Thierry Calandra, MD, PhD

Infectious Diseases Service Centre Hospitalier

Universitaire Vaudois1011 Lausanne

Switzerland

Innate Immune System in Host Defenses against Infections

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•• Microbes and microbial Microbes and microbial productsproducts

•• ATPATP

•• DNA DNA

•• Uric acidUric acid

•• Calcium pyrophosphateCalcium pyrophosphate

•• Ultraviolet BUltraviolet B

•• TrinitrochlorobenzeneTrinitrochlorobenzene

•• AlumAlum

•• Asbestos, silicaAsbestos, silica

•• NickelNickel

•• ……

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NucleusNucleus

TLRsTLRs

LRRLRR

CARDCARD NACHTNACHT

RIGRIG--11

RLRsRLRs

MAMPsMAMPs DAMPsDAMPsCC--type type llectinsectins

DAIDAIAIM2AIM2NLRsNLRs

InflammasomesInflammasomes

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STATs

IFNβ−dependent

chemokines(RANTES, IP-10)

TLR2

IFNβ-independent chemokines

(IL-8, MIP-1, MIP-2)

TLR6

MyD88ERK1/2JNK

NF-κBIRF3IRF7

IFNβ

MDA-5

IPSIPS-1NLRP3ASC

Pro-IL1β

Caspase-1

IL-1β

Pro-caspase 1

IFNR

Poxvirus (NYVAC, MVA, WR)

Delaloye J et al. PLoS Pathogens 2009; 5: e1000480

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•• Type I transmembrane proteinsType I transmembrane proteins

•• 3 domains:3 domains:

–– EctodomainEctodomain: leucine rich repeats : leucine rich repeats (LRR), mediates sensing of MAMPs(LRR), mediates sensing of MAMPs

–– Transmembrane domainTransmembrane domain

–– Intracellular domainIntracellular domain: Toll: Toll--Interleukin Interleukin 1 (IL1 (IL--1) (TIR) domain, required for 1) (TIR) domain, required for signal transductionsignal transduction

•• Homodimers or heterodimersHomodimers or heterodimers•• Human: 10 Human: 10 •• Mouse:Mouse: 1212•• Sea urchin: > 200Sea urchin: > 200

LRRLRR

TIRTIR

NN--terminusterminus

CC--terminusterminus

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•• Microbial patternsMicrobial patterns::

–– BacteriaBacteria: : LPS, lipoproteins, peptidoglycan LPS, lipoproteins, peptidoglycan fragments, fragments, lipoteichoic acid, flagellin, lipoteichoic acid, flagellin, lipoarabinomannan, DNA, RNA, lipoarabinomannan, DNA, RNA, ……

–– VirusVirus: : DNA, RNA, structural protein, DNA, RNA, structural protein, ……

–– FungiFungi: : zymosan, zymosan, ββ--glucan, mannan, DNA, RNA, glucan, mannan, DNA, RNA, ……

–– ParasitesParasites:: tGPItGPI--mutin, glycoinositolphospholipids, mutin, glycoinositolphospholipids, DNA, hemozoDNA, hemozoïïn, profilinn, profilin--like molecule, like molecule, ……

•• Synthetic compoundsSynthetic compounds::–– Acylated lipopeptides, CpG ODN, imiquimod, R848, Acylated lipopeptides, CpG ODN, imiquimod, R848, ……

•• HostHost--derived moleculesderived molecules::

–– HSP60, fibrinogen, HSP60, fibrinogen, ββ--defensin, hyaluronic acid defensin, hyaluronic acid fragments, annexin 2, HMGBfragments, annexin 2, HMGB--1, prothymosin 1, prothymosin αα, , tenascin C, tenascin C, ……

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Gain or loss

fonction

Severe Severe SepsisSepsis

HomeostasisHomeostasis

Loss or gain of

fonctionInfectionInfection

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Takeuchi O et al. J Immunol 2000;165:5392Takeuchi O et al. J Immunol 2000;165:5392--53965396

Rare TLR4 Mutations are Associated with Rare TLR4 Mutations are Associated with Susceptibility to Meningococcal SepsisSusceptibility to Meningococcal Sepsis

Smirnova I et al. Proc Natl Acad Sci USA 2003;100:6075Smirnova I et al. Proc Natl Acad Sci USA 2003;100:6075--8080

