U124

IRCL

JCD

U124

IRCL

JCD

Adenovirus: an eternal archetypeA short history

1953: first adenovirus isolation by Rowe et al., Proc. Soc. Exp.Biol. Med.

1962: oncogenic properties of HAdV species A (Ad12).

1953-1968: biochemical characterization

1970-1980: virion assembly

1978-1990: splicing and transcriptional control

1985-1995: viral oncogenes and association with tumor suppresseur

1995-200N: viral vector in gene therapy

Adenovirus

Replication Encapsidation

- DNA ds (~36 kb)

hexon

penton penton base

fibre

IX

IVa2 DNApol pTP

52K IIIa III pVII V pVI II 20K 100K pVIII E3 IV

72K E4

E1A E1B

[[

[

U124

IRCL

JCD

U124

IRCL

JCD

Adenoviruses uptake

1) Membrane attachment

RGD

hCAR

V integrins...2) Uptake

Modify adenovirus tropism :specific fiber modifications

U124

IRCL

JCD

U124

IRCL

JCD

Heparan sulfate

Caveolae

Non-caveolaenon-clathrinendocytosis

Clathrin-caoted pit endocytosis

Macropinocytosis

macropinosomeCCV

EE

LELY

4.5-5.0

6.5

5.5-6.0

Golgi

trans

Medial

Cis

ER

CaveosomeLysosomal degradation

A549HEK29HeLa

NL

S-m

achi

nery

hCARv3/5

NPC proteins

Central channel

Peripheral channel Nuclear basket

Outer membrane

Inner membraneNucleus

Cytoplasm

Viral DNA

Transgene expression

Endocytosis...

U124

IRC

L

JCD

U124

IRC

L

JCD

Structure of the fiber protein

Pentonbase

Shaft

-X--X-X-X-X-P--X--X-X-X-X--X-X-G-X-G--X--X-X-X

Tail Knob

IVa2IIIa

pVI pVIII

E4

ITR

Precursor RNA 1

DAdV-1Atadenovirus

Fiber

FAdV-1Aviadenovirus

Fiber 1 Fiber 2

HAdV2Mastadenovirus

E3Fiber

E4

E1AE1B

Adenoviridae

E3E4

Species Serotype % GC % homology species

Fiber length (nm)

oncogenicproperties

A 12, 18, 31 48 8-20 28-31 high

B 3, 7, 16 51 9-20 9-11 weak

C 1, 2, 5, 6 58 10-16 23-31 nul

D 8, 9, 10,13, 15, 19

58 4-17 12-13 nul (except Ad 9)

E 4 58 4-23 17 nul

F 40, 41 49 15 36 et 23 nul

Human adenoviruses

receptor

hCAR

hCAR

hCAR

hCAR

CD46

hCAR +X

Tropism modifications of adenovirus

• Association with antibodies

• Genetic modifications of the fiber

• Chimeric fiber

• transient expression• important immun response• wide tropism

Project and history

• 1984 cloning of the BAdV4 genome

• 1997 BAdV-4 based vector ?• no permissive cell line• bovine cell line obtaining …

