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 InstitutetDepartment of Laboratory MedicineDivision of Clinical Microbiology

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

April 24, 2023Fredrik Holmström 2


Three domains of NS5A

Domain I Important for

RNA replication Membrane anchoring

domain

Domain III Vital for virus

assembly RNA binding domain

Domain II Interfere with

PKR, CypA, NS5B RNA binding domain

Over all aim

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


Vaccine

Full length NS5A gene genotype 1b pVAX1 vector backbone

Codon optimized

Truncated plasmids pcDNA3.1

Tg mice NS5A

NS5A-EL-4 cells


Construct Amino acid sequence1 1-449 (full lenght)

2 1-163

3 1-215

4 1-326

5 1-378

6 31-449

7 105-449

8 211-449

9 302-449

pcDNA3 (vector)


Liver specific expression of HCV NS5A gt 1b (e.g. albumin promoter)

No spontaneous liver disease in transgenic mice C57BL/6J FVB/N

Stable NS5A-transgenic mice

6

Localization of NS5A in transgenic mouse livers

NS5A-TgWild-type

α-NS5A

Kriegs et al., JBC 2009

50 kDa

40 kDa

C57BL/6J FVB/N

Tg Tgwt wtM

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

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


Amino acids covering 327-449 of NS5A are required for priming strong NS5A IgG titres

8Fredrik Holmström April 24, 2023

NS5

A Ig

G ti

tre

Time after immunization (weeks)

F)E)D)

B) C)A)

I)H)G)

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


NS5A-DNA immunization primes strong IFNγ-production in C57BL/6J mice


pro

duci

ng S

FC/1

06

IFN

γ

Antigen and concentration

IL-2

***

Wildtype (non-immunized)Wildtype NS5A-Tg


***

*** **

Vacc Sac2 weeks

ELISpot51Cr release

NS5A-DNA immunization primes CD8+ T cells with lytic activity


E:T ratio

% N

S5A

spe

cific

lysi

s

NS5

A C

TL 1

NS5

A C

TL 2

p=NS



p=NS

Vacc Sac2 weeks

ELISpot51Cr release

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


NS5A-based vaccination protects mice against tumor growth

13

coNS5A vaccinated wtNS5A vaccinated

Fredrik Holmström April 24, 2023

Vacc Sac2 weeks

Inoculation2 weeks1x106 cells50μg DNA

Measure tumor volume

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

Construct Amino acid in NS5A

NS5A domain

1 (wtNS5A) 1-449 (full length)

2 1-163

3 1-215

4 1-326

5 1-378

6 31-449

7 105-449

8 211-449

9 302-449

coNS5A 1-449 (full length) codon optimized

1 2 3

Overview of NS5A-DNA vaccine constructs

April 24, 2023

16Fredrik Holmström

NSNS

NS

IFN

γ pr

oduc

ing

SFC

/106

Tum

or v

olum

e (m

m3 )

Days post tumor inoculation Antigen and concentration

p<0,05

p<0,01

p<0,01

p=NS

p<0,01

p<0,01

p=NS

p<0,05

p<0,01

************

NSNS

******

******

******

NSNS

1 2

3 4

5

7

9

6

8

April 24, 2023

17Fredrik Holmström

NSNS

NS

IFN

γ pr

oduc

ing

SFC

/106

Tum

or v

olum

e (m

m3 )

Days post tumor inoculation Antigen and concentration

p<0,05

p<0,01

p<0,01

p=NS

p<0,01

p<0,01

p=NS

p<0,05

p<0,01

************

NSNS

******

******

******

NSNS

1 2

3 4

5

7

9

6

8

April 24, 2023

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


Polyfunctional NS5A-specific immune responses after immunization

19

IFN

γ pr

oduc

ing

SFC

/106

Antigen and concentration

***

*** ***

NS

April 24, 2023Fredrik Holmström

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

Acknowledgement

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


Funding Swedish Research Council Swedish Society of Medicine Åke Wiberg Foundation Royal Swedish Academy of

Sciences Juhlin Foundation Karolinska Institutet

ChronTech Pharma AB

Date post:	19-Mar-2016
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Fredrik Holmström

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