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Fredrik Holmström

A synthetic codon-optimized HCV non-structural 5A DNA vaccine primes polyfunctional CD8+ T cell responses in wildtype and NS5A-transgenic mice. Fredrik Holmström. Karolinska Institutet Department of Laboratory Medicine Division of Clinical Microbiology. Background. NS5A

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Fredrik Holmström

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  1. A synthetic codon-optimized HCV non-structural 5A DNA vaccine primes polyfunctional CD8+ T cell responses in wildtype and NS5A-transgenic mice Fredrik Holmström Karolinska Institutet Department of Laboratory Medicine Division of Clinical Microbiology

  2. Background NS5A • Function/s not fully understood • Important for replication and assembly • Interfere with a variety of cellular proteins • Interact with both the innate & adaptive immune system • NS5A target for CTLs during resolution and acute HCV infections • Urbani et al, 2001, Hepatology Fredrik Holmström

  3. Three domains of NS5A Domain III • Vital for virus assembly • RNA binding domain Domain II • Interfere with • PKR, CypA, NS5B • RNA binding domain Fredrik Holmström Domain I Important for RNA replication Membrane anchoring domain

  4. Over all aim • To develop and evaluate HCV NS5A genotype 1b DNA plasmids as vaccine candidates for chronic HCV infections. Fredrik Holmström

  5. Vaccine • Full length NS5A gene • genotype 1b • pVAX1 vector backbone • Codon optimized • Truncated plasmids • pcDNA3.1 • Tg mice • NS5A • NS5A-EL-4 cells Fredrik Holmström

  6. Stable NS5A-transgenic mice • Liver specific expression of HCV NS5A gt 1b (e.g. albumin promoter) • No spontaneous liver disease in transgenic mice • C57BL/6J • FVB/N Tg wt Tg wt M 50 kDa 40 kDa C57BL/6J FVB/N • NS5A-Tg mice represents a host with tolerant or dysfunctional immune response e.g. mimics a chronic HCV individual • Evaluate vaccines in wt and NS5A-Tg mice • Localization of NS5A in transgenic mouse livers α-NS5A Wild-type NS5A-Tg Kriegs et al., JBC 2009 Fredrik Holmström 6

  7. Characterization of humoral immune responses • Aim: Characterization of humoral immune responses after DNA or protein immunization with full-length and truncated NS5A expression constructs. • Strategy: • Immunizations (monthly) • rNS5A (s.c.) • coNS5A (i.m. + EP) • wtNS5A (i.m. + EP) • wtNS5A truncated constructs (i.m. + EP) • Detection with ELISA Fredrik Holmström

  8. Amino acids covering 327-449 of NS5A are required for priming strong NS5A IgG titres A) B) C) D) E) F) NS5A IgG titre G) H) I) Time after immunization (weeks) Fredrik Holmström Fig. 1

  9. Identification of NS5A CTL epitopes • Aim: To identify NS5A CTL epitopes • Strategy: • 87 synthetic 20-peptides (15aa overlap) • Epitope predicted peptides (SYFPEITHI) • Result: Two NS5A CTL epitopes identified (named 1 & 2) • Aim: Is it possible to activate IFNγ-, IL-2 production and NS5A-specific lysis in wildtype and NS5A-Tg mice? • Strategy: • Immunizations of 50μg im EP coNS5A-pVAX1 • Mice sacrificed after 2 weeks • ELISpot & 51Cr release assay Fredrik Holmström

  10. *** *** *** ** NS5A-DNA immunization primes strong IFNγ-production in C57BL/6J mice Wildtype NS5A-Tg Wildtype (non-immunized) IFNγ producing SFC/106 Wildtype NS5A-Tg Wildtype (non-immunized) IL-2 Antigen and concentration ELISpot Vacc Sac 51Cr release 2 weeks Fredrik Holmström

  11. NS5A-DNA immunization primes CD8+ T cells with lytic activity Wildtype (non-immunized) Wildtype NS5A-Tg ELISpot p=NS Vacc Sac NS5A CTL 1 51Cr release 2 weeks % NS5A specific lysis Fredrik Holmström Wildtype NS5A-Tg Wildtype (non-immunized) p=NS NS5A CTL 2 E:T ratio

  12. Does NS5A-DNA immunization prime tumor-inhibiting immune responses? • Aim: Is it possible to activate T cells that can localize & kill the tumor cells? • Strategy: • Immunizations 50μg im EP coNS5A-pVAX1, wtNS5A-pcDNA3.1 • Inoculation of NS5A-EL-4 and EL-4 cells • Measurement of tumor growth Fredrik Holmström

  13. NS5A-based vaccination protects mice against tumor growth coNS5A vaccinated wtNS5A vaccinated Measure tumor volume Sac Vacc Inoculation 1x106 cells 50μg DNA 2 weeks 2 weeks Fredrik Holmström

  14. Is the full length NS5A needed to mediate protection against tumor growth? • Aim: Which regions of NS5A are important to mediate protection against tumor growth? • Strategy: • Immunizations 50μg im EP using NS5A truncated plasmids • Inoculation of NS5A-EL-4 • Measurement of tumor growth • Mice sacrificed after 2 weeks (spleen) • ELISpot Fredrik Holmström

  15. Overview of NS5A-DNA vaccine constructs 1 2 3        Fredrik Holmström

  16. NS NS NS *** *** *** *** *** *** *** *** *** NS NS NS NS ** * p<0,05 p=NS 1 2 p=NS p<0,01 3 4 IFNγ producing SFC/106 Tumor volume (mm3) p<0,01 p<0,01 5 6 p<0,01 p<0,01 7 8 p<0,05 9 Antigen and concentration Days post tumor inoculation Fredrik Holmström Fig. 5

  17. NS NS NS *** *** *** *** *** *** *** *** *** NS NS NS NS ** * p<0,05 p=NS 1 2 p=NS p<0,01 3 4 IFNγ producing SFC/106 Tumor volume (mm3) p<0,01 p<0,01 5 6 p<0,01 p<0,01 7 8 p<0,05 9 Antigen and concentration Days post tumor inoculation Fredrik Holmström Fig. 5

  18. Does NS5A prime polyfunctional T cell responses? • Aim: To characterize the cytokines secreated after NS5A-DNA immunization • Strategy: • Immunizations 50μg im EP coNS5A-pVAX1 • Mice sacrificed after 2 weeks (spleen) • ELISpot, Pentamer, ICS Fredrik Holmström

  19. *** *** Polyfunctional NS5A-specific immune responses after immunization IFNγ producing SFC/106 Antigen and concentration *** Fredrik Holmström NS

  20. Conclusion • NS5A-based DNA immunization activates: • Lytic CTLs • IFNγ- and IL-2 production • Expansion of T cell frequencies • Polyfunctional T cells • Tumor protective immune responses • Thus, the NS5A-based DNA vaccines activates a preferred type of T cell immunity • The coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections Fredrik Holmström

  21. Acknowledgement • Funding • Swedish Research Council • Swedish Society of Medicine • Åke Wiberg Foundation • Royal Swedish Academy of Sciences • Juhlin Foundation • Karolinska Institutet • ChronTech Pharma AB • Karolinska Institutet, Sweden • Lars Frelin • Gustaf Ahlen • Matti Sällberg • Anna Pasetto • Sepideh Alahyari • Paul Ehrlich Institut, Langen, Germany • Eberhard Hildt • University Medical Center Hamburg-Eppendorf, Hamburg, Germany • Malte Kriegs Fredrik Holmström

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