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