modulation of hepatitis c virus core dna vaccine immune responses by co-immunization with...

Modulation of hepatitis C virus core DNA vaccine immuneresponses by co-immunization with CC-chemokine ligand 20(CCL20) gene as immunoadjuvant

Christine Hartoonian • Zargham Sepehrizadeh • Mehdi Mahdavi •

Arash Arashkia • Yon Suk Jang • Maasoumeh Ebtekar • Mojtaba Tabatabai Yazdi •

Babak Negahdari • Azita Nikoo • Kayhan Azadmanesh

Received: 9 November 2013 / Accepted: 14 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Plasmid DNA vaccination is a promising vaccine

platform for prevention and treatment of infectious disease.

Enhancement of the DNA vaccine potency by co-inoculation

of immunoadjuvant has been shown to be an effective strat-

egy. Modulation of dendritic cells and T-cells locomotion and

trafficking to prime an immune response is mediated by dis-

tinct chemokines. The recent study was designed to elucidate

the adjuvant activity of plasmid expressing CC-chemokine

ligand 20 (pCCL20) in co-inoculation with hepatitis C virus

(HCV) core DNA vaccine immunization. pCCL20 was con-

structed and evaluated for its functional expression. Sub-

cutaneous inoculation of pCCL20 with HCV core DNA vac-

cine was performed via electroporation in BALB/c mice on

day 0 and 14 and a HCV core protein booster was applied on

day 28. On week after final immunization, both humoral and

cell-mediated immune responses were assessed by indirect

ELISA for core specific antibodies, lymphocyte proliferation,

cytokine ELISA/ELISpot and cytotoxic Grenzyme B (GrzB)

release assays. Mice were co-immunized with pCCL20

developed higher levels of core specific IFN-c/IL-4 ratio and

IL-2 release, IFN-c producing cells, lymphocyte proliferation

and cytotoxic Grenzyme B release in both draining lymph

nodes and spleen cells of immunized mice. The core-specific

serum total IgG and IgG2a/IgG1 ratio were significantly

higher when the pCCL20 was co-inoculated. These results

suggest the potential of CCL20 chemokine as vaccine adju-

vant to enhance Th1 mediated cellular and humoral immune

responses in HCV core DNA immunization.

C. Hartoonian � Z. Sepehrizadeh � M. T. Yazdi

Department of Pharmaceutical Biotechnology, Faculty of

Pharmacy, Biotechnology Research Center, Tehran University of

Medical Sciences, Enghelab Ave, 16 Azar Ave,

1417614411 Tehran, Iran

e-mail: [email protected]

