approaches to genetic vaccines britta wahren karolinska institutet riga 2013
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Approaches to genetic vaccines
Britta WahrenKarolinska Institutet
Riga 2013
Selection of immunogenDNA, RNA or (glyco)protein Whole genes to cover polymorphic MHC in humansAll genes to cover structural and regulatory genesSeveral subtypes for virus variability and polymorphic MHCVectors like vaccinia or adeno
Physical approachSmall total vector sizeIntracellular delivery (skin)- electroporationDelivery devicesTotal cell targets – repeated injectionsDivided delivery-avoid immunocompetitionAdditional adjuvant like cytokine gene, carrier compound
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
1 2 3 4 5
1 2 3 4 5
1 2 3 4 5
Minicircle1x
650 bp
Minicircle3x
1 900 bp
Plasmid
3 579 bp 1: untreated
2: linearized
3: UV-nicked
4: shot to empty vial
5: shot through mouse hide
construct blank shot hide shot
mc 3.1 2.4
mc3x 3.6 8.6
plasmid 3.7 23.4
% nicked
sc
sc
sc
nicked
nicked
Needle injection
Dermis
Electroporation after needle/jet stream injection
Jet stream injection
Intracellular delivery
Needle injection
Dermis
Electroporation after needle/jet stream injection
Jet stream injection
MHC II
Secreted antigens
1. Transfection of somatic and professional antigen presenting cells (APCs)
MHC I
MHC I
Somaticcell
APC
APC
2. Antigen presentation to and activation of lymphocytes in the draining lymph nodes
T cell receptorMHC II
YAPC
CD8 T cell
CD4 T cell
B cell
MHC IT cell receptor
Co-stimmulatory signals
3. Activated lymphocytes
Delivery device
Biojector
im
Left arm: Env/rev, 1 inj +/- GMCSF im
Right arm: Gag/RT, 1 inj
id
Left arm: Env/rev, 3 inj +/- GMCSF sc (needle)
Right arm: Gag/RT, 2 inj
spacer
Delivery device electroporator
Total length of pulse-train: 0,27 seconds
Time
Vo
ltage
2x450V
8x110V
Repeated injection of DNA, dispersed delivery
p17/p24B ELISA post 1st immunization
BJ-[high
]
BJ-repeated [lo
w]
BJ-[low]
EP-[high
]
EP-repeated [lo
w]
EP-[low]
100
101
102
103
104
105
106
107
End
poin
t IgG
tite
r
p17/p24B ELISA post 2nd immunization
BJ-[high
]
BJ-repeated [lo
w]
BJ-[low]
EP-[high
]
EP-repeated [lo
w]
EP-[low]
100
101
102
103
104
105
106
107
End
poin
t IgG
tite
r
Volume and concentration
• Comparison of different immunization protocols using ID EP
– BALB/c mice– Imm w 0, 4 and 8 with GagB– ID+EP (15ug) or IM (50ug)– IFNg ELISpot
1xEP
2xEP (4
w)3xE
P
2xEP (8
w)
2xIM+1xE
P
1xIM+1xE
P3xIM 1xIM naïve
0
2000
4000
6000
8000
Net
SFC
/106 s
plen
ocyt
es
needle (10ug in
10ul)
needle+EP (10ug i
n 10ul)
BJ (10ug i
n 100ul)
BJ+EP (10ug i
n 100ul)Naive
0
250
500
750
10002000
2250
2500
Gag p24B pep poolAMQMLKETI (MHC I restr pep)
Net
SFC
/106 p
oole
d PB
MCs
• Comparison of different immunization methods
– BALB/c mice– 1 imm with GagB– IFNg ELISpot
Divided delivery of env and gag genes, subtypes
A, B, C Vial 1
Envelope and rev plasmids
Vial 2
gag and RT plasmids
Vaccine intramuscularly deliveredVial 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
SMITTSKYDDSINSTITUTETSwedish Institute for Infectious Disease Control
SMI/KIBritta WahrenAndreas BråveMargaret LiuKarl LjungbergGunnel BiberfeldJorma HinkulaErik RollmanGunnel EngströmKristian HallermalmLindvi GudmundsdotterAndreas BobergSusanne JohanssonAnne KjerrströmMaria IsaguliantsCharlotta NilssonKatarina KarlénReinhold 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
Richard Stout
Pontus BlombergJenny EngerSofia Stenler
NIH/NIAID
Bernard Moss
Patricia Earl
Members of the AVIP ConsortiumUniversity of MunichVolker ErleGeorg GasteigerISS, RomeBarbara Ensoli
Bambino GesúPaolo RossiPaolo Palma
MUHAS, Dar es SalaamFred MhaluMuhammad BakariEligius Lyuamuya
CytoPulseRichard WaltersAnna-Karin Roos
Cellectis
Nils Carlin
Staffan PauliBartek Zuber
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