-
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
