Hybrid SFV VRP system

Eva Žusinaite, MD, PhD

Tartu University Institute of Technology

Estonia

VIII Annual Conference of New Visby Network on Hepatitis CVilnius, February 14-17, 2011

SFV expression vectors – basic studies

• Alphavirus replication– 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– 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

3’ Replicase genes nsP1234 Structural genes5’

5’ 3’ Negative strand

SG

3’ Replicase genes nsP1234 Structural genes5’

Structural genes 3’ 5’ Structural genes 3’ 5’

Structural genes 3’ 5’ Structural genes 3’ 5’

Structural genes 3’ 5’

nsP2

nsP3nsP1

nsP4Early replicase

nsP2

nsP3nsP1nsP4

Late replicase

CapsidE3

E2E1

6K

Structural proteins

Packaging of genomic RNA

Budding of virus

SFV replicon particle constructionHelper system

3’ Replicase genes nsP1234 Structural genes5’ Gene of interest5’ MarkerReplicase genes nsP1234

SFV replicon RNA

Structural genes5’ 3’ Helper RNA

Apoptosis

SFV replicon particle – infection of target cells

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

Bef

ore

infe

ctio

n24

h p

. i.

3’ Structural genes5’ MarkerReplicase genes nsP1234

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 nsP12345’ HCV sequenceRenillaluciferase

Replicase genes nsP1234

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 ofoligos into Huh7

cells bylipofection

24 hInfection with

SFV-HCVviral replicon

particles

12 h

Renilla

luciferasae

assay

Resultsanti-NS4B oligos (C-rich,

replacements with 5-hydroxycytosine)

Oligo 6 – no modificationsOligo 7 – 1 replacementOligo 8 – 5 replacementsOligo 9 – 10 replacementsOligo 10 – 12 replacements

SFV-NS3/4B infection 24 h post transfection

0

20000

40000

60000

80000

100000

120000

140000

Cells

Scram

bled

siRNA co

ntrol

Oligo6

Oligo7

Oligo8

Oligo9

Oligo1

0

RL

U

Replicase genes nsP12345’ HCV sequenceRenillaluciferase

Replicase genes nsP1234

Renilla

luciferaseReplicase genes nsP1234

Renillaluciferase

Replicase genes nsP1234

SFV markers expression profile

0

50000000

100000000

150000000

200000000

250000000

300000000

0 6 12 18 24 30 36

hours post infection

RL

U

SFV-stRluc

SFV-nsRluc

mock

Replicase genes nsP12345’ HCV sequenceRenillaluciferase

Replicase genes nsP1234

Renilla

luciferaseReplicase genes nsP1234

Conclusion 1SFV 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:

• 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

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 nsP12345’ HCV sequenceRenillaluciferase

Replicase genes nsP1234

Mechanism of immune response?

Hybrid SFV replicon system

• 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

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 cross-

priming

• 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

1

10

100

1000

10000

100000

1000000

10000000

100000000

2 6 12 18 24 48 72hours post infection

wt-5AB

wt-34B

RDR-5AB

RDR-34B

PG-5AB

PG-534B

RLU

SFV-HCV VRP expression profile in Huh7 cells

0

10000000

20000000

30000000

40000000

50000000

60000000

70000000

2 6 12 18 24 48 72

hours post infection

wt-5AB

wt-34B

RDR-5AB

RDR-34B

PG-5AB

PG-534B

RLU

Immunization approach – SFV-ZnT8 VRP

3’ ZnT8

Capsid5’ 3’

Envelope5’ 3’

BHK21 cells

3×106 iu i/p 2×107 iu i/p 2×107 iu i/p 1×108 iu i/p

1 month 2 months 4 days 4 days

Replicase genes nsP12345’

„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

Totalhybridomas ~1000

anti-SFVstructuralproteins

100

anti-ZnT8 - 1

anti-SFV ns proteins - 5