molecular Medicine, Lisbon • Examine role of ER-PV fusion in parasite survival and innate immune recognition. • Characterize involvement of Toxo-specific T cells during Toxo-induced ileitis (CD8 immunomodulatory functions ?). • Identify immunodominant T cell epitopes in a mouse model of cerebral malaria and analyze role of parasite-specific CD8 T cells in disease • Identified the first natural and immunodominant T cell epitope from Toxo • Expertise in T cell antigen identification by hybridoma generation and expression cloning • Unique tools to analyze antigen processing and host-parasite interactions from an immunological standpoint • Developping new genetically modified parasites • Strong collaborations strengthening our research on the parasite and cell biology sides Institut Thématique Microbiologie et Maladies Infectieuses Nicolas Blanchard, Ph.D Antigen processing and parasite immunity 32
  35. 35. Institut thématique Microbiologie et Maladies infectieuses Keywords • Malaria • Anopheles mosquitoes, • Plasmodium parasites • Host-parasite interactions, • Complement-like proteins Major Grants • ERC starting grant Inserm U963 - CNRS UPR9022 - Strasbourg University Strasbourg Antiparasitic responses of the malaria mosquito, Anopheles gambiae Not all mosquitoes transmit malaria parasites to humans. Our objective is to understand what are the genetic factors that render mosquitoes resistant to malaria parasites, and therefore unable to transmit the disease Stéphanie Blandin, Ph.D Anopheles gambiae is a major vector for Plasmodium falciparum, the parasite causing the most severe form of human malaria. With an estimated 250 million infected people every year and another 3.3 billion at risk, it is one of the biggest scourges of humanity. The ability of mosquitoes to transmit malaria parasites is highly variable between individuals, with some mosquitoes fully resistant to the parasites and therefore unable to transmit the disease. A large part of this variability is determined by genetic factors. We previously demonstrated that different forms (or alleles) of the gene encoding the complement-like protein TEP1 confer different degrees of resistance to the rodent malaria parasite Plasmodium berghei. Still, our data show that other genes are involved. The objective of our group is to decipher the genetic networks that sustain mosquito resistance to the rodent malaria parasite P. berghei and the human parasite P. falciparum. For this, we develop new tools based on next-generation sequencing and high-throughput genotyping to efficiently dissect the genetic basis of complex traits in A. gambiae. The contribution of the identified genes and networks to vector competence in natural mosquito populations will be further evaluated in malaria-endemic regions. Because resistance naturally occurs in mosquito populations, this project has implications for the design of novel strategies and/or for the improvement of existing ones to reduce malaria transmission. Selected publications: • Dissecting the genetic basis of resistance to malaria parasites in Anopheles gambiae. Blandin SA, Wang- Sattler R, Lamacchia M, Gagneur J, Lycett G, Ning Y, Levashina EA, Steinmetz LM. Science. 2009;326: 147- 150. • Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium. Fraiture M, Baxter RH, Steinert S, Chelliah Y, Frolet C, Quispe-Tintaya W, Hoffmann JA, Blandin SA, Levashina EA. Cell Host Microbe. 2009;5:273-284. • Antimalarial responses in Anopheles gambiae: from a complement-like protein to a complement-like pathway. Blandin SA, Marois E, Levashina EA. Cell Host Microbe. 2008;3:364-374. • Mosaic Genome Architecture of the Anopheles gambiae Species Complex. Wang-Sattler R, Blandin S, Ning Y, Blass C, Dolo G, Toure YT, Torre AD, Lanzaro GC, Steinmetz LM, Kafatos FC, Zheng L PLoS ONE. 2007;2:e1249. • Structural basis for conserved complement factor-like function in the antimalarial protein TEP1. Baxter RH, Chang CI, Chelliah Y, Blandin S, Levashina EA, Deisenhofer J. Proc Natl Acad Sci U S A. 2007;104:11615- 11620. • Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. Waterhouse RM, Kriventseva EV, Meister S, Xi Z, Alvarez KS, Bartholomay LC, Barillas-Mury C, Bian G, Blandin S, Christensen BM, et al. Science. 2007;316:1738-1743. 33
