Alternative to Antibiotics:Alternative to Antibiotics ... · Approaching the Antibiotic Resistance Era • Multidrug resistanceMultidrug resistance • Dry pipeline • Emergiing

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