Jean-Claude SirardChristophe CarnoyFiordiligie CasilagDelphine Cayet

ABIMMUNERepurposingdisusedantibioticswithimmunemodulatorsas

antimicrobialstrategyforrespiratorytractinfections

The partners of the ABIMMUNE project

The seminal idea of the ABIMMUNE project:Target innate immunity (host-directed therapy)

• innate immunity is rapidly activated, is self-

limiting, and involves a broad spectrum of

effectors, i.e. multiple killer cells and

antibacterial molecules

• ubiquitous and universal defense

mechanisms

• antibacterial activities of innate immunity are

mostly independent of antibiotics’ mode of

action

• it is difficult for the pathogen to develop

resistance to innate immunity since this latter

involves multiple mechanisms

• targeting host innate immunity may reinstate

some immune defense in vulnerable patients

Relativ

elevelofimmun

erespon

ses

Constitutiveinnatedefenses

Inducibleinnate

defenses

Time

Microbe

X

NF-kB Transient activation ofimmune effectors

LTALAM

zymosan ssRNACpGDNA

UropathogenicbacteriaProfilin

FlagellinLPS FlagellindsRNA 23S rRNA

TLR13

Toll-like receptors (TLR), receptors of microbes for stimulation of innate immunity

Ramos et al. Trends Microbiol (2004)

< >20 Å

< 240 Å >capprotein

NC

D0

D1

D2D3

Flagellin FliC fromSalmonella typhimurium

52 KDa

N

C

Flagellin is the main sructural protein of flagellum

TLR5-specificactivationmotif (89-96)èflagellinTLR5mut andTlr5-/- animals

N

C

Chemokines, cytokinesAnti-microbial compounds

MyD88

NF-kBMAPKactivation

Toll-likereceptor5(TLR5)

Flagellin activates Toll-like receptor 5 (TLR5) signaling

Flagellin induces an immediate and transient TLR5 signaling in the respiratory tract

0 2hours

Flagellin

4 18

Intranasalinstillation

TLR$signaling$pathway$

0h$

2h$

4h$

18h$

Cytokine9cytokine$receptor$interac

Epithelialcells

Neutrophils

phagocyte-specific chemokines

Inflammatory monocytes

TLR5

Mucosal flagellin stimulates innate immunity via epithelium

antimicrobial molecules

pathogen phagocytosis andclearance

2h

Lung RNA

0h

S. pneumoniae ± FliC∆174-400

0h

S. pneumoniae

6h 8h

Lung RNA

± FliC∆174-400

0h

S. pneumoniae

16h 18h

Lung RNA

± FliC∆174-400

0h

S. pneumoniae

24h 26h

Lung RNA

± FliC∆174-400

Porteetal.Antimicrob AgentsChemother (2015)

Flagellin stimulates lung innate immunity during Streptococcus pneumoniae pneumonia

Cxcl1

PB

S

FliC

PB

S

FliC

PB

S

FliC

PB

S

FliC

1

10

100

1000

* * * *

0h 6h 16h 24h

- - - -+ + + +flagellin

time oftreatment

rela

tive

mR

NA

leve

ls (v

ersu

s m

ock)

S.pneumoniae± flagellinintranasal

± flagellin

± flagellin

± flagellin

Amoxicillin

body weightbacterial load

histological analysis

ß-lactamin (aminopenicillin)

à amoxicillinisaveryeffectivepenicillin

throughoralroute

àmostcommonantibioticsprescribed

forpneumococcalinfection

amoxicillin250 µg/kg oral

12h 60h0h 42h

S. pneumoniae intranasal (i.n)

survival± flagellin 2.5µg i.n

Combination therapy with direct antimicrobial agent (antibiotic) and host-directed agent (TLR agonist)

± flagellin

-1 -1 -1 -1

80

90

100

110

**

FliC∆174-400

+ ++-

-+

--

amoxicillin

***

% in

itial

wei

ght

0 2 4 6 8 100

25

50

75

100

amoxicillin

FliC∆174-400

Untreated

amoxicillin + FliC∆174-400

days

% S

urvi

val

-1 -1 -1 -1

80

90

100

110

**

+ ++-

-+

--

**

AMXFliC∆174-400

***

% in

itial

wei

ght

Porteetal.Antimicrob AgentsChemother (2015)

Flagellin improves the therapeutic efficacy of amoxicillin during pneumococcal infection

+ flagellin

flagellin

lung spleen

Flagellin improves the therapeutic efficacy of amoxicillin during pneumococcal infection

flagellin flagellin

Porteetal.Antimicrob AgentsChemother (2015)WO2015011254; EP14307154.6

A

C D

B

Porte et al.FIG.2

PV PV PV PV

PV

PV

PVB

B

B

B B

S. pneumoniae+ amoxicillin+ flagellin

S. pneumoniae

S. pneumoniae+ amoxicillin

Mock

PV : pulmonary venule ; B : bronchiole

Flagellin combine with amoxicillin does not exacerbate lunginflammation

12h 42h 60h

bacterial load

0h

S. pneumoniaeSp1103CFUi.n amoxicillin + flagellin

d-7

IAV+_

2

3

4

5

6

7

8

9 ***

amoxicillin

FliC∆174-400

+ ++-

Log 1

0 C

FU /

lung

+ ++-

1

2

3

4

5

6

7 ***

amoxicillin

FliC∆174-400

Log 1

0 C

FU /

sple

en

lung spleen

Porteetal.Antimicrob AgentsChemother (2015);Sirard etal.EP14307154.6, 23 decembre 2014

Combination therapy improves treatment of influenza A virus/pneumococcal superinfection

flagellin flagellin

The general idea of the ABIMMUNE project:Combine ND-AB and approved immune modulators

• immune modulators and ND-AB may synergize to kill bacteria thereby allowing for

dose reduction of ND-AB and potentially reducing side effects of ND-AB (toxicity or

disruption of normal flora)

• using ND-AB may globally dampen the proportion of bacteria resistant to first-line

antibiotics, allowing their maintenance in clinics

• Design in vitro assays for the PK and activity evaluation of combination of

ND-AB and immune modulators (DAA and IAA)

• Test antibacterial activity on clinical strains and MDR strains

• Characterize how immune system and antibiotics cooperate for

killing/elimination of bacteria

The ABIMMUNE objectives

• The first objective is to screen combination therapies based on in vitroantibacterial efficacy, immunological studies and PK/PD modeling.

• The second objective is to demonstrate the proof-of-concept of increased efficacyof selected combination therapies compared to standalone treatments in

validated mouse models of bacterial pneumonia.

• The third objective is to assess the efficacy of combination therapies on clinicalisolates resistant to first-line antibiotic or MDR and the impact on emergence of

resistance to ND-AB and immune-modulators.

The disease and causing agents

ABIMMUNE will focus on pneumonia caused by bacteria.

The main agents are:

1. Pseudomonas aeruginosa2. Klebsiella pneumoniae3. Staphylococcus aureus4. Streptococcus pneumoniae

What are the immune targets?

1. thecentralreceptorsofinnateimmunity(TLRs)

2. phagocytes(macrophages/monocytesandneutrophils)

3. theautophagypivotalregulatormTOR (mammalianTargetOfRapamycin)

4. theinhibitionofinflammationviaPPARγ

What are the ND-AB?

1. Streptomycin(protein)

2. Fosfomycin (cellWall)

3. Colistin (membrane)

Targeting TLR4 with MPLA

MonoPhosphoryl LipidA[MPLA®],pleiotropicactivatorofimmunedefenses

Targeting TLR4 with MPLA