Transcript Hybrid SFV-HCV replicon system

Hybrid SFV VRP system
Eva Žusinaite, MD, PhD
Tartu University Institute of Technology
Estonia
VIII Annual Conference of New Visby Network on Hepatitis C
Vilnius, February 14-17, 2011
SFV expression vectors – basic studies
• Alphavirus replication
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Formation and functioning of replication complex
Viral proteins’ interactions
Interactions with host cell proteins
Host cell biosynthesis shut-off
Interference with viral replication
Superinfection exclusion……………..
• Alfavirus infection in hosts and vectors
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Viral entry
Pathogenesis of infection
Mechanisms of defence
Transmission of infection
Mechanisms of virulence………………...
SFV replicon system - applications
In vitro screening of
inhibitors of viral
replication:
- small chemical
compounds
- antisense
oligonucleotides
- RNA interfering
coumpound
- Plant derivatives
- ………….
Viral replicon particles
(VRP) as genetic
delivery system:
- Vaccine candidates
- Challenging vectors
- Gene therapy vectors
Tools for biotechnology:
- production of antibodies
- recombinant protein
production
Semliki Forest virus
• Alphaviridae
• Arthropod-borne virus
• ss+RNA ~11.5 kb
5’
Replicase genes nsP1234
Structural genes
3’
SG
3’
5’
nsP1 nsP2
nsP3 nsP4
Early replicase
nsP2
nsP3
nsP1
nsP4
Late replicase
Negative strand
5’
Replicase genes nsP1234
Structural genes
3’
5’
5’
Structural genes
3’
Structural genes
3’
Structural genes
5’
3’
Structural genes
5’
3’
Structural genes
5’
3’
Packaging of genomic RNA
Budding of virus
E3
Capsid
E2
6K
E1
Structural
proteins
SFV replicon particle construction
Helper system
SFV replicon RNA
5’
Gene
Structural
of interest
genes
Replicase
genes nsP1234
Marker
Replicase genes
nsP1234
5’
Helper RNA
5’
Structural genes
3’
3’
SFV replicon particle – infection of target cells
Apoptosis
SFV is cytotoxic!
• wt – translational and transcriptional shut off
• Cell death within 24-48 h post infection
• Cytotoxicity reducing mutations in the nsP2 region
– RRR  RDR – blocks nuclear localization signal
3638-3646: CGA CGC AGG  CGA GAC AGG
– PG – reduces cytotoxicity
3848-3850: CCC  GGA
5’
Replicase genes nsP1234
Marker
3’
Structural genes
Hybrid SFV-HCV system
• SFV replicase
• Marker – Renilla luciferase gene inserted in the SFV nsP3
• Antisense oligonucleotide target – parts of HCV replicase
under SFV subgenomic promoter:
– NS3-NS4A-NS4B
– NS5A-NS5B
• Cytotoxicity reducing mutation (PG) in the nsP2 region
Replicase
genes nsP1234
Renilla
Replicase genes
nsP1234
5’
luciferase
HCV sequence
Modifications of nucleic bases
Oligonucleotides
Anti-NS5B
G-rich
Number of
replacements:
1, 5, 10, 13
Anti-NS4B
C-rich
Number of
replacements:
1, 5, 10, 12
Experiment design
Transfection of
oligos into Huh7
cells by
lipofection
24 h
Infection with
SFV-HCV
viral replicon
particles
Renilla
luciferasae
assay
Oligo 6 – no modifications
Oligo 7 – 1 replacement
Oligo 8 – 5 replacements
Oligo 9 – 10 replacements
Oligo 10 – 12 replacements
Results
anti-NS4B oligos (C-rich,
replacements with 5-hydroxycytosine)
SFV-NS3/4B infection 24 h post transfection
140000
120000
80000
60000
40000
20000
lig
o1
0
O
lig
o9
O
lig
o8
O
lig
o7
O
lig
o6
O
nt
ro
co
A
N
siR
Sc
r
am
bl
ed
l
0
Ce
lls
RLU
100000
Replicase
genes nsP1234
Renilla
Replicase genes
nsP1234
5’
luciferase
HCV sequence
SFV markers expression profile
300000000
SFV-stRluc
SFV-nsRluc
mock
250000000
RLU
200000000
150000000
100000000
50000000
0
0
6
12
18
24
hours post infection
Renilla
luciferase
Replicase genes nsP1234
Replicase genes nsP1234
Renilla
luciferase
30
36
Replicase
genes nsP1234
Renilla
Replicase genes
nsP1234
5’
luciferase
Replicase genes nsP1234
Renilla
luciferase
HCV sequence
Conclusion 1
SFV VRP use in vitro
• Easy to manipulate at the cDNA level
• Easy to produce and concentrate VRPs
• Suitable for most mammalian cell types, including
“difficult-to-transfect” and non-dividing cells
• Highly reproducible results
• Enables testing/screening of inhibitory compounds at
the BSL2 conditions
• Perfect solution in the absence of in vitro virus model
Hybrid SFV replicon system
• In vitro screening of
inhibitors of viral
replication:
Viral replicon particles
(VRP) as genetic delivery
system:
• Small chemical
compounds
- Challenging vectors
- Vaccine candidates
- Gene therapy vectors
• Antisense oligonucleotides
• RNA interfering
coumpound
• Plant derivatives
• ………….
