Genetic Engineering of Live-attenuated Malaria Vaccines

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10 Δεκ 2012 (πριν από 4 χρόνια και 9 μήνες)

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Genetic Engineering of Live-attenuated
Malaria Vaccines
Stefan H.I. Kappe
The GAP consortium:
Seattle Biomedical Research Institute, USA
Walter and Eliza Hall Institute Australia
Walter Reed Army Institute of Research, USA
University of Maryland, USA
•Genetic-attenuation
–Introduce precise gene deletions that
cause a defined attenuation
–uniform genetic identity
–Reproducible and defined product
–Attenuation by design
–Optimization possible
Genetically Attenuated Parasites (GAP)
Clinical
Trials
Knockouts
In P.falciparum &
In VitroTesting
Infectivity, Immunization & Protection
Rodent model
Knockout
Rodent Malaria
Parasites
Sporozoite and Liver Stage transcriptome Data
of Rodent Malaria and P.falciparum
Genetically attenuated parasite (GAP)
vaccines: Research and development
Greenwood et al, JCI 2007
The huntfor infectivity-associated genes:
A simple observation
Sporozoite phenotype:
-Not infective for hepatocytes
-Infective for salivary glands
-No gliding motility
Sporozoite phenotype:
-Infective for hepatocytes
-Not Infective for salivary glands
-Gliding motility
Mosquito midgut with
oocyst sporozoites
Mosquito salivary glands
with sporozoites
Infectivity-associated genes in sporozoites
Mikolajczaket al., MCB 2008
LS24 (1138)
LS50 (1069)
LS40 (1085)
537
405
14254
301
309
237
LS24LS40LS50
oo
sg
40
50
sc
bs
I
II
III
IV
oo
sg
24
50
sc
bs
oo
sg
24
40
sc
bs
Liver stage-expressed genes
Tarun et al., PNAS 2008
5
P52myc
P52myc
P52myc
TRAPTRAPCSP
DAPIDAPIDAPI
P52-myc WT
Proteins involved in establishment of the
intracellular niche: P52 and P36
P52P36
Mikolajczak, unpublished
Labaied et al., I&I 2007
Attenuation by disrupting establishment of the
intracellular niche: p52(-)/p36(-) double knockout
p52(-)/p36(-)GAPs
are completely attenuated
Labaied et al., I&I 2007
p52(-)/p36(-)sporozoites cannot induce
blood stage patency
03100,000p52(-)
/p36(-)
0 4510,000p52(-)
/p36(-)
10 (3)1810,000wildtype
blood stage infectionMice
injected
Sporozoites
injected
Labaied et al., I&I 2007
COMPLETE
COMPLETE
10,000 / day 30
10,000 / day210
10,000 (day 7)
10,000 (day 14)
10,000
COMPLETEMosquito bite /
day 7
10,000 (day 7)
10,000 (day 14)
10,000
COMPLETE10,000 / day 710,000 (day 7)
10,000 (day14)
10,000p52(-)
/p36(-)
protectionChallenge /
Days After
Boost
Boosts
(Date given)
Primary
Dose
GAP
p52(-)/p36(-)GAPs
completely protect against infection
Labaied et al., I&I 2007
Attenuation by disrupting the liver stage/host
cell interface: UIS3 and UIS4
Overlay
10 μm10 μm
anti-CS
anti-UIS4
Mueller et al., PNAS 2005
Tarun et al., JID 2007
Jacobs Lorena et al., unpublished
Mueller et al.,Nature 2005
anti-HSP70
anti-UIS3myc
BA
PyUIS3 or PyUIS4
DHFR/ts
DHFR/ts
WT
T7TEST 1
TEST 2
PyUIS3 or PyUIS4
DHFR/ts
DHFR/ts
DHFR/ts
WT
T7TEST 1
TEST 2
plasmidPyuis3(-)
W
T
Te
s
t

