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Vaccine 28 (2010) 3913–3919

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

he delivery of HBcAg via Tat-PTD enhances specific immune response andnhibits Hepatitis B virus replication in transgenic mice

iaohua Chen, Jinglan Lai, Qingchun Pan, Zhenghao Tang, Yongsheng Yu, Guoqing Zang ∗

epartment of Infectious Disease, Shanghai No.6 People’s Hospital, College of Medicine, Shanghai JiaoTong University, Shanghai 200233, China

r t i c l e i n f o

rticle history:eceived 21 December 2009eceived in revised form 19 February 2010ccepted 26 March 2010vailable online 13 April 2010

eywords:at-PTDBV core antigen

a b s t r a c t

Recent studies have indicated that the therapeutic vaccine based on enhancement of HBV-specific cyto-toxic T-lymphocyte (CTL) activity may lead to viral clearance in chronically infected individuals. It isdemonstrated that protein transduction domains (PTD) from HIV-1-Tat protein is able to enter cellswhen combined with exogenous antigens and induce specific CTL responses. We have previously tes-tified that the expressed and purified fusion protein containing Tat-PTD47-57 and HBcAg could entercytoplasm of dendritic cells, and enhance T cells response to generate HBcAg-specific CTLs efficiently invitro. In the present study, we evaluated HBcAg-specific immune responses of PTD-HBcAg fusion proteinin BALB/c mice and antiviral immunity in HBV transgenic mice. The studies showed that PTD-HBcAg notonly induced significantly higher antibody responses, but also increased production of cytokine (IFN-�,

BV transgenic mouse

ytotoxic T-lymphocyteirus immunotherapy

IL-2, IL-4 and IL-10) compared to HBcAg alone and PBS. Moreover, PTD-HBcAg fusion protein increasedsignificantly the percentages of IFN-�+CD8+ T cells and HBcAg-specific (CTL) responses. Also, enhance-ment of immune response induced by fusion protein reduced HBV DNA and HBsAg levels and decreasedthe expression of HBsAg in liver tissue of HBV transgenic mice. In conclusion, PTD-HBcAg fusion proteincould enhance not only cell immune response but also humoral immune response, and induce robust

herap

specific CTL activity and t

. Introduction

Hepatitis B virus (HBV) infection is the leading cause of chronicepatitis, cirrhosis and hepatocellular carcinoma. Approximately% of the world’s population is chronically infected by HBV [1,2].BV is a noncytopathic, enveloped virus with a circular, double-

tranded DNA genome [3]. Interferon and nucleoside analoguesuch as lamivudine and adefovir have been widely used for thereatment of chronic HBV infection. Unfortunately, these agentsave not been able to attain complete eradication of the virusffectively and frequently [4,5]. Several studies have shown thatstrong CTL response can clear the virus and that viral persistence

n chronic HBV infection might be due to the failure in the develop-ent of HBV-specific cellular immunity [6]. Therefore, stimulation

nd expansion of specific CTLs represent a strategy to treat chronicepatitis B patients. Strategies to boost or broaden the weak virus-pecific T-cell response of patients with chronic hepatitis B were

roposed by means of various HBV vaccines [7–9].

It is reported that the Tat protein from human immunod-ficiency virus 1 (HIV-1) is able to enter cells when addedxogenously. A basic region of the Tat protein called protein trans-

∗ Corresponding author. Tel.: +86 21 64369181; fax: +86 21 64369181.E-mail address: zangguoqin@126.com (G. Zang).

264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2010.03.070

eutic effects in HBV transgenic mice.© 2010 Elsevier Ltd. All rights reserved.

duction domains (PTD), 9–11 amino acids in length, could cross thelipid bilayer of cells either alone or as a fused form to some polypep-tides or nucleotides [10,11]. However, the mechanism by which Tattraverses a membrane and the precise intracellular location of thiscourse have not been explicated. Several studies have shown thepotential of PTD in drug delivery and transduction of proteins aslarge as 110 kDa into different cells [12]. Meanwhile, the injectionof fusion proteins in vivo has demonstrated the effectiveness ofthe PTD in protein delivery [13]. Recently, It is demonstrated thatTat protein from HIV-1 is able to enter cells when combined withexogenous antigens and allow proteins to be used as immunogensfor major histocompatibility complex class I (MHC-I)-restricted CTLresponses. Some studies on pulsing of dendritic cells with PTD-fused antigens have shown robust induction of antigen-specific CTLresponse and Th1-mediated anti-tumor immunity in immunizedmice [14–17]. Based on the PTD properties, it may be a wise strategyto fuse PTD with antigen and elicit a robust immune response.