Tools for biotechnology:
- production of antibodies
- recombinant protein
production
Data from literature...
•
Induction of genome-encoded antigen-specific humoral
response upon SFV particle immunization appears to be
highly variable – from apparent absence to very strong
neutralizing response
•
Effect probably depends on a set of factors: antigen,
administration route, immunization regimens, etc...
•
Usually detected as a by-products of immunization
Replicase
genes nsP1234
Renilla
Replicase genes
nsP1234
5’
luciferase
HCV sequence
Mechanism of immune response?
Hybrid SFV replicon system
• In vitro screening of
inhibitors of viral
replication:
Viral replicon particles
(VRP) as genetic delivery
system:
• Small chemical
compounds
- Vaccine candidates
• Antisense oligonucleotides
- Gene therapy vectors
• RNA interfering
coumpound
• Plant derivatives
• ………….
- Challenging vectors
Tools for biotechnology:
- production of antibodies
- recombinant protein
production
Data from literature…
• Immunization with SFV VRP induces CTL-memory
that persists for a long time
• CTL responses can be induced upon administration of
as little as 102 iu of SFV VRP
• Mechanism of CTL response by SFV VRP is crosspriming
• The best administration routes for induction of strong
CTL response are i/v, i/p, and s/c
• Predominant T-helper response is Th1
Use of SFV vector against melanoma
Data provided by N. Jaanson, MD, MSc, Tartu University Institute of Technology, 2010
Conclusion 2
Use of hybrid SFV VRP is beneficial for
vaccination/challenging against pathogens or conditions
that are controlled by cell-mediated immunity
Thank you for your attention!
SFV-HCV VRP expression profile in Huh7 cells
RLU
100000000
10000000
1000000
100000
10000
wt-5AB
1000
wt-34B
RDR-5AB
100
RDR-34B
PG-5AB
10
PG-534B
1
2
6
12
18
24
hours post infection
48
72
SFV-HCV VRP expression profile in Huh7 cells
RLU
70000000
60000000
50000000
40000000
30000000
wt-5AB
wt-34B
20000000
RDR-5AB
RDR-34B
10000000
PG-5AB
PG-534B
0
2
6
12
18
hours post infection
24
48
72
Immunization approach – SFV-ZnT8 VRP
5’
Replicase genes nsP1234
3’
ZnT8
5’
3’
Capsid
5’
Envelope
3’
BHK21 cells
3×106 iu i/p
1 month
2×107 iu i/p
2 months
2×107 iu i/p
4 days
1×108 iu i/p
4 days
„Case study“ - ZnT8
How to obtain ZnT8-specific
antibodies?
Screening of hybridomas - ELISA
Expected antibodies
anti – SFV structural proteins
anti – SFV non-structural proteins
anti – ZnT8
Preliminary results – mouse 1
Total
hybridomas
~1000
anti-SFV
structural
proteins
100
anti-SFV ns proteins - 5
anti-ZnT8 - 1