Z. Sepehrizadeh


M. T. Yazdi


M. Mahdavi � A. Arashkia � B. Negahdari �K. Azadmanesh (&)

Department of Virology, Pasteur Institute of Iran, 69 Pasteur

Ave., Kargar Ave., 1316943551 Tehran, Iran


M. Mahdavi


A. Arashkia


Y. S. Jang

Department of Molecular Biology, Institute for Molecular

Biology and Genetics, Chonbuk National University,

Jeonju 561-756, Korea


M. Ebtekar

Department of Immunology, Faculty of Medical Sciences,

Tarbiat Modares University, Ale Ahmad and Chamran Highways

Cross, P.O. Box 1415-331, Tehran, Iran


B. Negahdari

Department of Medical Biotechnology, School of Advanced

Technologies, Tehran University of Medical Sciences, #88, Italy

Avenue, 1417755469 Tehran, Iran

A. Nikoo

Department of Pathology, Razi Hospital, Tehran University of

Medical Sciences, Vahdat Eslami St., Tehran, Iran


123

Mol Biol Rep

DOI 10.1007/s11033-014-3470-5

Keywords Hepatitis C virus � Core � DNA vaccine �CCL20 � Immunoadjuvant

Introduction

Approximately 130–170 million people worldwide are

chronically infected with hepatitis C virus (HCV) [1]. The

viral infection is followed by persistence of virus and

chronic liver disease, cirrhosis and hepatocellular carci-

noma in infected people [2, 3]. The conventional treatment

regimen by PEGylated Interferon-a/Ribavirin is effective

only at 20–60 % of patients depending on virus genotype,

viral load and age of patient [4]. Even though newly

described HCV protease inhibitors, VICTRELISTM

(Boceprevir) and INCIVEK (Telaprevir), showed promis-

ing therapeutic results in HCV genotype 1 infected

patients, the rapid selection for drug resistant HCV vari-

ants, drug induced side effects and the costly regimen of

therapy underlines the necessity of an effective HCV

vaccine preparation [5, 6]. Studies on clearance mecha-

nisms of HCV infection in chimpanzees and human have

revealed that both humoral and cellular immune responses

are of paramount importance in clearance of the virus [7,

8]. These observations put emphasis on potential capacity

of designed vaccines to induce robust humoral and cellular

immune responses, able to act as prophylactic/therapeutic

vaccine. DNA vaccines represent effective strategy to

enhance both humoral and cell-mediated immunity by

virtue of expressing antigens via MHC class I and II

molecules [9]. Considering the fact that HCV genome is

variable and evolution of virus quasi-species is one of the

main mechanism of evading immune system [10], devel-

oping an effective vaccine consisting of conserved

sequences as a broad-spectrum anti-HCV vaccine, would

be of vital importance in vaccine design. The core protein

of HCV is fairly conserved among genotypes and has been

identified as an immunogenic antigen in provoking T cell

responses [11]. The antibodies raised against core are the

first detectable antibodies in HCV infected patients [12].

Several studies have been conducted to evaluate the effi-

cacy of HCV core DNA vaccine [13–16]. In spite of the

advantages that have been attributed to DNA vaccines, the

immunogenicity in large animals and humans is poor [17].

There are several strategies to overcome this obstacle;

among them are considering application of immunoregu-

latory molecules e.g. chemokines [18]. The central dogma

in immune response generation is the proper interaction of

antigen presenting cells (APCs) and T cells [19]. In this

regard, regulation of APC and T cell trafficking via

immunoregulatory molecules e.g. chemokines to recruit

APCs to local site of immunization and consequently

increase antigen presentation to naı̈ve T cells, is among

feasible strategies to ameliorate priming of immune

responses [18, 20]. CCL-20/MIP3a (Macrophage Inflam-

matory Protein-3a) is a chemokine expressed at a low basal

level by epithelial cells, e.g. keratinocytes, and exerts its

function trough CCR6 receptor. CCL20 chemoattracts

immature dendritic cells (DCs), effector/memory T cells

and B cells [21, 22]. Likewise, it increases the ability of

DCs in capture and presenting of antigens. In the current

study, the efficacy of CCL20 to enhance HCV core DNA

vaccine immune responses was investigated with regards of

both cell-mediated and humoral immunity. Herein we

report that sub cutaneous (s.c.) administration of plasmid

expressing CCL20 via electroporation rapidly recruits

immune cells to the site of injection and consequently

enhances the local and systemic immune responses gen-

erated by HCV core DNA vaccine.

Materials and methods

Plasmid DNA constructs

Plasmid expressing HCV core gene (summarized here as

pCore) was precisely described before [13]. Murine CCL20

coding sequence into pGEM vector (pGEM/CCL20) was

provided by Cytokine Bank, South Korea. pGEM/CCL20

was digested by BamHI/KpnI in order to obtain CCL20

coding consequence and consequently inserted into

BamHI/KpnI sites of pcDNA3.1? plasmid (Invitrogen,

CA) summarized here as plasmid expressing CC-chemo-

kine ligand 20 (pCCL20). The construct was confirmed by

DNA sequencing. pcDNA 3.1? as mock plasmid was used

in all invitro experiments and invivo immunization as the

control plasmid. All plasmids were prepared in large scale

for immunization using the Endofree plasmid Giga Kit

(Qiagen, Germany).

Expression of CCL20

To confirm the expression of CCL20 by constructed plas-

mid, HEK 293T cells (National Cell Bank of Iran) were

cultured in DMEM (Gibco, Life technologies) supple-

mented with 10 % FBS (PAA, Germany), 1x GlutaMAX

(Gibco, Life technologies) and 1x Penicillin/Streptomycin

(Gibco, Life technologies). Cells were transfected with

2 lg of pCCL20 using Lipofectamine LTX (Invitrogen,

CA) transfection reagent according to the provider’s

instruction. Cell supernatant was assayed 3 days after

transfection using mouse CCL20 Duo Set ELISA kit (R&D

systems, Minneapolis, MN) following the provided

instruction.