The HBV nucleocapsid or core antigen (HBcAg) possessesunique immunologic features. During chronic HBV infection, HBcAgis the only antigen that elicits prominent immune responses

[18]. Patients who successfully clear the virus usually have effi-cient HBcAg-specific CTL responses; however, these responses arereduced or below detection levels in patients with chronic HBVinfection [19,20]. Therefore, specific CTLs generated by efficientHBcAg-stimulation may provide a useful immunotherapeutic strat-

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Fig. 1. Schematic diagram of fusion protein designed and humoral immuneresponses in immunized mice. (A) Schematic diagram of M-PTD-HBcAg, M-HBcAgand M-PTD fusion proteins. The HBcAg gene and Tat-PTD47-57 sequence werespliced by PCR and cloned into pMAL-c2x expression vector. The E.coli Rosetta-gamiTM 2 (DE3) cells were transformed with constructed vectors and the solublefusion proteins were expressed in the supernatants. (B) BALB/c mice were vacci-nated with emulsified M-PTD-HBcAg, M-HBcAg, M-PTD (10 �g, 20 �g and 40 �g)and PBS. Boosters were performed 3 weeks later. The anti-HBc IgM antibody titers

914 X. Chen et al. / Vacc

gy for virus elimination in chronic HBV carriers [21]. But theipophilic nature of biological membranes restricts the direct intra-ellular delivery of proteins and peptides. Usually, the antigens oreptides of HBV are all exogenous antigens for antigen presentingells (APCs), HBV-specific CD8+ T-cell responses induced by exoge-ous antigens are too weak to eradicate HBV infection ultimately.herefore, the properties of PTD may give an opportunity to thentigens and peptides of HBV, especially the HBcAg, to enter theytoplasm of APC and to be presented by MHC-I molecules.

Our former experiments have testified that Tat-PTD47-7(YGRKKRRQRRR)-HBcAg fusion protein could be highlyxpressed in E.coli Rosetta-gamiTM 2 (DE3) and purified viaffinity chromatography. Purified fusion protein could penetratehe plasma membrane into DCs and induce HBcAg-specific CTLs22]. In the present study, we investigated whether the PTD-HBcAgusion protein was able to enhance HBcAg-specific immuneesponses and antiviral immunity in mice. Our results showed thathe combination of PTD and HBcAg exhibited a dramatic increasen HBcAg-specific CTL responses and an impressive antiviral effect.

. Materials and methods

.1. Reagents, cells and fusion proteins

All of the fluorescent antibodies and isotype controls were pur-hased from eBioscience (USA). ELISA kits for IFN-�, IL-2, IL-4nd IL-10 were purchased from R&D Co., Ltd. (USA). Anti-HBcAgLISA kits were purchased from Kehua Biological Products Co.,td. (China). Hepatitis B surface antigen (HBsAg) was determineduantitatively using the IMX system (USA) according to the man-facturer’s instructions. The levels of HBV DNA were detectedy real-time fluorescent quantitation PCR assay kits (PG Biotech,hina). Phorbol 12-myristate 13-acetate (PMA), ionomycin andonensin, complete Freund’s adjuvant (CFA), incomplete Freund’s

djuvant (IFA) were obtained from Sigma (USA). HepG2.2.15 cells,hich had a 1.3-fold length of HBV genome (subtype ayw), were

ultured in DMEM containing 10% fetal calf serum (FCS) at 37 ◦Cnder a humidified condition of 5% CO2 and 100 U/ml penicillin,00 �g/ml streptomycin, and 380 �g/ml G418. HepG2 cells wereultured in DMEM containing 10% FCS and 100 U/ml penicillin,00 �g/ml streptomycin. All cells were plated into 6-well plates andllowed to adhere for 24 h before experiments. Soluble fusion pro-eins MBP-PTD-HBcAg (M-PTD-HBcAg), MBP-HBcAg (M-HBcAg)nd MBP-PTD (M-PTD) were purified and had undetectable endo-oxin level according to our former experiments (Fig. 1A) [22].

