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A Synthetic Codon-Optimized Hepatitis C
Virus Nonstructural 5A DNA Vaccine Primes
Polyfunctional CD8 + T Cell Responses in
Wild-Type and NS5A-Transgenic Mice
Fredrik Holmström, Anna Pasetto, Veronica Nähr, Anette
Brass, Malte Kriegs, Eberhard Hildt, Kate E. Broderick,
Margaret Chen, Gustaf Ahlén and Lars Frelin
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Copyright © 2013 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2013; 190:1113-1124; Prepublished online 2
January 2013;
doi: 10.4049/jimmunol.1201497
http://www.jimmunol.org/content/190/3/1113
The Journal of Immunology
A Synthetic Codon-Optimized Hepatitis C Virus
Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+
T Cell Responses in Wild-Type and NS5A-Transgenic Mice
Fredrik Holmström,* Anna Pasetto,*,† Veronica Nähr,* Anette Brass,* Malte Kriegs,‡
Eberhard Hildt,x Kate E. Broderick,{ Margaret Chen,† Gustaf Ahlén,* and Lars Frelin*
C
hronic liver disease caused by the hepatitis C virus (HCV)
is associated with development of fibrosis, cirrhosis and
hepatocellular carcinoma. It is estimated that 170 million
individuals are infected by HCV worldwide (1, 2). The HCV has
a positive sense ssRNA genome of ∼9.6 kb, which encodes 10
structural and nonstructural (NS) proteins. The current standardof-care therapy for HCV genotype 1 infection is based on a triple
combination composed of pegylated IFN-a, ribavirin, and a direct-acting antiviral (DAA) compound. Numerous DAAs are in
clinical development, and thus far, two NS3 protease inhibitors
*Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska
Institutet, Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden;
†
Department of Dental Medicine, Karolinska Institutet, S-141 04 Stockholm, Sweden; ‡Laboratory of Radiobiology and Experimental Radiooncology, University
Medical Center Hamburg-Eppendorf, D-20246 Hamburg, Germany; xPaul-EhrlichInstitut, Federal Institute for Vaccines and Biomedicines, D-63225 Langen, Germany; and {Inovio Pharmaceuticals, Blue Bell, PA 19422
Received for publication May 30, 2012. Accepted for publication November 21,
2012.
The work was supported by grants from the Swedish Research Council (K2012-99X22017-01-3 to L.F. and K2009-58X-20043-04-2 to M.C.), the Swedish Society of
Medicine (to L.F.), the Swedish Cancer Society (to M.C.), the Goljes Memorial Fund
(to L.F.), the Åke Wiberg Foundation (to L.F.), the Royal Swedish Academy of
Sciences (to L.F. and G.A.), the Ruth and Richard Julin Foundation (to L.F.), the
Lars Hierta Memorial Foundation (to G.A.), the Magnus Bergwalls Foundation (to
G.A.), and the Karolinska Institutet (to L.F.)/Södertörn University (postdoctoral grant
to G.A.).
Address correspondence and reprint requests to Dr. Lars Frelin, Division of Clinical
Microbiology, Department of Laboratory Medicine, F 68, Karolinska Institutet,
Karolinska University Hospital Huddinge, S-141 86 Stockholm, Sweden. E-mail
address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: co, codon-optimized; DAA, direct-acting antiviral;
DDS, DNA delivery system; EP, electroporation; HCV, hepatitis C virus; NS, nonstructural; SFC, spot-forming cell; SVR, sustained virological response; Tg, transgenic; wt, wild-type.
Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201497
have reached the market. Data show an improved sustained virological response (SVR) in patients treated with NS3 protease
inhibitors (3, 4). For HCV nongenotype 1 infections, the therapy is
composed of pegylated IFN-a and ribavirin. Individuals that clear
an acute HCV infection spontaneously or by standard-of-care
treatment more commonly have CD4+ and CD8+ T cells to multiple HCV proteins, whereas those who progress to a chronic infection lack these responses. One explanation may be that these
chronic carriers have dysfunctional T cells (5–9). Hence, one
approach to reactivate these dysfunctional T cells is therapeutic
vaccination (10–12). Until now, no vaccine is available for HCV,
although several candidates have been evaluated in clinical phase
I/II trials (11, 13–15). By using DNA-based vaccines, one may
activate the preferred type of immunity, including both humoral
and cellular immune responses, in small animals and humans. The
HCV NS5A is of particular interest because it is recognized by
cytotoxic T lymphocytes during natural infection and resolution of
acute HCV in humans (16). Thus, we decided to evaluate HCV
NS5A as a therapeutic DNA vaccine candidate for chronic HCV
infection, because relatively few studies have investigated NS5A
as a potential vaccine (17–19). The activation of a potent antiviral
T cell response is a prerequisite for a functional therapeutic vaccine in the presence of the homologous virus. We therefore use
NS5A-Tg mice in addition to wild-type (wt) mice, because they
better represent a model of an infected host with impaired or
dysfunctional immune response to NS5A (20). In addition, NS5A
is an interesting target for DAAs, because clinical evaluation has
shown promising results with improved SVRs (21–23). Importantly, these inhibitors seem effective across genotypes (24).
The function of NS5A is not completely understood, although
it is known to be an essential component of the HCV replication
complex (25, 26). The NS5A protein has no known enzymatic
activity, and it is divided in three distinct structural domains,
domain I, II, and III (27). Domain I is essential for RNA repli-
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The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both
innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this
study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5Atransgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell
responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic
expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >104 postimmunization. With respect to CD8+
T cell responses, the coNS5A gene primed more potent IFN-g–producing and lytic cytotoxic T cells in wt mice compared with NS5ATg mice. In addition, high frequencies of NS5A-specific CD8+ T cells were found in wt mice after a single immunization. To test the
functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the
coNS5A gene primed polyfunctional NS5A-specific CD8+ T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine
candidate for chronic HCV infections. The Journal of Immunology, 2013, 190: 1113–1124.
1114
NS5A-BASED GENETIC IMMUNOGENS PRIMES CYTOTOXIC T CELLS
Materials and Methods
Animals
Inbred C57BL/6J mice were obtained from a commercial vendor (Charles
River Laboratories, Sulzfeld, Germany). NS5A-Tg C57BL/6J mice were
generated by backcrossing NS5A-Tg FVB/N mice (20) with C57BL/6J
mice for .10 generations. Transgenic (Tg) mice were bred and maintained in-house at Karolinska Institutet, Division of Comparative Medicine
(AKM), Clinical Research Centre, Karolinska University Hospital
Huddinge (Stockholm, Sweden). Female mice were 6–16 wk of age at
the start of the experiments. All experimental protocols involving
animals were approved by the Ethical Committee for Animal Research
at Karolinska Institutet.
