Rir 10th june_2011_v2

Institut thématique
Microbiologie et
Maladies infectieuses
3RD INTERNATIONAL RESEARCH MEETING
MICROBIOLOGY & INFECTIOUS DISEASES - HÔTEL MARIGNY,
PARIS, JUNE 10TH, 2011

Microbiologie et
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

Microbiologie et
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

Microbiologie et
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.
• 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

Institut Thématique
Microbiologie et
Maladies Infectieuses
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

Microbiologie et
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

Microbiologie et
• 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

Microbiologie et
• 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

Microbiologie et
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
• 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

Microbiologie et
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
8

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)*
Microbiologie et
9

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-
)
Microbiologie et
10

Microbiologie et
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
• 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

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
Microbiologie et
12

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%
CD4+NKG2D+
CD4+NKG2D-
CDR3length(aa)
Immunopathology
Microbiologie et
13

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
Immunopathology
Microbiologie et
14

Microbiologie et
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.
• 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

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
Immunity – Cancer – Infection
Microbiologie et
16

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:
Microbiologie et
17

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)
Microbiologie et
18

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
Microbiologie et
A
19

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
Microbiologie et
20

Microbiologie et
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.
• 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

Microbiologie et
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

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
Microbiologie et
Thomas Baumert, M.D
23

Perspectives
• 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
Microbiologie et
Thomas Baumert, M.D
24

Microbiologie et
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.
• 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

Microbiologie et
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
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§¥
§¢
¢
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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

Microbiologie et
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
27

Microbiologie et
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
28

Microbiologie et
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).
• 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

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)
Microbiologie et
30

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
Microbiologie et
31

Perspectives
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
Microbiologie et
32

Microbiologie et
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.
• 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

Microbiologie et
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

Microbiologie et
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

Perspectives
Microbiologie et
• 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

Microbiologie et
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.
• 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

Microbiologie et
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

Microbiologie et
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

Microbiologie et
• 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
40

Microbiologie et
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.
• 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

Microbiologie et
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

Microbiologie et
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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