• 2001-200X vectors with chimeric fibers

Project: targeting vectors to B lymphocytes

• lost of hCAR interaction• add new target

Project: targeting vectors to B lymphocytes

Noreceptor

Lost of hCAR interaction Add new target

HAdV2HAdV5HAdV4

SAdV25HAdV17HAdV9HAdV15

HAdV12HAdV31

BAdV10HAdV41-LHAdV40-L

HAdV41-SHAdV40-SHAdV3HAdV7HAdV16

HAdV35BAdV-3CAdV-2MAdV-1

DAdV-1FAdV-1-LBAdV-4

OAdV-287Odocoileus

FrAdV1FAdV1-STAdV-3PAdV-4

FAdV-1-L

BAdV10

PAdV-4

HAdV12HAdV31HAdV17

HAdV9

HAdV2HAdV5HAdV4

SAdV-25HAdV41-L

HAdV40-L

HAdV40-SHAdV41-SHAdV7

HAdV3HAdV16HAdV35

FAdV-1-S

BAdV-3

MAdV-1

FrAdV-1TAdV-3

BAdV-4odocoileusOAdV-287DAdV-1

Siadenovirus

Atadenovirus

Mastadenovirusnon-human

Aviadenovirus

Mastadenovirus

Fiber

KnobPhylogenic

tree

Penton base binding sequence of fiber proteins

HAdV2 (1) --------MKRARPSEDTFNPVYPYDTETGPPT--------HAdV5 (1) --------MKRARPSEDTFNPVYPYDTETGPPT--------HAdV12 (1) -MKRSRTQYAEETEENDDFNPVYPFDPFDTS-D-------- HAdV31 (1) -MKRSRTQYAEEPEENDDFNPVYPFDPYDTA-H--------HAdV3 (1) --------MAKRARLSTSFNPVYPYEDESSS----------HAdV7 (1) --------MAKRARLSTSFNPVYPYEDESNL-Q--------HAdV16 (1) --------MAKRARLSSSFNPVYPYEDESSS----------HAdV9 (1) --------MSKRLRVEDDFNPVYPYGYARNQ-N--------HAdV15 (1) --------MSKRLRVEDDFNPVYPYGYARNQ-N--------HAdV17 (1) --------MSKRLRVEDDFNPVYPYGYARNQ-N--------HAdV35 (1) --------MTKRVRLSDSFNPVYPYEDESTS-Q--------HAdV4 (1) -------MSKSARGWSDGFDPVYPYDADNDRPC-------- SAdV-25 (1) -------MSKKRVRVDDDFDPVYPYDADNAP----------HAdV40-L (1) ---------MKRARFEDDFNPVYPYEHYNPL-D--------HAdV41-L (1) ---------MKRARLEDDFNPVYPYEHYNPL-D--------HAdV40-S (1) ---------MKRTRIEDDFNPVYPYDTSSTP-S--------HAdV41-S (1) ---------MKRTRIEDDFNPVYPYDTFSTP-S--------BAdV-1(1) ----------MKRSIPSDFDPVYPYGKRP------------BAdV-3 (1) ----------MKRSVPQDFNLVYPYKAK-------------BAdV-10 (1) ----------MKRSAASIFAPVYYNTKRR------------CAdV-2 (1) -------MKRTRRALPANYDPVYPYDAPGSS----------MAdV-1 (1) --------------MVEALNAVYPYDLALLPEDYEKTTAP-PAdV-4 (1) ----------MKRSVPSDFNPVYPYDYQPIS----------FAdV-1-L (48) NADPQTLAFNNSGALEVNLDPDGPWSKTATGIDLRLDPTTLFAdV-1-S (10) DPDAEAPTGKMARAGPGELDLVYPFWYQVAA----------DAdV-1 (1) -----------MKRLRLDPDPVYPFGTSETIP---------BAdV-4 (1) -------------MKRARWDPVYPFTEERLIP---------OAdV-287 (1) -------------MKRARWDPVYPFSEER------------odocoileus (1) -------------MKRARWDPVYPFSEDKLIP---------FrAdV-1 (1) MAQSSKRLRRDAEAAKSDLNLVYPFFAPKIN----------TAdV-3 (11) EPDQQTLKKSKQSDQSQGLNLAYPFDKITEF---------- Consensus FNPVYPY