The liver-specific expression of NS5A protein in C57BL/6J mice was
under the control of the mouse albumin enhancer and promoter, followed by
the rabbit b-globulin intron that enhances protein expression. A V5 tag and
a polyadenylation signal from SV40 were inserted downstream of the
NS5A gene.
All NS5A-Tg mice were genotyped by PCR to verify the transgene.
Extraction of DNA from the tail was performed using the DNeasy Blood
and Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer’s
instructions, and the NS5A transgene and the reference gene tubulin were
amplified by PCR.
Peptides and proteins
Peptides (87 synthesized 20-mer) corresponding to the complete NS5A
protein sequence with 15 aa overlap were synthesized by automated peptide
synthesis (37) (provided by ChronTech Pharma, Huddinge, Sweden). In
addition, the SYFPEITHI database, version 1.0 (38) (http://www.syfpeithi.
de), was used to predict H-2Db (9-mer) and H-2Kb (8-mer) epitopes. The
following NS5A MHC class I binding peptides were used: NS5A 2251–
2259 (VILDSFDPL, H-2Db) and 2252–2259 (ILDSFDPL, H-2Kb).
Recombinant NS5A (rNS5A) protein was obtained from Mikrogen
(Neuried, Germany) and GenScript (Piscataway, NJ).
Control Ags HCV NS3 (GAVQNEVTL, H-2Kb), HBcAg (MGLKFRQL,
H-2Kd), HCV NS5A (NKVVILDSF), and OVA CTL (SIINFEKL) were
provided by ChronTech Pharma. rNS3 protein was provided by Dr. D.L.
Peterson (Department of Biochemistry, Commonwealth University, Richmond, VA). PMA–ionomicyne and Con A were purchased from SigmaAldrich (St. Louis, MO).
Plasmids
A wt NS5A genotype 1b gene [GenBank accession number D16435,
http://www.ncbi.nlm.nih.gov/genbank (39)] and eight truncated wtNS5A
constructs were inserted into the pcDNA3.1 vector (Table I). A codonoptimized (co) NS5A genotype 1b gene was synthesized and inserted
into the pVAX1 vector (Retrogen, San Diego, CA). The wtNS5A and
coNS5A genes had 100% aa homology but differed by 23% in nucleotide
sequence. The GC content was reduced from 60 to 58%.
The plasmid DNAs used for in vivo immunization were grown in
competent Escherichia coli TOP10 cells and purified by using the Plasmid
DNA Purification and QIAfilter Plasmid Mega Kit (Qiagen), according to
the manufacturer’s instructions. The DNA was diluted in sterile PBS to 1
mg/ml, and the plasmid DNA concentration was determined spectrophotometrically (Biophotometer; Eppendorf, Hamburg, Germany). Restriction
enzyme digests were performed to ensure that the plasmid contained the
gene of interest with the correct size. All DNA plasmids were sequenced to
ensure correct nucleotide sequence (Eurofins MWG Operon, Ebersberg,
Germany).
Cell lines and primary cultures
The RMA-S (TAP deficient) and EL-4 (T cell lymphoma) cell lines were
cultured in RPMI 1640 medium supplemented with 5% FBS, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen,
Carlsbad, CA).
Primary cell cultures from spleen were cultured in complete CTL medium (RPMI 1640 medium supplemented with 10% FBS, 1 mM sodium
pyruvate, 10 mM HEPES buffer, 1 mM nonessential amino acids, 50 mM 2ME, 100 U/ml penicillin, and 100 mg/ml streptomycin [Invitrogen]). All
cells were incubated in humidified incubators at 37˚C with 5% CO2.
Stable transfection of EL-4 cells
The EL-4 suspension cells were stably transfected using Turbofect (Fermentas, Leon-Rot, Germany), according to manufacturer’s instructions. In
brief, 5 3 105 cells/well were seeded in a 24-well plate in 1 ml growth
medium 24 h prior to transfection. Linearized DNA (1 mg) was diluted in
100 ml serum-free medium, mixed with 2 ml TurboFect, and incubated at
room temperature for 20 min. The TurboFect/DNA mixture was then
added dropwise to the cells without removing the growth medium. The
cells were incubated for 48 h at 37˚C and 5% CO2. After transfection,
the EL-4 cells containing wtNS5A-pcDNA3.1 (aa 1–449) were grown in
the presence of 800 mg geneticin (G418)/ml (Invitrogen) for 48 h and then
single cell diluted for selection of single clones. This procedure was repeated twice. Expression of NS5A protein from the stably transfected EL-4
cells was analyzed by reverse transcription and PCR and Western blot.
RNA extraction and RT-PCR
Detection of NS5A expression in stably transfected NS5A-EL-4 cells was
performed by an RT-PCR to visualize NS5A mRNA. Total RNA was
isolated using the TRIzol reagent, according to manufacturer’s instructions.
In brief, stably transfected NS5A-EL-4 cells were pelleted (5 3 106) and
lysed using TRIzol reagent (Invitrogen). Phase separation was performed
with chloroform and RNA precipitation with 2-propanol. RNA was washed
and redissolved in 40 ml nuclease-free dH2O containing 1 ml RNasin (40
U/ml) (Promega, Madison, WI). The concentration of RNA was measured
spectrophotometrically (Biophotometer; Eppendorf). The extracted RNA
(1 mg) was then transcribed into cDNA by using Reverse Transcription
with Elimination of Genomic DNA using the Quantitative Real-Time PCR
kit (Qiagen), according to the manufacturer’s instructions. The cDNA was
amplified by PCR carried out in a final volume of 50 ml containing 22.5 ml
dH2O, 5 ml 103 PCR buffer, 4 ml 2.5 mM 29-deoxynucleoside 59-triphosphate mix, 3 ml 25 mM MgCl2, 0.5 ml Taq polymerase (Roche, Basel,
Switzerland), 5 ml NS5A forward (59-GCA GCA GAC GAT GTC GTC
GC-39) and reverse (59-GCA GAG ATC CTG CGA AAA TC-39) primers
(each 10 mM) (Invitrogen), or 5 ml tubulin forward (59-TCA CTG TGC
CTG AAC TTA CC-39) and reverse (59-GGA ACA TAG CCG TAA ACT
GC-39) primers (each 10 mM) (Invitrogen) and cDNA template. The following PCR program was used: denaturing at 94˚C for 5 min, 35 cycles of
denaturing at 94˚C for 30 s, annealing at 56˚C for 30 s, extension at 72˚C
for 1 min, followed by extension at 72˚C for 7 min. The amplified PCR
fragments were separated by gel electrophoresis, labeled with ethidium
bromide, and visualized under UV light in the Gene Genius apparatus
(Syngene, Frederick, MD). The expected band sizes were 550 bp for NS5A
and 350 bp for the mouse tubulin PCR product.