  36. 36. Institut Thématique Microbiologie et Maladies Infectieuses Stephanie Blandin, Ph.D Resistance to Malaria parasites in the mosquito Anopheles gambiae Objectives: • What makes mosquitoes resistant to malaria parasites? • What are the differences between mosquito responses to human and rodent malaria parasites? Tools: • Laboratory models of infections • Next-generation sequencing technologies • High throughput genotyping • Forward genetics / QTL mapping • Functional analysis by RNA interference • Molecular and cellular biology tools Susceptible (S) strain Resistant (R) strain Collins, Science 1986 Mosquito midguts, 8 days post infection with GFP expressing parasites Figure 1 : rasRNAi to assess the contribution of different alleles to a phenotypic trait Reciprocal allele-specific RNA interference (rasRNAi) can be used to specifically silence one or the other allele of a given gene in the same genetic context, in order to compare their contribution to a trait. rasRNAi can be applied to all organisms where RNAi is feasible to dissect complex phenotypes to the level of individual quantitative trait alleles. Allele-specific dsRNA probes: F1: R x S 34
  37. 37. Institut Thématique Microbiologie et Maladies Infectieuses Stephanie Blandin, Ph.D Resistance to Malaria parasites in the mosquito Anopheles gambiae Figure 2 : Polymorphisms in TEP1 confer resistance TEP1 is a mosquito complement-like protein with antiparasitic activity. Polymorphisms in the TEP1 gene affect parasite survival: mosquitoes expressing only the susceptible form of TEP1 (dsR group) carry more parasites than those expressing only the resistant form (dsS group). F1: R x S Figure 3 : Complement-like structure of TEP1 TEP1 protein structure is very close to that of human complement factor C3. Most differences between the resistant TEP1*R1 and susceptible TEP1*S3 alleles are located in the 3’ half, encoding the α-ring that mediates binding to pathogen surfaces in complement C3. 0 0.1 0.2 0 300020001000 4000 Ka Nucleotide Position (bp) *S3/*R1 Average divergence between alleles at non synonymous sites (Ka) TEP1*R1 vs. TEP1*S3 Human complement factor A. gambiae 35
  38. 38. Perspectives Unique Selling Points Institut Thématique Microbiologie et Maladies Infectieuses Stephanie Blandin, Ph.D Resistance to Malaria parasites in the mosquito Anopheles gambiae • Improved tools for genetic analysis in A. gambiae. • Identification of genetic networks controlling resistance to rodent and human parasites in laboratory conditions. • Genetic and environmental factors controlling resistance in field mosquitoes. • Mosquito-based strategies to limit malaria transmission • Strong expertise in laboratory models of infection of A. gambiae by Plasmodium parasites • New insectary to be built in Strasbourg, including facilities for P. falciparum infection (Plan Campus) • Strong expertise in immunology and insect immunity + common environment with groups studying Drosophila immunity • Access to imaging and proteomics platforms on site, and to next generation sequencing and structure analyses through collaborations • From laboratory models to the field: access to field mosquitoes and experimental infections in Cameroon through collaboration with IRD laboratory 36
  39. 39. Institut thématique Microbiologie et Maladies infectieuses Keywords • Q fever • Coxiella burnetii • Transposon mutagenesis • Cellular microbiology • Automated microscopy Major Grants • ATIP-Avenir Team • Programme de mécénat partenariat Aviesan/Sanofi Aventis CNRS UMR5236 Montpellier Centre d'études d'agents Pathogènes et Biotechnologie pour la Santé (CPBS) Bacterial infections The aim of our research is to investigate and understand Coxiella infections, to understand how this pathogen subverts host functions, design new therapies to cure infections, develop diagnostic tools and vaccines Matteo Bonazzi, Ph.D Coxiella burnetii is an obligate intracellular gram-negative