Mastadenovirus

Aviadenovirus

Atadenovirus

Siadenovirus

Conserved the Nter of HAdV fiber protein

1HAdV2 (1) ---NDDKLTLWTTPDPSPNCRIHSDN----DCKFTLVLTKC-GSQVLATVAALAVS-HAdV3 (1) -------NTLWTGPKPEANCIIEYGKQNP-DSKLTLILVKN-GGIVNGYVTLMGASDHAdV4 (1) GNKDYDKLTLWTTPDPSPNCQILAEN----DAKLTLCLTMC-DSQILATVSVLVVRSHAdV5 (1) ---NDDKLTLWTTPDPSPNCRIHSDN----DCKFTLVLTKC-GSQVLATVAALAVS-HAdV7 (1) -------NTLWTGPKPEANCIIEYGKENP-DSKLTLILVKN-GGIVNGYVTLMGASDHAdV9 (1) ----EDKRTLWTTPDTSPNCKIDQDK----DSKLTLVLTKC-GSQILANVSLIVVDGHAdV12 (1) SNTPYDPLTLWTTPDPPPNCSLIQEL----DAKLTLCLTKN-GSIVNGIVSLVGVKGHAdV15 (1) --KKEDKRTLWTTPDTSPNCKIDQDK----DSKLTLVLTKC-GSQILANVSLIVVDGHAdV17 (1) --PKYDTRTLWTTPDTSPNCRIDKEK----DSKLTLVLTKC-GSQILANVSLIVVSGHAdV31 (1) IATPYDPLTLWTTPDPPPNCTLRQEL----DAKLTLCLTKN-ESIVNGIVSLIGVKGHAdV35 (1) ------INTLWTGINPPPNCQIVENTNTN-DGKLTLVLVKN-GGLVNGYVSLVGVSDHAdV40-L (1) QTRSVTSLTTIWSISPTPNCSIYETQ----DANLFLCLTKN-GAHVLGTITIKGLKGHAdV40-S (1) SPTTTTPTTLWTTADPSPNATFYESL----DAKVWLVLVKC-NGMVNGTISIKAQKGHAdV41-L (1) SPTTTTPTTLWTTADPSPNATFYESL----DAKVWLVLVKC-NGMVNGTISIKAQKGHAdV41-S (1) RTRSVPSLTTIWSISPTPNCSIYETQ----DANLFLCLTKN-GAHVLGTITIKGLKGBAdV-3 (1) TAPNMVTETLWTGTGSNANVTWRGYTAP--GSKLFLSLTRFSTGLVLGNMTIDSNASMAdV-1 (1) LQAPQYELTTNGMREITFSFNSTGGLEQPAPVTYWGALDPPPTAKAAEIENQKRVKKFAdV-1-L (1) -----------------------NTN---AAKVAAYCRLAAAGGVVSGTIQVTSYAGFAdV-1-S (1) -------TGNFVSSSNNPFNGSYFLQQINTMGMLTTSLYVKVDTTTMGTRPTGAVNEBAdV-4 (1) ----FEVLNDNLALGLNNSLTVESGK---LAVKTAGPIQSTEQGINLSVANPFSISSDAdV-1 (1) ----------------------PQNK---AAKVAGYVYLTSVGGLVHGTIQIKATAGFrAdV-1 (1) AVSPPFSSTGGLSLLLDPSLRLISGALGLKPSSASCIVSSP-AGVELKTGKGLGVTGTAdV-3 (1) --GIRLAPNSGLQITPNGLAVSVNAVQILSSPLITAASIGPPTTMVTGTVSPGRATN

Consensus (1) TLWTT DPSPNC I DAKLTLVLTK GSIVLGTVSLLGV G

A B C

Sequence homology of fibers knob domain

3D structure of the HAdV5 fiber knob

BAdV-4

HAdV5

HAdV3

HAdV4

HAdV15

HAdV12

DAdV-1

HAdV40-S

HAdV40-L

BAdV-3

FAdV-1-L

OAdV-287

CAdV-2

MAdV-1

FrAdV-1

FAdV-1-S

TAdV-3

PAdV-4

HAdV35

A B C D E F G H I J

Putative secondary structure of the fibers knob domain.

Construction of modified genomes

Homologous recombination in E.coli

Swa I (1)

HAdV5 WT fiber

ITR

Swa I (37523)CMV

ITR

lacZ

Chimeric fiber

Backbone plasmid

Shuttle plasmids

transgene

ClaI PacI

Production of recombinant vectors without hCAR interaction.