Immunizations
Mice were immunized i.m. in the tibialis cranialis muscle with 50 mg
plasmid DNA [coNS5A-pVAX1, wtNS5A-pcDNA3.1, or truncated wtNS5A
constructs in pcDNA3.1 (Table I)] in a volume of 50 ml. Immediately
following administration of the plasmid DNA, tibialis cranialis muscles
were subsequently electroporated (EP) using the MedPulser DNA delivery
system (DDS; Inovio Pharmaceuticals, Blue Bell, PA) (40). The mice were
boosted at monthly intervals and were immunized one to three times.
Protein-based immunizations were performed by a s.c. injection of 50 mg
rNS5A protein (Mikrogen) diluted in PBS mixed with Freund’s (prime:
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cation and has a zinc-binding motif, and the domain is a dimer as
determined by crystallography (27). Domains II and III are less
conserved and natively unfolded (28–30). Domain III is required
for proper assembly of viral particles (31) but is not needed for
RNA replication (32). NS5A has been described to interact both
with cellular proteins like Raf-1, p53, PI3K, and Grb2, which are
important for host cell signaling (20, 33), but also to interfere with
components of innate immunity by inhibiting PKR and cyclophilin A (33–35). Hence, NS5A may affect IFN signaling pathways and production of proinflammatory cytokines. In addition,
we have shown that NS5A interferes with adaptive immunity by
protecting hepatocytes from cytolytic killing (20). In contrast,
long-term intrahepatic NS5A protein expression does not induce
spontaneous liver disease or cancer (20, 36). In this study, truncated forms of NS5A genes were investigated along with the fulllength NS5A to define the immunogenicity of different domains of
this viral protein.
The aim of this study was to develop and evaluate HCV NS5A
genotype 1b DNA vectors as potential vaccine candidates for
chronic HCV infection. We initiated a study to characterize the
immunogenic properties of NS5A-vaccine candidates in wt and
immunologically tolerant NS5A-Tg mouse models.
The Journal of Immunology
complete, boost incomplete) adjuvant 1:1 to a final volume of 100 ml that
was administered at the base of the tail.
In vivo challenge with tumor cells stably expressing NS5A
In vivo challenge with NS5A stable expressing EL-4 lymphoma cells was
performed in naive and immunized mice 2 wk postimmunization using 1–
1.5 3 106 tumor cells. The cells were washed, resuspended in 200 ml PBS,
and inoculated s.c. in the right flank of the mouse. The kinetics of the
tumor growth was determined by measuring the tumor volumes through
the skin using a sliding caliper every second or third day, and the volume
was calculated by using the formula: 0.5 3 (tumor length 3 tumor diameter2) (41).
Detection of NS5A-specific Abs using ELISA
Serum from immunized C57BL/6J mice was collected at indicated time
points via retro-orbital bleeding of Isofluran anesthetized mice. Detection
of mouse IgG Abs to NS5A was performed as described previously (42).
In brief, plates were coated with 1 mg/ml rNS5A protein (Mikrogen for
protein- and GenScript for DNA-immunized mice). Mouse serum was
added in serial dilution (starting dilution of 1:60 and then diluted 1:6). OD
was read at 405 nm with a 620-nm background. The cutoff value was set to
three times the OD value of a negative serum sample.
Liver biopsies (100 mg) were homogenized using sonication (Vibra Cell
Homogenizator; Chemical Instruments, Lidingö, Sweden) in 1 ml radioimmunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris-HCl [pH
7.4], 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS, 2
mM PMSF, 0.25 mM DTT, and 10 mM Na3VO4 (Sigma-Aldrich]). The
liver homogenates were incubated on ice for 10–20 min and centrifuged at
12.000 3 g for 2 min at 4˚C, and the supernatant was transferred to a new
vial and stored at 280˚C until analysis. A total of 60 mg protein was
separated on an SDS-PAGE 4–12% Bis-Tris gel. For running and blotting
of protein gels, the XCell SureLock Mini-Cell system and iBlot equipment
(Invitrogen) were used following the manufacturer’s protocol and as described previously (43). Abs targeted to rabbit anti-V5 (diluted 1:1000) and
goat anti-rabbit HRP (diluted 1:5000) were purchased from Sigma-Aldrich
and DakoCytomation (Glostrup, Denmark), respectively. Proteins were
visualized using the ECL Plus Western blotting reagent (GE Healthcare,
Uppsala, Sweden), according to the manufacturer’s protocol.
Detection of IFN-g– and IL-2–producing CTLs and Th cells by
ELISPOT assay
Secretion of IFN-g and IL-2 by splenocytes from pooled groups mice was
detected using a commercial ELISPOT assay according to manufacturers
protocol (MabTech; Nacka Strand, Sweden) and as described previously
(10). The number of spots was measured using the Aid ELISPOT reader
system (version 2.6) with the ELISPOT 3.2.3 software (Autoimmun
Diagnostika, Strausberg, Germany). Results are shown as the mean number
of IFN-g– or IL-2–producing spot-forming cells (SFCs) per 106 cells of
triplicate values.
Detection of NS5A-specific CTLs by cytotoxicity assay
(e.g., [51Cr] release assay)
Detection of NS5A-specific lytic cytotoxic T cells was performed using a [51Cr]
release assay as described previously (44). In brief, splenocytes from coNS5ApVAX1–immunized and nonimmunized mice were harvested and resuspended
in complete CTL medium. The splenocytes were restimulated in vitro for
5 d in 25-ml flasks (e.g., T25) containing 25 3 106 spleen cells and 25 3 106
irradiated (2000 rad) syngenic splenocytes. The restimulation was performed
in presence of the 9- or 8-mer peptides corresponding to the NS5A protein
sequence. After 5 d of in vitro restimulation, a standard [51Cr] release assay was
performed in 96-well V-bottom plates. The radioactivity was detected using
a 1450 Microbeta Trilux scintillation counter, with the Microbeta Workstation
Software 4.0 (PerkinElmer, Waltham, MA). The percentage of specific [51Cr]
release was calculated according to the formula: ([experimental release 2
spontaneous release]/[maximum release 2 spontaneous release]) 3 100.
