Genetic Immunization with Recombinant Lentivector

Yukai He, MD/PhD

Assistant Professor

Departments of Dermatology and Immunology, University of Pittsburgh,

School of Medicine

Vaccines

• Conventional vaccines– Attenuated organisms: Oral Polio vaccine

(Sabin vaccine)

– Inactivated organisms: Injected polio vaccine (Salk Polio vaccine)

– Subunit protein vaccines: HBV vaccine

Immune correlates: Neutralizing Ab

B cell vaccines

Salk and Youngner At University of Pittsburgh

T cell vaccines

• HIV, Malaria, TB, Tumor• Therapeutic vaccines: Chronic HBV infections

Attenuated organismsGenetic vaccines

DNA vaccine: Naked DNA, gene gunViral vectors: Adenovector, alpha viral vector, vaccinia vector, lentivector, AAV

Recombinant Lentivector

CMV-p-Globin-intron

Gag-pol RRE Globin-pApLP1

pRSV Rev HIV-pApLP2

CMV-p-Globin-intron

VSV-G Globin-pApLP/VSV-G

A: Packaging plasmids

3rd Generation of Lentivector

Trip-EGFP

SD SA

pRSV/5LTR

RRE U3/HIV 3’-LTRpSV40EM7BlasticidinTRIP CMV-EGFP

B: Transfer Plasmids

Comparison between Adenovector and Lentivector

• Adenoviral vector– High titer– High transduction

efficiency– Non-intergation– Short term gene

expression– Long term Ag

presentation– Immune dominant

antivector immunity– Pre-existing antivector

immunity

• Lentiviral vector– High titer– High transduction

efficiency– Intergration or Non-

integration– Long term gene

expression and Ag presentation

– Low or no immune dominant antivector immunity

– No pre-existing antivector immunity

T cell immunity elicted by lentivector immunization

Ex vivo Approach

In vivo Approach

Ex vivo Approach (He et al., 2005, JI)

1. Prepare BMDCs and recombinant lentivector

2. Transduction of BMDCs ex vivo

3. Immunize mice with BMDCs

4. Monitor the T cell immunity by in vivo killing assay and intracellular staining of IFN

5. Examine the antitumor effect

Transduction of BMDCs ex vivo by Lentivector

PBS-DC EGFP-lv-DC

PBS-DC EGFP-lv-DC

CD

11c

B7.

2

GFP

IL-12 p70

0

1000

2000

3000

4000

5000

6000

Control LPS CpG

different treatment

PBS-DC

EGFP-lv-DC

B7.2

0

50

100

150

200

250

300

350

Control LPS CpG

different treatment

PBS-DC

EGFP-lv-DC

pg/

mill

ion

cells

MF

I

IFN-gamma produced by allogenic T cells

0

100

200

300

400

500

600

700

800

1:50 1:100 1:200 1:400

DC:responder ratios

pg/m

l

PBS-DC

OVA-lvv-DC

MLR

0

20

40

60

80

100

120

140

160

180

1:50 1:100 1:200 1:400 1:800 1:1600

DC: responder ratios

cpm

(X

10-3

)

PBS-DC

OVA-lvv-DC

Lentivector does not change the intrinsic properties of transduced BMDCs

Naive PBS-DC Pulsed DC Transduced DC

49.6550.35 48.8251.18 4.995.11 0.0999.91

CFSE

In vivo killing assay

CD

8C

D4

IFN-

0 per 100K 68 per 100K 493 per 100K 1895 per 100K

4 per 100K 30 per 100K 160 per 100K 150 per 100K

IC-staining of IFN

Lentivector Transduced BMDCs induce strong T cell immune responses

Antitumor Therapy

0

50

100

150

200

250

300

350

3 5 8 11 13 15 17 19 22 24

Days after tumor inoculation

Pulsed DC

Transdued DC

Ctrl

Tum

or

are

a (m

m)

(0/10)

(0/10)

(10/10)

Antitumor effect of lentivector mediated genetic immunization

In vivo approach (He et al., 2006 Immunity)

