Upload
others
View
12
Download
0
Embed Size (px)
Citation preview
Alternative to Antibiotics:Alternative to Antibiotics:Alternative to Antibiotics:Alternative to Antibiotics:Selective Modulators of Innate Selective Modulators of Innate
I iI iImmunityImmunity
R.E.W. (Bob) HancockUniversity of British Columbia,
Vancouver Canada
Approaching the Antibiotic Resistance EraApproaching the Antibiotic Resistance Era• Multidrug resistanceMultidrug resistance• Dry pipeline
i• Emerging problems• Animal Uses
IDSA ‐“Bad BugsBad Bugs No Drugs”
There is an urgent need for newThere is an urgent need for newThere is an urgent need for new There is an urgent need for new strategies to treat infectionsstrategies to treat infections
Cationic PeptidesCationic PeptidesBi d d i b d i
I t t h t d f h i i ll
Birds do it, bees do it, even educated fleas do it.... Cole Porter
Important host defense mechanism in all complex species of life.
>1000 peptides known. Diverse amino acid sequences and structures.
12 to 40 (or more) amino acids. Net charge +2 to +9 (Lys; Arg). Amphipathic.
Role in Innate Immunity involves both antimicrobial and immunomodulatoryyactivities; “Host Defence” peptides.
Peptide Arrays and Machine Learning Peptide Arrays and Machine Learning (QSAR) creates improved 9(QSAR) creates improved 9--mer peptidesmer peptides( ) p( ) p p pp p Relates activity to structure (based on primary sequence and
physical and inductive “descriptors”); using Neural Networks. Trained on tested peptides; predicts activity of virtual peptides Trained on tested peptides; predicts activity of virtual peptides Very effective and quite accurate for 100,000 peptide library
MIC (M) Bacterium
Bac2A HHC-10
HHC-36
MX-226 Tobramycin Imipenem Ceftazidime Ciprofloxacin
Protects in mice (IP and IV) S10 36 226 y p p
P. aeruginosa H103 (wild type) 48 0.8 0.7 19 1.1 6.7 3.6 0.4 9 (Brazil MDR) 192 5.9 5.7 76 >274 428 >231 386 198 (Brazil MDR) 95 1.5 1.4 153 >274 107 231 97 213 (Brazil MDR) 192 3 5.7 38 >274 428 >231 193 LES400 (Liverpool MDR) 95 5.9 11 76 8.6 3.3 58 1.5 H1027 (Liverpool MDR) 12 0.8 0.3 38 69 27 231 3.0MICs = 0.8 to 6 g/ml 1.0×108
1.0×109
vs. S. aureus
( p ) 0.8 0.3 H1030 (Liverpool MDR) 95 3 1.4 76 17 27 58 0.8 P. maltophilia ATCC13637 3 0.4 1.4 19 8.6 428 0.5 0.8 E. cloacae 218R Class C -lactamase 24 3 11 9.6 1.1 0.4 58 0.4 E. coli 63103 (ESBL) 24 1.5 5.4 38 137 0.4 231 >386
MICs 0.8 to 6 g/ml vs. most Superbugs
10X better than 1.0×106
1.0×107
CFU
/ml
64771 (ESBL) 24 1.5 2.7 38 274 0.4 >231 >386 K. pneumonia 61962 (ESBL) 192 25 174 153 69 0.4 >231 0.4 63575 (ESBL) 192 6.2 22 76 34 0.4 >231 193 S. aureus ATCC25923 24 3 2.9 9.6 0.5 0.4 29 0.4 C623 (MRSA) 24 1.5 1.4 19 >274 0.4 115 6.0
MX-226; Overall better than 4
leading antibiotics line
C-10 C-36
1.0×104
1.0×105
Cherkasov et al. 2009. ACS Chemical Biol. 4:65-74.
E. faecalis ATCC29212 12 12 43 76 34 1.7 231 0.8 W61950 (VRE; VanA) 48 99 >174 >153 >274 6.7 >231 97 f43559 (VRE;VanB) 12 3.1 10.8 153 69 6.7 >231 97 E. faecium mic80( VRE; VanA) 3 1.5 1.3 38 274 428 >231 97 t62764 (VRE; VanB) 48 49 174 76 >274 >428 >231 386
leading antibiotics Sali
HHC-
HHC-
Treatment Group
New Therapeutic PossibilitiesNew Therapeutic Possibilities--AntiAnti--Biofilm ActionBiofilm ActionPeptides killPeptides kill bacteria in biofilms
AAC 56:2696-704, 2012
Peptides work vs. MDR Gram positive and Gram negative bacteria & AMP gresistant bacteria: IC50 < 0.25 g/ml vs. Pseudomonas
Many natural antimicrobial peptides Many natural antimicrobial peptides have very weak direct antimicrobial activity have very weak direct antimicrobial activity y yy y
e.g. Human LLe.g. Human LL--37 vs. 37 vs. S. aureus S. aureus 10
Staph aureus106
mL Sodium phosphate
buffer
8Staph. aureus
Infection Blood Counts
(x 104)104
105
10
102
CFU
/m bufferTissue culture medium serum
4
6103
10
102
2
4
101
0 20 40 60 80 100 120
LL-37 concentration (g/mL) 0 LL-37Control
p < 0.05100
Ability to stimulate innate immunity may be more importantDawn Bowdish, Monisha Scott
Immune Modulation ApproachesImmune Modulation Approaches Successful antimicrobial
therapy requires assistance from the host:assistance from the host: immune response
I d l ti i Immune modulation is highly used in antiviral and anticancer therapyand anticancer therapy
IDSA “Bad Bugs No Drugs”
Hamill, P., K. Brown, H. Jenssen and R.E.W. Hancock. 2009. Novel anti-infectives: is host defence the answer? Curr. Opin. Biotechnol. 19:628-636.