Maximum release was calculated from supernatants of cells that were lysed
by addition of Triton X-100 (Sigma-Aldrich). Spontaneous release was determined from supernatants of cells incubated without effector cells. Results
are shown as the mean percent-specific lysis of triplicate values.
with 5 mg/ml NS5A2251–2259 (VILDSFDPL) peptide, 5 mg/ml NS5A2252–2259
(ILDSFDPL) peptide, 5 mg/ml OVA CTL peptide, PMA-ionomicyne
(100 ng/ml and 1 mg/ml, respectively), and medium control. After stimulation, the cells (0.5 3 106 cells/well in 96 V-bottom plate) were washed
and resuspended in FACS buffer (PBS/1% FBS) and then incubated with
Live and Dead (Invitrogen) in dark for 30 min on ice and blocked with
Fc block (anti-mouse CD16/32; BD Biosciences, Franklin Lakes, NJ).
Staining for cell surface markers with Abs against mouse CD107a-PE
(1D4B) and CD8-PerCP (53-6.7) proceeded for 15 min on ice. The cells
were then fixed with Cytofix/Cytoperm (BD Biosciences) for 20 min on
ice, washed with permwash (BD Biosciences), and subsequently stained
intracellularly with Abs against mouse CD3-Pacific Blue (17A2), IFNgFITC (XMG1.2), IL-2-allophycocyanin (JES6-5H4) and TNFa-PE-Cy7
(MP6-XT22) in permwash for 30 min on ice. All Abs were purchased
from BD Biosciences. Approximately 50,000 living lymphocytes were
counted per sample with a Fortessa flow cytometer (BD Biosciences) and
analyzed with the FlowJo software (Tree Star, Ashland, OR).
Splenocytes from individual naive and immunized mice were harvested
and used directly ex vivo for NS5A-specific CD8+ T cell quantification. A
total of 1 3 106 cells/well in 96 V-bottom plate were washed and resuspended in PBS/1% FBS (FACS buffer) and incubated 15 min in the dark at
22˚C with R-PE–labeled H-2Kb Pro5 pentamer refolded with HCV
NS5A2251–2259 (VILDSFDPL) or HBV core (MGLKFRQL) from ProImmune (Oxford, U.K.). After labeling, the cells were blocked with Fc block
(anti-mouse CD16/32) for 15 min on ice, washed in FACS buffer, and
stained with cell surface markers against mouse CD19-PE-Cy5 (clone
6D5) and CD8-FITC (clone KT15) (ProImmune, Oxford, UK) for 20 min
on ice. The cells were then fixed in 2% paraformaldehyde in PBS. A total
of 150,000 events were subsequently acquired from each sample using
a FACSCalibur flow cytometer (BD Biosciences) and analyzed using the
CellQuest software. From a live lymphocyte gate, CD19+ events were
excluded, and the remaining cells were gated for CD8 expression. Frequency of NS5A H-2Kb (VILDSFDPL)-positive events within this population was determined.
RMA-S stabilization assay
The following peptides were evaluated for ability to stabilize the MHC
class I molecule: NS5A2251–2259 (VILDSFDPL), NS5A2252–2259 (ILDSFDPL),
NS3 CTL (GAVQNEVTL), HBcAg CTL (MGLKFRQL), and NS5A2248–2256
(NKVVILDSF) in five different concentrations (10, 5, 1, 0.5, and 0.1 mg/ml).
RMA-S cells were incubated overnight at 26˚C with the different peptides
(2 3 106 cells for each peptide concentration). After the incubation, the
cells were washed and resuspended in FACS buffer and then stained in two
separate tubes (1 3 106 cells for each peptide concentration) with Abs
against mouse H-2Db-FITC and H-2Kb-PE for 30 min on ice. A total of
30,000 events from each sample were counted on a FACSCalibur flow
cytometer (BD Bioscience, Franklin Lakes, NJ) using the CellQuest software
and analyzed using the FlowJo software (Tree Star, Ashland, OR).
Statistical analysis
All comparisons were performed using GraphPad InStat 3, Macintosh
(version 3.0b, 2003; GraphPad Software, San Diego, CA) and Microsoft
Excel 2008, Macintosh (version 12.2.8; Microsoft, Redmond, WA). The
kinetic of measurements was compared using the area under the curve
(Excel). Parametrical data were compared using the ANOVA (InStat 3), and
nonparametrical data were compared with Mann–Whitney U test (InStat 3).
Results
Characterization of full-length and truncated NS5A expression
constructs
To determine the immunogenic properties of the different domains
of NS5A, we immunized mice with one wt full-length, one co fulllength, and eight wt truncated NS5A constructs for their ability
to activate NS5A-specific immune responses (Table I) (45). All
constructs were verified by restriction enzyme digest, sequencing,
and an in vitro transcription and translation assay for proper NS5A
protein expression prior to immunization (data not shown).
Flow cytometry
Humoral immune responses after DNA or protein
immunization with full-length and truncated NS5A expression
constructs
Splenocytes from naive and vaccinated mice were stimulated overnight at
37˚C (0.5 3 106 cells/well in 96 V-bottom plate) in complete CTL medium
To investigate the immunogenic properties of different NS5A-DNA
constructs or protein, groups of C57BL/6J (H-2b) mice were im-
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Sample preparation and Western blot analysis
1115
1116
NS5A-BASED GENETIC IMMUNOGENS PRIMES CYTOTOXIC T CELLS
Table I. An overview of NS5A-based DNA expression plasmids used
for immunogenicity evaluation
NS5A Domain
Construct
Amino Acid in NS5A
1 (wtNS5A)
2
3
4
5
6
7
8
9
coNS5A
1–449 (full-length)
1–163
1–215
1–326
1–378
31–449
105–449
211–449
302–449
1–449 (full-length)
codon optimized
For each plasmid the amino acid and NS5A domain coverage is indicated. ❖, The
location of the herein identified CTL epitopes.