0 200 400 600 80001234567

Mut

atio

n Fr

eque

ncy

(%)

Mut

atio

n Fr

eque

ncy

(%)

Controls Controls (n=532)(n=532)

Meningococcal Sepsis Meningococcal Sepsis (n=355)(n=355)

D299GD299GT399IT399I R763HR763H

D299GD299G

T399IT399IR763HR763H

0 200 400 600 80001234567

G9EG9E

Y46CY46C C281YC281Y

E474KE474K

K694RK694R ��VGTVGT

14 rare mutations (10 separate alleles) : P<1014 rare mutations (10 separate alleles) : P<10--55

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Wurfel M et al. AJRCCM 2008;178:210Wurfel M et al. AJRCCM 2008;178:210--2020

TLR1 TLR1 --7202 A/G7202 A/G: elevated TLR1: elevated TLR1--mediated NFmediated NF--kB activity, kB activity, cytokine production (Pamcytokine production (Pam33CSK4) and TLR1 expressionCSK4) and TLR1 expression

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Barreiro et al. Barreiro et al. PLoS Genetics PLoS Genetics

20092009

158 healthy individuals from Africa, Europe, East Asia158 healthy individuals from Africa, Europe, East Asia

•• Membrane TLRsMembrane TLRs: high rates of damaging mutations; redundancy: high rates of damaging mutations; redundancy•• Intracellular Intracellular TLRs TLRs (3,7(3,7--9): 9): strong purifying selection; essential, strong purifying selection; essential,

nonnon--redundant role in host survival redundant role in host survival

LPSLPS

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MyD88MyD88TIRAP/MalTIRAP/Mal

MAPK, NFMAPK, NF--κκBB

IFNIFN--ββChemoChemokineskines

TRIF/TRIF/TITICAMCAM--11TRAMTRAM

CytokinesCytokines CytokinesCytokines

NFNF--κκBB TBK1TBK1

TLR4TLR4

MDMD--22CD14CD14

LBPLBP

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elat

ive

Ligh

t Uni

t (x

10R

elat

ive

Ligh

t Uni

t (x

10-- 55

))

Shimazu R et al. Shimazu R et al. J J Exp Med 1999;1777Exp Med 1999;1777

Lipid A (ng/ml)Lipid A (ng/ml)00 0.10.1 11 1010 100100 10001000

00

22

44

66

88

1010

TLR4TLR4

TLR4+MDTLR4+MD--22

Nagai Y et Nagai Y et al. al. Nat Immunol 2002;3:667Nat Immunol 2002;3:667

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Ohto et al. Science 2007;316:1632Ohto et al. Science 2007;316:1632--44Kim et al. Cell 2007;130:906Kim et al. Cell 2007;130:906--1717

Park et al. Nature 2009; 458:1191Park et al. Nature 2009; 458:1191--66

TLR2 -/-

TLR4 TLR4 --//--

MyD88MyD88--//--

00 11 22 33 44 55 6600

2525

5050

7575

100100

Days postDays post--infectioninfection

Sur

viva

l (%

)S

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val (

%)

WildWild--typetype

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WTWT

TLR2

TLR2--

//--

TLR4

TLR4--

//--

MyD88

MyD88

--//--00

44

88

1212

16162020

TNF

(ng/

ml)

TNF

(ng/

ml)

E. coli 018: E. coli 018: 2x102x1099 cfu/mouse i.p.cfu/mouse i.p.Antibiotics (ceftriaxone + gentamicin)Antibiotics (ceftriaxone + gentamicin)

Roger T et al. Roger T et al. Proc Natl Acad Sci USA Proc Natl Acad Sci USA 2009,106:23482009,106:2348--5252

Anti-TLR4-Fc Antibodies

mouse TLR4mouse TLR4--FcIgGFcIgG

TLR4 +/+TLR4 +/+

TLR4 TLR4 --//--

MacrophagesMacrophages

AntiAnti--TLR4TLR4

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30 min with Abs (100 µg/ml)stimulation for 4 h

ControlControl

00 11 100100

LPS (ng/ml)LPS (ng/ml)

10101010

PGN PGN ((µµg/ml)g/ml)

7.57.5

00

2.52.5

55

TNF

(ng/

ml)