ITR lacZ ITR

modified fiber gene

transfection amplification

wt fiberHEK-293

wt fiber

Virus with modified

fiber

Modified fiber

HEK-293

HEK-293 cells expressing HAdV2 fiber

- Modification of the sequence around AUG

- 5’ UTR: TPL exons 1/2/3

HEK-293

- non modified : GCC AUG AAA- consensus : GCC AUG GNG (Kozak) - modified : ACC AUG GCG AAA

in vitro transcription/translation

G418 selectionnorthern blot

western blot

HEK-293-247E

pOM2477208 bp

polyA / intron

polyA- intron

SV40 promoter

CMV

ColE1

neo

AmpR

Fibre Ad2

TPL 2/3

EcoR I (2079)

Hind III (663)

SalI (2600)

NotI (831)

MluI (2)

MluI (991)

NcoI (384)

NcoI (840)

NcoI (3601)

NcoI (4336)

pC3Fb

modified ATG - - + - + -

TPL exons - 1/2/3 1/2/3 2/3 2/3 1/2/3(wt)

62 kDa

6 LSIATKGP ITVSDGK7 LALQTSAP LSGSDSDT

FAdV-1-L GK21 LEVNTDP SGPLTSGNNG22 LSLAAVTP LAVSSAG

DAdV-1-14 IVLNLQDP LGVIDGK5 LGVKLGPG VHINGEGA

DAdV-1-213 LGLTEPAKP LKLADGK14 LGVNVGLG LAVSNGS

FAdV-1-S GK 9 VAAFTSGT IGLSSPTGN10 FVSSSNNP FNGSYF L

HAdV2/BAdV-4

HAdV2

HAdV2/FAdV-1-L

HAdV2/FAdV-1-S

HAdV2/DAdV-1-1/2

7 LVITSPELP LSTTPEGK8 LFIQLSNCF ATDNGK

Chimeric fiber proteins

K LAAR CTNAAG CTT

HindIII

in vitro trimerization

DD N NHAdV2 HAdV2/BAdV-4

trimer

in vivo trimerization

HAdV

2

HAdV

2/FA

dV-1

-L

HAdV

2/FA

dV-1

-S

HAdV

2/D

AdV

-1-1

HAdV

2/D

AdV

-1-2

HAdV

2/BAdV

-4

trimer

Chimeric fibers trimerization

MluI HindIII SalI

pFIB/HAdV2/XXX

31041 32788

PacI

NotI XhoI

HAdV5 Fiber

lITR rITR

SwaI

BstBI

SwaI

AscI

p5Shut/HAdV2/XXX

pRECad

KpnI

PacIlacZ

Viral genome with chimeric fiber gene

Homologous recombination in E. coli Top10F’

Production of recombinant vectors with chimeric fibers.

ITR lacZ ITR

Chimeric fiber gene

transfection amplification

wt fiberHEK-293-247E

wt fiber

HEK-293

Virus with modified

fiber

Modified fiber

HAdV2

-ß-G

alHAd

V2/D

AdV-

1-1

HAdV2

/DAd

V-1-

2

HAdV2

/BAd

V-4

62kDa

Patent N° B0151WOHAdV2/XXX : 2x1012PP/ml

Vectors with knob domain of L- or S- FAdV-1 fiber

In vitro trimerization analysisA549

20

60

100

0.5 1.0 2.0 4.0 8.0

10-3 PP/cell

HAdV2

FAdV-1-L% p

osi

tive

cells

HAdV2/FAdV-1-S

HAdV2/FAdV-1-L No virus

HAdV2

FAdV-1

-S

Tropism of the HAdV2/DAdV-1 and HAdV2/BAdV-4 vectors

A549 cells

0

30

60

90

0.5 1.0 2.0 4.0

103 PP/cell

% p

osi

tive c

ells

HAdV2 62.0 kDa

HAdV2/BAdV-4 58.2 kDa

HAdV2/DAdV-1-1 73.0 kDa

HAdV2/DAdV-1-2 56.5 kDa

HAdV2/DAdV-1-2

HAdV2/DAdV-1-1

Shaft repeats

30

21

HAdV2/BAdV-4 is the best prototype

Tropism of the HAdV2/BAdV-4 vector

HepG2

0

800

1600

2400

1 3

HAdV2

HAdV2/BAdV-4RFI

X 103 PP/cell

1 3X 103 PP/cell

0

40

100

% p

osi

tive c

ells

HEK

0

40

% p

osi

tive c

ells

Daudi Jurkat

5.0

1.103 PP/cell

100 101 102 103 104

Relative Fluorescence Intensity

Cel

l Num

ber

HAdV2

84

21

0.50

84

21

0.50

HeLa cells

PP

(10

3 )

