A synthetic codon-optimized HCV non-structural 5A DNA vaccine primes polyfunctional CD8+ T cell resp...
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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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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 vaccine primes polyfunctional CD8+ T cell responses in wildtype and NS5A-transgenic mice

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 vaccine primes polyfunctional CD8+ T cell responses in wildtype and NS5A-transgenic mice

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 vaccine primes polyfunctional CD8+ T cell responses in wildtype and NS5A-transgenic mice

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

Fredrik Holmström


Vaccine
Vaccine vaccine primes polyfunctional CD8+ T cell responses in wildtype and NS5A-transgenic mice

  • Full length NS5A gene

    • genotype 1b

    • pVAX1 vector backbone

  • Codon optimized

  • Truncated plasmids

    • pcDNA3.1

  • Tg mice

    • NS5A

  • NS5A-EL-4 cells

Fredrik Holmström


Stable NS5A-transgenic mice vaccine primes polyfunctional CD8+ T cell responses in wildtype and 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 vaccine primes polyfunctional CD8+ T cell responses in wildtype and NS5A-transgenic mice

  • 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 priming strong NS5A IgG titres

  • 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

*** priming strong NS5A IgG titres

***

***

**

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 responses?

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 responses?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 responses?constructs

1

2

3

Fredrik Holmström


NS responses?

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


NS responses?

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? 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

*** responses?

***

Polyfunctional NS5A-specific immune responses after immunization

IFNγ producing SFC/106

Antigen and concentration

***

Fredrik Holmström

NS


Conclusion
Conclusion responses?

  • 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 responses?

  • 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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