To evaluate cellular immune responses primed by NS5A–DNA
expression constructs we needed to identify NS5A MHC class I
CTL epitopes in C57BL/6J (H-2b) mice since there are no NS5A
MHC class I epitopes identified in the H-2b haplotype. A set of 87
synthetic 20-mer peptides (15 aa overlap) spanning the full-length
NS5A gt1b gene was made for this purpose. In addition, MHC
class I epitopes were also predicted using the SYFPEITHI database version 1.0 (38) (http://www.syfpeithi.de). Screening for
MHC class I NS5A epitopes was performed by harvesting spleens
from NS5A–DNA-immunized wt C57BL/6J mice and restimulating cultures for 36 h in the presence of the 87 individual
peptides in two concentrations (10 and 1 mg/ml) and thereafter
determining IFN-g production by ELISPOT assay. Identified and
predicted peptides were tested for the stabilization of surface
expression of MHC class I molecules on the TAP2–deficient
RMA-S cell line (46, 47). Two NS5A peptides were identified that
bound H-2Db and H-2Kb molecules with high affinity. Further
fine-mapping revealed two candidate peptides, one with high affinity for the H-2Db molecule (sequence VILDSFDPL, aa 2251–
2259) and one for the H-2Kb molecule (sequence ILDSFDPL, aa
2252–2259) (Fig. 2A, 2B). It was noted that these two epitopes
share the same amino acid sequence but differ in their MHC
restriction. To verify whether the newly identified peptides are
processed and are immunogenic in vivo, we used two approaches
to analyze NS5A epitope–specific immune responses. First, wt
C57BL/6J mice were immunized with 50 mg coNS5A-pVAX1
i.m. in combination with EP. Second, NS5A-Tg mice (20) backcrossed to a C57BL/6J (H-2b) background were immunized with
the same dose of vaccine. The NS5A-Tg C57BL/6J mouse strain
is a newly generated mouse lineage with constitutive hepatic
NS5A protein expression (Fig. 2C) (20). Two weeks postimmunization, mice were sacrificed, and splenocytes were
restimulated with the two peptides and analyzed for IFN-g and IL2 production by ELISPOT assay and for lytic activity by a [51Cr]
FIGURE 1. Ab responses primed by NS5A–DNA- or protein-based immunogens. All mice were primed and boosted at week 0, 4, and 8. Mice were
immunized with 50 mg coNS5A-pVAX1 followed by in vivo electroporation or with 50 mg rNS5A protein (A) or truncated NS5A plasmids (see Table I). As
a positive control we used full-length wtNS5A-pcDNA3.1 (aa 1–449) and as negative control we used an empty pcDNA3.1 plasmid (B–I). Values are given
as mean anti-NS5A IgG titers and SD in groups of five wt C57BL/6J mice. Arrows indicates time point of immunization.
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munized i.m. with 50 mg plasmid DNA in combination with
in vivo EP using the MedPulser DDS apparatus (40) or s.c. with
50 mg rNS5A protein at the base of the tail. Analysis of anti-NS5A
IgG titers after immunization revealed that DNA immunization
induced an efficient Ab response, although it appeared later and
had a lower magnitude than the NS5A protein immunization (Fig
1A). NS5A IgG titers reached the plateau around week 6 after the
first immunization (Fig. 1). Interestingly, immunization of mice
with the different truncated and full-length DNA constructs
showed that plasmids containing NS5A amino acid sequences
corresponding to aa 327–449 were able to induce equally strong
Ab responses as the full-length wt NS5A construct (Fig. 1E–I). In
contrast, constructs containing amino acid sequences corresponding
to aa 1–326 did not prime any detectable NS5A IgG Ab titers
detectable in full-length NS5A (Fig. 1B–D). This suggests the
humoral immune responses are mainly focused toward the Cterminal part of NS5A.
Cellular immune responses after DNA immunization with
full-length NS5A expression constructs
The Journal of Immunology
1117
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FIGURE 2. Characterization of NS5A-specific cellular immune responses in wt and NS5A-Tg mice. A peptide stabilization assay was used to identify
epitopes binding to H-2Db (A) and H-2Kb (B). Results are presented as the lowest binding peptide concentration in mg/ml. Peptides below the detection
limit are marked with ♦. (C) Detection of NS5A protein in NS5A-Tg C57BL/6J mice (lanes 1, 2), and a non-Tg wt C57BL/6J mouse (lane 3). Size marker
(M), Magic Mark (Invitrogen). Quantification of NS5A-specific IFN-g– and IL-2–producing T cells per 106 splenocytes determined by an ELISPOT assay.
Wt C57BL/6J mice (D, G), NS5A-Tg (E, H), and nonimmunized wt C57BL/6J mice (F, I) (n 5 5/group) were immunized i.m. once with 50 mg coNS5ApVAX1, followed by in vivo electroporation. Data have been given as SFCs/106 splenocytes and SD. Cutoff has been set as 50 SFCs/106 splenocytes.
NS5A-specific lytic CTL activity was determined by [51Cr] release assay in wt C57BL/6J (J, M), NS5A-Tg (K, N), and nonimmunized wt C57BL/6J (L, O)
mice. Groups of five mice were i.m. immunized once with 50 mg coNS5A-pVAX1 or left untreated. Splenocytes from individual mice were restimulated in
presence of NS5A peptides (NS5A2251–2259 or NS5A2252–2259), and lytic activity was determined by using peptide-loaded RMA-S cells. Values are presented as percent NS5A peptide–specific lysis. Values are given for E:T ratio of 60:1, 20:1, and 7:1. The presence of a statistical (Figure legend continues)
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NS5A-BASED GENETIC IMMUNOGENS PRIMES CYTOTOXIC T CELLS
In vivo protection against growth of NS5A-expressing tumor
cells
One way to evaluate the functionality of a vaccine-primed T cell
response in vivo is to challenge immunized mice with syngeneic
tumor cells expressing the vaccine Ag. Thus, determination of protection against tumor growth is a direct measure of in vivo T cell
functionality (48, 49). We and others have shown that the protection against in vivo tumor growth is mainly dependent on cytotoxic T cells (CTLs) (44, 48). In this study, we used this tumor
model to characterize in vivo functional immune responses to
NS5A. To perform the proposed studies, we generated an EL-4
lymphoma cell line with a constitutively expressed NS5A protein
by stable DNA transfection using the wtNS5A-pcDNA3.1 plasmid. The presence of HCV NS5A RNA in total RNA extracts from
transfected EL-4 cells was analyzed by RT-PCR. Only extracts
from the NS5A-EL-4 cell line were positive for NS5A by RT-PCR
(Supplemental Fig. 1A). The NS5A-EL-4 and the parental EL-4
tumor cell models were evaluated in naive C57BL/6J mice to
compare tumor growth between these two cell lines. No difference
in growth properties between the NS5A-EL-4 and the parental EL4 cell lines were seen (p = NS; Supplemental Fig. 1B). To determine the specificity of protection against tumor growth,
C57BL/6J mice were immunized once with 50 mg coNS5ApVAX1 or wtNS5A-pcDNA3.1 and challenged with NS5A-EL-4
or the parental EL-4 cells. We found that mice immunized with
the co or wt NS5A plasmids could mediate protection against
tumor growth (p , 0.001 and p , 0.05, respectively; Fig. 4A, 4B).