Modulating Immunity for Therapeutic Modulating Immunity for Therapeutic BenefitBenefitBenefitBenefit
Agonist orAntagonist
TLRPRR
Microbial Signature
Antagonist
This is an
Pathway Pathway
IDRIDR Adjuvant Therapy -
ModulationModulationpy
Designed to work with
Output
Antibiotics
Signal Integration(e.g. TFs, Hubs)
Output(Protection vs. Inflammation)
Hamill P, et al. 2009. Novel anti-infectives: is host defence the answer? Curr. Opin. Biotechnol. 19:628-636.
New immunomodulatory peptides show New immunomodulatory peptides show broad protection in Mouse Model Infectionsbroad protection in Mouse Model Infectionspp
Also protects vs. TB, E. coli, Salmonella, MRSA, VRE, P.
aeruginosa, IBD, CF,Cerebral malaria, SterileCerebral malaria, Sterile
inflammation, etc.Safety demonstrated in
i● PBS■ IDR-1018
In mice
Phase I trialsThese peptides developed independently for Grand
& pigsindependently for Grand
Challenges Program.
Lars Steinstraesser, Louis Schofield, Sandra Pillat, Lisa Thorsen, Sarah Mullaly, Bruce Vallance, Brett Finlay
In pig model no effect on infectious loadTime (days)
Therapeutic IDRTherapeutic IDR--1018 protects in conjunction with 1018 protects in conjunction with AntiAnti--Malarial Malarial TherapyTherapy
80
90
100Therapeutic IDR-1018 +
Anti-malarial10 6 PbA
p = 0 034
60
70
80
Anti-malarial
Only via IV No effect on Parasite load A ti i fl t
d 04 days (Par 3-6%)
P i th i +
p 0.034
40
50
% S
urvi
val Anti malarial
Control Peptide Anti-inflammatoryPyrimethamine + Chloroquine indrinking water
20
30%
1018 or IDR-1d 4, 5, 6
0
10
0 1 2 3 4 5 6 7 8 9 10 11 12 13
No Treatmentd 4, 5, 6
0 1 2 3 4 5 6 7 8 9 10 11 12 13Days post-infection
Louis Schofield, Ariel Achtman, Sandra Pilat, WEHI. Science Transl. Med,. 2012. 4:135ra64
Systems BiologySystems BiologyNetwork analysisNetwork analysis –– IDR peptidesIDR peptidesNetwork analysis Network analysis IDR peptidesIDR peptides
InnateDB N t k Interactions between
IDR1 treatment of
Network Construction
Interactions between differentially expressed Genes
Human Monocytes
Top HubspTRAF2; CRK; HSPA1B; RELA
(MAPK & Chemokine Induction)TNFAIP3; SMAD3; CUL 1
NB. RNANB. RNA--Seq Seq delivers 897 genesdelivers 897 genes
688 nodes2895 Edges
Highly connected Nodes (Hubs)
TNFAIP3; SMAD3; CUL-1(Anti-Inflammatory & TGF)
CREBBP1; IRF7; AKT2; GRB2 & Bottlenecks
; ; ;(JAK-STAT & IFN)
Monocytes/Macrophages are essential and Monocytes/Macrophages are essential and recruitment is enhanced in infection modelrecruitment is enhanced in infection modelrecruitment is enhanced in infection modelrecruitment is enhanced in infection model
Nij ik l 2010Nijnik et al. 2010. J. Immunol.184:2539-50.