0.5

1.0

2.0

4.0

8.0

PP

(10

3 )

0.5

1.0

2.0

4.0

8.0HAdV2/BAdV-4

Reduction in cell binding of the HAdV2/BAdV-4 vector

0

50

100

% IF

Infection (ß-galactosidase)

HAdV2 HAdV2/BAdV-4

Uptake

HAdV2

HAdV2/BAdV-4

A549 cells

hCAR receptor and HAdV2/BAdV-4 infection

• no competition between HAdV2 fiber and HAdV2/BAdV-4 vector.

• hCAR independent infection of CHO cells.

CHO cells CHO-hCAR cells

% p

osit

ive

cell

s

0

20

40

60

80

100

1 3 1 3 x 103 PP/cell

Chinese Hamster Ovaries: hamster and/or ovarian cell receptor(s) ?

HAdV2

HAdV2/BAdV-4

Ovarian cell tropism of HAdV2/BAdV-4 vector

0

40

100

0 0.5 1 2 4 8

% p

osi

tive c

ells

SKOV3

HAdV2

HAdV2/BAdV-4

A549 cells

0

30

60

90

0.5 1.0 2.0 4.0

103 PP/cell

% p

osi

tive c

ells

103 PP/cell

20

60

100

HELA CHO OVCAR3 IGROV1

% p

osi

tive c

ells

SKOV3

B-CLL specific receptor ?B-CLL specific receptor ?

EDPGFFNVEEDPGFFNVE

Epstein-Barr virus: Binding to CD21 receptor Epstein-Barr virus: Binding to CD21 receptor on B lymphocytes surface using GP 350/220on B lymphocytes surface using GP 350/220

(Nemerow (Nemerow et al.,et al., Cell, 1989 ) Cell, 1989 )

0

40

80

Raji Daudi HSB2 Jurkat% o

f po

siti

ve c

ells Isotype

CD21

Daudi

Jurkat

Raji

HSB2

[K]7EDPGFFNVE [K]7EDGGFFNVE

Daudi

Jurkat

Targeting of CD21 on B-lymphocytes

LB

HI loopEDPGFFNVEIPEFPF

Penton base

FiberCD21

?

Genetic modifications of adenovirus fiber

Endosomolysis

CC

V

6.5

Clathrin-caoted pit endocytosis

Non-modified HAdV

EE

LE

CC

V

6.5

Fiber-modified HAdVs ??

Clathrin-caoted pit endocytosis

LY

HAdVCD21 conserved its membrane lysis ability

Early endosomes

0

20

40

5,0 5,5 6,0 6,5 7,0 7,5

% 3 H

rel

ease

pH

HAd5Vfiber wt

HAdV3HAdVCD21

1) Membrane attachment

X0.5

0

100

HAdV5-FAM

HAdV-CD21HI-FAM

A549 HSB2 Raji Daudi

X5.3X3.7

X0.3

% o

f po

siti

ve c

ells

50

Targeting of CD21 on B-lymphocytes

Copies of viral genome per cell

A549

HeLa

Jurkat

HSB2

HAdV5 HAdV5 CD21HI

80 11

18 1

80 3

81 2

2) Infection (-galactosidase)

0

40

80

120

A549 Daudi Raji

% o

f po

siti

ve c

ells

HAdV5

HAdV-CD21HI

X5.5X10.4

X0.3

MOI : 300

Enhance positive cells

weak-gal level

Altered intracellulartrafficking ?

Interaction betweenHAdVs and

hematopoïetic cells: New therapeutic ways ??

0

40

80

100

0 15 30 60 150 300 600

% o

f po

siti

ve c

ells

HAdV5gal

HAdV5-CD21HIgal

0

60

120

0 15 30 60 150 300 600

Flu

ores

cenc

e In

tens

ity

A549 : MOI 30FI : 4000

Targeting of CD21 on B-lymphocytes

MOI MOI

ND: not determined

Cell type

Binding

hCAR

CD18(2)

CD11a(L)

Uptake

HEK293

A549 HeLaReh Daudi Raji B C U266HSB2JurkatK562

ND ND ND ND

ND

Epithelial cells B cell lineages T cells

-gal

0 50 100 150 200 250

HEK293

A549

Reh

Daudi

Raji

B

C

U266

HSB2

K562

%/HEK293

B lineage

- trypsin

+trypsin

Attach. Upt.. Upt. Eff.