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Chemotaxis assay (migration assay)

The biological activity of CCL20 was determined by

migration towards a concentration gradient of chemoat-

tractant in polycarbonate 8 lm pore 24-well transwells (SPL

Life Sciences Inc., Korea). Briefly, 1 9 106 cells of MT2

(Human Lymphoblastoid T cell line (National Cell Bank of

Iran) expressing CCR6 chemokine receptor [23] was resus-

pended in 100 ll RPMI 1640 supplemented with 1 % BSA

(chemotaxis medium) and applied to the upper chamber.

600 ll of the supernatant of cells transfected with pCCL20

(described earlier) was added to the lower compartment.

After 4 h of incubation at 37 �C, the migrated cells to the

lower chamber were collected, and counted utilizing hem-

ocytomer slide. Percentage of migrated cells was calculated

according to the provided formula:

% migrated cells

¼�number of migrated cells to the lower chamber=

number of of total cells applied to the transwellÞ � 100

Blockage of CCL20 induced chemotaxis by neutralizing

antibody

Supernatant of pCCL20-transfected cells (described ear-

lier) was pre-incubated with mouse CCL20/MIP-3a MAb

(Clone 114906), Rat IgG1 (R&D systems, Minneapolis,

MN) in concentration of 30 lg/ml for 20 min at room

temperature. 600 ll of prepared mix was added to the

lower compartment of transwell and migration assay was

performed as described. An irrelevant IgG1 antibody was

used as the isotype control.

Histopathological analysis

6–8 weeks old female BALB/c mice were anesthetized.

100 lg of pCCL20 was dissolved in 20 ll of Endotoxin

free saline and injected s.c. into both front foot pads by

31 g needle syringe. Electroporation was performed

immediately after injection with field strength of 50 V/cm

(constant), 6 pulses for 200 ms each by electroporator

(ECM 830 BTX; BTX/Harvard Apparatus, MA, USA)

apparatus. At 12, 24 and 48 h post inoculation, the injec-

tions loci were resected, fixed with 10 % formal saline,

embedded in paraffin and 6 lm sections were stained with

hematoxylin and eosin for light microscopy observations.

HCV core synthetic peptides and protein

Two H2D–restricted epitopic peptides corresponding to

HCV core (core39–48; RRGPRLGVRA) [24] and

(core133–142; LMGYIPLVGA) [25], were synthetized with

95 % purity (GL Biochem, Shanghai, China) and applied

for in vitro stimulation in immunoassays. HCV nucleo-

capsid protein (Abcam, UK) was purchase for protein

booster immunization, serological examinations and

in vitro stimulation in cell based assays. A cocktail of

peptide/protein consisting of 3 lg/ml of each peptide and

nucleocapsid protein at 1 lg/ml dilution was prepared and

applied in all in vitro cell stimulations. An H2D–restricted

epitope of HCV NS3 protein was used as an irrelevant

peptide control [26].

Mice and immunization

Naı̈ve female 6–8 weeks old BALB/c (H2D) mice were

purchased from animal care center of Pasteur Institute of

Iran (Karaj, Iran) and were allowed to acclimate for 1 week

prior to the experiments. Mice were anesthetized and

injected with 100 lg of pCore in the presence or absence of

100 lg of pCCL20 in a total of 50 ll Endotoxin-free

normal saline following electroporation with field strength

of 50 V/cm (constant), 6 pulses for 200 ms each by elec-

troporator (ECM 830 BTX; BTX/Harvard Apparatus, MA,

USA) apparatus twice at a 2 weeks interval. For protein

booster, 10 lg of recombinant HCV nucleocapsid protein

(Abcam, UK) was formulated in 70 % Montanide ISA 206

(Seppic, France) according to the manufacturer’s instruc-

tion and injected 1 week after second DNA immunization

in all mice previously received pCore with or without

pCCL20. All injections were performed via 31 g needle

syringe s.c. into both front foot pads.