1
Te
s
t

2
Te
s
t

1
T7
W
T
Te
s
t
1
Te
s
t

2
T7
wtplasmidPyuis4(-)
WT
T
e
s
t

1
T
e
s
t

2
T
e
s
t

1
T
7
WT
T
e
s
t

1
T
e
s
t

2
T
7
Marker
(kb)
2
1
0.5
Marker
(kb)
2
1
0.5
wt
plasmidPyuis3(-)
W
T
Te
s
t

1
Te
s
t

2
Te
s
t

1
T7
W
T
Te
s
t
1
Te
s
t

2
T7
wtplasmidPyuis4(-)
WT
T
e
s
t

1
T
e
s
t

2
T
e
s
t

1
T
7
WT
T
e
s
t

1
T
e
s
t

2
T
7
Marker
(kb)
2
1
0.5
Marker
(kb)
2
1
0.5
wt
Gene deletion strategy
Tarun et al., JID 2007
uis3(-)and uis4(-)GAPs form a PV and then
arrest in development
WT
uis3(-)
Overlay
10 μm
CS
10 μm
Overlay
10 μm
Overlay
10 μm
anti-CS
anti-CS
Overlay
10 μm
anti-CSanti-UIS4
anti-UIS4
anti-UIS4
uis4(-)
Tarun et al., JID 2007
Tarun et al., JID 2007
Mueller et al., PNAS 2005
COMPLETE10,000 / day 18010,000 (day 14)
10,000 (day 28)
10,000
COMPLETE10,000 / day 710,000 (day 14)
10,000 (day 28)
10,000
COMPLETE10,000 / day 3050,000
COMPLETE10,000 / day 750,000 uis4(-)
protectionChallenge /
Days After Boost
Boosts
(Date given)
Primary
Dose
uis3(-)and uis4(-)GAPs completely protect
against infection
12 hr and 48 hr
12 hr and 48 hr
Primaquine
COMPLETE
10,000 / day 14 (iv)
10,000 (day 14)
10,000 (day 28) (iv)
10,000 (iv)
uis3(-)
NO
10,000 / day 14 (iv)
-
-
-
protectionChallenge /
Days After
Boost
Boosts
(Date given)
Primary
Dose
GAP
Persistent liver stage GAPs are not
necessary for protection
•primaquine was administered 12 hr and 48 hrs after the primary dose
and each boost
What are the immune effectors?
50K Pyuis4(-
)
-
0
20
40
60
80
100
d2d3d4d5d6
% Protection
50K Pyuis3(-)
0
20
40
60
80
100
d2d3d4d5d6
% Protection
Na•ve
Rat I
g
G
Anti-CD4
Anti-CD8
Anti-IFN-
g
in vivodepletion
: Complete CD8 and partial IFN-γdependency
Wang Lab, SBRI unpublished
PVMPPM
Membrane
lipids
Unique
fatty acids ??
ELO
FAD
ER
Bulk fatty acids
Fatty acylcoA
Octanoyl-ACP
-
FAS II
Acetyl-CoA
LipA
Acetate
Apicoplast
Pyruvate
ACP
C10-C14
fatty acids
host
fatty acids
Complex
lipids
PDH
ACS
LipB
FabZ
FabG
FabB/F
FabI
FabH
FabD
ACCase
Host cytoplasmParasite
PVMPPM
Membrane
lipids
Unique
fatty acids ??
ELO
FAD
ER
Bulk fatty acids
Fatty acylcoA
Octanoyl-ACP
-
FAS II
Acetyl-CoA
LipA
Acetate
Apicoplast
Pyruvate
ACP
C10-C14
fatty acids
host
fatty acids
Complex
lipids
PDH
ACS
LipB
FabZ
FabG
FabB/F
FabI
FabH
FabD
ACCase
Host cytoplasmParasite
Attenuation by disrupting metabolic pathways: Fatty
acid biosynthesis (FASII)
Tarun et al., PNAS 2008
Fatty acid synthesis enzymes are up-regulated
in the liver stages
E2
E3
fabb/f(-)is not necessary for
blood stage replication
-2
0
2
4
6
8
10
12
048121620
days post-injection
% parasitmemia
----- wildtype
----- fabb/f
-
-2
0
2
4
6
8
10
12
048121620
days post-injection
----- wildtype
----- fabb/f
-
% parasitemia
1,000 iv (n=4) 1,000,000 iv (n=4)
Vaughan et al., Cell Microbiol, 2009
fabb/f(-)sporozoites cannot induce
blood stage patency
010100,000fabb/f(-)
01610,000fabb/f(-)
10 (3)1010,000wildtype
blood stage infectionMice
injected
Sporozoites
injected
Vaughan et al., Cell Microbiol, 2009
in vivoliver stage development
of fabb/f(-)parasites