HEK293

K562

HSB2

U266

C

B

Raji

Daudi

Reh

A549

19

246

100

59

27.5

73

147

20.4

140

93.4

18.3

152

100

9.3

1.3

25.6

47

1.5

126

53

96

62

100

16

535

32

790

57

Deficiency of HAdV2 uptake into B-lymphocytes

Deficiency of Clathrin coated pit endocytosis...

HE

K

HeLa

A549

Reh

Raji

Daudi

Jurk

at

K562

HS

B2

U266

C D

Clathrin192 kDa

Epithelial cells Hematopoietic cells

Total cell extract

Clathrin192 kDa

HE

K

HeLa

A549

Reh

Raji

Daudi

Jurk

at

K562

HS

B2

U266

C D

Epithelial cells Hematopoietic cells

Membrane Fraction

0

4

8

12

16

A549 RehDaudi Raji C D U266HSB2 Jurkat

Rela

tive F

luore

scen

ce In

ten

sity

Weak Transferin uptake

Control

Transferin

Clathrin coated pit endocytosis of transferin

A549

U266

Raji

Is Caveolae endocytosis an alternative

to clathrin coated pit endocytosis deficiency ?

Associated- membrane receptors

Transmembrane receptors

Caveolin

CAVEOLAE

Lipid rafts

Lipid raft Caveolin-1

A549

U266

C

D

Daudi

HSB2

Jurkat

Raji

A549

U266

HSB2

Jurkat Daudi

D

C

Raji

Top

Bottom

Dot-Blot anti-Caveolin-1

U266

A549

A-

B-

0

50

100

A549 HEK293 HeLa U266

% p

osit

ive

cell

s

mU

nits

-g

al/m

g of

pro

tein

(x10

3 )

0

2

4

6

8

2 5 10200

ethylcyclodextrine (mM)

A549

HeLa

U266

2 5 10200 2 5 10200

0

50

150

250

A549 HeLa HEK293

Chlorpromazine (g/ml)

Upt

ake

(% o

f no

n tr

eate

d)

0 20

0

0.4

0.8

1.2

0 1 2 3 4

Chlorpromazine (g/ml)

U266

mU

nits

-g

al/m

g of

pro

tein

(x10

3 )

0 2 5 10

ethylcyclodextrin (mM)

20

0 20

U266 (up to 90 %) A549

Colocalization caveolin-1/ HAdV5

Is Caveolae endocytosis an alternative to clathrin coated pit endocytosis deficiency ?

Dominant négatif CAV-1-GFP

U266

?A549

U266

Non-caveolaenon-clathrinendocytosis

Clathrin-caoted pit endocytosis

Macropinocytosis

macropinosomeCCV

EE

LELY

4.5-5.0

6.5

5.5-6.0

Golgi

trans

Medial

Cis

ER

CaveosomeLysosomal degradation

A549HEK29HeLa

NL

S-m

achi

nery

hCARv3/5

NPC proteins

Central channel

Peripheral channel Nuclear basket

Outer membrane

Inner membraneNucleus

Cytoplasm

Viral DNA

Transgene expression

Endocytosis...?

Plasmocyte?

Caveolae

Conclusion: targeting vectorsto B lymphocytes and ovarian cells

Noreceptor

Lost of hCAR interaction Add new target

XOvarian cell

specific receptor andreduction of

hCAR interaction

In vitro

- CD21+ Lineage

CD21 targeting

- Interaction hCAR

- Primary B-LLC cells

Other receptors ?

New therapeutic ways

Caveolae/phagocytose ?(Primary plasmocytes)

In vivoEx vivo

- Primary B-LLCcells

ex vivo assays...

doses cytotoxicity/immunogenicity

Non-specificdissemination

Biodistribution

transgene

HSV-tk, CD154 ?

Immune response

Cellular antibody

Conclusion