Isolation of splenocytes and draining lymph nodes

(DLN)

Spleens and DLNs (brachial and axillary LNs) were iso-

lated and smashed in a cell homogenizer, washed and

resuspended in PBS. RBCs were lysed with ACK lysis

buffer (NH4Cl 0.15 M, KHCO3 0.15 M, EDTA.2Na

0.1 mM) for 5 min and washed twice with PBS. Single cell

suspension of spleen or lymph node cells was prepared in

Advanced RPMI 1640 (Gibco, Life Technologies) sup-

plemented with 5 % FBS Gold (PAA, Austria), 1x Gluta-

MAX (Gibco, Life Technologies), penicillin/streptomycin

1X (PAA, Austria) and cells were counted by trypan blue

exclusion method using hemocytometer. Singe cell con-

centration was adjusted to 2 9 106 cell/ml to be applied in

cell based assays. All cell based experiments were per-

formed utilizing the aforementioned culture medium

formula.

Cytokine release assay

Single cell suspension of splenocytes and lymph nodes

(2 9 106 cells/ml) were cultured in 24 well plates, recalled

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123

by peptide/protein cocktail and incubated at 5 % CO2 and

37 �C for 72 h. Supernatants were collected and stored at

-80 �C until being tested. IFN-c, IL-2 and IL-4 levels

were measured by Duo set ELISA kit (R&D sys-

tems,Minneapolis,MD) according to the manufacturer’s

protocol as described previously in detail [13].

IFN-c ELISpot assay

Mouse IFN-c ELISpot assay was performed utilizing pre-

coated IFN-c ELISpot kit (MabTech, Sweden) according to

the manufacturer’s instruction. Briefly, 5 9 105 cell/well

of splenocytes or lymph nodes were cultured and stimu-

lated by peptide/protein cocktail and incubated for 48 h at

37 �C and 5 % CO2. Wells containing unstimulated cells

were considered as negative controls. Plates were then

washed 5 times to remove cells and then the mAb directed

to the mouse IFN-c was applied to wells for 2 h at room

temperature. Washing steps were repeated 5 times, fol-

lowed by addition of streptavidin-conjugated HRP and

incubation for 1 h at room temperature. Spots were

developed by addition of precipitating TMB substrate and

incubated for 30–45 min in the dark. Plates were washed

with distilled water and dried at 4 �C. The wells were

photographed using a dissection stereo microscope (Nikon,

Japan) and the pictures were used for counting spot

forming cells. The number of specific IFN-c producing

cells was calculated by subtracting the number of spots in

un-stimulated wells from stimulated ones and graphed as

spot forming cell (SFC) in 1 9 106 cells.

Proliferation assay

To evaluate lymphocyte proliferation, non-radioactive cell

proliferation ELISA, BrdU (colorimetric) kit (Roche, Ger-

many) was utilized according to the manufacturer’s

instruction. Briefly, 3 9 105 cells/200 ll of lymph nodes

and splenocytes suspension were cultured in 96-well flat

bottom tissue culture plate (Nunk, Denmark). Peptide/pro-

tein cocktail (1 lg/ml of each) was added and incubated for

72 h at 37 �C and 5 % CO2. Un-stimulated cells were con-

sidered as negative control. Cells were labeled by addition of

20 ll of BrdU labeling solution (100 lM) and incubated for

18 h at 37 �C and 5 % CO2. Plates were centrifuged for

10 min at 300 g, labeling medium was removed by suction

and plates were dried. Anti-BrdU-POD was applied to wells

and incubated for 1 h at RT. Substrate was applied to mea-

sure incorporation of BrdU into cell DNA by colorimetric

analysis at A450 nm by Microplate reader ELx800 (BioTek,

USA). Stimulation Index (SI) for each sample was calculated

according to the following formula: OD450 nm of stimulated

well/OD450 nm of un-stimulated well. All experiments were

performed in triplicate.