.2. Animals and immunization

Female BALB/c mice, 6–8 weeks old, were purchased fromhanghai SLAC Laboratory Animal Co., Ltd. (China) and maintainedn the experiment animal centre of Shanghai No.6 hospital underpecific pathogen-free conditions. All experiments were approvedy the laboratory animal ethical commission of Shanghai Jiaotongniversity. Mice were divided into 10 groups, with 6 mice in eachroup. The fusion proteins M-PTD-HBcAg, M-HBcAg, M-PTD (10 �g,0 �g and 40 �g) and PBS were emulsified with equal volume inomplete Freund’s adjuvant (CFA) and incomplete Freund’s adju-ant (IFA), respectively. Mice were immunized subcutaneously athe tail base one time at 3-week intervals with 100 �l of mixedmulsion including CFA. Booster immunizations were identically

erformed with 100 �l of mixed emulsion including IFA 3 weeks

ater. Mice were sacrificed, and serum samples and splenocytesere collected at day 7 after the second immunization.

The HBV transgenic mouse lineage was initially produced on aALB/c background and the transgene was consisted of 1.3 copies

in sera from immunized mice were detected by ELISA. Results are the means forsix mice/group within one experiment. Anti-HBc IgM antibody titers in the M-PTD-HBcAg group are significantly higher than those in the M-HBcAg groups (*p < 0.01).

of the complete genome of HBV (subtype ayw), which starts justupstream of the X promoter and enhancer I and ends downstreamof the unique HBV polyadenylation site. High level of HBsAg andHBV DNA in the sera could be detected in the HBV transgenic mice[23]. Mice were immunized intramuscularly in the left tibialis ante-rior muscle three times at 1-week intervals with PBS, M-PTD-HBcAg(50 �g, 100 �g), M-HBcAg (50 �g). Mice were sacrificed, and serumsamples, splenocytes and livers were collected at day 7 after thethird immunization.

2.3. Detection of anti-HBcAg antibodies

The titer of anti-HBcAg IgM antibodies was determined withanti-HBc ELISA kit (Kehua Bio-engineering Co., Ltd., ShanghaiChina). Following manufacture’s instructions, 50 �l of seriallydiluted sera (starting from a dilution 1:100 in PBS with 5% skimmedmilk) and 50 �l enzyme conjugant were incubated in microtiterplates for 30 min at 37 ◦C. After extensive washing, 50 �l of sub-strate were added to each well and incubated for 15 min at 37 ◦C,and the enzyme reaction was terminated by adding 50 �l/well ofstop solution. Absorbance was read at 450 nm (reference wave-length 630 nm). Antibody titers are presented as the reciprocal ofthe highest dilution showing a positive reaction. Mouse sera weretested individually.

2.4. Cytokines release assay

Splenocytes (2 × 106 cells/ml) were cultured in 24-well platesat 37 ◦C in the presence of 10 �g/ml M-HBcAg. After a 72 h incu-bation, culture supernatants were harvested and the presence ofcytokines, including IFN-�, IL-2, IL-4 and IL-10, were tested with

commercial mouse cytokine immunoassay ELISA kits according tothe manufacturer’s instructions. The concentrations of cytokines inthe samples were determined from the standard curves. Data areexpressed as pg/ml.

X. Chen et al. / Vaccine 28 (2010) 3913–3919 3915

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ig. 2. Cytokines production in the supernatant of splenocytes harvested from imm-PTD-HBcAg group is significantly higher than in the M-HBcAg or M-PTD groups. D-PTD groups and PBS groups (*p < 0.01, **p < 0.05).

