fredrik holmström
DESCRIPTION
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 - PowerPoint PPT PresentationTRANSCRIPT
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
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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.
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Vaccine
Full length NS5A gene genotype 1b pVAX1 vector backbone
Codon optimized
Truncated plasmids pcDNA3.1
Tg mice NS5A
NS5A-EL-4 cells
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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)
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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
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Amino acids covering 327-449 of NS5A are required for priming strong NS5A IgG titres
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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
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NS5A-DNA immunization primes strong IFNγ-production in C57BL/6J mice
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pro
duci
ng S
FC/1
06
IFN
γ
Antigen and concentration
IL-2
***
Wildtype (non-immunized)Wildtype NS5A-Tg
Wildtype (non-immunized)Wildtype NS5A-Tg
***
*** **
Vacc Sac2 weeks
ELISpot51Cr release
NS5A-DNA immunization primes CD8+ T cells with lytic activity
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E:T ratio
% N
S5A
spe
cific
lysi
s
NS5
A C
TL 1
NS5
A C
TL 2
p=NS
Wildtype (non-immunized)Wildtype NS5A-Tg
Wildtype (non-immunized)Wildtype NS5A-Tg
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
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NS5A-based vaccination protects mice against tumor growth
13
coNS5A vaccinated wtNS5A vaccinated
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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
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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
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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
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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
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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
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Polyfunctional NS5A-specific immune responses after immunization
19
IFN
γ pr
oduc
ing
SFC
/106
Antigen and concentration
***
*** ***
NS
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Conclusion
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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
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Funding Swedish Research Council Swedish Society of Medicine Åke Wiberg Foundation Royal Swedish Academy of
Sciences Juhlin Foundation Karolinska Institutet
ChronTech Pharma AB