GrzB release assay

GrzB release assay was performed to measure the ability of

lymphocytes isolated from mice to release cytotoxic GrzB

molecule after in vitro re-stimulation by cognate antigen/

peptide. Briefly, splenocytes or LN cells were cultured in

96-well culture plates (Nunc, Denmark) with or without

peptide cocktail. After 36 h incubation at 37 �C and 5 %

CO2, supernatants were collected and objected for mouse

GrzB ELISA (R&D systems, Minneapolis, MN) according

to the manufacturer’s protocol. Specific GrzB release was

measured as: specific GrzB release (pg/ml) = (specific

lysis (pg/ml) - spontaneous lysis (pg/ml)).

Serological assays

Anti-core specific IgG was determined by an optimized

indirect ELISA as described previously [13] with minor

modifications. Briefly, 0.5 lg of nucleocapsid protein

(Abcam, UK) in 100 ll carbonate coating buffer (CCB)

was coated in Maxisorb 96 well plates (Nunc, Denmark)

overnight at 4 �C. After 3 washing steps with washing

buffer (0.05 % Tween 20 in 1x PBS), blocking buffer (3 %

BSA in 1x PBS) was applied and incubated for 2 h at

37 �C while shaking. After washing steps, sera diluted in

blocking buffer were applied to wells and incubated for 2 h

at 37 �C. Washing steps were repeated 5 times and 100 ll

of 1:7,000 diluted HRP-conjugated anti-mouse IgG

(Sigma) incubated for 1 h at 37 �C followed by 5 washing

steps. TMB was added to wells in order to develop color-

imetric reaction and optical density measured at 450 nm

with Microplate reader ELx800 (BioTek, USA) following

addition of 2 N H2SO4 as stop solution. In order to deter-

mine anti-core specific IgG subtypes, IgG1 and IgG2a

subclasses were measured utilizing goat anti-mouse IgG1

and IgG2a secondary antibodies (Sigma) according to the

same protocol.

Statistical analysis

The statistical analysis software SPSS version 16 (SPSS

Inc., Chicago, IL, USA) was used to perform analysis

procedure. Student t test or one-way ANOVA was per-

formed for data comparison and p \ 0.05 was considered

as statistically significant difference.

Results

Expression and biological activity of CCL20

Murine CCL20 coding sequence was sub cloned into

pcDNA3.1? and expression was confirmed by transfection

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123

of HEK 293 T cells. Cell supernatant was subjected to

CCL20 sandwich ELISA 72 h post transfection and CCL20

expression was confirmed by ELISA (Fig. 1a). The bio-

logical activity of pCCL20 expression product to attract

target MT2 cells was evaluated in chemotaxis transwell

assay. As shown in Fig. 1b, supernatant of pCCL20

transfected cells potently stimulated MT2 cell migration. In

order to determine whether the induced chemotaxis is

specifically mediated by CCL20, chemokine function was

neutralized by neutralizing antibody against CCL20. As

shown in Fig. 1c, chemotaxis was significantly hampered

in the presence of neutralizing antibody indicating the

specific CCL20 mediated chemotaxis.

Histopathological analysis

In order to assess the kinetic of CCL20 responsive

immunocytes recruitment after s.c. injection of pCCL20,

the inoculation loci was histologically examined after H&E

staining in 3 time points. Population of inflammatory cells,

well-defined as mononuclear cells mostly plasma cells and

lymphocytes, were found in sections 12 h post injection.

After 24 h, the aforementioned populations were reduced

drastically and were not detectable at 48 h. In contrast,

mock plasmid injection did not increase cellular infiltration

in any time point (Fig. 2). Results indicated rapid recruit-

ment of immunocytes during 12 h post s.c. injection of

pCCL20 convincing the co-injection of pCCL20 and pCore

as adequate time point for chemokine plasmid

administration.