• Compare the efficacy of in vivo and ex vivo approach

• Immunize mice with recombinant lentivector

• Monitor T cell immunity

• Examine the antitumor effect

Na

ive

Na

ke

d D

NA

Ge

ne

Gu

n

OV

A-l

vv

OV

A-a

d

OV

A-V

V0

20

40

60

80

Immunization methods

% s

pe

cif

ic l

ys

is

Na

ive

Na

ked

DN

A

Ge

ne

-Gu

n

OV

A-lvv

OV

A-A

d

OV

A-V

V

0

500

1000

1500

2000

Immunization methods

No

. o

f IF

N- +

ce

llsp

er

0.1

mill

ion

CD

8 T

ce

lls

Lentivector induce strongest CD8 T cell immunity

0 10 20 30 40 50 600

25

50

75

100

125

OVA-lvv

OVA-VV

Days after immunization

% s

pe

cif

ic l

ys

is

Lentivector induce persistent and potent CD8 T cell immunity

0 10 20 30 400

100

200

300

400

500 OVA-lvv

EGFP-lvv

Control

days after tumor inoculation

tum

or a

rea

(mm

) (M

ean

SE

M)

0 10 20 30 40 500

25

50

75

100

OVA-lv

EGFP-lv

Control

days after tumor inoculation

Per

cent

sur

viva

l

Antitumor effect of lentivector mediated genetic immunization

HBsAg specific CTL activity following genetic immunization

Naive

50.4949.51 8

144

20491CD

8

IFN

DNA

50.1049.94 51.9848.06

IM IM+Electroporation

20

91

27365

CD

8

IFN

12

119

21329C

D8

IFN

HBS-lvv

10.0689.95

IM

0.3699.64

S.C

In vivo killing assay

211

112

27644

CD

8

IFN

803

165

29214

CD

8

IFN

IC-staining

In vivo CTL activity

Naive EP1 EP2 IM HBS-lv-IM HBS-lv-SC

-25

0

25

50

75

100

Immunization procedure

% o

f sp

eci

fic ly

sis

Comparative efficacy of different immunization approaches

IC-staining

Naive HBS-lv-IM HBS-lv-SC0

1000

2000

3000

Immunization procedure

% o

f IF

N-

+ c

ells

/pe

r 0

.1 m

illio

n C

D8

T c

ells

Specific lysis after vaccination with different HBS-lv and different routes16hr in vivo killing assay

-20

0

20

40

60

80

100

120

% o

f spe

cific

lysi

s

Series1 -0.007968065 35.16373152 95.61707259 99.26842 99.2728774

Naïve HBS-lv-IM HBS-lv-FPHBS-Ig2aFC-

lv-IMHBS-Ig2aFC-

lv-FP

Intra cellular staining of IFN-gamma

0

500

1000

1500

2000

different vaccination routes

per

100k

CD

8+ c

ells

HBS-lvv

HBS-Ig2aFC-lvv

HBS-lvv 233.6650801 921.3124001

HBS-Ig2aFC-lvv 1038.905209 1525.989509

IM FP

Mechanism of T cell priming in lentivector mediate genetic

immunization

• He et al., 2006 Immunity

1

sDC

2

Infection/Danger Signal

sDC Paradigm: Direct Priming

1: Resting sDC2: Activated sDC

TT

TT

TT

Skin Ag

• Tissue derived DCs:– LC– Dermal DCs– Other tissue DCs

• Blood derived DCs:– CD8+– CD4+– CD8-CD4-– pDCs

DC Network

Heath and Carbone groups indicate that LCs are not directly involved with priming of naïve T cells after skin HSV infection (Allan et al, Science 2003; 301:1925)

CD8+ LN resident DCs prime naïve T cells via cross priming after HSV, Influenza A, vaccinia virus, LCMV, and Listeria Monocytogene infection(Allan et al, Science 2003; Belz et al., 2004; Smith et al., 2003; Belz et al., 2005 )

1

sDC

2

Infection/Danger Signal

Paradigm Shift: Cross Priming

T

T

TT

TT

Skin Ag

Ag

1. Resting sDC2. Activated sDC3.Resident CD8 DC

(Carbone et al., 2004; Heath et al., 2004; Serbina and Pamer, 2003)

1.How to efficiently Ag transfer and faithfully transfer the environmental cues from sDCs to LN resident DCs

2. Is this a generalized truth or restricted to the few viruses studied? 1.Cytopathic virus or with well-described

mechanism for immune evasion (Bosnjak et al., 2005; Engelmayer et al., 1999; Larsson et al., 2004; Sevilla et al., 2003).

2.Non-cytopathic vectors such as lentivector that was shown not interfere with the APC function of transfected DCs (He et al, 2005) are able to directly prime T cells.