Moreover, IFN-g production was also determined in immunized
mice that were challenged with NS5A-EL-4 or the EL-4 tumor
cells (Fig. 4C‑G). Similar levels of IFN-g production were ob-
served in the different immunized groups, except in mice immunized with coNS5A-pVAX1 and challenged with NS5A-EL-4,
which showed a higher postchallenge cytokine production against
NS5A (p , 0.01 for 2251–2259 and p , 0.001 for 2252–2259;
Fig. 4C). Thus, the endogenous production of NS5A protein from
the tumor cells boosts the vaccine-primed immune response to
NS5A in vivo. Interestingly, this was only evident for mice immunized with coNS5A-pVAX1. This could be explained by the
fact that coNS5A-pVAX1 primes stronger NS5A-specific immune
responses as compared with wtNS5A-pVAX1. Nonimmunized
mice did not show any IFN-g production. One important question
to address is whether coNS5A-DNA immunization can protect
NS5A-Tg mice against NS5A-EL-4 tumor growth. Therefore, wtand NS5A-Tg mice were either immunized once with 50 mg
coNS5A-pVAX1 or left nonimmunized. Two weeks later, they
were challenged with NS5A-EL-4 tumor cells and monitored for
tumor growth. We found that immunized wt mice were protected
against tumor growth, whereas nonimmunized wt mice developed
tumors (p , 0.001; Fig. 4H). Moreover, immunized NS5A-Tg
mice showed partial protection against tumor growth, whereas
nonimmunized NS5A-Tg mice were not protected (p , 0.05; Fig.
4I). There was no statistical significant difference in tumor volumes between immunized wt and NS5A-Tg mice. Next, we
wanted to know whether different regions of NS5A differed in
their priming of cellular immune responses. This has important
implications for the design of NS5A-based genetic vaccines because it will allow us to exclude unwanted areas or regions of
NS5A. Groups of C57BL/6J mice were immunized once with 50
mg full-length or truncated NS5A-DNA–based vaccine constructs
(Table I). Only groups of C57BL/6J mice immunized with fulllength wt NS5A plasmids or truncated constructs that contained
the NS5A sequence between aa 216 and 449 were protected
against NS5A-EL-4 tumor challenge. Moreover, only these groups
had detectable NS5A2251–2259- and NS5A2252–2259-specific IFN-g
production (Fig. 5A, 5B). As expected, only plasmids containing
the MHC class I NS5A epitopes (aa 281–289) induced IFN-g
production (Fig. 5B). In addition, mice immunized with NS5A
region aa 302–449 were also protected against tumor growth (p ,
0.05; Fig. 5A), even though this region does not contain the
identified epitopes. This indicates the presence of additional CTL
epitopes within NS5A in the H-2b haplotype.
Expansion and polyfunctionality of the activated NS5A-specific
T cells
Because high quality polyfunctional CD8+ T cells are a hallmark
of highly efficient human vaccines, we analyzed the polyfunctionality of T cell responses in wt and NS5A-Tg mice immunized
once with 50 mg coNS5A-pVAX1 i.m. and EP. Two weeks later,
the animals were sacrificed and immune responses were analyzed by ELISPOT, pentamer- and intracellular cytokine staining. The results revealed IFN-g production in both wt and NS5ATg mice, although a statistically significantly higher number of
SFCs was evident in the wt group (p , 0.001; Fig. 3A, 3B). The
control groups were negative for IFN-g production (Fig. 3C,
3D). Direct ex vivo pentamer staining was used to quantify the
number of NS5A-specific CD8+ T cells after immunization.
Quantifiable levels of NS5A-specific CD8+ T cells were detected
in vaccinated wt and NS5A-Tg mice but not in nonimmunized
groups. It was noticed that pentamer staining in NS5A-Tg mice
was generally weaker, presumably because of the low frequency
difference (e.g., wt mice compared with NS5A-Tg mice) has been indicated as follows: ***p , 0.001, **p , 0.01 using area under the curve (AUC) and
ANOVA.
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release assay using peptide-loaded RMA-S cells (Fig. 2D–O).
These results revealed that both MHC class I NS5A peptides were
processed and properly presented in vivo to induce T cell
responses that were able to produce high levels of IFN-g and
IL-2 in wt mice (Fig. 2D, 2G). Interestingly, IFN-g production
was also detected in immunized mice with constitutive liverspecific NS5A protein expression, although at a lower magnitude
(Fig. 2E, 2H). The NS5A-specific IFN-g and IL-2 production was
statistically significantly higher in immunized wt compared with
NS5A-Tg mice (p , 0.001 and p , 0.01, respectively). This
indicates that the NS5A-Tg mice have a partially impaired T cell
response to NS5A. Moreover, we were only able to detect low
levels of IFN-g production by splenocytes stimulated with rNS5A
in wt mice but none in NS5A-Tg mice (Fig. 2D–F). No IL-2
production was detected from splenocytes stimulated with
rNS5A (Fig. 2G–I). Moreover, the cytotoxicity potential of the
NS5A-specific T cells could be demonstrated in a [51Cr] release
assay on peptide-loaded RMA-S target cells. Functional testing
revealed that both peptides presented on MHC class I can be
recognized and killed by in vivo primed NS5A-specific CTLs (Fig.
2J–O). There was no statistical significant difference between the
lytic activity of the two peptides and between wt and NS5A-Tg
mice, although the percent-specific lysis of target cells was
somewhat lower in the NS5A-Tg mice as compared with wt mice.
Because direct ex vivo quantification of NS5A-specific CD8+ T
cells revealed a statistical significant difference between wt and
NS5A-Tg mice (p , 0.001; Fig. 3E–I), the reason for the similar
level of lytic activity detected in wt and NS5A-Tg mice can be
explained by the 5-d in vitro restimulation with the peptides.
The Journal of Immunology
1119
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FIGURE 3. NS5A–DNA-based immunization primes NS5A-specific polyfunctional T cell responses in both wt and NS5A-Tg mice. Wt and NS5A-Tg
C57BL/6J mice were immunized i.m. once with 50 mg coNS5A-pVAX1, followed by in vivo electroporation. Two weeks postimmunization, mice were
sacrificed, and quantification of NS5A-specific IFN-g–producing T cells per 106 splenocytes was determined by ELISPOT assay (A–D). Data have been
given as SFCs/106 splenocytes and SD. Cutoff has been set as 50 SFCs/106 splenocytes. Also shown is the expansion of NS5A-specific CTLs in wt and
NS5A-Tg mice as determined by direct ex vivo pentamer staining, with data given as representative dot plots (E–H) and as the percentage pentamer-positive
CD8+ T cells with SD (I). In (J) and (K), bars illustrate the percentage of CD8+CD107a+TNFa/IL-2/IFNg–producing cells after 12 h ex vivo Ag stimulation.