.5. Measurement of interferon-� production by intracellularytokine staining

To assess the number of IFN-� secreting cells at the individualell level of mouse splenocytes, single cell suspensions from spleensarvested from immunized mice were analyzed by flow cytom-try. Splenocytes were stimulated in the presence of 10 �g/ml-HBcAg for 6 h. After incubation for 3 h, 25 �g/ml of phorbol 12-yristate 13-acetate (PMA), 1 �g/ml of ionomycin and 1.7 �g/ml

f monensin were added and incubation continued for anotherh. After washing with PBS, cells were stained with saturatingoncentrations of FITC conjugated anti-CD8� McAb. After fixedith Fix and Perm reagent A and B (BD Biosciences, USA), the

ells were incubated for 20 min with PE-labeled anti-interferon-�IFN-�) McAb, followed by washing twice with PBS and analyz-ng by flow cytometry. Fluorescence analyses were performed onOULTER EPICS XL Flow Cytometer (Beckman) using Expo32-ADCoftware.

.6. Cytotoxic T-lymphocyte (CTL) assay

Mouse splenocytes isolated from immunized mice with M-PTD-BcAg, M-HBcAg, M-PTD (40 �g) were cultured with 10 �g/ml-HBcAg and murine recombinant IL-2 (at a final concentra-

ion of 25 IU/ml) for 5 days in vitro and were used as effectorells. The effector cells were cocultured with HepG2.2.15 cellsr HepG2 cells (1 × 104 cells/well, at effector/target ratios of 5:1,0:1 or 20:1) at 37 ◦C under 5% CO2 for 4 h [24]. The specificTL activity was measured by a lactate dehydrogenase (LDH)elease assay using HepG2.2.15 cells and HepG2 cells as target

ells according to the manufacturer’s instructions of CytoTox 96®

on-Radioactive Cytotoxicity Assay (Promega, USA). The percentf cytotoxicity was calculated as [(Experimental release − Effectorpontaneous release − Target spontaneous release)/(Target maxi-um release − Target spontaneous release)] × 100.

ed mice after in vitro re-stimulation. Secretion of IFN-�, IL-2, IL-4 and IL-10 in theepresent the means ± SD (n = 6). However, there is no statistical difference between

2.7. Serological analysis and quantitation of HBV DNA in serum ofHBV transgenic mice

Venous blood was collected from eye sockets of HBV trans-genic mice at day 7 after the last immunization. Serum HBsAg werequantified by Abbott kits. Sera from HBV transgenic mice were sub-jected to detection of HBV DNA by the fluorescent quantitative PCRmethod using a commercial PCR kit according to the manufacturer’sinstructions [25].

2.8. Measurement of IFN-� production by intracellular cytokinestaining to splenic T cells

Cells staining and flow cytometry were performed accordingto protocols by the reagent manufacturers. The splenocytes fromthe HBV transgenic mice were stimulated in vitro for 6 h in cRPMI1640 medium with M-HBcAg, PMA, ionomycin and monensin atthe concentration described above. The cells were then harvestedfor the detection of intracellular cytokines by flow cytometry as theabove-mentioned.

2.9. Histological analyses and immunohistochemical analysis ofthe livers

For histological analysis, liver tissue was fixed in 10% forma-lin, embedded in paraffin, sectioned (3 �m), and stained withhematoxylin and eosin. Briefly, paraffin-embedded sections inPBS, pH 7.4, were treated for 10 min at 37 ◦C with 3% hydro-gen peroxide and washed with PBS. After the sections wereblocked with normal goat serum for 30 min at room tempera-

ture, a goat anti-HBsAg polyclonal antibody (Novus Biologicals,USA) was applied overnight at 4 ◦C following three times ofwashing in PBS, sections were incubated for 30 min with biotiny-lated secondary antibody (Boster, China) at 37 ◦C then for 30 minwith streptavidin–biotin-peroxidase complex before revealed by

3916 X. Chen et al. / Vaccine 28 (2010) 3913–3919

Fig. 3. Intracellular cytokine expression in spleen cells and HBcAg-specific CTL response induced by M-PTD-HBcAg. (A) The mice from each group were sacrificed andsplenocytes were isolated after last immunization, and then were re-stimulated in vitro. And the whole cell population was doubly stained with fluorescent material labeledu nd anS rvestec centar

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sing FITC-CD8� and PE-IFN-� antibodies. The doubly stained cells were counted apecific CTL activity was measured by LDH release assay. The splenocytes were haultured with HepG2.2.15 or HepG2 cells. CTL activity is indicated as the mean perepeated three times with similar results.

iaminobenzidin (DAB) and counterstained with hematoxylin23].