TNF

(ng/

ml)

RAW 264.7

00

LPS (ng/ml)LPS (ng/ml)

1010 0.10.1100100

CpG CpG ((µµM)M)

2020

00

2255

11

1515

1010

TNF

(ng/

ml)

TNF

(ng/

ml)

Whole blood

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00

1010

2020

3030

4040

5050

Control Control IgGIgG

Anti-TLR4IgG

Mortality (%)Mortality (%)

00

1.01.0

2.02.0

3.03.0

20.020.021.021.0

TNFTNF--αα (ng/ml)(ng/ml)

Control Control IgGIgG

AntiAnti--TLR4 TLR4 IgGIgG

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E. coli 018: 2x10E. coli 018: 2x1099 cfu/mouse cfu/mouse i.p; antibioticsi.p; antibiotics

Sur

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l (%

)S

urvi

val (

%)

Days postDays post--infectioninfection00 11 22 33 44 55 66

00

2525

5050

7575

100100

ControlControl

AntiAnti--TLR4TLR4

ProphylacticProphylacticAbAb: : -- 4h, 4h, --30 min and +4h30 min and +4h

P<0.0001P<0.0001

TherapeuticTherapeuticAbAb: +1h and +4h: +1h and +4h

Sur

viva

l (%

)S

urvi

val (

%)

Days postDays post--infectioninfection00 11 22 33 44 55

00

2525

5050

7575

100100

Control AbControl Ab

AntiAnti--TLR4TLR4

P=0.02P=0.02

Roger T et al. Roger T et al. Proc Natl Acad Sci USA Proc Natl Acad Sci USA 2009,106:23482009,106:2348--5252

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00

2525

5050

7575

100100

0 1 2 3

Control AbControl Ab

P=0.03P=0.03

AntiAnti--TLR4TLR4

E. coli 018: E. coli 018: 2x102x1055 cfu/mouse cfu/mouse i.p; antibioticsi.p; antibioticsTherapeuticTherapeutic

AbAb: : +13h+13h

Roger T et al. Roger T et al. Proc Natl Acad Sci USA Proc Natl Acad Sci USA 2009,106:23482009,106:2348--5252

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Eritoran (E5564, Eisai Co., Ltd)Eritoran (E5564, Eisai Co., Ltd)•• synthetic analogue of lipid A portion of LPS, synthetic analogue of lipid A portion of LPS,

antagonist of TLR4antagonist of TLR4TAK 242 (Takeda)TAK 242 (Takeda)•• cyclohexene derivativecyclohexene derivative•• inhibitor of TLR4 signal transduction pathway inhibitor of TLR4 signal transduction pathway

(cytokines and NO production) (cytokines and NO production)

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Kim HM et al. Cell 2007;130:906Kim HM et al. Cell 2007;130:906--1717

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Phase II studyPhase II study293 patients with severe 293 patients with severe

sepsis (US centers)sepsis (US centers)3 groups: 3 groups:

•• Placebo Placebo •• Eritoran 45 mgEritoran 45 mg•• Eritoran 105 mgEritoran 105 mgq12h i.v. over 6 daysq12h i.v. over 6 days

Adverse events: Adverse events: •• Phlebitis 6.7%Phlebitis 6.7%

33.3 32.0

26.9

0

10

20

30

40

Placebo E 45 E 105

Tidswell J et al. Crit Care Med Tidswell J et al. Crit Care Med 2010; 38:722010; 38:72–– 8383

Day 28 MortalityDay 28 Mortality

•• DoubleDouble--blind, placeboblind, placebo--controlled controlled •• Patient population:Patient population:

–– Severe sepsis (infection+SIRS+Organ failure)Severe sepsis (infection+SIRS+Organ failure)

–– Baseline APACHE II of 21Baseline APACHE II of 21--3737

–– Less than 12h between onset of 1Less than 12h between onset of 1stst qualifying organ qualifying organ failure and study drug administrationfailure and study drug administration

•• Sample size: 2000 subjectsSample size: 2000 subjects•• 2:1 randomization (eritoran vs placebo)2:1 randomization (eritoran vs placebo)•• Treatment: 6 days (11 doses)Treatment: 6 days (11 doses)•• Primary endpoint: 28Primary endpoint: 28--day all cause mortalityday all cause mortality•• Key secondary endpoint: mortality at 1 yearKey secondary endpoint: mortality at 1 year

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Dear Investigator,

Based on the preliminary findings from the ACCESS trial, Eisai Inc. announced today that the company will not submit marketing authorization applications for eritoran to regulatory authorities in the United States, the European Union and Japan by the end of the fiscal year (year ending March 31, 2011), as previously planned. This decision was based on the fact that the study did not meet its primary endpoint of reduction in 28-day all-cause mortality in patients with severe sepsis.

