Transcript PowerPoint-presentation

Approaches to genetic
vaccines
Britta Wahren
Karolinska Institutet
Riga 2013
Selection of immunogen
DNA, RNA or (glyco)protein
Whole genes to cover polymorphic MHC in humans
All genes to cover structural and regulatory genes
Several subtypes for virus variability and polymorphic MHC
Vectors like vaccinia or adeno
Physical approach
Small total vector size
Intracellular delivery (skin)- electroporation
Delivery devices
Total cell targets – repeated injections
Divided delivery-avoid immunocompetition
Additional adjuvant like cytokine gene, carrier compound
Minicircle1x
650 bp
1
2
3
4
5
% nicked
sc
Minicircle3x
1 900 bp
1
2
3
4
construct
blank shot hide shot
mc
3.1
2.4
mc3x
3.6
8.6
plasmid
3.7
23.4
5
nicked
sc
Plasmid
3 579 bp
1
2
3
4
5
nicked
sc
1: untreated
2: linearized
3: UV-nicked
4: shot to empty vial
5: shot through mouse hide
Small minicircles were more robust when exposed to
shearing forces than the corresponding plasmid. Also as
splice correcting vectors, they exhibited a more long-term
expression as compared to plasmids in vivo.
3
Intracellular delivery
Needle injection
Dermis
Jet stream injection
Electroporation after needle/jet
stream injection
Needle injection
Jet stream injection
Electroporation after
needle/jet stream injection
Dermis
1. Transfection of
somatic and professional
antigen presenting cells
(APCs)
Secrete
d
antigens
Somatic
cell
MHC I
APC
MHC II
MHC I
APC
2. Antigen presentation to
and activation of lymphocytes
in the draining lymph nodes
CD4
T cell T cell
receptor
MHC II
APC
MHC I
T cell
receptor
CD8
T cell
Costimmulatory
signals
B cell
3. Activated
lymphocytes
Delivery device
Biojector
im
id
spacer
Left arm: Env/rev, 1 inj
+/- GMCSF im
Left arm: Env/rev, 3 inj
+/- GMCSF sc (needle)
Right arm: Gag/RT, 1 inj
Right arm: Gag/RT, 2 inj
Delivery device electroporator
Voltage
2x450V
8x110V
Time
Total length of pulse-train: 0,27 seconds
Repeated injection of DNA,
dispersed delivery
p17/p24B ELISA post 2nd immunization
10 7
10 6
10 6
End point IgG titer
10 7
10 5
10 4
10 3
10 2
10 1
10 5
10 4
10 3
10 2
10 1
EP
-re
pe
]
]
w
EP
-[l
o
w
at
ed
[lo
ig
h]
]
EP
-[h
w
lo
]
w
[lo
d
at
e
pe
BJ
-[
h]
ig
-[h
BJ
-re
]
]
EP
-[l
o
w
w
BJ
EP
-re
pe
at
ed
[lo
ig
h]
EP
-[h
w
lo
]
d
at
e
pe
BJ
-[
w
[lo
ig
-[h
BJ
-re
]
10 0
h]
10 0
BJ
End point IgG titer
p17/p24B ELISA post 1st immunization
Volume and concentration
•
BALB/c mice
Imm w 0, 4 and 8 with GagB
ID+EP (15ug) or IM (50ug)
IFNg ELISpot
–
–
–
2500
2250
750
10
0u
in
Na
iv
e
l)
l)
10
0u
(1
0u
g
0u
g
e+
EP
ed
l
ed
l
in
in
n
ug
i
e
(1
0
1x
10
ul
)
10
ul
)
ïv
e
0
na
IM
1x
P
IM
3x
xE
+1
250
IM
xE
P
)
+1
IM
2x
2x
EP
(8
w
EP
3x
w
EP
(4
1x
EP
)
0
500
(1
2000
Gag p24B pep pool
AMQMLKETI (MHC I restr pep)
2000
1000
6
4000
BALB/c mice
1 imm with GagB
IFNg ELISpot
BJ
+E
P
Net SFC/10 pooled PBMCs
6000
2x
6
Net SFC/10 splenocytes
8000
ne
–
–
–
–
Comparison of different
immunization methods
BJ
(1
0u
g
Comparison of different immunization
protocols using ID EP
ne
•
Divided delivery of env and
gag genes, subtypes A, B, C
Vial 1
Vial 2
Envelope
and rev
plasmids
gag and
RT
plasmids
Vaccine intramuscularly delivered
Vial 1 in the left arm, Vial 2 in the right
DNA vaccines, licenced and
clinical
Licenced
• West Nile virus for horses
• Hematopoietic necrosis (rhabdovirus) for salmon
• Melanoma (tyrosine kinase) for dogs, humans?
• Pig hormonal treatment
Clinical small trials
• HCV for chronic disease, Sällberg et al, SE
• Prostate heterologous antigen, Pisa et al, SE
• CEA heterologous and homologous antigen for gi cancer
Mellstedt, Wahren et al, SE
• HIV for healthy individuals, Ho et al, USA and China,
• HIV for healthy or chronic disease, Wahren et al, SE, IT,
TZ, MC; Pantaleo et al, CH, Robinson et al US
Acknowledgements: The volunteers
SMI/KI
Britta Wahren
Andreas Bråve
Margaret Liu
Karl Ljungberg
Gunnel Biberfeld
Jorma Hinkula
SMITTSKYDDSINSTITUTET
Erik RollmanSwedish Institute for Infectious Disease Control
Gunnel Engström
Kristian Hallermalm
Lindvi Gudmundsdotter
Andreas Boberg
Susanne Johansson
Anne Kjerrström
Maria Isaguliants
Charlotta Nilsson
Katarina Karlén
Reinhold Benthin
Karolinska/SöS
Eric Sandström
Bo Hejdeman
Lars Eriksson
Viveca Holmberg
Walter Reed Army Institute of
Research
Josephine Cox
Mary Marovich
Nelson Michael
Merlin Robb
Deborah Birx
NIH/NIAID
Bernard Moss
Patricia Earl
Members of the AVIP Consortium
University of Munich
Volker Erle
Georg Gasteiger
ISS, Rome
Barbara Ensoli
Bambino Gesú
Paolo Rossi
Paolo Palma
MUHAS, Dar es Salaam
Fred Mhalu
Muhammad Bakari
Eligius Lyuamuya
Pontus Blomberg
Jenny Enger
Sofia Stenler
Nils Carlin
Richard Stout
CytoPulse
Richard Walters
Anna-Karin Roos
Cellectis
Staffan Pauli
Bartek Zuber