dna-immunogen based on a consensus integrase of hiv-1 subtype a
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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
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