1. 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

2. 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………………...

3. 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

4. Semliki Forest virus Alphaviridae Arthropod-borne virus ss+RNA ~11.5 kb 3’ Replicase genes nsP1234Structural genes 5’ 3’ Negative strand SG 3’ Replicase genes nsP1234Structural genes 5’ Structural genes 3’ 5’ Structural genes 3’ 5’ Structural genes 3’ 5’ Structural genes 3’ 5’ Structural genes 3’ 5’ nsP2 nsP3 nsP1 nsP4 Early replicase nsP2 nsP3 nsP1 nsP4 Late replicase Capsid E3 E2 E1 6K Structural proteins Packaging of genomic RNA Budding of virus

5. SFV replicon particle construction Helper system 3’ Replicase genes nsP1234 Structural genes 5’ Gene of interest 5’ Marker Replicase genes nsP1234 SFV replicon RNA Structural genes 5’ 3’ Helper RNA

6. Apoptosis SFV replicon particle – infection of target cells

7. 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 Before infection24 h p. i. 3’ Structural genes 5’ Marker Replicase genes nsP1234

8. 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 5’ HCV sequence Renilla luciferase Replicase genes nsP1234

9. Modifications of nucleic bases

10. Oligonucleotides Anti-NS5B G-rich Number of replacements: 1, 5, 10, 13 Anti-NS4B C-rich Number of replacements: 1, 5, 10, 12

11. Experiment design Transfection of oligos into Huh7 cells by lipofection 24 h Infection with SFV-HCV viral replicon particles 12 h Renilla luciferasae assay

12. Results anti-NS4B oligos (C-rich, replacements with 5-hydroxycytosine) Oligo 6 – no modifications Oligo 7 – 1 replacement Oligo 8 – 5 replacements Oligo 9 – 10 replacements Oligo 10 – 12 replacements

13. Replicase genes nsP1234 5’ HCV sequence Renilla luciferase Replicase genes nsP1234

14. Renilla luciferase Replicase genes nsP1234 Renilla luciferase Replicase genes nsP1234

15. 5’ HCV sequence Renilla luciferase Replicase genes nsP1234 Renilla luciferase Replicase genes nsP1234

16. 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

17. 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

18. 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 5’ HCV sequence Renilla luciferase Replicase genes nsP1234

19. Mechanism of immune response?

20. 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

21. 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 10 2 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

22. Use of SFV vector against melanoma Data provided by N. Jaanson, MD, MSc, Tartu University Institute of Technology, 2010

23. Conclusion 2 Use of hybrid SFV VRP is beneficial for vaccination/challenging against pathogens or conditions that are controlled by cell-mediated immunity

24. Thank you for your attention!

25. RLU

27. Immunization approach – SFV-ZnT8 VRP 3’ ZnT8 Capsid5’3’ Envelope 5’ 3’ BHK21 cells 3×10 6 iui/p 2×10 7 iu i/p 2×10 7 iu i/p 1×10 8 iu i/p 1 month2 months4 days Replicase genes nsP1234 5’

28. „Case study“ - ZnT8 How to obtain ZnT8-specific antibodies?

29. Screening of hybridomas - ELISA Expected antibodies anti – SFV structural proteins anti – SFV non-structural proteins anti – ZnT8

30. Preliminary results – mouse 1 Total hybridomas ~1000 anti-SFV structural proteins 100 anti-ZnT8 - 1 anti-SFV ns proteins - 5