bacterium responsible of the zoonosis Q fever, a disease that manifests as an acute flu-like illness. Coxiella infects domestic ruminants, pets and arthropods resulting in usually asymptomatic infections but it can lead to miscarriages and stillbirths. The main source of infection for human hosts is contaminated aerosols, as a consequence population at risk of contamination includes farmers, veterinarians, and slaughterhouse workers. Although acute Q fever is not associated with a high mortality rate (2% approximately) it provokes acute disabling disease and it can lead to chronic infections that have fatal complications such as endocarditis pneumonia and hepatitis. As many as 65% of the patients affected by chronic Q fever may die of the disease. Due to its high infectivity it has been classified as a class B biothreat and is responsible of severe outbreaks with a very high economic impact on rural areas. Despite the scientific and economic interest that Coxiella infections raise, its obligate intracellular nature has hampered the research activity due to the impossibility of genetic manipulation and growth in broth. The mechanisms of subversion of host functions remain therefore obscure and the number of Coxiella virulence factors identified is to date very limited. The recent characterization of a specific growth medium that allows axenic growth of Coxiella opens the way to genetic engineering of the bacterium. The laboratory of Cell Biology of Bacterial Infections is a newly set up unit at the CPBS in Montpellier. The aim of this project is the large-scale identification of Coxiella burnetii virulence factors by generating a bank of mutants by transposon mutagenesis. This will be coupled to the set up of robust high throughput screens to identify phenotypes that will allow the characterization of virulence factors. Genes of particular relevance in the infectious cycle of Coxiella will be then sorted and analysed in detail by common cellular microbiology approach. Our study will contribute significantly to the understanding of Coxiella pathogenesis and will serve the development of alternative therapeutic strategies and animal vaccines to prevent future outbreaks. Selected publications: • Entrapment of intracytosolic bacteria by septin cage-like structures. Mostowy, S. Bonazzi, M. et al. Cell Host Microbe. 2010;8:433-444. • Listeria monocytogenes internalin and E-cadherin: from bench to bedside. Bonazzi, M., Lecuit, M. Cossart, P. Cold Spring Harb Perspect Biol. 2009;1:a003087. • Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes. Bonazzi, M., Veiga, E., Pizarro-Cerdá, J. Cossart, P. Cell Microbiol. 2008;10:2208- 2222. • Invasive and adherent bacterial pathogens co-Opt host clathrin for infection. Veiga, E., Guttman, J., Bonazzi, M. et al. Cell Host Microbe. 2007;2:340-351. 37
  40. 40. Institut Thématique Microbiologie et Maladies Infectieuses Matteo Bonazzi, Ph.D Large scale identification of Coxiella burnetii virulence factors Objectives: • Understand the cell biology of Coxiella infections • Identify virulence factors that regulate Coxiella replication within host cells. • Identify novel host factors involved in the intracellular cycle of Coxiella Tools: • Library of Coxiella mutants generated by transposon mutagenesis • Imaging-based high throughput screens • Eukaryotic genome-wide siRNA libraries High-throughput screen of putative Coxiella burnetii virulence factors Gram negative bacterium Obligate intracellular Genome sequenced in 2003 World-wide spread Causative agent of the zoonosis Q fever (acute and chronic phase) Coxiella burnetii a closer look 38
  41. 41. Institut Thématique Microbiologie et Maladies Infectieuses Matteo Bonazzi, Ph.D Large scale identification of Coxiella burnetii virulence factors Gram negative bacterium Obligate intracellular Genome sequenced in 2003 World-wide spread Coxiella burnetii a closer look Causative agent of the zoonosis Q fever (acute and chronic phase) Inhibition of apoptosis No virulence factors identified to- date High-throughput screen of putative Coxiella burnetii virulence factors Growth curve by fluorescence intensity MultimodePlateReader Cm+ RFP Axenic growth Infection of host cells in 96-wells plates Step-by-step analysis of Coxiella intracellular cycle •Hits validation •Protein tagging •Protein purification •Antibody production •Y2H screens •Interactor/s analysis •Manipulation of host functions In depth characterization of a gene of interest 39