Cytokine release assay and IFN-c ELISpot

IFN-c as markers of Th1 and IL-4 as a marker of Th2

response were measured in order to characterize the mag-

nitude of generated immune responses. As shown in

Fig. 3a, IFN-c productions was increased upon injection of

mice with core DNA/DNA/protein immunization in both

spleen and DLNs while co-inoculation of pCCL20 signif-

icantly enhanced IFN-c production (p \ 0.05). IL-4 pro-

duction was not affected by injection of either pCore or co-

administration of pCore ? pCCL20 in comparison with the

Fig. 1 Expression and

bioactivity of CCL20.

a Expression of CCL20 was

confirmed by ELISA and

quantified utilizing standard

curve. The mock plasmid

(pcDNA 3.1?) was utilized as

control in transfection

experiments. b, c The

competence of expression

product of pCCL20 to stimulate

MT2 (CCR6 receptor

expressing cells) migration in

transwell chemotaxis assay

evaluated in the presence (c) or

absence (b) of 100 lg/ml of

neutralizing CCL20 neutralizing

monoclonal antibody

(mAbCCL20). All experiments

have been performed in

duplicate and repeated three

times. Results are expressed as

mean percentage of migrated

cells to the lower chamber of

transwell ± standard deviation

(SD) and p \ 0.05 was

considered as significant

difference. All experiments

were performed in triplicate.

Asterisks depict the statistically

significant differences

Mol Biol Rep

123

control groups (Fig. 3c). IL-2 was also elicited in pCore

group and further enhanced by co-inoculation of pCCL20

(Fig. 3b). These results demonstrated a predominant Th1

biased immune response following co-administration of

pCCL20. As IL-2 has T lymphoproliferative properties,

these results are in accordance with proliferative responses.

In order to further evaluate the effect of pCCL20 admin-

istration on breadth of IFN-c producing cells, IFN-c ELI-

Spot was performed to analyze the frequency of IFN

producing cells in both spleen and DLNs. As depicted in

Fig. 3d, co-immunization with pCCL20 resulted in the

greatest enhancement of SFC with an increase from

78 ± 26 SFC in 106 cells in pCore group to 325 ± 68 at

pCore ? pCCL20 group in splenocytes and 65 ± 30 to

304 ± 62 in DLNs (p \ 0.05). These results were in

accordance with IFN-c release ELISA assay indicating that

pCCL20 co-administration enhanced the population of

IFN-c producing cells and in depth indicated that the

increased amount of IFN-c is predominantly due to the

elevated number of IFN-c secreting cells in an antigen

specific manner.

Proliferation assay

To evaluate lymphocyte proliferation response, splenocytes

and DLN were re-called in vitro for 72 h with peptide/

protein cocktail and proliferation was detected utilizing

BrdU ELISA. As shown in Fig. 4a, immunization of mice

with or without CCL20 enhanced lymphocyte proliferation.

The increase in proliferation by pCore alone was 4.8 and

6.5 fold in spleen and DLNs, respectively compared to the

control group (p \ 0.05), while co-injection with CCL20

gene enhanced proliferative responses by almost twofold

(p \ 0.05) in comparison with pCore alone indicating the

CCL20 as a marginal inducer of lymphocyte-proliferative

responses.

Fig. 2 Histopathological analysis. 50 lg of pCCL20 or control

pcDNA3.1? in 20 ll normal saline was s.c. injected once in front

footpad of BALB/c mice (n = 3) via electroporation. Maximum

infiltration of inflammatory cells was observed 12 h post-injection

(p.i.). a Left panel is representative of infiltration loci 12 h p.i. (910)

and the right panel shows a small region of area at a higher

magnification (940). b The same pattern of magnification is shown in

backbone plasmid injected specimens. Scale bars represent 300 lm

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123

GrzB release assay

Measurement of released GrzB following in vitro peptide/

antigen re-stimulation can be a specific indicator of CTL

activity. Administration of pCore marginally induced the

release of GrzB (p \ 0.05) and pCCL20 coadministration

enhanced the quantity of released GrzB up to 2.5 and 2 fold

in spleen and DLNs, respectively. These observations

illustrated that spleen and DLN cells from pCore immu-

nized mice had possessed the ability to release GrzB in

reaction to stimulation by cognate antigen and that of

CCL20 co-administration had an arousing effect on GrzB

release in both splenocytes and DLNs (Fig. 3b).

Humoral immune responses

Groups of mice were immunized with pCore with or

without pCCL20 according to the immunization platform.