.10. Statistical analysis

Data are expressed as the mean values ± SD. The statisticalignificance of the differences was determined by the unpairedwo-tailed Student’s t-test. Differences were considered statisti-ally significant if p < 0.05.

. Results

.1. M-PTD-HBcAg enhances the antibody response andtimulates the secretion of cytokine

To analyze the immunogenicity of M-PTD-HBcAg and M-HBcAgn mice, serum antibody titers were detected by ELISA at day 7fter the second immunization. As shown in Fig. 1B, anti-HBc IgMntibodies were strongly enhanced in mice immunized with M-TD-HBcAg, and no specific anti-HBc antibodies were detected inBS- or M-PTD-immunized mice. The anti-HBc antibody titers inice immunized with M-PTD-HBcAg were significantly higher than

hose immunized with M-HBcAg (p < 0.01), and also were relatedlosely with the does of M-PTD-HBcAg.

Splenocytes from immunized animals were assayed for theecretion of the cytokines IFN-�, IL-2 (Th1 like) and IL-4, IL-10 (Th2ike) upon re-stimulation with M-HBcAg. As shown in Fig. 2, spleno-ytes from animals immunized with 40 �g M-PTD-HBcAg producedigher levels of IFN-� (294.50 pg/ml), IL-2 (503.74 pg/ml), IL-4

ig. 4. Intracellular cytokine expression in splenocytes of in HBV transgenic mice immuow cytometry. Results are representative of three separate experiments.

alyzed by flow cytometry. The data are the mean ± SD from six mice per group. (B)d in mice immunization with 40 �g M-PTD-HBcAg, M-HBcAg and M-PTD and PBS,ge of specific lysis (±SD) at different effector:target (E:T) ratios. Experiments were

(128.34 pg/ml) and IL-10 (152.51 pg/ml). However, the productionsof above cytokines were very low and there were no significantdifference in mice immunized with M-PTD or PBS (p > 0.05).

3.2. M-PTD-HBcAg induces production of IFN-� from CD8+ T cellsin the spleen

We next measured the intracellular levels of IFN-� in T cells onan individual cell level by flow cytometry. The number of doublystained positive cells was counted by flow cytometry after incu-bation. As shown in Fig. 3A, significantly higher percentages ofHBcAg-specific IFN-�+CD8+ T cells were observed in mice immu-nized with M-PTD-HBcAg than M-HBcAg, M-PTD and PBS. Resultsdemonstrated that delivery of HBcAg via Tat-PTD enhanced thegeneration of specific CTLs in vivo.

3.3. Enhancement of CTL activity pulsed with M-PTD-HBcAg

To confirm the modulating role of M-PTD-HBcAg fusion pro-tein in cell-mediated immune responses, we further analyzed theability of HBcAg-specific CTL response in different groups of immu-nized mice. As shown in Fig. 3B, higher levels of CTL responseswere detected in M-PTD-HBcAg-immunized mice compared withM-HBcAg-immunized, M-PTD or PBS-immunized mice. The specific

cytolysis percentages for 40 �g M-PTD-HBcAg-immunized micewere 47.93%, 32.17% and 16.5% for ratios of effector/target (E/T) of20:1, 10:1 and 5:1, respectively. In contrast, specific cytolysis per-centages were 25.45%, 20.17% and 11.10% in 40 �g M-HBcAg groupsfor all effector/target (E/T) ratios. However, the CTL killing activity

nized with M-PTD-HBcAg. The doubly stained cells were counted and analyzed by

X. Chen et al. / Vaccine 28 (2010) 3913–3919 3917

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ig. 5. Histopathologic changes and immunohistological analysis of HBsAg in HBV-PTD-HBcAg. Then, liver sections were stained with hematoxylin and eosin, and ex

rrow). Representative photographs are presented (original magnifications: 400×). (hotographs are presented (original magnifications: 400×).

etween M-PTD and PBS had no significant difference. Meanwhile,e found that splenocytes from mice immunized by different stim-li were no significant CTL response to HepG2 cells which werenrelated target cells (data not shown). These results suggestedhat M-PTD-HBcAg induces specific CTL activities, which is consis-ent with the high level of IFN-� expressed in CD8+ T cells.