  42. 42. Institut Thématique Microbiologie et Maladies Infectieuses Matteo Bonazzi, Ph.D Large scale identification of Coxiella burnetii virulence factors • Pioneering the cell biology of Coxiella infections in Europe • Strong expertise in Bacteria-host interactions • Innovative approaches for high-throughput screening and high-content data analysis • Extended network of collaborations with leaders in the field of Cell Biology and Cellular Microbiology • Access to cutting-edge technology Unique Selling Points 40
  43. 43. Institut thématique Microbiologie et Maladies infectieuses Major Grants • 2010-2015: ERC-2010- StG-Proposal nº260901 • 2006-2010: Inserm Avenir Keywords • Cellular Microbiology • Chemical genomics • Mycobacterium tuberculosis • Macrophages • Automated confocal imaging Inserm U1019 - CNRS UMR8204 - Institut Pasteur Lille - Lille-Nord de France University Lille Center for Infection and Immunity of Lille Cellular Microbiology and chemical genomics of Mycobacterium tuberculosis colonization into host cells We developed type of assays based on the visualization of mycobacterium replication within host cells and applied it to large scale genome wide screen for the identification of compounds and genes that are involved in intracellular bacterial growth and persistence Priscille Brodin, Ph.D Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis bacillus that results in millions of deaths annually, and an increasing number of drug resistant cases are being reported each year. New drugs – and new drug targets – are urgently needed. M. tuberculosis persists and replicates within macrophages (i.e., professional phagocytic cells) using a variety of mechanisms, including inhibition of phagosome maturation, escape to the host cell cytosol, induction of host macrophage apoptosis, and resistance to killing by oxygenated metabolites. Host-pathogen cross-talk is then established, leading to a balance between M. tuberculosis virulence factors and the macrophage antibacterial response, to create a niche favourable to the infection. Detailed elucidation of the manner in which the host macrophage initiates innate immune responses upon infection by pathogenic mycobacteria, and how the latter escapes immune surveillance, will contribute to a better understanding of tubercle bacillus persistence and latency. To this end, we have been taking unbiased, three-dimensional, large scale approaches using visual phenotypic assays (relying on monitoring by automated confocal fluorescence microscopy) of the trafficking and replication of M. tuberculosis inside macrophages. Screening of an 8,000 member small interfering RNA (siRNA) library, an 11,000 member M. tuberculosis mutant library and 200,000 small chemical molecules has led to the identification of key host and mycobacterial genes involved in the trafficking and replication of M. tuberculosis in mammalian macrophages, as well as chemicals able to prevent bacterial intracellular growth. Using this set of results, together with automated confocal fluorescence microscopy and cellular microbiology techniques, our project is to further explore the signalling pathways used specifically by M. tuberculosis. On the pathogen side, we will focus on the in depth study of bacterial protein effectors belonging to the ESX and PPE families. On the host side, we will focus on understanding how host cell protein promotes intracellular mycobacterial survival. Lastly, chemicals that target cellular partners of M. tuberculosis could constitute a new starting point for the development of drugs able to counteract host response manipulation without directly targeting the pathogen, thereby overcoming the issue of the emergence of drug-resistant