Questions raised

Experimental Design

Luc-lvvOVA-lvv

DLN DC subsetsBy Cell sorting

Co-culture with

OT-I cells

2 days

Analysis byFlow cytometry

3H incorporation

3 days

Luciferase activityRT-PCR

58.18%

41.83%

CD8-B220-DC

95.76%

4.27%

CD8+B220-DC

96.95%

3.09%

pDCs99.28%

0.74%

OT-I alone

CFSE

CD8-B220- CD8+B220- pDC OT-I alone0

2000

4000

6000

8000

CP

M

CD11c+ cells

CD8

B2

20

CD11c+CD8-B220- prime naïve CD8 T cells

CD8+CD11b- CD8-CD11b- CD8-CD11b+0

10000

20000

30000

DC Subsets

pg/m

l

CD8+CD11b- CD8-CD11b- CD8-CD11b+ background0

1000

2000

3000

4000

5000

DC Subsets

CPM

CD11c+ Cells

CD

8-P

E

CD11b

CD11c+CD8-CD11b+ prime naïve CD8 T cells

CD8-/loDEC205+ CD8-DEC205- CD8+DEC205-/lo background0

2000

4000

6000

8000

10000

DC Subsets

CPM

CD11c+ Cells

CD8

DE

C2

05

CD8-/loDEC205+ CD8-DEC205- CD8+DEC205-/lo background0

1000

2000

3000

4000

5000

6000

DC Subsets

CP

M

CD11c+ Cells

CD8

DE

C2

05

CD11c+CD8-/loDEC205+ sDCs prime CD8 T cells

LV

VV

CD8+DEC205-/lo CD8-/loDEC205+ DN-DC0

20

40

60

80

DC Subsets

RLU

/Milli

on c

ells

CD11c+ Cells

CD8

DE

C20

5

CD8+ B220- CD8-B220- pDCs0

10

20

30

40

DC Subsets

RL

U/M

illio

n c

ells

CD11c+ Cells

B22

0

CD8

Transgene expression is only found in sDC

OVA-lvv

7 hours 20 hours 46 hours

CD8+ DEC205+ CD8+ DEC205+ CD8+ DEC205+

OVA

S15

OVA-VV

OVA

S15

7 hours 20 hours 46 hours

CD8+ DEC205+ CD8+ DEC205+ CD8+ DEC205+

Immunization with lentivector showed prolonged in vivo Ag presentation

8.19%91.81%5.34%94.71%

11.20%88.88%27.09%73.13%

43.38%56.65%

7.71%92.29% 4.73%95.31% 65.32%34.90% 97.35%2.69%53.56%46.74%

Day 1-4 Day 2-5 Day 5-8 Day 12-15 Day 21-24

Naïve FITC + PT

FITC

175 491856

FITC

CD

11

c-P

E

Inhibition of skin DC migration by injecting pertusis toxin

FITC+ DCs in the DLN migrate from skin

FITC- FITC+ OT-I alone0

5000

10000

15000

20000

25000

DC Subsets

CP

M

FITC

CD

11c

CD8

DE

C20

5

FITC-

CD

11c

FITC

FITC+

FITC

CD

11c

CD8

DE

C20

5

Summary: Paradigm Found1.In contrast to previous studies using HSV,

IAV, and VV, skin derived DCs appear to play a dominant role in priming naïve T cells after LV immunization.

2.Classical Paradigm is restored in this LV mediated immunization system

3. Immunization with non-cytopathic LV result in potent effector and memory CD8 T cell responses underscored by extended time of direct Ag presentation.

4. It remains to be determined if LC, DDC, or both play the of APC after LV immunization

Acknowledgements:

Jiying Zhang

Cara Donahue

Louis D. Falo, Jr., MD, PhDChairman of the Department of DermatologyUniversity of Pittsburgh

Dr. Jonathan Yewdell of NIH

for providing VV-OVA

This research is supported the grant from NIAMS to Dr. Yukai He.

• Introduction to Lentivector

• T cell immune responses induced by lentivector immunization

• Mechanism of lentivector mediated genetic immunization

Ad-vector Lenti-vector0

25

50

75

100108 pfu or TU

107 pfu or TU

None

% o

f spe

cific

lysi

s

Lentivector is less immunogenic in inducing antivector immunity

0 10 20 30 40 50 600

25

50

75

100

125

OVA-lvv

OVA-VV

Days after immunization

% s

pe

cif

ic l

ys

is

Elicitation of potent effector and memory CD8 T cell response