Total anti-core IgG antibody responses are depicted in

Fig. 5a. In general, antibody response was increased in

pCore group indicating that DNA/DNA/protein immuni-

zation of core protein induces specific antibody. CCL20

co-expression exhibited enhancing effect in the induction

of antigen-specific IgG. As the effect of pCCL20 co-

administration in cellular immune responses fits nicely

with a Th1 biased immune response, the subclasses of

vaccine induced IgG were further analyzed utilizing IgG1

and IgG2a secondary antibodies. As shown in Fig. 5b,

immunization with pCore according to the vaccination

regimen, induced IgG2a indicative for a Th1 type response

while the IgG1 did not changed significantly. CCL20 gene

co-administration resulted in higher level of specific IgG2a

when compared to pCore group whereas the IgG1 level did

not changed. The IgG2a/IgG1 ratio significantly increased

by pCCL20 co-inoculation (p \ 0.01) which denotes the

ability of CCL20 to enhance the shift of immune response

to a Th1 pattern.

Discussion

HCV is associated with significant mortality and morbidity

globally [2]. The burden of HCV-related disease is asso-

ciated with significant costs to the health system resulting

Fig. 3 Cytokine expression magnitude in splenocytes and DLNs of

vaccinated mice. One week after final immunization, spleen and

DLNs were isolated. Culture supernatant of splenocytes and DLNs of

representative mice were collected following 72 h of recall by core

protein/peptide cocktail and subjected for IFN-c (a), IL-2 (b) and IL-4

(c) ELISA. Data are representative of mean cytokine concentration

(pg/ml) ± SD of six mice per group. Asterisks indicate the significant

difference among groups at p \ 0.05. d IFN-c secretion further

assessed by ELISpot. The frequency of spot-forming cells after 48 h

of in vitro recall was expressed as the number of spots per 106 cells.

Negligible amount of spots were detected in the control groups (data

not shown). Clustered columns depict means of core specific SFCs for

IFN-c in each group (n = 6 mice). All experiments were performed

in triplicate

Mol Biol Rep

123

from the need to manage chronic illness and the demand on

services for treatment of advanced liver disease. As the

prevalence of advanced liver disease increases [27], these

costs will continue to escalate, therefore highlight the need

for increased investment in effective preventive interven-

tions, notably safe and efficacious vaccines [28]. As it has

been postulated by Halliday et al. [29] based on the viral–

host interactions, a successful HCV preventive/therapeutic

vaccine shall likely bear aforesaid characteristics alike: a)

targeting relatively conserved viral regions as a pan-

genotype vaccine, b) capable of induction of robust T cell

and humoral immunity. HCV core DNA vaccine has been

broadly investigated as a proper vaccine candidate [12, 15,

16], but in DNA vaccine studies, the weak immunogenicity

still remains an initial barrier to be overcome. Application

of genetic adjuvants has been amongst the successful

approaches to augment HCV core DNA vaccine-elicited

immune responses [13, 15, 17]. Chemokines as proinfla-

matory molecules play a major role in leukocyte migration

and activation and their potential as genetic adjuvant to

enhance DNA vaccine-elicited immune responses have

been explored in some previous studies [30–34]. Consid-

ering the aforementioned criteria, this study was designed

to utilize CCL20 chemokine gene as an immunoadjuvant to

enhance immune responses of HCV core DNA immuni-

zation protocol. Of note we performed vaccination by s.c.

route considering the high level of resident immature

Langerhans APCs arsenal in the skin, in order to benefit

their migration to the higher concentration zone of CCL20.

The rational of employing a protein booster was that HCV

core DNA vaccine administration, even by boosting with

the same construct, did not raise the core specific IgG while

after protein booster specific IgG rose rapidly [16]. Also in

our previous study we showed that heterologous HCV core

DNA prime/protein boost raise up core specific antibody

responses [13].

In this report, it was indicated that established immunity

following administration of pCCL20 with pCore enhanced

cell mediated immunity and Th1 type responses by the

increase in lymphocyte proliferative responses, enhanced

production of IL-2 and IFN-c and elevated levels cytolytic

GrzB release in both DLNs and splenocytes of immunized

mice. We also observed that mice co-immunized with

pCCL20 had higher IgG responses and this higher antibody

was due to the increased production of IgG2a subtype

which extends findings of cellular immunity that elucidate

induced shift to Th1 type cellular responses. Bitagyn et al.