Number of mice used in experimental setting: immunized wt mice (n = 10), immunized NS5A-Tg mice (n = 9), nonimmunized wt mice (n = 10), and
nonimmunized NS5A-Tg mice (n = 8). The presence of a statistical difference (e.g., wt mice compared with NS5A-Tg mice) has been indicated as follows:
***p , 0.001, **p , 0.01, *p , 0.05 using area under the curve (AUC) and ANOVA (A, B) and a Mann–Whitney U test (e.g., all groups compared, I).
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NS5A-BASED GENETIC IMMUNOGENS PRIMES CYTOTOXIC T CELLS
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FIGURE 4. NS5A-DNA–based immunization primes NS5A-specific CTLs that protects against in vivo tumor growth. Mice were immunized i.m. once
with 50 mg coNS5A-pVAX1 (A) or wtNS5A-pcDNA3.1 (B), followed by in vivo electroporation. Two weeks postimmunization, mice were challenged with
NS5A-expressing EL-4 cells or with the parental EL-4 cells (1 3 106 cells s.c. in the right flank/mouse). Tumors were measured every second to third day
with start at day 6 postinoculation. Values are presented as mean tumor volumes 6 SE in groups of nine mice. Quantification of NS5A-specific IFN-g–
producing T cells per 106 splenocytes determined by an ELISPOT assay. Mice were immunized i.m. once with 50 mg coNS5A-pVAX1 (C, E), wtNS5ApcDNA3.1 (D, F), or left nonimmunized (G). Two weeks postimmunization, mice were challenge using NS5A-EL-4 (C, D) or EL-4 (E, F) cells, and NS5Aspecific IFN-g–producing T cells were detected at the end of the experiment (n = 9). Data have been given as SFCs/106 splenocytes and SD. Cutoff has been
set as 50 SFCs/106 splenocytes. Mice were immunized i.m. once with 50 mg coNS5A-pVAX1, followed by in vivo electroporation or left nonimmunized in
wt mice (H, n = 10 mice/group) or NS5A-Tg mice (I, n = 8 mice per group). Two weeks postimmunization, mice were challenged with NS5A-expressing
EL-4 cells (1.5 3 106 cells s.c. in the right flank/mouse). Tumors were measured at days 5, 7, and 9 postinoculation. Values are (Figure legend continues)
The Journal of Immunology
found in this group. Our results also indicate that the frequency of
NS5A-specific CD8+ T cells was statistically significantly higher
in wt mice (range, 0.7–3.6%) compared with NS5A-Tg mice
(range, 0.2–0.7%) (p , 0.001; Fig. 3E–I). The polyfunctionality
of NS5A-specific T cells was determined by flow cytrometry (Fig.
3J, 3K). Immunization of wt mice primed NS5A-specific T cells
producing mainly combinations of IFN-g/TNF-a/CD107a specific
for the NS5A CTL epitopes. The expression pattern was similar in
NS5A-Tg mice, albeit at lower levels. Interestingly, we could
detect quadrifunctional T cells producing IFN-g/IL-2/TNFa/CD107a in both wt and NS5A-Tg mice. Thus, the NS5A-DNA
immunization was highly efficient in activating NS5A-specific
T cells to produce cytokines that enabled expansion and broadened the polyfunctionality of the T cell response.
Discussion
and IL-2 production in splenocytes from wt and NS5A-Tg mice.
Before evaluating NS5A-specific immune responses in H-2b
NS5A-Tg mice, we first confirmed liver-specific NS5A protein
expression by Western blot analysis. The m.w. of the NS5A protein band was identical to that previously shown in NS5A-Tg
FVB/N mice (20). We discovered that NS5A-specific immune
responses could be activated in both wt and NS5A-Tg mice,
although the Tg mice were tolerant as evidenced by significantly lower levels of IFN-g and IL-2 produced after immunization. Importantly, the vaccine-primed T cells were able to kill
peptide-pulsed target cells in a cytotoxicity assay. This proves that
the activated T cells are functional. Similar results have been
described in wt and NS3/4A-Tg mice immunized with a NS3/4Abased DNA vaccine (10). To further evaluate the in vivo functionality of NS5A vaccine–primed immune responses, we
established a homologous in vivo tumor challenge model in which
NS5A is stably expressed in the EL-4 T cell lymphoma cell line.
Parental EL-4 and NS5A-transfected EL-4 tumor cells had
comparable growth properties in wt mice. To use this tumor
challenge model, we first determined the specificity of tumor
protection by challenging immunized mice with the transfected
tumor cells or the parental cell line and comparing in vivo protection. We showed that only NS5A-immunized groups of mice
were protected against tumors expressing the NS5A protein.
Similar specificity of protection has been shown in other tumor
challenge models (44). In addition, the protection is mainly confirmed by CD8+ T cells and to a lesser extent CD4+ T cells (44).
Interestingly, protection against tumor growth in immunized
NS5A-Tg mice was not complete although significant compared
with nonimmunized NS5A-Tg mice. This demonstrates that even
in a tolerogenic environment, as in NS5A-Tg mice, dysfunctional
T cells can be activated by NS5A–DNA immunization and mediate protection. Hence, a single NS5A–DNA immunization in
NS5A-Tg mice can prime T cells that inhibit NS5A-expressing
tumor cells. Moreover, analysis of immune responses in immunized and NS5A-EL-4 tumor challenged mice revealed an improved immunogenicity as compared with immunization and EL-4
tumor challenge. Thus, the improved immunogenicity may be
explained by a boost from the NS5A protein expressed in the
tumor cells. We have previously seen similar results in a hepatitis B virus tumor model (55). We were also interested in the
importance of the different regions and domains of NS5A. Several studies have shown that the three domains of NS5A have
important functions in the viral life cycle (27–32). Simultaneously, we were interested whether the different regions and
domains also had different immunogenic properties. This was
investigated by testing eight truncated NS5A constructs for
priming protective immunity against tumor challenge using EL-4
cells stably expressing the full-length NS5A-protein. This revealed that region aa 216–449 was important for protection
against NS5A-EL-4 tumor growth. Analysis of immune responses
in the same mice showed that all constructs containing the NS5A
CTL epitopes elicited IFN-g production. In addition, one construct
that did not contain the NS5A CTL epitopes also elicited protection against tumor growth, indicating that protection is not
solely dependent on immune responses to the identified CTL
epitopes. Hence, our results indicate that, at least in mice, the
minimum region of NS5A needed to mediate protection against
tumor growth and activate NS5A-specific T cell responses is the
presented as mean tumor volumes 6 SE. The presence of a statistical difference has been indicated as follows: ***p , 0.001, **p , 0.01, **p , 0.05 using
area under the curve (AUC) and ANOVA. In (C)–(F), groups were compared as follows: (C) compared with (E), and (D) compared with (F).