strains. Altogether, our results will contribute to a better appreciation of the host manipulation exerted by the tubercle bacillus for its successful escape from immune surveillance. Selected publications: • Ethionamide boosters: Synthesis, Biological Activity and Structure-Activity Relationship of a series of 1,2,4- oxadiazole EthR inhibitors. Flipo M, Desroses M, Lecat-Guillet N, Dirie B, Carette X, Leroux F, Piveteau C, Demirkaya F, Lens Z, Rucktooa P, Villeret V, Christophe T, Jeon HK, Locht C, Brodin P, Deprez BP, Baulard A, Willand N. J Med Chem. 2011 Mar 21. • High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose- containing glycolipids involved in phagosome remodeling. Brodin P, Poquet Y, Levillain F, Peguillet I, Larrouy- Maumus G, Gilleron M, Ewann F, Christophe T, Fenistein D, Jang J, Jang MS, Park SJ, Rauzier J, Carralot JP, Shrimpton R, Genovesio A, Gonzalo-Asensio JA, Puzo G, Martin C, Brosch R, Stewart GR, Gicquel B, Neyrolles O. PloS Pathogens. 2010 Sep 9;6(9). pii:e1001100. • High-content imaging of Mycobacterium tuberculosis-infected macrophages: an in vitro model for tuberculosis drug discovery. Christophe T, Ewann F, Jeon HK, Cechetto J, Brodin P. Future Medicinal Chemistry. 2010;2(8):1283-1293. • High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors. Christophe T, Jackson M, Jeon HK, Fenistein D, Contreras-Dominguez M, Kim J, Genovesio A, Carralot JP, Ewann F, Kim EH, Lee SY, Kang S, Seo MJ, Park EJ, Skovierová H, Pham H, Riccardi G, Nam JY, Marsollier L, Kempf M, Joly-Guillou ML, Oh T, Shin WK, No Z, Nehrbass U, Brosch R, Cole ST, Brodin P. PloS Pathogens. 2009 Oct; 5(10):e1000645. • Benzothiazinones Kill Mycobacterium tuberculosis by Blocking Arabinan Synthesis. Makarov, V.; Manina, G.; Mikusova, K.; Mollmann, U.; Ryabova, O.; Saint-Joanis, B.; Dhar, N.; Pasca, M. R.; Buroni, S.; Lucarelli, A. P.; Milano, A.; De Rossi, E.; Belanova, M.; Bobovska, A.; Dianiskova, P.; Kordulakova, J.; Sala, C.; Fullam, E.; Schneider, P.; McKinney, J. D.; Brodin, P.; Christophe, T.; Waddell, S.; Butcher, P.; Albrethsen, J.; Rosenkrands, I.; Brosch, R.; Nandi, V.; Bharath, S.; Gaonkar, S.; Shandil, R. K.; Balasubramanian, V.; Balganesh, T.; Tyagi, S.; Grosset, J.; Riccardi, G. Cole, S. T. Science. 2009 May 8;32455928-.801-4. 41
  44. 44. Institut Thématique Microbiologie et Maladies Infectieuses Priscille Brodin, Ph.D Chemical Genomics Approaches of Intracellular Mycobacterium tuberculosis Objectives: • How does the intracellular replication and survival of Mycobacterium tuberculosis contribute to tuberculosis pathogenesis? • What are the molecular and the cellular mechanisms used by virulent M. tuberculosis to survive inside the macrophage? • Can small molecules that selectively interfere with intracellular replication of M. tuberculosis enrich the antituberculosis drug regimen? Tools: • High content imaging and automated confocal microscopy in Biosafety Level 3 (BSL-3) • Mycobacterium tuberculosis and clinical isolates • Small animal studies • Chemical and genetic libraries Figure 1: High content assay monitoring the replication of M. tuberculosis inside host macrophages M. tuberculosis parasitizes and replicates within host macrophages Invasion Survival Replication Day 0 1 2 3 5 6 Red- Macrophages Infected with Green M. tub Non-infected %Infected cells Cell number Use of %Infected cells as read out for M. tuberculosis intracellular replication 42
  45. 45. Institut Thématique Microbiologie et Maladies Infectieuses Priscille Brodin, Ph.D Chemical Genomics Approaches of Intracellular Mycobacterium tuberculosis Figure 2 : Quantification of early trafficking of M. tuberculosis inside host macrophages Attenuated Red- M. tub Wild type M. tub Subcellular localisation within Lysosomes Lysotracker signal positive Cell nucleus in blue 1 2 3 4 Use of Lysotracker signal as a positive read out for M. tuberculosis trafficking into the lysosomes 0 100 200 300 400 500 0 500 1,000 1,500 2,000 Cell nuclei number Surfaceoflysosomes proximaltocellnuclei P55C04 P55D03 Mean Mean + 3s.d. 43

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