[35] reported that CCL20 enhanced the immunogenicity of

an evolutionary conserved 37 kDa immature lamininre-

ceptor protein (OFA-Ilrp), which was non immunogenic

embryonic antigen expressed in different tumors. CCL20

significantly induced both specific anti IgG1 and IgG2a

antibodies and CTL responses. Co-immunization of HIV

Gag plasmid (pGag) with CCL20 expressing plasmid

(pCCL20) raised a Th1 type immune response while

slightly decreased the levels of Gag specific antibody

responses [34]. Such controversies in humoral immune

response with results of current study might be due to the

nature of antigen and/or immunization regimen. The dif-

ference of our approach was that we utilized a protein

booster to increase anti-core specific IgG which results in

humoral immune response enhancement. Chaven et al.

[36] found co-expression of choriomeningitis virus

Fig. 4 Lymphocyte proliferation and cytotoxic GrzB release assays.

a Lymphoproliferative activity of splenocytes and DLNs isolated

from experimental groups after immunization with pCore with or

without pCCL20 in DNA/DNA/protein boost strategy. One week after

final booster mice were sacrificed and single cell suspension of spleen

and DLNs in vitro recall was performed for 3 days as described.

Incorporation of BrdU in proliferative lymphocytes post recall was

determined by ELISA and OD was read at 450 nm. OD ratio of

stimulated cells to the same sample non-stimulated ones were

depicted as stimulation index (SI) in order to compare proliferation

level. b Acquisition of cytotoxic function by CTLs post vaccination:

The capacity of immunized spleen and DLN cells to release cytolytic

GrzB after in vitro re-stimulation with corresponding peptide/protein

was determined by GrzB ELISA. Antigen specific GrzB release was

calculated as described in ‘‘Materials and methods’’. Data represents

the mean ± SD of 6 mice per group in triplicate. The Asterisks

indicate the groups which are statistically different (p \ 0.05)

Mol Biol Rep

123

nucleoprotein (NP) antigen with CCL20 in a Modified

Vaccinia Ankara (MVA) virus resulted in two to fourfold

increase in cell mediated immune responses and 6–17-fold

increase in humoral immune responses. Guo et al. [37]

found that fusion of GFP as a model weak antigen to

CCL20 and administration as DNA vaccine significantly

enhanced humoral immunity with a predominant IgG2a/

IgG1 response and cellular DTH responses which are in

consistence with our findings.

The precise mechanism of CCL20 function as immuno-

adjuvant has yet to be further elucidated. Song et al. showed

that in mice tumor transduced with an adenoviral vector

expressing CCL20, regression of tumor was observed by local

accumulation of DCs into tumor and induced anti-tumor

immunity mediated by tumor-specific CTL response. The

elicited immunity was effective enough to protect mice from

challenge with the identical tumor cells following syngeneic

adoptive transfer of splenocytes of animals receiving this

treatment [38]. Rapid inflammatory immunocytes recruit-

ment in injection loci was observed 12 h post pCCL20

injection (Fig. 2). Since CCL20 chemoattracts immature

DCs, this might emphasize the DNA vaccine uptake by APCs

especially resident Langerhans cells of skin and consequently

augment antigen specific immune responses.

The potential improvements for the vaccination regimen

described in this paper can be considered to achieve more

robust specific immune induction by several approaches

e.g. co-expression of molecules to induce chemoattraction

and directed locomotion of varied populations of leuko-

cytes (GM-CSF, CCL-19, CCL21) [20, 34, 39].

Overall, in this study, we demonstrated that co-admin-

istration of CCL20 expressing plasmid is a promising

strategy for modulation of HCV core specific immune

responses. Correspondingly, these results are in further

agreement with the claims of purposeful harnessing of the

adjuvant properties of chemokines in modulating vaccine

induced immune responses.

Acknowledgments We thank Dr. Mahsa Rasekhian and Dr. S.M.

Hossein Etemadzadeh for their technical assistance. Financial support

was provided by Faculty of Pharmacy, Tehran University of Medical

Sciences and partly was supported by Grants of Virology Department

of Pasteur Institute of Iran.

Conflict of interest The authors declare any conflict of interest and

financial disclosure.

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