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The NS5A protein of HCV is an essential component of the replication complex (25, 26). The protein is subdivided into three
distinct parts, domains I, II, and III, where domains I and II have
been shown to be essential for RNA replication, whereas domain
III is important for secretion of infectious virions (27). In addition,
several studies have shown that NS5A may be a target for antiviral
therapy. Results from clinical evaluation of NS5A-specific DAAs
revealed that the candidate drugs had potent antiviral activity (21–
23). The encouraging results suggest that NS5A-specific DAAs
may be part of future antiviral therapy for HCV. Interestingly,
combination treatment of HCV genotype 1 infection using NS3and NS5A-specific DAAs achieved SVR without IFN-a and ribavirin, although the addition of IFN-a and ribavirin produced
superior responses (50). With this in mind, we decided to pursue a
detailed characterization of NS5A as a genetic vaccine candidate
for chronic HCV infection. Because of the limited knowledge of
the immunogenic properties of NS5A, we sought to study its intrinsic properties. The limited number of studies of the intrinsic
properties of NS5A as a genetic immunogen has demonstrated
poor immunogenicity (17–19). Moreover, only a limited number of
HLA-A2 CD8+ T cell–restricted epitopes have been identified
(16, 51, 52). We therefore cloned full-length and truncated NS5A
genotype 1b genes into expression vectors and evaluated the immunogenicity in wt and NS5A-Tg mice. In this study, we demonstrated that the carboxyterminal domains II and III of NS5A are
important for priming NS5A-specific humoral immune responses.
It is known that this region of NS5A contains several human B cell
epitopes (53, 54). Our results show that a rNS5A protein was more
immunogenic in priming NS5A-specific IgG Abs compared with
NS5A as a DNA plasmid. Although, the ability of NS5A DNA
to elicit NS5A-specific IgG Ab titers of .104 postimmunization
highlights that NS5A is intrinsically immunogenic and may be
suitable in vaccine compositions. To be able to characterize cellular immune responses to NS5A in H-2b mice, we first had to
identify MHC class I epitopes within NS5A. Several MHC class
I epitopes are described in other mouse haplotypes (17–19), but
to our knowledge, none is described for H-2b. We therefore used
overlapping peptides covering the complete NS5A gene combined
with epitope predictions. By fine-mapping of epitopes, we were
able to identify two NS5A MHC class I epitopes sharing the same
sequence but with different MHC restrictions (H-2Kb and H-2Db).
Identified epitopes were tested for recognition by vaccine-primed
CD8+ T cells and whether MHC-TCR recognition induced IFN-g
1121
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NS5A-BASED GENETIC IMMUNOGENS PRIMES CYTOTOXIC T CELLS
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FIGURE 5. Immunization with truncated NS5A-DNA immunogens mediates protection against in vivo tumor growth. Mice were immunized i.m. once
with 50 mg truncated wtNS5A expression plasmids (see Table I), followed by in vivo electroporation. Two weeks postimmunization, mice were challenged
with NS5A-expressing EL-4 cells (1 3 106 cells s.c. in the right flank/mouse). Tumors were measured every second to third day with start at day 5
postinoculation (A). Values are presented as mean tumor volumes 6 SE in groups of seven mice. Quantification of NS5A-specific IFN-g–producing T cells
per 106 splenocytes determined by an ELISPOT assay at the end of the experiment (B). Data have been given as SFCs/106 splenocytes and SD. Cutoff has
been set as 50 SFCs/106 splenocytes. Truncated constructs 1–163, 1–215, and 302–449 do not contain the sequences for the identified CTL epitopes and
was therefore negative for IFN-g production in response to peptide stimulation. The presence of a statistical difference (e.g., NS5A-based plasmid
compared with pcDNA3.1 empty vector) has been indicated as follows: ***p , 0.001, **p , 0.01 using area under the curve (AUC) and ANOVA.
region connecting domains I and II, and the amino terminal part of
domain II.
One important feature for a therapeutic vaccine candidate is to
prime high frequencies of lytic T cells that can also produce mul-
tiple cytokines. We therefore analyzed the polyfunctionality of
T cells primed in wt and tolerant NS5A-Tg mice. We showed that
NS5A vaccination in wt and NS5A-Tg mice primed NS5A-specific
T cell responses with frequencies of ∼1.5% NS5A-specific CD8+
The Journal of Immunology
Acknowledgments
We thank Dr. David R. Milich (Vaccine Research Institute of San Diego,
CA) for critical reading of the manuscript. We thank ChronTech Pharma
AB for providing synthetic peptides. We also thank Inovio Pharmaceuticals
for providing the Medpulser DDS apparatus for in vivo EP.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Disclosures
22.
L.F. and G.A. are paid consultants to ChronTech Pharma AB. L.F. owns
patent/patents pending for HCV Tg mouse models and HCV therapeutics.
K.E.B. is an employee of Inovio Pharmaceuticals. The other authors have no
financial conflicts of interest.
23.
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T cells in wt mice compared with 0.5% in NS5A-Tg mice. This
further highlights the tolerant status of these Tg animals but also
shows that vaccination can activate an immune response. Importantly, when determining the pattern of cytokines produced after
immunization, we found a similar expression profile in wt and
NS5A-Tg mice that was weaker in the Tg group. Notably, the
detected frequencies of NS5A-specific CD8+ T cells were lower
compared with the cytokines produced by peptide-stimulated
CD8+ T cells in the immunized NS5A-Tg mice. This may be
explained by the fact that functionally competent T cells can bear
TCRs with affinities for cognate ligands below the threshold
required for peptide–MHC pentamer engagement. Thus, CD8+
T cells may exhibit effector functions in the absence of detectable
pentamer binding. In addition to intrinsic affinity, variations of
TCR density, differences in membrane lipid organization, the state
of T cell activation, and differentiation status may also influence
pentamer binding (56–60). Therefore, peptide stimulation used
in functional assays enhances the detection of pentamer-negative
functional T cells. We demonstrated that NS5A-based DNA
vaccines were able to prime specific T cell responses of suitable
phenotype. Therefore, NS5A may be considered as a therapeutic
vaccine candidate for chronic HCV infection. Moreover, even
though NS5A may interfere with cellular signaling pathways
including molecules and cells involved in innate and adaptive
immunity (20, 34, 35), long-term intrahepatic NS5A protein
expression does not induce any liver disease (20, 36).
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