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Hybrid SFV VRP 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 1 4 -17, 2011. SFV expression vectors – basic studies. Alphavirus replication

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Hybrid SFV VRP system

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  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 • Viral replicon particles (VRP) as genetic delivery system: • Vaccine candidates • Challenging vectors • Gene therapy vectors In vitro screening of inhibitors of viral replication: • small chemical compounds • antisense oligonucleotides • RNA interfering coumpound • Plant derivatives • …………. • Tools for biotechnology: • production of antibodies • recombinant protein production

  4. Replicase genes nsP1234 Replicase genes nsP1234 Structural genes Structural genes 3’ 3’ 5’ 5’ nsP2 nsP1 Early replicase nsP3 SG nsP4 Negative strand 3’ 5’ nsP2 nsP3 Late replicase nsP1 nsP4 5’ 5’ 5’ 5’ 5’ Structural genes Structural genes Structural genes Structural genes Structural genes 3’ 3’ 3’ 3’ 3’ 6K E3 Capsid E1 Structural proteins E2 Packaging of genomic RNA Budding of virus Semliki Forest virus • Alphaviridae • Arthropod-borne virus • ss+RNA ~11.5 kb

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

  6. Apoptosis SFV replicon particle – infection of target cells

  7. Before infection 24 h p. i. 5’ Replicase genes nsP1234 Marker 3’ Structural genes 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

  8. Replicase genes nsP1234 Renilla luciferase 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

  9. Modifications of nucleic bases

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

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

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

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

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

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

  16. 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

  17. Hybrid SFV replicon system • Viral replicon particles (VRP) as genetic delivery system: • Vaccine candidates • Challenging vectors • Gene therapy vectors • In vitro screening of inhibitors of viral replication: • Small chemical compounds • Antisense oligonucleotides • RNA interfering coumpound • Plant derivatives • …………. • Tools for biotechnology: • production of antibodies • recombinant protein production

  18. Replicase genes nsP1234 Renilla luciferase 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

  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 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

  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

  26. RLU

  27. 3’ ZnT8 BHK21 cells 5’ 3’ 5’ Envelope Capsid 3’ 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 Immunization approach – SFV-ZnT8 VRP 5’ Replicase genes nsP1234

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

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