Eisai will continue an analysis of the data and determine next steps. …

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•• While waiting for the complete data set of the While waiting for the complete data set of the ACCESS trial, many possible explanations:ACCESS trial, many possible explanations:–– Traditional issues with phase III sepsis trials Traditional issues with phase III sepsis trials

(heterogeneity: patients, microorganisms, site of (heterogeneity: patients, microorganisms, site of infection, treatment pratices, infection, treatment pratices, ……))

–– Patient population: not sick enough, too sick ?Patient population: not sick enough, too sick ?

–– Timing and dosing of EritoranTiming and dosing of Eritoran

–– Is LPSIs LPS--LBPLBP--CD14CD14--MDMD--22--TLR4 pathway an TLR4 pathway an appropriate target ?appropriate target ?

–– Better antiBetter anti--TLR4 therapy (mAbs, inhibitors of TLR4 therapy (mAbs, inhibitors of signalling, signalling, …… ) ?) ?

–– Combined antiCombined anti--TLR treatment strategies, antiTLR treatment strategies, anti--PRR ?PRR ?

–– ……

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•• Innate immune system: Innate immune system: plays an essential role in the plays an essential role in the sensing of dangersensing of danger--associated molecular patterns and in associated molecular patterns and in the pathogenesis of infection and sepsisthe pathogenesis of infection and sepsis

•• LigandsLigands:: microbial macromolecules microbial macromolecules (endotoxin, (endotoxin, peptidoglycan, lipoproteins, DNA, RNA, peptidoglycan, lipoproteins, DNA, RNA, ……), endogenous ), endogenous or exogenous proteins (ATP, DNA, uric acid, nickel, or exogenous proteins (ATP, DNA, uric acid, nickel, ……) )

•• FonctionFonction:: Activation of innate and adaptive immune Activation of innate and adaptive immune responses eradication of pathogensresponses eradication of pathogens

•• Human genetic studies: Human genetic studies: provided a proof of principle that provided a proof of principle that infectious diseases may result from inborn errors of innate infectious diseases may result from inborn errors of innate immunity (for example TLR mutations) immunity (for example TLR mutations)

•• PRR pathwaysPRR pathways: are a reasonable target for development : are a reasonable target for development of new treatment strategies against severe sepsisof new treatment strategies against severe sepsis

Infectious Diseases Infectious Diseases Service Service CHUVCHUV, UNIL, UNILCCééline Rondiline Rondi--FroidevauxFroidevauxThierry RogerThierry RogerJulie DelaloyeJulie DelaloyeDidier Didier Le RoyLe RoyMarlies KnaupMarlies Knaup--ReymondReymond

Division of Immunology and Division of Immunology and Allergy, CHUV Allergy, CHUV & UNIL& UNILGiuseppe PantaleoGiuseppe PantaleoAlexandre HarariAlexandre HarariIsabelle MiconnetIsabelle MiconnetKim EllefsenKim Ellefsen--LavoieLavoieGonzalo TapiaGonzalo TapiaMaud ZuchuatMaud Zuchuat

Dpt of Biochemistry, UNILDpt of Biochemistry, UNILJJüürg Tschopprg TschoppVirginie PetrilliVirginie PetrilliManuele RebsamenManuele Rebsamen

Institute of MicrobiologyInstitute of MicrobiologyCHUV & UNILCHUV & UNILAmalio TelentiAmalio TelentiDarius MoradpourDarius MoradpourAngela CiuffiAngela CiuffiMiguel MunozMiguel Munoz

Biotechnology Biotechnology Centrer, Centrer, MadridMadridMariano EstebanMariano EstebanCarmen GomezCarmen GomezBeatrix PerdigueroBeatrix Perdiguero

Swiss National Science FoundationSwiss National Science Foundation