.4. Cellular immunity elicited by M-PTD-HBcAg in HBVransgenic mice

Then we investigated whether M-PTD-HBcAg fusion proteinsere able to prime specific T-cell immune response in HBV trans-

enic mice. For this, the intracellular expression of IFN-� in CD8+ Tells in BALB/c HBV transgenic mice immunized with various fusionroteins was analyzed by flow cytometry. When splenocytes wereonspecifically stimulated with PMA and ionomycin in vitro, it washowed that 50 �g or 100 �g M-PTD-HBcAg caused higher levels ofFN-�+CD8+ T cells than 50 �g M-HBcAg or PBS vaccination, respec-ively (Fig. 4). A week after the last treatment, the HBcAg-specificntracellular IFN-� assay showed that M-PTD-HBcAg fusion proteinaused higher levels of IFN-�-producing CD8+ T cells than M-HBcAgr PBS immunization (Fig. 4). Thus, HBcAg fused with PTD couldnduce more efficiently T-cell immune response than HBcAg alonen vivo.

Liver sections were evaluated primarily in terms of hepato-yte degeneration and necrosis, and inflammatory cell infiltration.here were few infiltrations of lymphocytes in the liver of micemmunized with PBS. However, the amounts of lymphocytes werencreased significantly in the liver of mice treated with M-HBcAgnd M-PTD-HBcAg. A larger number of lymphocyte infiltration, bal-ooning degeneration and spotty necrosis appeared in the liverf mice immunized with M-PTD-HBcAg (Fig. 5A). Furthermore,he results suggested these histological changes in the liver wereelated with the intracellular expression of IFN-� in CD8+ T cells.

.5. M-PTD-HBcAg reduces efficiently the titers of serum HBsAgnd HBV DNA in HBV transgenic mice

We evaluated whether M-PTD-HBcAg immunization couldeduce HBsAg expression and the viral load in serum of HBV trans-

enic mice livers. (A) Mice were treated with PBS, 50 �g M-HBcAg, 50 �g or 100 �gd by light microscopy. Some lymphocytes appeared in the liver of mice (indicated byer sections were subjected to immunohistological analysis of HBsAg. Representative

genic mice. Serum HBsAg and HBV DNA were monitored at day7 after the third immunization by ELISA and quantitative PCRassay, respectively. The inhibition to serum HBsAg or viral DNAin HBV transgenic mice immunized by HBcAg alone or HBcAgfused with PTD showed a significantly difference (Fig. 6). Theseresults indicated that M-PTD-HBcAg immunization more efficientlysuppresses the expression of serum HBsAg and HBV DNA than M-HBcAg and PBS in HBV transgenic mice. Serum HBsAg level from themice immunized with 50 �g M-PTD-HBcAg decreased markedly ascompared with the mice immunized with 50 �g M-HBcAg or PBS(p < 0.01). The titer of HBV DNA in sera was significantly reducedfrom the mice immunized with 50 �g M-PTD-HBcAg comparedwith50 �g M-HBcAg or PBS (p < 0.01). It was worth noting that100 �g M-PTD-HBcAg immunization decreased remarkably thelevel of HBsAg and HBV DNA in sera than that with 50 �g M-PTD-HBcAg did (p < 0.01).

To further confirm the in vivo anti-HBV activity of fusionproteins in transgenic mice, immunohistological analysis was per-formed in livers from the above various treatment groups. A largenumber HBsAg were detected (stained brownish yellow) in thecytoplasm of hepatocytes in mice treated with PBS. M-PTD-HBcAgimmunization reduced the HBsAg level in a dose dependent man-ner, and HBsAg expression was nearly undetectable with 100 �gM-PTD-HBcAg treatment (Fig. 5B).

