DNA-IMMUNOGEN BASED ON A CONSENSUS INTEGRASE OF HIV-1

SUBTYPE A

St.-Petersburg, 2012 г.

Krotova Olga1,2,3

1Engelhardt Institute of Molecular Biology, Moscow (Russia);2MTC, Karolinska Institutet, Stockholm (Sweden);3Ivanovsky Institute of Virology, Moscow (Russia).

HIV EPIDEMICS

CONSENSUS INTEGRASE

34 full-length amino acid integrase sequences

from treatment naïve patients

isolated on the territory of the former Soviet Union

(Belarus, Estonia, Georgia, Russia, Ukraine, and Uzbekistan)

IN_a

BioEdit

1 50 212 288

64

D

1 50 212 288

64

D

1 50 212 288

64

V

1 50 212 288

64

V

1. IN_a

3. IN_in

2. IN_a_e3

4. IN_in_e3

+ H51Y, E92Q, S147G, E157Q, K160Q

+ H51Y, E92Q, S147G, E157Q, K160Q

INTEGRASE DESIGN

AMINO ACID SEQ

NUCLEOTIDE SEQ

• codon usage (OPTIMIZER) • secondary mRNA structure (UNAFold)

Optimization of IN nucleotide sequences for expression:

pET15b

(prokaryotic)

pVAX1

(eukaryotic)

ProteinYield±SD, mg/L of

culture

IN_a 11,7±1,2

IN_a_e3 18,0±1,8

IN_in 13,2±1,2

IN_in_e3 11,7±1,2

PROKARYOTIC EXPRESSION

anti-IN

• SDS-PAGE analysis with subsequent staining by Coomassie blue

• Western blotting analysis using ployclonal anti-IN antibodies

WB analysis

• Purification by Ni-NTA agarose chromatography

 

3'-processing efficiency, %

Strand transfer efficiency, %

measured relative measured relative

IN_subt_B 34±4 100 6,1±6 100

IN_a 51±5 150±15 7,8±8 128±15

IN_a_e3 8,4±0,9 24,7±2,5 1,3±0,2 3,8±0,4

In vitro INTEGRASE ACTIVITY TESTS

U5 = 32P-U5B + U5A

3’-processing Strand transfer

Unspecific exo- activity

U5-2 = 32P-U52 + U5A

0,00

0,10

0,20

0,30

0,40

0,50

0,60

0,70

0,80

0,90

IN_a IN_a_e3 IN_in IN_in_e3

IN [

ng

/ce

ll] HeLa

HEK293

NIH3T3

EUKARYOTIC EXPRESSION

HeLa cells

IN variants were also expressed in:

• HEK293

• NIH3T3

anti-IN

anti-Actin

IN variant Half-life, h

IN_a 4,7

IN_in 11,3

HALF-LIFE OF ACTIVE AND INACTIVE CONSENSUS IN(cycloheximide-chase)

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10

Time, h.

Am

ou

nt

of

pro

tein

le

ft,

%

IN_a

IN_in4,7 h

11,3 h

anti-IN

anti-Actin

PROTEASOMAL DEGRADATION PATHWAY

0,0

1,0

2,0

3,0

4,0

5,0

6,0

IN_a IN_a_e3 IN_in IN_in_e3

Rel

ativ

e am

ou

nt

of

pro

tein

s

MG132

Epoxomycin

FLUOROSPOT experiments

IFNγ

IL2

IFNγ/IL2

0

200

400

600

800

1000

1200

1400

1600

1800

IFN

g s

fu/m

ln s

ple

no

cy

tes

IN_a

IN_in

IN_in_e3

vector

0

100

200

300

400

500

600

700

800

900

1000

IL-2

sfu

/mln

sp

len

oc

yte

s

IN_a

IN_in

IN_in_e3

vector

0

100

200

300

400

500

600

IFN

g/I

L-2

sfu

/mln

sp

len

oc

yte

s

IN_a

IN_in

IN_in_e3

vector

Intracellular cytokine staining (ICCS)

0,00

0,05

0,10

0,15

0,20

0,25

0,30

IN_a IN_in IN_in_e3 vector

Pool_MIN

% C

D4+

cel

ls s

ynth

esiz

ing

cyt

oki

nes

in

res

po

nse

to

M

IN p

oo

l

IFNg

IL2

IL4

TNFa

0,00

0,50

1,00

1,50

2,00

2,50

3,00

IN_a IN_in IN_in_e3 vector

Pool_MIN

% C

D8+

cel

ls

syn

thes

izin

g c

yto

kin

es i

n r

esp

on

se t

o

MIN

po

ol

IFNg

IL2

IL4

TNFa

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

IN_a IN_in IN_in_e3 vector

Pool_MIN

% C

D4

+ c

ell

s

sy

nth

es

izin

g c

yto

kin

es

in r

es

po

ns

e

to P

oo

l_M

IN

IFNg+ IL2+ IL4+

IFNg+ IL2+ TNF+

IFNg+ IL4+ TNF+

IL2+ IL4+ TNF+

IFNg+ IL2+ IL4+ TNF+

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

IN_a IN_in IN_in_e3 vector

Pool_MIN

% C

D8

+ c

ell

s

sy

nth

es

izin

g c

yto

kin

es

in r

es

po

ns

e

to P

oo

l_M

IN

IFNg+ IL2+ IL4+

IFNg+ IL2+ TNF+

IFNg+ IL4+ TNF+

IL2+ IL4+ TNF+

IFNg+ IL2+ IL4+ TNF+

• CD4+

• CD8+

• IFNγ

• IL2

• IL4

• TNFα

CONCLUSIONS

• Consensus INs whose genes were optimized for eukaryotic expression are expressed at high levels both in eukaryotic and prokaryotic cell lines;

• Active consensus integrase is even more active (2-fold) than HXB2 integrase of clade B, while mutation of inactivation totally inhibits integrase activities;

• Inactivation mutation was shown to enhance protein half-life 3-fold;

• Active consensus integrases degrade via proteasomal pathway, while inactive forms do not;

• All IN genes are immunogenic and revealed IFN-gamma/IL2/TNFa profile of cytokine expression according to in vitro T-cell stimulation tests (Fluorospot and ICCS).

• Inactive variants can be used as components of the multi-gene vaccine against HIV-1;• The consensus gene approach can be applied to other variable viruses, as HCV.

RESUME

ACKNOWLEDGEMENTS

Engelhardt Institute of Molecular Biology, Moscow (Russia)Starodubova ElizavetaKarpov Vadim

MTC, Karolinska Institutet, Stockholm (Sweden)Petkov StefanViklund AleciaKostic LindaHallengärd DavidIsaguliants Maria

Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow (Russia)Agapkina JuliaGottikh Marina

Ivanovsky Institute of Virology, Moscow (Russia)Latyshev Oleg

Academic Medical Center of the University of Amsterdam, Department of Medical Microbiology, Amsterdam (Netherlands)Lukashov Vladimir

1,00E+04

1,00E+05

1,00E+06

1,00E+07

4 9 15 21

Day post injection

Bio

lum

ine

sc

en

ce

in

ten

sit

y (

p/s

ec

/cm

^2

/sr)

pVAX1

IN_a

IN_in

IN_in_e3

In vivo assessment of immune response: IVIS