Vaughan et al., Cell Microbiol, 2009
Attenuatedin vivoliver stage development
of fabb/f(-)parasites late in the liver
DAPIMSP1Hep17DICmerge
DAPIMSP1Hep17DICmerge
fabb/f-
WT
WT
fabb/f-
44 hours
DAPIMSP1Hep17DICmerge
DAPIMSP1Hep17DICmerge
52 hours
fabb/f(-)parasites:
no exoerythrocytic merozoites
Scale bar: 5 μm
52 hour liver stage
Vaughan et al., Cell Microbiol, 2009
fabb/f(-)GAPs completely
protect against infection
COMPLETE10,000 /
day 210
COMPLETE10,000 /
day 120
COMPLETE10,000 /
day 30
10,000 (day 14)
10,000 (day 28)
10,000
Rechallenge/
Days After 1st
Challenge
COMPLETE
protection
COMPLETE
COMPLETE
COMPLETE
COMPLETE
protection
10,000 /
day 210
Rechallenge/
Days After 1st
Challenge
10,000 /
day 210
10,000 /
day 30
10,000 /
day 7
10,000 /
day 7
Challenge /
Days After
Boost
50,000 (day 14)
50,000 (day 28)
50,000
none50,000
none10,000
10,000 (day 14)
10,000 (day 28)
10,000
protectionBoosts
(Date given)
Primary
Dose
Vaughan, unpublished
•p52(-)p36(-)
–Hepatocyte entry, no PV formation.
Very early liver stage arrest
•sap1(-)
–Hepatocyte entry, PV formation?
Very early liver stage arrest
•uis3(-)
–Hepatocyte entry, PV formation.
Early liver stage arrest (~14 hrs)
•uis4(-)
–Hepatocyte entry, PV formation.
Early liver stage arrest (~14 hrs)
•Fabb/f(-) and pdhe1
α
(-)
–Hepatocyte entry, PV formation, growth.
Late liver stage arrest (~40 hrs)
Attenuation by design
Generating P. falciparumGAPs
Simultaneous deletion of P36and P52
5’3’
NF54 recipient line
Dela Vegaet al., PNAS, in press
Pre-clinical testing of P. falciparumGAPs
Mice with
humanized liver
Human hepatocytes
(HC-04) in vitro
Sattabongkot et al., 2006
Sacci et al., 2006
P. falciparum p52-/p36-
parasites
show a severe defect in liver stage development
Dela Vegaet al., PNAS, in press
P. falciparum p52-/p36-
parasites
infect a humanized mouse model
with a chimeric liver but do not grow and do not persist
Dela Vegaet al., PNAS, in press
Phase 1 Safety (Spring 2010)
•6 volunteers receive bites of 5 Pf GAP-
infected mosquitoes
•Same 6 volunteers receive bites of200 Pf
GAP-infected mosquitoes
•Go if no breakthrough.
Phase1/2a proof of concept safety and
efficacy (summer 2010)
•20 volunteers each receive bites of 6 X 200
Pf GAP-infected mosquitoes
•Challenge with 5 Pf WT-infected
mosquitoes 2 weeks after last immunization
•Efficacy readout
•Re-challenge of all protected volunteers 6
month after first challenge
Acknowledgements
Financial supportFNIH, B&MGF (GCGH project)
NIH
Ellison Medical Foundation
SBRI

Patrick Duffy
•Ruobing Wang
•Brant Murdock
•Amy Asawachaicharn WEHI

Alan Cowman
•Julie Healer
•Matt O’Neill
•ThuanPhuong
•Monica Brown
University of Maryland

John Sacci
University of
Heidelberg

Kai Matuschewski
WRAIR
•Gray Heppner
•Mark Polhemus
•Chris Ockenhouse
•Lisa Ware
•Jack Williams
•Jittawadee Murphy
•Michele Spring
•Urszula Krzych
•Patricia De La Vega
•Joanne Lumsden
•Mike O’Neil
Conventus

Steve Kradjian
•Gabrielle Petrosian
SeerPharma

Steve Williams
•Daniel Flewellen
•EceSmerdelj