4. Discussion

The past decade has witnessed tremendous advancement in thefield of protein transduction to produce a given protein molecule byrecombinant DNA technology for in vivo therapeutic applications.Nevertheless, it still remains a challenge to deliver the recombi-nant proteins to desired targets in vivo, although the ability ofsmall molecules or peptides to cross cellular membranes have beensuccessfully demonstrated via adeno-associated virus (AAV), ade-

novirus, lentivirus, herpes virus (HSV) vectors and so on [26,27].Nonetheless, viral vectors can integrate with the host chromatinmaterial and may have consequences from long term effects onhost genetic systems, and therefore, safety remains a serious con-cern for their ultimate clinical application [28]. A new approach that

3918 X. Chen et al. / Vaccine 28 (2010) 3913–3919

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ig. 6. In vivo inhibitory effect on HBsAg and HBV DNA in HBV transgenic mice immuice were immunized with PBS, 50 �g M-HBcAg, 50 �g or 100 �g M-PTD-HBcAg th

nd fluorescent quantitative PCR method, respectively. The HBsAg (A) and HBV DNAhose in 50 �g M-PTD-HBcAg group. Data represent the means ± SD (n = 5) (*p < 0.0

ppears to be the safest is to produce recombinant proteins exoge-ously and then deliver them systemically or by localized injections

nto the target cells or tissues. Discovery of the HIV-Tat proteinransduction domain (PTD) has opened avenues for directing initro and in vivo delivery of proteins into cells.

It is well known that CTLs recognize short amino acid sequenceserived from the intracellular processing of viral antigens in asso-iation with MHC-I molecules on the surface of the infected cells.ecently, some researchers demonstrated that DC pulsed with aecombinant PTD fusion proteins are effective on eliciting potentumor-specific CTL responses both in human blood mononuclearells and in mice model. Although the mechanism for internal-zation and processing of Tat-PTD-containing antigen by DC hasot yet been defined, these data suggested that Tat-PTD-containingntigen might efficiently transduce DC, prolong processing by pro-easomes for MHC class I restricted to CTL by DC, and inducenti-tumor immunity [29,30]. Immunization with HBcAg prefer-ntially induces Th1-type cellular immune responses [31]. TheBV-specific CTLs play an important role in chronic HBV infection

herapy, and the properties of PTD can facilitate the entrance ofhe antigens and peptides of HBV, especially the HBcAg, into theytoplasm of APC.

In the present study, we expressed HBcAg gene included theomplete amino acid sequences which were fused with Tat-TD. Meanwhile, our previous results showed that recombinantTD-HBcAg could penetrate into DCs cytoplasm while recombi-ant HBcAg was detected on the surface of cells. Furthermore,e showed that DCs pulsed with M-PTD-HBcAg more efficiently

nduced IFN-� producing T cells than DCs pulsed with M-HBcAg,nd efficiently elicited CTLs than with others [22]. Base on theechanism, we used PTD as a tool for the efficient delivery of

ntigen to APCs and to induce more HBV-specific CD8+ T cells.We further investigated whether M-PTD-HBcAg could elicit cell-

ediated immune responses in vivo. Our results suggested that-PTD-HBsAg enhanced the percentages of HBcAg-specific IFN-producing CD8+ T cells in the spleen, enhanced the cytokine

ecretion (IFN-�, IL-2, IL-4 and IL-10) and HBcAg-specific CTL activ-ty. Meanwhile, MBP, certain fusion partners, has been shown to

e generally advantageous in improving solubility, proper proteinolding, protection against proteolysis, and overall purification viaffinity chromatography [32]. Specificity for HBcAg was confirmedy the fact that T-cell immune responses could not be elicited

n M-PTD fusion protein or PBS-immunized mice. Although cel-

with M-PTD-HBcAg. The data are the mean ± SD of 5 mice per group. HBV transgenices at 1-week intervals, and serum HBsAg and HBV DNA were quantitated by ELISA

vel in sera of mice treated with 100 �g M-PTD-HBcAg was significantly lower than

lular immune responses are a major contributor to controllingintracellular infections, humoral immune responses are essentialfor controlling extracellular infections. Therefore, the integratedactivation of both the cellular and humoral immune responses isnecessary for the host to control infections [33]. Our study showedthat vaccination with M-PTD-HBsAg resulted in much higher anti-HBc IgM antibody production than vaccination with M-HBcAg orM-PTD. M-PTD-HBcAg increased significantly the production ofanti-HBc IgM antibody in a does-dependent manner. Moreover, theantibody response of fusion proteins was testified by measuringthe level of anti-HBc IgG in our experiment. The titers of anti-HBcIgM were significant higher than anti-HBc IgG in the mice immu-nization with same method in our preliminary experiments (datanot shown). This indicated that HBcAg via Tat-PTD transductionalso could be efficiently captured, processed and presented to Tcells, and then mediated stronger humoral immunity. However,the anti-HBc IgG isotype response was not further measured in ourexperiments, and there were some shortcomings in testifying theantibody response of fusion proteins in its entirety.

