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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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slide1

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
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

three domains of ns5a
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

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

Fredrik Holmström

vaccine
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

slide6

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

characterization of humoral immune responses
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

amino acids covering 327 449 of ns5a are required for priming strong ns5a igg titres
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

identification of ns5a ctl epitopes
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

ns5a dna immunization primes strong ifn production in c57bl 6j mice

***

***

***

**

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

ns5a dna immunization primes cd8 t cells with lytic activity
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

does ns5a dna immunization prime tumor inhibiting immune responses
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

ns5a based vaccination protects mice against tumor growth
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

is the full length ns5a needed to mediate protection against tumor growth
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

overview of ns5a dna vaccine constructs
Overview of NS5A-DNA vaccine constructs

1

2

3

Fredrik Holmström

slide16

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

slide17

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

does ns5a prime polyfunctional t cell responses
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

polyfunctional ns5a specific immune responses after immunization

***

***

Polyfunctional NS5A-specific immune responses after immunization

IFNγ producing SFC/106

Antigen and concentration

***

Fredrik Holmström

NS

conclusion
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

acknowledgement
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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