Protective Protective Immunity Immunity Mechanism in animal modelsMechanism in animal models
BA
Harmful Harmful Inflammation Inflammation
e ce
ll co
unt
300
400
300
4004x107
CFU
/ml
2x107
Peptide protects with pre and post treatment (A)
Pro-inflammatory Mac
roph
age
0
100
200
0
100
200
010S.
aur
eus
010
cytokines TNF (C) and IL6 reduced. Anti-inflammatory cytokineIL-10 increased (D)
Vehicle - 24 h +4 hVehicle - 24 h +4 h
250250C
+4 hVehicle - 24 hVehicle - 24 h +4 h
3
D
3 IL 10 increased (D)
Macrophages increase at the site of infection (B)
M h (li lNF-
pg/m
l
100
150
200
100
150
200
2
3
IL-1
0 ng
/ml
2
3
Macrophages (liposomal chlodronate) but not neutrophils or lymphocytes are essential for protection
TN
Vehicle - 24 h +4 h0
50
-0
50
0
1
Vehicle0
1
Vehicle IDR-1 p
Scott et al. Nature Biotech 25:465-472, 2007Balancing of Inflammation in vivoBalancing of Inflammation in vivo
Summary: Summary: Normal immunity protects but Normal immunity protects but can lead to potentially harmful inflammationcan lead to potentially harmful inflammation
Bacteria
Woundcan lead to potentially harmful inflammationcan lead to potentially harmful inflammation
LP /LTBacteria LPS/LTA
Stimulation ofInnate
Effector Mechanisms
ToK ll M
PotentiallyHarmful
I fl ImmunityKill Microorganisms Inflammatory ResponsesInfections cause 33% of all
deaths worldwide; 100,000 Americans die from antibiotic resistant infections
Blood Vessel
resistant infections
Sepsis kills >200,000 people in USA annually& 5 million worldwide
Summary: Peptides Boost Innate Immunity Summary: Peptides Boost Innate Immunity and Block Harmful Inflammationand Block Harmful Inflammation
Bacteria
Wound
IDR-1018
and Block Harmful Inflammationand Block Harmful InflammationSelective boosting
of innate Bacteria IDR 1018IncreasedStimulation
of
immunity represents a new
adjunctive approach to of
Innate Immunity
Effector Mechanisms
ToKill
Muted Inflammatory
Responses
approach to treating
infections: Increasing
it t f Kill Microorganisms
Responses recruitment of immune cells while
suppressing inflammation.
Blood Vessel
f mm .Much needed as
antibiotic resistance rises.
Triple adjuvant formulation (TAF) gives the Triple adjuvant formulation (TAF) gives the potential for single dose protection vs. pertussispotential for single dose protection vs. pertussis. O i th R dbl k f M lti l D i
40,000High Titre
Mixed Th1 Th2
Overcoming the Roadblock of Multiple Dosing
30,000
40,000 Mixed Th1, Th2Protective
Pigs, Cattle, MiceWorks in Neonates
20,000
ImmunizeWorks in NeonatesPTdPTd with TACwith TAC
PTd &PP &CpG
10,000 Vaccination Site Vaccination Site Ivory CoastIvory CoastPTd & HH2
PTd & PP
PTd & CpG
0
Day 0 Day 22Vaccine 27:2055-2064, 2009Vaccine 27:4662-4671, 2009
PTd (pertussis toxoid alone)
CpG ODN Host DefencePeptide (HH2)Polyphosphazene; PP
Volker Gerdts, Lorne Babiuk, Bob Hancock and many others
Duration of immunity > 2 years (mice)Duration of immunity > 2 years (mice) Comparison to Alum (2 different Comparison to Alum (2 different Features of the Adjuvant FormulationFeatures of the Adjuvant Formulation
>10 months in pigs>10 months in pigs doses)doses)
1.0×105
1.0×106
*
y Ti
tre
1.0×105
1.0×106
y Ti
tre
1.0×103
1.0×104 *
gG2a
Ant
ibod
y
1.0×102
1.0×103
1.0×104
IgG
2a A
ntib
ody
2 4 6 8 10 12 14 16 191.0×102
Weeks
Ig
2 4 8 12 16 19 22 26 30 34 38 42 52 61 68 76 85 90
1.0×101
Weeks
Protective Protective –– Neonatal pigsNeonatal pigs Works mucosally at very low dosesWorks mucosally at very low doses
150000
200000
250000
lung
lesi
on
*
No Maternal I t f
0
50000
100000
cfu
per
tota
l l Interference
Contro
l
Quadr
acel
Tripl
ecom
bo
0
OVA + 0.6 g CpG, 1.2 g HDP, 0.6 g Polyphosphazene Volker Gerdts, VIDO
AcknowledgementsAcknowledgementsL b LL b L JJ M MM MLab: Laure Lab: Laure JanotJanot, Matt Mayer, , Matt Mayer,
Chris Chris FjellFjell, Ana , Ana NijnikNijnik, , NeelofferNeeloffer MookherjeeMookherjee, , jj ,,Jason Jason KindrachuckKindrachuck, ,
Reza Reza FalsafiFalsafi, ,
Collaborators: Collaborators: Volker Volker GerdtsGerdts; ; Bruno Rivas, Rogelio Hernandez; Bruno Rivas, Rogelio Hernandez; EdieEdie Dullaghan and CDRD; Louis SchofieldDullaghan and CDRD; Louis Schofield; Brett Finlay; Brett FinlayEdie Edie Dullaghan and CDRD; Louis SchofieldDullaghan and CDRD; Louis Schofield; Brett Finlay; Brett Finlay