One central goal of current immunotherapy and vaccine devel-opment is to enhance antigen presentation to induce potent andbroad immune responses. Studies have demonstrated that CTLsplay a critical role in the control and termination of HBV infec-tion. CTLs are thought to contribute to HBV clearance by killinginfected hepatocytes and secreting antiviral cytokines. Acutelyinfected patients characteristically elicit a vigorous, polyclonal, andmulti-specific CTL response that is usually sufficient to clear theinfection, while persistently infected patients elicit weak or unde-tectable HBV-specific CTL responses. Therefore, the therapeuticvaccine based on enhancement of HBV-specific CTL may lead toviral clearance in chronically infected individuals.

HBV transgenic mice are immunologically tolerant to HBV-encoded antigens and represent a model of chronic HBV infectionsuitable to assess the antiviral potential of immunotherapeuticstrategies to break tolerance and terminate persistent HBV infec-tion. Meanwhile, these mice do not develop chronic hepatitisbecause they are immunologically tolerant to viral antigens. Severalstudies have also illustrated that activated CTLs and the cytokines

they secrete might effectively down-regulate HBV gene expres-sion and even control viral replication [34]. At one week after theadministration of the last treatment to HBV transgenic mice, thehepatocytes appeared swelling and there were lymphocyte infil-tration, hyperemia and necrosis in the liver tissue of mice from

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X. Chen et al. / Vacc

-PTD-HBcAg fusion protein group. In contrast, these histolog-cal changes were not apparent in the mice from other fusionrotein groups, although there was also minimal lymphocyte infil-ration in the mice from PBS and M-HBcAg fusion protein groups.

-PTD-HBcAg fusion protein also enhanced the percentages ofFN-� producing CD8+ T cells in the spleen of HBV transgenic

ice. The result indicated the inflammatory reaction in livers wasonsistent with specific CTL activity induced by M-PTD-HBcAg.urthermore, our results showed that the fusion peptide couldecrease HBV DNA and HBsAg level in serum as well as HBsAgxpression in liver tissue. This reduction was related closely withhe does of M-PTD-HBcAg, which suggested the parallel therapeu-ic effects were associated with the enhanced immune responses.o address whether the specific CTLs brought the hepatocellularnjury, ALT and AST activities were measured by biochemical anal-sis and the results demonstrated that there was no significantifference in the immunized HBV transgenic mice groups (data nothown). These data indicated that HBV-specific CTLs could abolishiral replication in HBV transgenic mice by noncytopathic mecha-isms involving the degradation of viral RNA, perhaps by degradingiral nucleocapsid and replicative intermediates without killinghe cells [35]. Interestingly, the anti-HBV effect of M-PTD-HBcAgusion protein, lasted for approximately 14 days after terminationf treatment in transgenic mice, indicating the anti-HBV effect ofTD-HBcAg fusion proteins was limited by immunity period. Theseesults suggested that the acquisition of the therapeutic effectsere benefited from the facilitative delivery of antigens by HIV-

at47-57.In summary, our results demonstrate that vaccination with sol-

ble M-PTD-HBcAg fusion protein can induce robust specific CTLctivity and therapeutic effects in HBV transgenic mice. In addition,-PTD-HBcAg can not only induce specific cell immune responses,

ut also enhance remarkably humoral immune responses. Theres no apparent toxic reaction in mice immunization with soluble

-PTD-HBcAg fusion protein and this result provides the safetyor further clinical application on anti-HBV therapy. Therefore, this

ay be a potential therapeutic strategy for eradicating virus inatients with chronic HBV.

cknowledgements

We thank Prof. Ningli Li (Institute of Immunology, Shanghai Jiao-ong University) for the expert technical assistance. This study wasupported by the grants from the National Natural Science Foun-ation of China (No. 30571669).

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